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Posts Tagged ‘Embryonic stem cell’


New Liver Tissue Implants Showing Potential

Reporter: Irina Robu,PhD

To develop new tissues, researchers at the Medical Research Council Center for Regenerative Medicine at the University of Edinburgh have found that stem cells transformed into 3-D liver tissue can support liver function when implanted into the mice suffering with a liver disease.

The scientists stimulated human embryonic stem cells and induced pluripotent stem cells to mature pluripotent stem cells into liver cells, hepatocytes. Hepatocytes are the chief functional cells of the liver and perform an astonishing number of metabolic, endocrine and secretory functions. Hepatocytes are exceptionally active in synthesis of protein and lipids for export. The cells are grown in 3-D conditions as small spheres for over a year. However, keeping the stem cells as liver cells for a long time is very difficult, because the viability of hepatocytes decreases in-vitro conditions.

Succeeding the discovery, the team up with materials chemists and engineers to detect appropriate polymers that have already been approved for human use that can be developed into 3-D scaffolds. The best material to use a biodegradable polyester, called polycaprolactone (PCL).PCL is degraded by hydrolysis of its ester linkages in physiological conditions (such as in the human body) and it is especially interesting for the preparation of long term implantable devices, owing to its degradation which is even slower than that of polylactide. They spun the PCL into microscopic fibers that formed a scaffold one centimeter square and a few millimeters thick.

At the same time, hepatocytes derived from embryonic cells had been grown in culture for 20 days and were then loaded onto the scaffolds and implanted under the skin of mice.Blood vessels successfully grew on the scaffolds with the mice having human liver proteins in their blood, demonstrating that the tissue had successfully integrated with the circulatory system. The scaffolds were not rejected by the animals’ immune systems.

The scientists tested the liver tissue scaffolds in mice with tyrosinaemia,a potentially fatal genetic disorder where the enzymes in the liver that break down the amino acid tyrosine are defective, resulting in the accumulation of toxic metabolic products. The implanted liver tissue aided the mice with tyrosinaemia to break down tyrosine and the mice finally lost less weight, had less buildup of toxins in the blood and exhibited fewer signs of liver damage than the control group that received empty scaffolds.

According to Rob Buckle, PhD, Chief Science Officer at the MRC, “Showing that such stem cell-derived tissue is able to reproduce aspects of liver function in the lab also offers real potential to improve the testing of new drugs where more accurate models of human tissue are needed”. It is believed that the discovery could be the next step towards harnessing stem cell reprogramming technologies to provide renewable supplies of liver tissue products for transplantation.

SOURCE

https://www.rdmag.com/article/2018/08/new-liver-tissue-implants-showing-promise?et_cid=6438323

 

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First Haploid Human Stem Cells

Reported: Irina Robu, PhD

Most of the cells in our body are diploid, which indicate they carry two sets of chromosomes—one from each parent. So far, scientists have only succeeded in generating haploid embryonic stem cells—which comprise a single set of chromosomes in non-human mammals such as mice, rats and monkeys. Nevertheless, scientists have tried to isolate and duplicate these haploid ESCs in humans, which would allow them to work with one set of human chromosomes as opposed to a mixture from both parents.

Scientists from Hebrew from The Hebrew University of Jerusalem, Columbia University Medical Center (CUMC) and The New York Stem Cell Foundation Research Institute (NYSCF) were successful in generating a new type of embryonic stem cells that has a single copy of the human genome, instead of two copies which is typically found in normal stem cells.

This landmark was finally obtained by Ido Sagi, working as a PhD student at the Hebrew University of Jerusalem which was successful in isolating and maintaining haploid embryonic stem cells in humans. Unlike in mice, these haploid stem cells were capable to differentiate into various cell types such as brain, heart and pancreas, although holding a single set of chromosomes. Sagi and his advisor, Prof. Nissim Benvenisty showed that this new human stem cell type will play an important role in human genetic and medical research.  This new human cell type cell type will aid in understanding human development and it will make genetic screening simpler and more precise, by examining a single set of chromosomes.

Based on this research, the Technology Transfer arm of the Hebrew University, started a new company New Stem, which is developing a diagnostic kit for predicting resistance to chemotherapy treatments. By gathering a broad library of human pluripotent stem cells with various genetic makeups and mutations. The company is planning to use this kit for personalized medication and future therapeutic and reproductive products.

SOURCE

https://medicalxpress.com/news/2017-06-haploid-human-stem-cells-medical.html#jCp

Other related articles published in this Open Access Online Scientific Journal include the following:

Ido Sagi – PhD Student @HUJI, 2017 Kaye Innovation Award winner for leading research that yielded the first successful isolation and maintenance of haploid embryonic stem cells in humans.

Reporter: Aviva Lev-Ari, PhD, RN

Ido Sagi – PhD Student @HUJI, 2017 Kaye Innovation Award winner for leading research that yielded the first successful isolation and maintenance of haploid embryonic stem cells in humans.

 

 

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Immunopathogenesis Advances in Diabetes and Lymphomas

Larry H Bernstein, MD, FCAP, Curator

LPBI

 

 

 Science team says they’ve taken another step toward a potential cure for diabetes

Wednesday, January 27, 2016 | By John Carroll
Building on years of work on developing new insulin-producing cells that could one day control glucose levels and cure diabetes, a group of investigators led by scientists at MIT and Boston Children’s Hospital say they’ve developed a promising new gel capsule that protected the cells from an immune system assault.

Dr. Jose Oberholzer, a professor of bioengineering at the University of Illinois at Chicago, tested a variety of chemically modified alginate hydrogel spheres to see which ones would be best at protecting the islet cells created from human stem cells.

The team concluded that 1.5-millimeter spheres of triazole-thiomorphine dioxide (TMTD) alginate were best at protecting the cells and allowing insulin to seep out without spurring an errant immune system attack or the development of scar tissue–two key threats to making this work in humans.

They maintained healthy glucose levels in the rodents for 174 days, the equivalent to decades for humans.

“While this is a very promising step towards an eventual cure for diabetes, a lot more testing is needed to ensure that the islet cells don’t de-differentiate back toward their stem-cell states or become cancerous,” said Oberholzer.

Millions of diabetics have effectively controlled the chronic disease with existing therapies, but there’s still a huge unmet medical need to consider. While diabetes companies like Novo ($NVO) like to cite the fact that a third of diabetics have the disease under control, a third are on meds but don’t control it well and a third haven’t been diagnosed. An actual cure for the disease, which has been growing by leaps and bounds all over the world, would be revolutionary.

Their study was published in Nature Medicine.

– here’s the release
– get the journal abstract

 

Long-term glycemic control using polymer-encapsulated human stem cell–derived beta cells in immune-competent mice

Arturo J Vegas, Omid Veiseh, Mads Gürtler,…, Robert Langer & Daniel G Anderson

Nature Medicine (2016)   http://dx.doi.org:/10.1038/nm.4030

The transplantation of glucose-responsive, insulin-producing cells offers the potential for restoring glycemic control in individuals with diabetes1. Pancreas transplantation and the infusion of cadaveric islets are currently implemented clinically2, but these approaches are limited by the adverse effects of immunosuppressive therapy over the lifetime of the recipient and the limited supply of donor tissue3. The latter concern may be addressed by recently described glucose-responsive mature beta cells that are derived from human embryonic stem cells (referred to as SC-β cells), which may represent an unlimited source of human cells for pancreas replacement therapy4. Strategies to address the immunosuppression concerns include immunoisolation of insulin-producing cells with porous biomaterials that function as an immune barrier56. However, clinical implementation has been challenging because of host immune responses to the implant materials7. Here we report the first long-term glycemic correction of a diabetic, immunocompetent animal model using human SC-β cells. SC-β cells were encapsulated with alginate derivatives capable of mitigating foreign-body responses in vivo and implanted into the intraperitoneal space of C57BL/6J mice treated with streptozotocin, which is an animal model for chemically induced type 1 diabetes. These implants induced glycemic correction without any immunosuppression until their removal at 174 d after implantation. Human C-peptide concentrations and in vivo glucose responsiveness demonstrated therapeutically relevant glycemic control. Implants retrieved after 174 d contained viable insulin-producing cells.

Subject terms: Regenerative medicine  Type 1 diabetes

Figure 1: SC-β cells encapsulated with TMTD alginate sustain normoglycemia in STZ-treated immune-competent C57BL/6J mice.close

(a) Top, schematic representation of the last three stages of differentiation of human embryonic stem cells to SC-β cells. Stage 4 cells (pancreatic progenitors 2) co-express pancreatic and duodenal homeobox 1 (PDX-1) and NK6 homeobox 1…

 

Potential Cure for Diabetes Discovered  
http://www.rdmag.com/news/2016/01/potential-cure-diabetes-discovered   01/27/2016

Two new scientific papers published on Monday demonstrated tools that could result in potential therapies for patients diagnosed with type 1 diabetes, a condition in which the immune system limits the production of insulin, typically in adolescents.  See —

Bubble Technique Could Create Type 1 Diabetes Therapy

http://www.dddmag.com/news/2016/01/bubble-technique-could-create-type-1-diabetes-therapy

Two new scientific papers published on Monday demonstrated tools that could result in potential therapies for patients diagnosed with type 1 diabetes, a condition in which the immune system limits the production of insulin, typically in adolescents.

Previous treatments for this disease have involved injecting beta cells from dead donors into patients to help their pancreas generate healthy-insulin cells, writes STAT. However, this method has resulted in the immune system targeting these new cells as “foreign” so transplant recipients have had to take immune-suppressing medications for the rest of their lives.

The first paper published in the journal Nature Biotechnology explained how scientists analyzed a seaweed extract called alginate to gauge its effectiveness in supporting the flow of sugar and insulin between cells and the body. An estimated 774 variations were tested in mice and monkeys in which results indicated only a handful could reduce the body’s response to foreign invaders, explains STAT.

The other paper in the journal Nature Medicine detailed a process where scientists developed small capsules infused with alginate and embryonic stem cells. A six-month observation period revealed this “protective bubble” technique “began to produce insulin in response to blood glucose levels” after transplantation in mice subjects with a condition similar to type 1 diabetes, reports Gizmodo.

Essentially, this cured the mice of their diabetes, and the beta cells worked as well as the body’s own cells, according to the researchers. Human trials could still be a few years away, but this experiment could yield a safer alternative to insulin injections.

 

Combinatorial hydrogel library enables identification of materials that mitigate the foreign body response in primates

Arturo J Vegas, Omid Veiseh, Joshua C Doloff, et al.

Nature Biotechnology (2016)    http://dx.doi.org:/10.1038/nbt.3462

The foreign body response is an immune-mediated reaction that can lead to the failure of implanted medical devices and discomfort for the recipient1, 2, 3, 4, 5, 6. There is a critical need for biomaterials that overcome this key challenge in the development of medical devices. Here we use a combinatorial approach for covalent chemical modification to generate a large library of variants of one of the most widely used hydrogel biomaterials, alginate. We evaluated the materials in vivo and identified three triazole-containing analogs that substantially reduce foreign body reactions in both rodents and, for at least 6 months, in non-human primates. The distribution of the triazole modification creates a unique hydrogel surface that inhibits recognition by macrophages and fibrous deposition. In addition to the utility of the compounds reported here, our approach may enable the discovery of other materials that mitigate the foreign body response.

 

Video 1: Intravital imaging of 300 μm SLG20 microcapsules.

Video 2: Intravital imaging of 300 μm Z2-Y12 microcapsules.

Video 3: NHP Laparoscopic procedure for the retrieval of Z2-Y12 spheres.

 

Clinical Focus on Follicular Lymphoma: CAR T-Cells Active in Relapsed Blood Cancers

MedPage Today

CAR T-Cells Active in Relapsed Blood Cancers

Complete responses in half of patients

by Charles Bankhead

Patients with relapsed and refractory B-cell malignancies have responded to treatment with modified T-cells added to conventional chemotherapy, data from an ongoing Swedish study showed.

Six of the first 11 evaluable patients achieved complete responses with increasing doses of chimeric antigen receptor (CAR)-modified T-cells that target the CD19 antigen, although two subsequently relapsed.

Five of the six responding patients received preconditioning chemotherapy the day before CAR T-cell infusion, in addition to chemotherapy administered up to 90 days before T-cell infusion to reduce tumor-cell burden. The remaining five patients received only the earlier chemotherapy, according to a presentation at the inaugural International Cancer Immunotherapy Conference in New York City.

“The complete responses in lymphoma patients despite the fact that they received only low doses of preconditioning compared with other published data surprised us,” Angelica Loskog, PhD, of Uppsala University in Sweden, said in a statement. “The strategy of both providing tumor-reductive chemotherapy for weeks prior to CAR T-cell infusion combined with preconditioning just before CAR T-cell infusion seems to offer promise.

CAR T-cells have demonstrated activity in a variety of studies involving patients with B-cell malignancies. Much of the work has focused on patients with leukemia, including trials in the U.S. B-cell lymphomas have proven more difficult to treat with CAR T-cells because the diseases are associated with higher concentration of immunosuppressive cells that can inhibit CAR T-cell activity, said Loskog. Moreover, blood-vessel abnormalities and accumulation of fibrotic tissue can hinder tumor penetration by therapeutic T-cells.

Each laboratory has its own process for modifying T-cells. Loskog and colleagues in Sweden and at Baylor College of Medicine in Houston have developed third-generation CAR T-cells that contain signaling domains for CD28 and 4-1BB, which act as co-stimulatory molecules. In preclinical models, third-generation CAR T-cells have demonstrated increased activation and proliferation in response to antigen challenge. Additionally, they have chosen to experiment with tumor burden-reducing chemotherapy, a preconditioning chemotherapy to counter the higher immunosuppressive cell count in lymphoma patients.

Loskog reported details of an ongoing phase I/IIa clinical trial involving patients with relapsed or refractory CD19-positive B-cell malignancies. Altogether, investigators have treated 12 patients with increasing doses (2 x 107 to 2 x 108 cells/m2) of CAR T-cells. One patient (with mixed follicular/Burkitt lymphoma) has yet to be evaluated for response. The remaining 11 included three patients with diffuse large B-cell lymphoma (DLBCL), one with follicular lymphoma transformed to DLBCL, two with chronic lymphocytic leukemia, two with mantle cell lymphoma, and three with acute lymphoblastic leukemia.

All of the patients with lymphoma received standard tumor cell-reducing chemotherapy, beginning 3 to 90 days before administration of CAR T-cells. Beginning with the sixth patient in the cohort, patients also received preconditioning chemotherapy (cyclophosphamide/fludarabine) 1 to 2 days before T-cell infusion to reduce the number and activity of immunosuppressive cells.

Cytokine release syndrome is a common effect of CAR T-cell therapy and occurred in several patients treated. In general, the syndrome has been manageable and has not interfered with treatment or response to the modified T-cells.

On the basis of the data produced thus far, the investigators have proceeded with patient evaluation and enrollment. They have already begun cell production for the next patient that will be treated with autologous CAR T-cells.

Although laboratories have their own cell production techniques, the treatment strategy has broad applicability to the treatment of B-cell malignancies, said Loskog.

“The results using different CARs and different techniques for manufacturing them is very similar in the clinic, in terms of initial complete response,” she told MedPage Today. “By using 4-1BB as a co-stimulator in the CAR intracellular region, it seems possible to achieve long-term complete responses in some patients. However, preconditioning of the patients with chemotherapy to reduce the regulatory immune cells seems crucial for effect.”

In an effort to manage the effect of patients’ immunosuppressive cells, the investigators have begun studying each the immune profile before and after treatment. Preliminary results suggest that the population of immunosuppressive cells increases over time, which has the potential to interfere with CAR T-cell responses.

“Especially for lymphoma, it may be crucial to deplete such cells prior to CAR infusion,” said Loskog. “It may even be necessary with supportive treatment for some time after CAR T-cell infusion. A supportive treatment needs to specifically regulate the suppressive cells while sparing the effect of CARs.”

The immunotherapy conference is jointly sponsored by the American Association for Cancer Research, the Cancer Research Institute, the Association for Cancer Immunotherapy, and the European Academy of Tumor Immunology.

 

PET-CT Best for FL Response Assessment

PET-CT associated with better progression-free and overall survival rates in follicular lymphoma.

Kay Jackson

PET-CT (PET) rather than contrast-enhanced CT scanning should be considered the new gold standard for response assessment after first-line rituximab therapy for high-tumor burden follicular lymphoma (FL), a pooled analysis of a central review in three multicenter studies indicated.

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Optical Neurons

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Umbilical Cells Help Eye’s Neurons Connect

Factor released by cells helps connections, not longevity

“By learning more about how these cells work, we are one step closer to understanding the disease states in which these cells should be studied,” said Cagla Eroglu, an assistant professor of cell biology and neurobiology at the Duke University Medical Center, who led the research.
Umbilical cord tissue-derived cells (hUTC) differ from umbilical cord blood cells in that they are isolated from cord tissue itself, rather than the blood. The Duke
team used an established cell culture system to determine whether and how the hUTCs might affect the growth of neurons isolated from the retinas of rat eyes.
In an experimental setup that allowed the two types of cells to bathe in the same fluid without coming into physical contact, retinal neurons in a bath with hUTCs formed new connections between neurons called synapses, and they sprouted new ‘neurites’ — tiny branches that lead to additional connections.
These cells also survived longer than rat neurons placed in a bath lacking the umbilical cord tissue-derived cells.
Something present in the fluid surrounding the neurons in the bath with the hUTCs was apparently affecting the neurons. Through a series of experiments, the researchers determined that relatively large molecules, thrombospondin 1, 2 and 4, were primarily responsible for the effect.
Blocking thrombospondins was found to reduce new connections among neurons. By genetically inhibiting the individual members of the thrombospondin family, the researchers found that TSP1, TSP2, and TSP4 in particular were required to create both neurites and new connections.
“It’s exciting that thrombospondins had a really strong effect on neurite outgrowth,” said Eroglu, who is also a member of the Duke Institute for Brain Sciences (DIBS). She added that making neurites and forming new connections between them are crucial for helping neurons grow when faced with injury and neurodegenerative diseases.
However, blocking TSP1, 2 and 4 did not affect neuron survival, suggesting that there is some other factor in the UTC cells that promotes cell longevity. Her group is now searching for those molecules.
Eroglu’s earlier work has shown that thrombospondins are released by brain cells called astrocytes and boost new synapse formation between neurons in the brain.
Eroglu said there may be deficiencies in thrombospondin signaling in neurodegenerative disease, and the group is actively pursuing this hypothesis in animal studies.
Postdoctoral fellow Sehwon Koh is the lead author of this study and a member of the Eroglu lab. Other authors include Namsoo Kim and Henry H. Yin from Duke’s department of psychology and neuroscience. This research was supported by a research agreement with Janssen Research & Development, LLC.
CITATION: “Human Umbilical Tissue-Derived Cells (hUTC) Promote Synapse Formation and Neurite Outgrowth via Thrombospondin Family Proteins,” Sehwon Koh, Namsoo Kim, Henry H. Yin, Ian R. Harris, Nadine S. Dejneka, and Cagla Eroglu. Journal of Neuroscience, November 25, 2015.    http://dx.doi.org:/10.1523/JNEUROSCI.1364-15.2015
ScienceDaily
Cells isolated from the human umbilical cord have been shown to produce molecules that help retinal neurons from the eyes of rats grow, connect and survive. The findings implicate one family of molecules in particular — thrombospondins – that may have therapeutic potential for the treatment of degenerative eye diseases.

The findings, which appear Nov. 25 in the Journal of Neuroscience, implicate one family of molecules in particular — thrombospondins — that may have therapeutic potential for the treatment of degenerative eye diseases.

“By learning more about how these cells work, we are one step closer to understanding the disease states in which these cells should be studied,” said Cagla Eroglu, an assistant professor of cell biology and neurobiology at the Duke University Medical Center, who led the research.

Umbilical cord tissue-derived cells (hUTC) differ from umbilical cord blood cells in that they are isolated from cord tissue itself, rather than the blood. The Duke team used an established cell culture system to determine whether and how the hUTCs might affect the growth of neurons isolated from the retinas of rat eyes.

Something present in the fluid surrounding the neurons in the bath with the hUTCs was apparently affecting the neurons. Through a series of experiments, the researchers determined that relatively large molecules, thrombospondin 1, 2 and 4, were primarily responsible for the effect.

Blocking thrombospondins was found to reduce new connections among neurons. By genetically inhibiting the individual members of the thrombospondin family, the researchers found that TSP1, TSP2, and TSP4 in particular were required to create both neurites and new connections.

However, blocking TSP1, 2 and 4 did not affect neuron survival, suggesting that there is some other factor in the UTC cells that promotes cell longevity. Her group is now searching for those molecules.

Golgi Cells Have Active Dendrites
Stephanie Rudolph, Court Hull, and Wade G. Regehr
The Journal of Neuroscience, Nov 25, 2015 • 35(47):i • i    (see pages 15492–15504)
The cerebellum coordinates multijoint movements and contributes to motor learning. These functions require precise spike timing in Purkinje cells, the cerebellar output neurons. Purkinje cell spiking is driven partly by granule cells, which receive information about ongoing movements from mossy fibers, and the timing and spatial extent of granule cell output is determined largely by inhibitory input from spontaneously active interneurons called Golgi cells.
Golgi cell spiking is modulated by excitatory input from both mossy fibers and granule cells. How these inputs are integrated in Golgi cell dendrites remains poorly understood. Finding no evidence for active conductances in Golgi cell dendrites, Vervaeke et al. (2012, Science 30: 1624) hypothesized that dendritic gap junctions enable granule cell inputs to influence Golgi cell activity. Although gap junctions likely do contribute to dendritic processing in Golgi cells, Rudolph et al. now show that Golgi cell dendrites also express voltage-gated channels.
If dendrites lacked active conductances, one would expect signals to decay with distance from the soma. But calcium imaging in rat cerebellar slices revealed that action potentials caused uniform calcium elevation throughout Golgi cell dendrites. Moreover, applying a voltage-gated sodium channel (VGSC) blocker selectively to dendrites reduced spike-associated calcium elevation in distal dendrites. In addition, blocking T- and R-type voltage-gated calcium channels (VGCCs) attenuated calcium elevation selectivelyin distal dendrites,while blocking N-type channels reduced calcium elevation only in proximal dendrites.
Blocking voltage-gated channels also had functional consequences. Blocking N-type channels decreased the amplitude of the spike afterhyperpolarization and increased the spike rate of Golgi cells. In contrast, T-type channel blockers had little effect on baseline firing frequency. Nonetheless, blocking T-type channels attenuated rebound spiking after hyperpolarization and reduced the amplitude of EPSPs evoked by stimulation of granule cell axons.
These experiments suggest that VGSCs help depolarize distal dendrites to enhance activation of T-type VGCCs, which in turn amplify responses to granule cells and promote rebound bursting. Meanwhile, N-type VGCCs located near the soma appear to be tightly coupled to calcium-activated potassium channels, which regulate the spontaneous spike rate of Golgi cells. Thus, Golgi cell dendrites have multiple types of voltage-sensitive channels that are differently distributed and serve distinct roles in ensuring the precise timing of cerebellar output.

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Jmjd3 and Cardiovascular Differentiation of Embryonic Stem Cells

Author and Curator: Larry H Bernstein, MD, FCAP

and

Curator: Aviva Lev-Ari, PhD, RN

This article is a presentation of recently published work on the basis for control of mesodermal and cardiovascular differentiation of embryonic stem cells, which has taken on increasing importance in the treatment of cardiovascular disease, with particular application to heart failure due to any cause, but with particular relevance to significant loss of myocardium, as may occur with acute myocardial infarction with more than 60% occlusion of the left anterior descending artery, near the osteum, with or without adjacent artery involvement, resulting in major loss of cardiac contractile force and insufficient ejection fraction. The article is of high interest and makes the following points:

  1. Ablation of Jmjd3 in mouse embryonic stem cells does not affect the maintenance of pluripotency and self-renewal
  2. Ablation of Jmjd3 in mouse embryonic stem cells compromised mesoderm and subsequent endothelial and cardiac differentiation 
  3. Jmjd3 reduces H3K27me3 marks at the Brachyury promoter and facilitates the recruitment of β-catenin
  4. β-catenin s critical for Wnt signal–induced mesoderm differentiation. 

Jmjd3 Controls Mesodermal and Cardiovascular Differentiation of Embryonic Stem Cells

K Ohtani, C Zhao, G Dobreva, Y Manavski, B Kluge, T Braun, MA Rieger, AM Zeiher and S Dimmeler

I The Institute of Cardiovascular Regeneration, Centre for Molecular Medicine, University of Frankfurt, Frankfurt, Germany (K.O., C.Z., Y.M., B.K., S.D.); Max-Planck-Institute for Heart and Lung Research, Bad Nauheim, Germany (G.D., T.B.); Department of Hematology/Oncology, Internal Medicine
II LOEWE Center for Cell and Gene Therapy, University of Frankfurt, Frankfurt, Germany (M.A.R.); and Department of Cardiology, Internal Medicine
III University of Frankfurt, Frankfurt, Germany (A.M.Z.).
This manuscript was sent to Benoit Bruneau, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
Correspondence to Stefanie Dimmeler,  Institute of Cardiovascular Regeneration, Centre for Molecular Medicine, University of Frankfurt, Frankfurt, Germany. E-mail dimmeler@em.uni-frankfurt.de
 Circ Res. 2013;113:856-862;  http://dx.doi.org/10.1161/CIRCRESAHA.113.302035   http://circres.ahajournals.org/content/113/7/856  

Abstract

Rationale: The developmental role of the H3K27 demethylases Jmjd3, especially its epigenetic regulation at target genes in response to upstream developmental signaling, is unclear.

Objective: To determine the role of Jmjd3 during mesoderm and cardiovascular lineage commitment.

Methods and Results: Ablation of Jmjd3 in mouse embryonic stem cells does not affect the maintenance of pluripotency and self-renewal but compromised mesoderm and subsequent endothelial and cardiac differentiation. Jmjd3 reduces H3K27me3 marks at the Brachyury promoter and facilitates the recruitment of β-catenin, which is critical for Wnt signal–induced mesoderm differentiation.

Conclusions: These data demonstrate that Jmjd3 is required for mesoderm differentiation and cardiovascular lineage commitment. (Circ Res. 2013;113:856-862.)

  • Key Words: Brachyury protein ■ embryonic stem cells ■ epigenomics ■ Jmjd3 protein, mouse ■ mesodermn  –  Wnt signaling pathway

Introduction

Post-translational modifications of histone proteins represent essential epigenetic control mechanisms that can either allow or repress gene expression.1 Trimethylation of H3K27 is mediated by Polycomb group proteins and represses gene expression.2 The JmjC domain–containing proteins, UTX (ubiquitously transcribed tetratricopeptide repeat, X chromosome) and Jmjd3 (jumonji domain–containing protein 3, Kdm6b), not only act as demethylases to remove the repressive H3K27me3 marks, but also exhibit additional demethylase-independent functions.3–6 Jmjd3 is induced and participates in Hox gene expression during development,7 neuronal differentiation,8,9 and inflammation,5,10–12 and recent data suggest that Jmjd3 inhibits reprogramming by inducing cellular senescence.13 Because previous studies suggest that H3K27me3 regulates endothelial gene expression in adult proangiogenic cells,14 we addressed the function of Jmjd3 in cardiovascular lineage differentiation of embryonic stem cells (ESCs).

Methods

A detailed description of the experimental procedure is provided in the Online Data Supplement.  The online-only Data Supplement is available with this article at   http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA. 113.302035/-/DC1.

Cell Culture

Plasmid Construction and Stable Transfection

The full-length Jmjd3, the mutants, and Brachyury were cloned into pEF1 vector (Invitrogen). The linearized plasmids were transfected in Jmjd3−/− ESCs using the Amaxa nucleofection system (Lonza).

Chromatin Immunoprecipitation

Nonstandard Abbreviations and Acronyms

EB                       embryoid body

ESC                    embryonic stem cell

WI                        wild type

Results

Jmjd3 knockout ESCs were generated by 2 rounds of gene targeting (Online Figure IA and IB). We obtained 7 Jmjd3−/− ESC clones, which lacked Jmjd3 mRNA and protein expression. All of the clones showed slightly increased global H3K27me3, but the expression of pluripotency genes, the morphology, the growth kinetic, and survival was indistinguishable from wild-type (WT) ESCs (Figure 1A–1C; Online Figure IC–IF). No significant changes of repressive H3K27me3 marks at the promoters of pluripotency genes were detected in Jmjd3−/− compared with WT ESCs (Online Figure IH). When

  • spontaneous differentiation was induced by leukemia inhibitory factor withdrawal,
  • Jmjd3 expression increased in WT ESCs (Figure 1D).
  • EBs derived from Jmjd3−/− ESCs were slightly smaller in size compared with WT EBs (Figure 1E).

mRNA expression profiling of Jmjd3−/− and WT ESC clones at day 4 after induction of differentiation showed

  • a distinct expression pattern of lineage-specific genes (Online Figure IIA).

Gene ontology functional analyses revealed a significant repression of genes that are involved in mesoderm development (Figure 1F; Online Figure IIB). Moreover,

  • repressed gene sets in Jmjd3−/− EBs were shown to be related to cardiac and vascular development,
  • consistent with impaired mesoderm differentiation (Figure 1F; Online Figure IIB).

Validation of the microarray results showed a similar reduction of pluripotency gene expression after leukemia inhibitory factor withdrawal in Jmjd3−/− compared with WT ESCs (Figure 1G). However, depletion of Jmjd3 substantially compromised the induction of mesodermal genes (Figure 1G). Especially, the pan-mesoderm marker, Brachyury, and the early mesoendoderm marker, Mixl1, were profoundly increased at day 4 of differentiation in WT ESCs, but not in Jmjd3−/− ESCs (Figure 1G). Moreover, the mesoendodermal marker, Eomes, and endodermal markers, such as Sox17 and FoxA2, were significantly suppressed, which is consistent with a very recent study showing that Jmjd3 is required for endoderm differentiation.19 Ectodermal markers were not significantly changed in Jmjd3−/− ESCs when using the spontaneous differentiation protocol (Figure 1G).

Because Jmjd3−/− ESCs showed a prominent inhibition of mesodermal markers after leukemia inhibitory factor withdrawal, we next questioned whether this phenotype can also be observed when directing differentiation of mesoderm using 2 different protocols. Consistent with our findings,

  • Jmjd3−/− ESCs showed a reduced expression of mesodermal marker genes when using the protocol for mesoderm differentiation described by Gadue et al20 (data not shown). Moreover,
  • mesoderm differentiation was significantly suppressed when Jmjd3−/− ESCs were cultured on OP9 stromal cells, which support mesodermal differentiation21 (Figure 2A).

Whereas WT ESCs showed the typical time-dependent increase in Brachyury+ cells, Jmjd3−/− ESCs generated significantly less Brachyury+ mesodermal cells (Figure 2B). Moreover, fluorescence activated cell sorting analysis revealed that fetal liver kinase (Flk)1+ vascular endothelial-cadherin−mesodermal cells were generated in WT ESCs but were reduced when Jmjd3−/− ESCs were used (Figure 2C). Interestingly, the formation of vascular endothelial-cadherin+ Flk+ cells was also significantly reduced by 96±1% and 88±3% in the 2 Jmjd3−/− ESC clones compared with WT ESCs (P<0.01), prompting us to explore the role of Jmjd3 in vascular differentiation further.
Endothelial differentiation was induced by a cytokine cocktail18 and was associated with a significant upregulation of Jmjd3 expression (Online Figure IIIA). Jmjd3−/− ESCs showed a marked reduction of endothelial differentiation as evidenced by

  • significantly reduced mRNA levels of the endothelial marker vascular endothelial-cadherin and endothelial-specific receptor tyrosine kinase Tie2 (Figure 3A).
  • The formation of endothelial marker expressing vascular structures after induction of endothelial differentiation was abolished in Jmjd3−/− ESCs (Figure 3B; Online Figure IIIB).
  • The impaired endothelial differentiation of Jmjd3−/− cells was partially rescued by the overexpression of Brachyury (Online Figure IIIC and IIID), suggesting that the inhibition of mesoderm formation, at least in part, contributes to the impaired endothelial commitment.

Because genes involved in heart development and morphogenesis were significantly downregulated in Jmjd3−/− ESCs on differentiation (Figure 1F; Online Figure II), we additionally determined the capacity of Jmjd3−/− ESCs to generate cardio-myocytes by inducing cardiac differentiation.17

  • Expression of cardiac progenitor cell markers, Mesp1 and Pdgfra, was inhibited in Jmjd3−/− ESCs compared with WT ESCs (Figure 3C).

Moreover, after plating on gelatin-coated dishes,

  • the Jmjd3−/− ESCs showed an impaired formation of EBs and
  • only 20% of EBs were contracting (Figure 3D).

Consistently, expression of the cardiac transcription factor Mef2c, the marker of working myocardium Nppa, and cardiac structural proteins TnT2 and α-myosin heavy chain were downregulated in Jmjd3−/− ESCs (Figure 3E and 3F; Online Figure IIIE).

  • Next, we addressed whether the impaired mesoderm differentiation observed in Jmjd3−/− ESCs might be mediated by an increase of repressive H3K27me3 marks at the promoters of developmental regulators. Of the various promoters studied, only Brachyury and Mixl1 showed a significant augmentation of H3K27me3 marks in Jmjd3−/− ESCs on differentiation (Figure 4A; Online Figure IVA). Consistently, the recruitment of RNA polymerase II to the transcription start sites of the promoters of Brachyury and Mixl1 was also significantly reduced (Online Figure IVC). In addition, Jmjd3 deficiency repressed polymerase II recruitment to the Flk1 and Mesp1 promoter but the inactivation of these promoters was not associated with changes in H3K27me3 marks (Figure IVA and IVC). These data were confirmed using protocols
  • that induce mesoderm differentiation by addition of Wnt3a (data not shown).20 Under these conditions,
  • Jmjd3−/− ESCs showed a 1.81±0.23-fold (P<0.05) enrichment of H3K27me3 marks at the Brachyury promoter compared with WT ESCs.

To determine whether the demethylase activity of Jmjd3 controls Brachyury expression by reducing repressive H3K27me3 marks during differentiation, we overexpressed full-length Jmjd3, the carboxyl-terminal part, including the JmjC-domain (cJmjd3: amino acids, 1141–1641), and a carboxyl-terminal mutant construct, which includes a point mutation (cJmjd3H1388A) to inactivate demethylase activity. Overexpression of full-length Jmjd3 and the carboxyl-terminal part of Jmjd3 in Jmjd3−/− ESCs partially rescued the expression of Brachyury on differentiation (Figure 4B and 4C). Howver, the inactive carboxyl-terminal part of Jmjd3 failed to rescue the impaired Brachyury expression in Jmjd3−/− ESCs (Figure 4C), suggesting that

  • the demethylase activity of Jmjd3 is required for the activation of the Brachyury promoter.

Because canonical Wnt signaling regulates the expression of Brachyury during development22,23 and Wnt/B-catenin–dependent genes were suppressed in Jmjd3−/− EBs compared with WT EBs (Online Figure V), we further explored whether Jmjd3 might interact with B-catenin signaling. Indeed,

  • B-catenin recruitment to the Brachyury promoter was significantly suppressed in Jmjd3−/− ESCs (Figure 4D) and
  • was rescued by Jmjd3 overexpression (Figure 4E).

Similar results were obtained when using the protocol for direct mesoderm differentiation described by Gadue et al20 (data not shown). To determine whether Jmjd3 might interact with B-catenin, we performed coimmunoprecipitation studies and showed that

  • Jmjd3 interacts with B-catenin in human embryonic kidney 293 cell and differentiated EBs (Figure 4F; Online Figure VI).

To assess a direct effect of Jmjd3 on B-catenin responsive promoter activity, we used a luciferase reporter assay. Coexpression of lymphoid enhancer binding factor 1 and the constitutive active form of B-catenin harboring a nuclear localization signal resulted in the activation of lymphoid enhancer binding factor 1 luciferase reporter activity in WT ESCs, but

  • this transcriptional activation was markedly impaired in Jmjd3−/− ESCs (Figure 4G).

Discussion

The data of the present study demonstrate that

  • deletion of Jmjd3 in ESCs does not affect self-renewal but
  • significantly impairs the formation of mesoderm on induction of differentiation.

The findings that Jmjd3 is not required for ESC maintenance are consistent with the dispensability of the Polycomb complex and the related demethylase UTX for self-renewal.1

  • The requirement of Jmjd3 for mesoderm differentiation was shown in spontaneous differentiation, as well as
  • when more specifically inducing mesoderm differentiation by the OP9 coculture system or under serum-free conditions followed by Wnt3a stimulation.
  • Jmjd3 deficiency profoundly suppressed the expression of Brachury, which is essential for mesoderm differentiation.

In the absence of Jmjd3,

  • repressive H3K27me3 marks at the Brachyury promoter are significantly increased, and
  • the recruitment of B-catenin, which is a prerequisite for Wnt-induced mesoderm differentiation, is impaired.
  • In addition, Jmjd3 is interacting with B-catenin and is contributing to B-catenin– dependent promoter activation.

This is consistent with the recent findings that cofactors can form a complex with B-catenin/ lymphoid enhancer binding factor 1

  • at Tcf/lymphoid enhancer binding factor 1 binding sites
  • at B-catenin–dependent promoters and
  • synergize with canonical Wnt signaling.24

Interestingly, a demethylase-independent regulation of B-catenin–dependent gene expression was recently described for UTX.25 However, our data provide evidence that

  • Brachyury expression in Jmjd3−/− ESCs is only rescued by catalytically active Jmjd3, which has maintained the demethylase activity.

On the basis of these findings, we propose a model in which Jmjd3 is recruited to the Brachury promoter to remove repressive H3K27me3 marks and on Wnt stimulation additionally promotes B-catenin–dependent promoter activation (Figure 4H). Such a model is similar to the recently described function of Jmjd3 in endoderm differentiation, whereby Jmjd3 associates with Tbx3 and is recruited to the poised promoter of Eomes, to mediate chromatin remodeling allowing subsequent induction of endoderm differentiation induced by activin A.19 The present study additionally demonstrates that

  • Jmjd3 contributes to endothelial and cardiac differentiation.
  • endothelial differentiation was profoundly impaired,

a finding that is consistent with previous findings in adult progenitor cells, showing a high H3K27me3 at endothelial genes.14 The relatively modest inhibition of cardiomyocyte differentiation in Jmjd3−/−  ESCs may be, in part, explained by a compensatory effect of UTX which was shown to regulate cardiac development.26 Together, our study provides first evidence for the regulation of B-catenin–dependent Wnt target genes by Jmjd3 during differentiation of ESCs.  However, the in vivo relevance of the findings is still unclear. The Jmjd3−/− mice that we have generated out of the ESCs, used in the present study, showed embryonic lethality before E6.5, suggesting a crucial role of Jmjd3 in early embryonic development.

Conclusions

Novelty and Significance

What Is Known?

•            Cell fate decisions require well-controlled changes in gene expression that are tightly controlled by epigenetic modulators.

•            The post-transcriptional modifications of histone proteins epigeneti-cally regulate gene expression.

•            Trimethylation of lysine 27 at histone K3 (H3K27me3) silences gene expression and can be reversed by the demethylase Jmjd3.

What New Information Does This Article Contribute?

•            The histone demethylase Jmjd3 is required for mesoderm differentiation and cardiovascular lineage commitment of mouse embryonic stem cells.

•            This effect is partially mediated by a silencing of the mesodermal regulator Brachyury.

•            Ablation of Jmjd3 further reduces β-catenin recruitment to the Brachyury promoter, which interferes with Wnt signaling that is required for proper mesoderm differentiation.

The differentiation of stem cells to specific lineages requires a well-defined modulation of gene expression programs, which is often controlled by epigenetic mechanisms. Although several epi-genetically active enzymes and complexes have been described, the function of the histone demethylase Jmjd3 for cardiovascu¬lar lineage commitment was unknown. Using mouse embryonic stem cells as a model, we now show that the demethylase Jmjd3 is required for mesoderm differentiation and for the differentia¬tion of embryonic stem cells to the vascular and cardiac lineage. We further identified the mechanism and showed that ablation of Jmjd3 resulted in a silencing of the Brachyury promoter that is associated with an increase in H3K27me3 marks. In addition, Jmjd3 was shown to facilitate the recruitment of β-catenin to the Brachyury promoter, which contributes to the Wnt-dependent ac-tivation of mesoderm differentiation. Together these data describe a novel epigenetic mechanism that controls cell fate decision.

Supplemental Methods

Generation of Jmjd3 knockout ES cell lines

Mouse genomic DNA encompassing the murine Jmjd3 gene region were isolated by PCR amplification and used to generate short (1.6kb) and long (6.2kb) arms of homology. The targeting vectors were constructed by inserting a loxP site together with an FRT flanked neomycin selection cassette within the intron 5 and a single distal loxP within the intron 3. This targeting strategy results in the deletion of 600bp coding sequences encoding for the ATG methionine codon and produces a frame shift of JmjC domain existing exon 19-21 required for Jmjd3 demethylase activity. The targeting vector was electroporated in 129Sv ES cells. G418 resistant ES cell clones were screened for homologous recombination by PCR analysis and targeting was verified by Southern blot analysis. Homozygous Jmjd3lox/lox ES cells were generated by electroporation of heterozygous Jmjd3lox/+ ES cells with the same targeting vector as above except that the neomycin resistance gene was replaced by puromycin gene using the Nucleofector (Lonza). Double-allele-recombined ES cells were selected for puromycin (1.3µg/mL, Invitrogen). Correct targeting of homozygous Jmjd3lox/lox ES clones were determined by PCR. To obtain Jmjd3-/- ES cells, Jmjd3lox/lox ES cells were electroporated with Cre-recombinase plasmid vector and loss of targeting cassettes was evaluated by loss of resistance of G418 and puromycin. Correct targeting of homozygous Jmjd3-/- ES cells was determined by PCR.

Reporter gene assays

3xLEF1 reporter plasmid, LEF1 expression construct and NLS-13-catenin were kind gifts from Rudolf Grosschedl. Mouse ES cells were seeded (5×104) on gelatin coated 24-well. After 24 hours of plating, 3xLEF1 reporter plasmid, LEF1, and NLS-13-catenin plasmids were transiently transfected with FugeneHD (Promega). 13-galactosidase plasmid was co-transfected for normalization of transfection efficiency. Each group was transfected in triplicates. 48 hours after transfection, cells were harvested. Cell lysis and luciferase assay were performed following the protocol of Luciferase Reporter Assay System (Promega). 13-galactosidase assays were performed using CPRG (Sigma) as substrate and the absorbance at 600nm was measured. Luciferase activity was normalized to 13-galactosidase activity.

Manuscript References

  1. Shen, X. et al. EZH1 mediates methylation on histone H3 lysine 27 and complements EZH2 in maintaining stem cell identity and executing pluripotency. Mol Cell 32, 491-502 (2008).
  2. Sargent, C.Y., Berguig, G.Y. & McDevitt, T.C. Cardiomyogenic differentiation of embryoid bodies is promoted by rotary orbital suspension culture. Tissue engineering. Part A 15, 331­342 (2009).
  3. Gadue, P., Huber, T.L., Paddison, P.J. & Keller, G.M. Wnt and TGF-beta signaling are required for the induction of an in vitro model of primitive streak formation using embryonic stem cells. Proc Natl Acad Sci U S A 103, 16806-16811 (2006).
  4. Ohtani, K. et al. Epigenetic regulation of endothelial lineage committed genes in pro-angiogenic hematopoietic and endothelial progenitor cells. Circ Res 109, 1219-1229 (2011).
  5. Yamaguchi, T.P., Takada, S., Yoshikawa, Y., Wu, N. & McMahon, A.P. T (Brachyury) is a direct target of Wnt3a during paraxial mesoderm specification. Genes Dev 13, 3185-3190 (1999).

Selected Figures   (Figure may not be shown)

Figure 1. Aberrant differentiation of Jmjd3−/− embryonic stem cells (ESCs). A, Quantitative polymerase chain reaction analysis of Jmjd3 in wild-type (WT) and Jmjd3−/− ESCs. B, Western blot analysis of Jmjd3 and Histone marks in WT and Jmjd3−/− ESCs. Histone H3 is used as a loading control. Quantification is shown in the right (n=3–5). C, Top, Morphology of WT and Jmjd3−/− ESCs on feeder cells. Bottom, Alkaline phosphatase staining of undifferentiated WT and Jmjd3−/− ESCs. D, Western blot analysis of Jmjd3 and Oct4 in WT ESCs during differentiation. α-Tubulin is used as a loading control. E, Bright field image of embryoid bodies at day 5. Scale bar, 200 μm. F, Gene ontology analysis for >2-fold repressed genes in Jmjd3−/− ESCs compared with WT ESCs 4 days after differentiation. The most highly represented categories are presented with ontology terms on the y-axis and P values for the significance of enrichment are shown on the x-axis. G, Gene expression changes of pluripotency and lineage-specific markers in WT and Jmjd3−/− ESCs after spontaneous differentiation by leukemia inhibitory factor withdrawal (n=4). Flk indicates fetal liver kinase.

Figure 2. Jmjd3−/− embryonic stem cells (ESCs) show an impaired ability to differentiate into mesoderm. A, Schematic illustration of the experimental protocol. Differentiation of ESCs (wild-type [WT] and 2 Jmjd3−/− ESCs clones) on OP9 feeder cells was analyzed. B, Left, Representative fluorescence activated cell sorting (FACS) plots showing Brachyury expression of ESC-derived cells. Right, Quantification of FACS analyses (n=3). C, Left, Representative FACS plots showing fetal liver kinase 1 (Flk1) and vascular endothelial-cadherin expression on ESC-derived cells. Right, Quantification of FACS analyses in Flk1+ cells (n=3).

Figure 3. Jmjd3 is required for embryonic stem cells (ESCs) differentiation to the endothelial and cardiac lineage. A, mRNA expression of endothelial markers at day 7 of endothelial differentiation (n=3). B, Platelet endothelial cell adhesion molecule (Pecam)-1 staining of wild-type (WT) and Jmjd3−/− ESCs at day 8 of endothelial differentiation. Phalloidin is used to stain F-actin. Nuclei are stained with 4′,6-diamidino-2-phenylindole (blue). Scale bar, 20 pm. C, Gene expression of cardiac progenitor markers at day 3 of cardiac differentiation. D, Number of beating embryoid bodies (EBs) at day 10 of cardiac differentiation (n=8). E, Gene expression of cardiac markers at day 7 of cardiac differentiation (n=6). F, α-Myosin heavy chain staining of WT and Jmjd3−/− ESCs at day 9 of cardiac differentiation. Nuclei are stained with Hoechst (blue). Scale bar, 20 pm. *P<0.05, **P<0.01, and ***P<0.001

Online Figure I. Generation and characterization of Jmjd3 ESCs  (A) Targeting strategy to generate Jmjd3 mutant ESCs by homologous recombination. Primers used for PCR are shown. (B) Genotyping of Jmjd3 ESCs by using 2 different primers. (C) Oct4 and Nanog staining in WT and Jmjd3−’− ESCs. Scale bar indicates 10µm. (D) Expression of Oct4 and Nanog in WT and Jmjd3 ESCs. Data are presented as fold changes compared with day 0 WT ESCs. N=6. (E) Tunel staining (green) of WT and Jmjd3 ESCs. Nuclei are stained with Hoechst (blue). Scale bar indicates 20µm. (F) Growth curves of WT and Jmjd3 ESCs. N=6-8. (G) H3K27me3 staining in WT and Jmjd3 ESCs. Nuclei are stained with Hoechst (blue). Scale bar indicates 20µm. (H) ChIP assay of undifferentiated WT and Jmjd3 ESCs for H3K27me3. ChIP enrichments are normalized to Histone H3 density and represented as fold change relative to WT. N=3. Data represent mean ± SEM

Online Figure II. Jmjd3 ESCs show an impaired mesoderm differentiation. (A) Microarray gene expression heat map depicting expression of representative pluripotency and lineage markers 4 days after differentiation in Jmjd3 ESCs versus WI ESCs. Coloring illustrates log2 fold changes between Jmjd3 ESCs and WI ESCs. Green and red colors represent down-regulation and up-regulation, respectively. (B) Gene ontology analysis for more than 2-fold altered genes in Jmjd3 ESCs compared to WI ESCs 4 days after differentiation. Red and green colors represent down-regulation and up-regulation, respectively.

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English: This diagram shows the chromosomes of...

This diagram shows the chromosomes of Drosophila melanogaster approximately to scale. Chromosome sizes were based on basepair lengths given on the NCBI map viewer, and A. B. Carvalho, 2002. Curr. Op. Genet. & Devel. 12:664-668. Centimorgan distances were derived from selected loci listed in the NCBI website. (credit  Wikipedia)

Introduction

Generally speaking sexually reproducing species are composed of individuals of two complementary mating types or sexes.  An essential aspect of the developmental history of each individual is thus sex determination and differentiation. There exist two sex determination mechanisms, somatic and germline, that based on the chromosomal mechanism in the Drosophila melanogaster.  In the somatic sex determination mechanism, each individual assesses the ratio of X-chromosomes to autosomal chromosome sets), the X:A ratio provides the primary sex-determining signal   (reviewed by Cline and Meyer, 1996).  When X:A=1, female differentiation ensues (Bridges, 1925), along with the male-mode of X-chromosome dosage compensation.  The X:A ratio is calculated within each cell of the developing embryo, 2 hrs after fertilization. The X:A ratio determines the sex in Drosophila (Bridges, 1916, 1921, 1925) in a somatic-cell-autonomous manner that occurs early in embryonic development (Baker and Belote, 1983; Baker, 1989). Females possess two X-chromosomes, and males possess one X-chromosome and one Y-chromosome.   The Y-chromosome is required only for spermatogenesis (Lindsley and Tokuyasu 1980; Bridges 1986), and will not be considered further.  The number of X-chromosomes is counted through a mechanism involving positive-acting X-chromosome-encoded transcription factors, termed X-numerator elements (Cline, 1988), negative-acting autosome-encoded transcription factors or denominators, and signal transduction factors provided maternally.  Among the X-numerators are sisterless-a, sisterless-b (sis-b), sisterless-c, and runt (Schurpbach, 1985; Cline, 1986, 1988; Steinmann-Zwicky et al., 1989; Parkhurst et al., 1990; Ericson and Cline, 1991, 1993; Estes, 1995; Hoshijima et al., 1995; reviewed by Cline, 1993).

The best candidate for a denominator gene is the deadpan (dpn) locus.  Both daughterless (da) and extramacrochaete (emc) fulfill the role of maternally contributed transduction loci (Cline, 1976; Cronmiller et al., 1988).  Both in vitro biochemical evidence and in vivo genetic evidence support the idea that transcription factors of the basic-helix-loop-helix (bHLH) family are able to form homo- and hetero-dimers; thus the X:A ratio counting mechanism seems to involve the relative affinities and chromosome-dependent stoiciometries of the bHLH proteins SIS-B, DA, EMC, and DPN.  When X:A=1, sufficient SIS-B protein is synthesized so that it can effectively compete with the EMC and DPN proteins for binding to DA protein.  DA:SIS:B heterodimers then bind to so-called establishment promoter (Pe) elements of the SXL gene and activates its transcription, resulting in an early burst of SXL protein that sets splicing and dosage compensation in to female-specific modes.  When X:A=0.5, too little SIS-B is produced, and DA protein remains sequestered with EMC and DPN.  The Sxl Pe remains inactive, and splicing and dosage compensation enters male-specific modes. In response to X:A ratio=1, an embryo specific promoter of the gene called Sex-lethal (Sxl) is activated (Keyes et al., 1932).

Sxl protein that acts as a master gene for the somatic germline sex determination, has three somatic functions. First, Sxl protein carries out autoregulation at the level of pre-mRNA splicing.  Second, Sxl controls female-specific differentiation at the level of pre-RNA splicing and polyadenylation at least two genes that code for transcription factors that effect terminal differentiation. Third, Sxl protein negatively regulates X-chromosome dosage compensation.  It does so in two ways, by alternative RNA splicing of a normally male-specific gene, and by translation-level regulation of many X-chromosomal transcripts during embryogenesis. In the male, with Sxl in the off state, male differentiation occurs because tra is in the off state and therefore the differentiation-effector transcription factors are produced in alternative male-specific modes.  Dosage compensation is active, and the male X-chromosome is decorated by a minimum of four proteins and two RNA molecules that form a complex along the entire chromosome (reviewed by Cline and Meyer, 1996).  Transcription of the male X-chromosome is elevated two-fold, and it produces the same amount of RNA per template as found in females.

Germline pathway for sex determination and dosage compensation is different than the somatic sex determination mechanism.  (Figure 1) Figure 1: Sex determination of D. melanogaster (1998)The vast majority of somatic sex determination loci have no function in germline cells.  For example, none of the X-chromosome numerators is required for proper oogenesis (Granadino et al., 1989, 1992; Steinmann-Zwicky 1991), despite the fact that proper oogenesis requires that X:A =1 in the germline (Schupbach, 1982, 1985) nor are tra, tra-2, and dsxF required for oogenesis.  Sxl and snf have germline functions but the former is not a binary switch gene between oogenesis and spermatogenesis (Despande et al., 1996; Bopp et al., 1993, 1995; Hager et al., 1997). Systematic screens for female-sterile mutations have identified a large number of genes required for normal oogenesis (e.g. Gans et al., 1975; Mohler, 1977; Perrimon et al., 1986; Schupbach and Wieschaus, 19889, 1991).  Female-sterility can arise in diverse ways, but one interesting class of mutations is germline-dependent and causes an “ovarian tumor” phenotype.  “Ovarian tumor” mutations cause under-developed ovaries, in which egg chambers and ovarioles are filled with an excess of undifferentiated germ cells that have adopted male-like characteristics that include a prominent spherical nucleus, assembly of mitocondria around the nucleus, and mis-expression of male-specific marker genes (Oliver et al., 1988, 1990, 1993; Steinmann-Zwicky, 1988, 1992; Bopp et al., 1993; Pauli et al., Wei et al., 1994).  Among the “ovarian tumor” class of genes are ovo, ovarian tumor (otu), fused, and two genes with somatic phenotypes, namely snf and Sxl. Strong mutations at the ovo and otu loci result in ovaries totally devoid of germ cells (King and Killey, 1982; Busson et al., 1983; Oliver et al., 1987; Mevel-Ninio et al., 1989; Rodesh et al., 1995), Weaker mutations at both loci result in viable germline cells that have abnormal male-like splicing at the Sxl gene (Oliver et al, 1993). The overall conclusion is that oogenesis requires a chromosomally female germline is wild type for ovo, otu, Sxl, and snf.  If one of these genes is defective, either the germline will die or male-like differentiation and tumor formation ensure.

However, there are soma-germline interactions for a normal sex determination. (Figure 2) Figure 2: Somatic-Germline Interactions. (1998)Unlike the somatic regulatory hierarchy, which genetic mosaic experiments clearly showed functions in cell-autonomous fashion, sexual differentiation of the germline requires inductive signaling from somatic cells.  This was shown by use of pole cell transplantation, the method of making mosaics in which germline cells surgically transferred from donor embryos  (Schubach. 1985; Steinmann-Zwicky et al., 1989).  These experiments show that proper germline differentiation requires a combination of germline-autonomous chromosomal cues and proper signaling from the soma.  Evidence with tra and dsx mutant somatic hosts indicates these soma-germline interactions have detectable effects by larval stages (Steinmann-Zwicky., 1996).

The ovo gene is genetically complex.  At least three transcripts are produced from the ovo region (Mevel-Ninio et al, 1991, 1995, 1996; Garfinkel et al., 1992, 1994).  Two of these are germline-specific and correspond to the ovo function, while the third corresponds to the somatic-epidermal, non-sex-specific shavenbaby (svb) function.  (For a schematic of the gene map please refer to Figure3) 

 The ovo function is transcribed from two closely spaced germline-specific promoters, ovo a and ovob, give rise to 5-kb mRNAs (Mevel-Ninio et al., 1991, 1995; Garfinkel et al., 1992, 1994).   First identified  promoter was ovob  Garfinkel et al., (1994)  and the leader exon it forms is called Exon 1b, 1028-codon-long open reading frame that contains four Cys2-His2 fingers at the carboxy terminus; protein MW of 110.6 kD.  A second germline promoter, ovoa, was identified by Mevel-Ninio et al (1995), 1400 codons long, and predicts a 150.8-kD protein.  This Exon 1a contains an in-frame AUG upstream of the translation start in Exon 2 utilized by the OvoB open reading frame.  The OvoB mRNA isoforms is predominant during adult life, with the OvoA isoforms only appearing during Stage 14 of oogenesis (Mevel-Ninio et al., 1991, 1996; Garfinkel., 1994).  The ovo zinc finger domain binds to its own germline promoter regions, to the otu promoter region (Garfinkel et al., 1997; Lee, 1998; Lee and Garfinkel 1998).  This is consistent with ovo playing an important role in a sex determination hierarchy operating in germline cells that involves these other genes. The svb function is transcribed from an incompletely characterized somatic promoter that forms a 7.1 kb poly(A)+ mRNA (Garfinkel et al., 1994).  This transcript accumulates 9-12-hr post-fertilization, in the somatic tissues that later in embryogenesis form the cuticular structures affected by svb mutations.  Wieschaus et al. (1984) observed that ventral denticle belts and dorsal hairs are defective in svb mutations; hence the name, and svb mutations are polyphasic larval lethals. Exons and exon segments that are found in all mRNA forms coded by the region correspond to genomic DNA where so-called svb-ovo- mutations map (Mevel-Ninio et al., 1989; Garfinkel 1992).  Finally, somatic-specific exons, exon segments, and transcriptional regions correspond to region mutable to the svb- ovo- phenotype.  Since al known mRNA forms utilize the same splice junctions to join Exon3 to Exon4, all protein forms coded by the locus are believed to contain the same four zinc fingers at the carboxy terminus.   A wide variety of evidence points to ovo playing a critical role in germline sex determination.  High-level of ovo transcription in germline cells, as detected with Xgal staining of ovo promoter-lacZ constructs requires that they have a female karyotype (Oliver et al., 1994).  Chromosomally male germline cells have low levels of ovo transcription even if the soma is transformed towards female through the use of hs-traF cDNA minigenes.  Likewise, chromosomally female germline cells have high levels of ovo transcription even if the soma is anatomically male through the action of tra loss-of-function mutations.  This argues that high-level of ovo transcription is a germline X: A ratio-autonomous property, and stands in contrast to related experiments with otu.  In the case of otu, there is evidence that chromosomally male germline cells, which normally have no need of otu+ function at all, require otu- for proliferation when they are in a female host (Nagoshi et al., 1995). The D. melanogaster ovo gene is required for cell viability and differentiation of female germ cells, apparently playing a role in germline sex determination.  While female X: A ratio in germline cells is required for high levels of ovo germline promoters.  Therefore we undertook to identify trans-acting regulatory regions of the X-chromosome, with a particular interest in identifying candidate germline X-chromosome numerator elements. In this study, I screened  X-chromosome using 45 deficiency strains, I found that these trans-regulating regions were grouped into 12 loci based on overlapping cytology.  Five regions were trans-regulating activators, and seven were trans-regulating repressors; extrapolating to the entire genome, this result predicts nearly 85 loci.  A subset of the dozen X-chromosomal regions correlated with previously identified E(ovoD) and Su(ovoD) loci (Pauli et al., 1995).  

Materials and Methods

 

Fly Strains and Growth Flies were maintained on standard yeast/cornmeal medium and kept at 25oC and 18oC unless otherwise indicated.  Mutants are described in Lindsley and Zimm (1992).  The ovo3U21 and ovo4B8 were obtained from Brian Oliver of NIH;  OvoD1rS1 FM3 is from the Garfinkel lab collection.  The remaining stocks were obtained from the Bloomington Stock Center (see Table 2.1 for the list of stocks that had been used and Figure 2.1 for their location on the X Chromosome). 

Outcrosses Outcrosses were designed to create transgenic flies so that screening of the X chromosome for trans-regulators of ovo in the germline can be done.   Virgin female flies were collected 14 hour long windows at 18oC or 8 hour long windows at 25oC, during which newly emerged males remained immature.  Collected females were kept 3-5 days to make sure they are virgin before outcrossing them.  Heterozygous virgin females (5-7), carrying deficiency X-chromosomes balanced over first chromosome balancers were mated with males homozygous for either of two P-element transformation constructs of a lacZ reporter gene fused to the ovo promoter.  Both events were inserted on third chromosome.  They were grown at 25oC unless otherwise noted. The control class of F1 progeny has a complete X-chromosome pair, whereas the experimental class has one complete and one deficient X chromosome in its genome.  The [ovo::lacZ constructs] were designed by Oliver et al., (1994).  In this study two of their strains, ovo4B8 (pCOW+1.9) and ovo3U21 (pCOW-2.1) respectively, were used to determine the ovo promoter activity.

Outcrosses to Remove Duplications Several X-chromosome deficiencies in the Bloomington collection are carried in males, with compensatory duplications of X material on an autosome.  These had to be crossed to eliminate the duplications (Fig 2.4).  This was done as follows:  FM3/FM7a virgin flies were mated to Df/Y; Dp males.  Among the F1 progeny, half of the Df/(FM3 or FM7a) daughters will carry the unwanted duplication, and half will be free of the duplication.  In some cases, presence of the duplication could be determined from the females’ phenotypes.  In other cases, up to twenty individuals virgin Df(FM3 or FM7) F1 progeny were backcrossed to FM7a/Y males to establish stocks.  In the F2, absence of the duplication could be established by examining sons; in all cases, the Df is male-lethal unless “rescued” by the duplication.  Also FM3 is itself male lethal.  Thus, single-female stocks that produce only FM7a sons had the desired genotypes and were kept for experiments.

X-Gal Staining In this assay ovaries from two-day-old adults were dissected in Drosophila Ringer’s solution (182 mM KCl, 46 mM NaCl, 3 mM CaCl2, 10mM TrisHCl, pH 6.8).  Then, these tissues were transferred to a microtiter plate and fixed in 1% gluteraldehyde, 50mM Na-cacodylyte acid solution for 15 minutes. After rinsing the tissues, three times for 5 minutes each staining buffer (7.2 mM Na2HPO4, 2.8 mM NaH2PO4, 1.0 mM MgCl2, 0.15 mM NaCl), they were transferred to incubation buffer (staining buffer, 5 mM Fe2 (CN)3, 5 mM Fe3 (CN)2, 0.2% X-Gal) for an hour at 37oC.  Next, tissues were washed three times 5 minutes each in washing buffer, which is a 1 mM EDTA, added PBS (130 mM NaCl, 7 mM Na2HPO4*2H2O, 3 mM NaH2PO4*2H2O, pH 7.0) solution.  Finally, the tissues were dehydrated in ethanol solutions of increasing concentrations (50%, 75%, 95%) and mounted on a slide in Permount.  Preparate concentrations were examined under a compound microscope to make correlations between staining and gene activity. Although it was easy to determine positive and negative controls, but this assay wasn’t sensitive enough to see subtle differences due to effects of deleted regions on ovo promoters driving LacZ.

Histochemical Assay of LacZ Activity This method allowed us to make quantitative measurements of lacZ activity due to ovo promoter function in animals heterozygous for X-chromosome deletions.  Emerging F1 flies were collected and aged for two days before dissecting ovaries under a dissecting microscope.  For each soluble assay, 10 flies were dissected.  This is repeated at least seven assays (N, sample number) completed per stock for each construct.  Ovaries from ten dissected outcrossed flies were out into eppendorf tubes containing 100ml of Assay Buffer (50 mM K-phosphate, 1 mM MgCl2 at pH 7.8) and homogenized about 20 strokes.  For each dissected pair of ovaries 100 ml  of assay buffer was used and the volume was completed to appropriate amount.  After centrifuging for one minute, 20 ml of the supernatant was transferred into 980 ml of assay buffer (Simon and Lis, 1987; Ashburner, 1989) to make 2mM chlorophenol red-beta-D-galactopyranoside (CPRG).  Absorbance at 574 nm was measured at half hour time intervals starting from zero to two hours hydrolysis of CPRG by chlorophenol (red CPRG).  CPR has a molar extinction coefficient of 75,000 M-1 cm-1 (Boehringer-Manheim data sheet) and this is a very easily detected product of b-galactoside enzyme activity. Range finding experiments showed that 2mM of CPRG gives linear data for 2-3 hours often, color changes could be seen with the unaided eye. Two controls are shown in Figure 2.8 that validates CPRG for this work.  Ovaries from a non-transformed strain (y w RD) were used to prepare soluble extracts.  A near zero-absorbance at 574 nm was observed that did not appreciably change over several hours.  In contrast, ovarian extracts from the ovo promoter-lacZ transformant strain ovo3U21 and ovo4B8 (Oliver et al, 1994) showed a steep linear increase in A 574 during the same period.  The slopes of these lines were proportional to the amount of ovo3U21 and ovo4B8 extract added.

Bradford (1976) Assay For Protein This protein determination method is based on the binding of Coomasie Brilliant Blue G-250 to the protein.  Preparation of protein reagent was done according to Bradford (1976).  After 100 mg of Coomasie Brilliant Blue G-250 was dissolved in 50 ml 95% ethanol, and then 100 ml 85% (w/v) phosphoric acid was added.  The resulting solution was diluted to a final volume of 1 liter [final concentrations in the reagent were 0.01% (w/v) Coomasie Brilliant Blue G-250, 4.7% (w/v) ethanol, and 8.5% (w/v) phosphoric acid].  20ml of prepared soluble extract from the dissected tissues were used.  This volume is diluted to 0.1ml with ddH2O, then 5ml of protein reagent was added to the test tube and contents were mixed.  The absorbance at 595nm was measured after 2 min and before 1 hr in 3 ml cuvettes against a reagent blank prepared from 0.1 ml of the appropriate buffer and 5 ml of protein reagent.  A standard curve using known quantities of bovine serum albumin (BSA) was constructed.  Soluble extract absorbances were plotted on the standard curve and protein amount interpolated.

Statistical Analysis Average specific activity is calculated as nanomoles of substrate used per hour per nanogram protein expressed (nmole CPRG liberated /ng / hr).  Sample number (N) always exceeded seven.  Mean specific activity and standard error of the mean (SEM) were calculated for each experimental and control class.  The F test was used to determine whether variances were equal, and therefore,, which type of student’s t-test calculation was appropriate.  A significant difference between experimental and control values was identified by a P < 0.05 for the t-test score.

RESULTS

In this study and ovo mechanism study, the X-chromosome was screened, using 56 different deficiency strains    Table 1: List of Stocks for X-chromosome Screening (1998)Table 2: Stocks Made in This Study for X-Chromosome Screening Table 1: Stocks for Negative Autoregulation of ovo (1998)  to identify transregulation of ovo Table 3: LacZ Specific Activities Obtained by Screening X-Chromosome with ovo3U21Table 4: LacZ Specific Activities Obtained by Screening X-Chromosome with ovo4B8 (Results)

The results are given in three sections: X chromosome deficiency screening, negative autoregulation of ovo exhibited by deficiencies removing ovo, and gene dose analysis using P element transformants carrying extra copies of ovo.

X Chromosome Screening The presence of polytene chromosomes in the salivary glands, which have distinctive, banding patterns allows the map positions of genes to be correlated with physical features of the chromosomes.  Breakpoint locations rearrangements, and the locations of cloned sequences can be easily established.  Each of the major chromosome arms is divided into 20 numbered segments, except chromosome 4, which is divided into 4 regions.  Each numbered region is then divided into six consecutive lettered regions, and each lettered region into numbered bands, for example 4E1. The precise relationship between physical length and the numbering scheme depends on local topography (Lefevre, 1976).  In the summary tables, each deficiency listed according to cytological positions. The map of the X chromosome, including the deficiencies used in this study is given in Materials and Methods (Fig 1). Figure 1: Sex determination of D. melanogaster (1998) In Drosophila melanogaster germ cells, ovo has a primary role in female sex specific cell viability, proliferation and differentiation.  Ovo responds to the number of X-chromosomes as assessed by high level expression (Oliver et al., 1994).  Thus, the ovo promoter may be dependent upon X germline numerator elements.  To identify possible trans-regulators of the ovo germline promoter (and, I hope, to identify germline numerators) I undertook deficiency screen for quantitative effects on ovo::lacZ reporter constructs.  Determination of trans-regulation effect by any of the deletion mutant, was based on two general rules.  If the excised part of the X chromosomes has any genes with the positive regulatory effects on ovo gene activity, then the levels of LacZ reporter gene function will be reduced in experimentals compared to control siblings.  If the experimental class results in the elevation of the LacZ activity by producing high levels of enzyme compared to controls, the elevated region having removed a repression locus. Significant effects were determined by statistical analysis, which using a student’s t-test P value is less than or equal to 0.05.  X-chromosome screening results are presented in Table 3.1 and 3.2.  The entire X-chromosome deficiency set was tested twice: once with a 3.3kb ovo promoter fragment driving LacZ (strain ovo3u21), and separately with a 3.1kb ovo promoter (ovo4B8).  Of  45 deficiencies that represent about 70% of the X-chromosome 17 deficiencies had significant effects in both ovo3U21 and ovo4B8 reporter activity, 1 deficiency had significant effects on only ovo3U21 and only 1 deficiency effect on ovo4B8.  Some of these deficiencies partly overlap, allowing the identification of 11 regions that apparently contain trans-acting modifiers of ovo promoter activity six are positive regulators and five are negative.

Region 1-4.  This region covers the eight overlapping deficiency lines, Df(1) BA1, Df(1)sc14, Df(1)64c18, Df(1)JC19, Df(1)dm75e19, Df(1)N8, Df(1)A113, DF(1)JC70.  For three of them, Df(1)A113, Df(1)JC70, and Df(1)BA1, the student’s t-test probabilities show a significant difference between control and experimental siblings.  The remaining strain has no significant trans-regulation effect on ovo gene activity.  Df(1)BA1 enhanced the ovo gene expression activity about 20% when either ovo3U21 or ovo4B8 is used.  It was suggested that a suppressor of ovoD (1F-2B+ locus) maps within 1E3-4 to 2B3-4 because of the dramatic gene dose effect of this region on the development of ovoD2/+ ovaries (Pauli et al, 1995).  In contrast, it was found that Df(1)A113 and Df(1)JC70 have repressing effects on ovo expression.  Df(1)A113 (3D6-E1; 4F5) removes several genes beside ovo, showed a very significant repression effect in outcrosses, about 82% and 47% (e/C), in ovo3U21 and ovo4B8 respectively.  That data obtained in Df/+ females has a particular quantitative significance, which implies that the missing loci have the complementary effect. It was shown that this region is contains a gene or genes resulting in genetic unbalance (Cline et al., 1987).  Also, Oliver et al., (1988) show that in deficiency lines, which they have used, strains removing both ovo and snf together are reducing viability of the progeny, that is, there is a synergistic interaction between ovo and snf.  

Region 5-8.  Twelve overlapping deletions have been tested in this region.  Two deletions Df(1)N73 (5C3-5;5E-8) and Df(1)Lz90b24 (8B-D) caused very significant repressing effects, implying the presence of trans-activating loci, one deletion Df(1)RA2 (7D10;8A4-5) resulted in heterozygous experimentals with significant elevation in LacZ compared to siblings, implying a trans-repressor locus.  It has been reposted that Df(1)RA2 strongly enhances ovoD  phenotypes due to the function of otu+ in germline sex determination (Pauli et al., 1993).  However, since out protein is cytoplasmic, it is unlikely that the Df(1)RA2 effect on ovo::lacZ promoter activity is due to changing dosage of otu.  It is also suggested that there is a synergistic interaction between ovo and lozenge, eye phenotype, which is deleted by Df(1)Lz90b24, and here the data showed a trans-activating effect due to this deletion.  The other deletions do not cause any significant effect on gene activity.

Region 9-10.  In this cytological position nine deficiency lines had been tested.  Since this region was very dense for putative trans-regulation repressors, it was group in a small region.  Among nine of the deficiencies were used six of them showed a repressor effect.  These effective regions were: Df91)vL15, Df(1)N110, Df(1)HC133, Df(1)vL11, Df(1)KA7, and Df(1)N71.  This region seems to have a very important effect on ovo, since in the 9Bto 10F interval there are various levels of repressor effect.  Two common overlapping regions were found; one was from 9C4 to 9D1-2, and the other was from 10A to10F6.  Other repressor effects from strongest to weakest was Df(1)vL11 (9C4;10A1-2), Df(1)HC133 (9B9-10;9E-F), Df(1)N110 (9B3-4;9D1-2), and Df(1)v-L15 (9B1-2;10A1-2), Df(1)KA7 (10A9;10F6-7) breakpoint was outside the first loci in the examined region.  Df(1)Ka7 and Df(1)vL15 show about 20% increase in the heterozygous siblings, the longest and the shortest breakpoints, respectively.  Three out of five repressing effect intervals, Df(1)v-L11 (9C4; 10A1-2), Df (1)HC133 (9B9-10; 9E-F), Df(1) N110 (9C4; 10A1-2) is the strongest of all in Df/+ and bearing the common region among the five strains, which is 9C4; 10A1-2.  

Region 11-13.     Eight deficiency lines were in this region, Df(1)JA26, Df(1)HF368, Df(1)N12, Df(1)C246, Df(1)g, Df(1) RK2, Df(1)RK4, and Df(1) sd 72b   .  It has been found that this region involves five overlapping deletions that gave rise to repressing effect on ovo gene expression.  According to common regions of the cytological positions, these overlapping deletions were grouped into three loci.  These three common regions, which are responsible from trans-regulation activity of ovo, reside on 11D0F; 12B-D, and 13F-B regions of the X-chromosome.  Df(1)N12 (11D12;11F1-2) and Df(1)C246 (11D-E; 12A1-2) were in the 11D-F loci, Df(1)g (12B;12E8) and Df(1)RK2 (12D2-E1; 13A2-5) were in the 12B0D region, and Df(1)sd72B (13F1-14B1) in the 13B-14B loci, all of which in this examined region showed a repressor activity. The strongest effect among the X-chromosome screening was located in 11D1-11F1-2 excised region of X-chromosome, this deletion corresponds to Df(1)N12 strain, which shows a significant effect as well as high gene activity repression, Around 140% to 240% E/C in Df/+ flies for both ovo::LacZ constructs.  In addition, it has been reported that reduced dose of the 11D-F region results in synergistic mutant phenotypes with a number of somatic sex determination genes (Belote et., 1985).  Furthermore, Flybase reports that this region seems to include locus involved in early sex determination examined by Scott and baker (1986). However, ambiguities in deficiency breakpoint assignments complicate interpretation.  For example, first loci, which includes Df(1)N12 and Df(1)C246 due to uncertainty at the distal end breakpoints of Df(1)C246 (12D-e; 12A1-2); the trans-acting repressor of ovo maybe located in 11E-F rather than 11D-F. Similarly, for the second loci in this region ambiguity at the distal breakpoint of Df(1)RK2 also cause a dilemma about the location of the trans-acting repressor, since the question was the common region between Df(1)g and Df(1)RK2 was whether in the 12D-E or in the 2E1-2E8 of X-chromosome. On the other hand, the last loci were determined by the only one deficiency strain.  In this case, the problem was whether determination of the loci was accurate enough, or whether another locus is involved in repressing of ovo reporter activity which Df(1)sd72b (13F114B1) may have a common region with.  This deficiency removes several lethal mutations, Myb, sd (scalloped), shi (shibiri), and exd (extradenticle).  Two genes previously cloned in the 13F cytological region are the Drosophila c-myb oncogene homolog (Katzen et al, 1985) and a G protein b-subunit (Yarfitz et al 1988).  It has been suggested that the sd+ gene might be associated with more than one product (perhaps a differential processing) or it might reflect differential tissue and/or temporal regulation (Campbell et al., 1991).

Region 14-20.   In this region eight deficiency strains, Df(1)4b18, Df(1)rD1, Df(1)B, Df(1)N19, Df(1)JA27, Df(1)HF396, DF(1)DCB1, and Df(1) A-209, were tested.  According to measured specific activities Df(1)4b18 (14B8; 14C1) and DF(1) B (15F9=16A6-7) showed significant activating effect on ovo promoter, activity of the former was weaker than that of latter.  Since there is no common region between these two putative trans-acting activators, interpretations of the results gave rise to two loci, 14B8-14C1 and 15F-16A1; 16A6-9. In addition, the Flybase report for Df(1) shows that 70 deletion that breaks within the second exon of the non A (no on or transient A) gene from Stanewsky et al (1993). As a result of X-chromosome screening, 45 deficiency strains were tested and found 17 regions were trans-regulating ovo promoter.  These regions were classified into 12 loci according to their overlapping common regions.  Among these, six, of which were showing trans-acting activator effect, and seven, of which were responsible for trans-acting repressor effect on ovo promoter.   Furthermore, one deficiency strain, Df(1)sc14, showed a significant trans-acting repressor effect in only ovo4B8 strain but not in ovo3U21 strain.  This maybe explained by position effect of P[ovo::LacZ] construct due to landing on P element transposase onto insertion site or by difference between the size of the ovo::LacZ constructs, e.g. ovo3U21 carries 200 bp longer than ovo4B8 at the N-terminal end that may cause a better translation product.  Consequently, among the X-chromosome screening data, it was found that two of the deficiency lines. Df(1)A113 and Df(1)JC70, which are removing ovo and snf along with the several genes due to deletions, and correspond to one loci acting as an repressor, were taking into more detailed investigations.  These results suggested a negative autoregulation mechanism in the ovo promoter.  Therefore, negative autoregulation of ovo was examined with three approaches: ovo point mutations, more defined deficiency strain, and downstream genes.

DISCUSSION

  The sex determination involves complex set of mechanisms.  The fly is chosen to be studied since Drosophila is inexpensive to rear, generates large numbers of progeny, and has nearly a century of accumulated data upon which to design experiments.  Mutational analysis of cell biological and developmental process is relatively simple, even if the resulting mutations are organism-lethal when homozygous.  This is decided advantage over mammalian genetics, in which lethal mutations often die in utero, which complicates the ability to examine and interpret mutant phenotypes. The Drosophila genome is one-twentieth the size of the mammalian genome, making insertional mutagenesis and positional cloning much less difficult.  Additionally, mammalian genetics lacks genetic tools such as balancers that make the maintenance of sterile and lethal-mutations nearly trouble free in Drosophila.  Nematodes have many of the same conveniences as Drosophila, with the added advantage of a highly stereotyped pattern of embryonic (and post-hatching) cell lineages.  The more-regulative character of Drosophila development induces complications lacking from worm genetics, with respect to cellular level analysis of mutant phenotypes.  Perhaps, the most compelling reason to take advantage of the specialized properties of Drosophila, is the extent to which prior studies have shown that genes, proteins, and developmental pathways and processes are conserved among metazoan groups.  We can, with high confidence, study sex determination in Drosophila with a reasonable confidence that what we learn can be extrapolated to other species, including man and his clinical diseases.

  The deletion mapping technique was used to identify the locations of genes that are required for ovo trans-regulation.  Each deficiency line removes several to many genes from the genome.  A sufficiently complete set of overlapping deletions can allow, potentially, every individual trans-acting gene to be localized. Seventeen deficiencies that have effects on the ovo germline promoters are shown in Table 4.1.  Twelve deficiencies showed repressor effects, and five deficiencies showed activator effects.  Deleted regions may affect any of several processes, such as numerator elements, cell viability and differentiation, dosage compensation, and response to inductive signals from soma.  Determination of which gene within a specific region is responsible for the effect on ovo requires more defined deletions or having null alleles for each gene. Estimation of the Number of Trans-Regulators.  Among the seventeen deficiencies in Table 4.1, overlapping common regions identify seven that function as trans-acting repressor loci, and five that function as trans-acting activator loci.  Thus, the entire euchromatic X-chromosome may have as many as ≈10 repressor genes and ≈7 activator genes for the ovo germline promoters.  If these results were extrapolated to the entire fly genome, ≈50 repressors and ≈35 activators of ovo transcription are predicted.  These are underestimates from the data, since any given deleted common region need not remove exactly one relevant gene. Is it reasonable for nearly 85 genes to be involved in regulating the ovo germline promoters?  Precedents from other developmental control systems suggest this is not an implausibly high number.

Regulation of the master sex determination gene Sxl is complex.  To establish somatic sex determination in the early embryo, nine genes are required to activate the Sxl early promoter.  These are sis-a, sis-b, sis-c, run, da, emc, gro, dpn, and her.  In biochemical terms, most are DNA-binding proteins.  In genetic terms, some are positive and are others are negative regulators.  Maintenance of Sxl expression involves positive autoregulation at the level of pre-mRNA alternative splicing.  At least five genes are known to play specific roles in this process: Sxl itself, snf, vir, her, and fl(2)d.  Function of Sxl in the germline is regulated in several ways.  Germline-specific transcriptional control of Sxl is still conjectural, but it is clear that the somatic functioning numerator elements play no role in the germline.  It is possible that ovo may play an important role in germline transcriptional control of Sxl (e.g., Lee. 1998); certainly it has an indirect role (e.g., Oliver et al., 1993).  Splicing-level autoregulation of Sxl is active in the female germline, and it involves the same genes that function in this process in somatic cells.  Once Sxl protein is produced in female germline cells, the otu protein plays an important role in this relocalization into the nucleus.  Thus, a minimum of sixteen genes is required for proper regulation of Sxl.

Establishment of the body plan in Drosophila is also under complex transcriptional control.  Maternally localized RNA and protein molecules establish the gross body axes: anterior-posterior and dorsal-ventral.  Hierarchically organized sets of zygotically activated genes are transcribed, and their protein products serve to refine the body axes into progressively finer-grained structures.  The metameric anterior-posterior body axis is specified by so-called gap genes, pair rule genes, and segment polarity genes, which create the segment-sized repeating units of the body.  Homeotic genes encoded by the Antennapedia Complex (ANT-C) and bithorax Complex (BX-C) then confer position-specific identities upon each segment. During the cellular blastoderm stage, gap genes and maternal coordinate genes regulated the activation of primary pair rule genes such as even-skipped (eve).  These are expressed in seven one-segment-wide stripes that alternate with on-segment-wide regions of non-expressing cells.  For example, the second stripe of eve expression is positively regulated by hunchback and bicoid, and negatively regulated by giant and Kruppel.  All four proteins directly bind to a 500-bp-long “eve-stripe 2 enhancer.”  Binding have giant and Kruppel is competitive with binding of hunchback  and bicoid, and vice versa.  Thus, spatially controlled concentrations of giant, Kruppel, bicoid, and hunchback proteins result in spatially restricted activation or repression of the eve stripe 2 enhancer.  The remaining six stripes of eve expression are similarly controlled by other DNA-binding proteins, which are acting another discrete stripe-specific enhancers. Ectopic expression of homeotic genes can have disastrous effects on development.  Thus, a special heterochromatin-like mechanism functions to ensure that ANT-C and BX-C genes are inactive in cells and tissues that do not require their expression.  Stable repression is mediated by the Polycomb class of proteins, which number over forty. Each of these examples illustrates that developmental control of individual gene transcription is mediated by both positive and negative effectors, and that sometimes the number of such upstream regulators numbers between one and several dozen.  Thus, our estimate of 85 regulators of the ovo germline promoters is not out of line with other developmentally regulated systems.

Evaluation of Candidate Loci Within Common Regions.   Based overlapping cytology, seventeen deficiencies that affected the ovo germline promoter fell into twelve common regions.  Each of these will be discussed in turn below. Of particular interest was the relationship each of our trans-acting may have with Su(ovoD) and E(ovoD) loci identified in a generic screen by Pauli et al. (1995).  In general, it is not straightforward to suggest identities between Su(ovoD) or E(ovoD) loci and our trans-acting repressor or activator loci because of the dissimilar means of assaying these gene-dose-sensitive interactions.  We use quantitative measures of LacZ reporter activity as a proxy for ovo transcription, while Pauli et al. (1995) use semi-quantitative measures of vitellogenesis.

Region 1 (polytene bands 1A1; 2A1-4):  The distal region of the X-chromosome showed a trans-regulating activator effect on the ovo promoters.  This region includes the acheate-scute complex (AS-C), home of the X-chromosome numerator element sis-b (Cline, 1988; Parkhurst and Ish-Horowicz, 1990), also known as scute-T4.  This numerator has no function in the female germline (Granadino et al., 1989).  Pauli et al., (1995), using other deficiency strains affecting this section of the X-chromosome, identified a strong Su(ovoD) locus in the polytene region 1E3-4; 2B3-4 that may correspond with our trans-activator.  Flybase indicates that this region contains over 100 genes, among them 23 unassigned open reading frames, 33 genes defined by apparent visible mutations, 53 lethal genes,, and two female sterile loci.

Region 2 (polytene bands 4C15-16; 4F15):  This region includes the ovo and snf loci, and was identified by Pauli et al., (1995) as a strong E(ovoD) due to the effects of these loci.  Further discussion is deferred to mechanism of ovo autoregulation, which deal with ovo negative regulation. Region 3 (polytene bands 5C3-5; 5E8):  This region has a trans-regulatory activation effect on the ovo germline promoters.  Deficiency for this region showed no interaction with ovoD in the vitellogenesis assay (Pauli et al., 1995).  Examination of Flybase records for this region reveals over twenty genes, and no strong candidates that may account for the interaction with the ovo promoters.

Region 4 (polytene bands 7D10; 8A4-5):  Results  showed that this region contains a transacting-repressor of ovo germline promoter activity.  This region reported by Pauli et al. (1995) to contain a strong E(ovoD) locus, which was identified as the ovarian tumor gene (Pauli et al., 1993, 1995).  It is virtually certain that the repressor-of-ovo is distinct from otu.  First, the otu protein is cytoplasmic and plays a role in egg chamber cytoskeletal function (Nagoshi et al., 1997).  Second, the ovo protein binds to the otu promoter in vitro (Garfinkel et al., 1997; Lee, 1998, Lee and Garfinkel 1998; Lu et al., 1998).  Third, under certain conditions, in vivo activity of the otu promoter is dependent upon ovo protein production (Hager and Cline, 1997; Lu et al., 1998).  Examination of Flybase reveals that this region contains fifty genes mutable to lethal, visible, or female-sterile phenotypes, but none appear to be a strong candidate for the repressor-of-ovo locus.

Region 5 (polytene bands 8B5-8; 8DE):  This region also has an apparent repressor of ovo germline promoter activity.  Deficiency for this region showed no interaction with ovoD mutations in the Pauli et al. (1995) vitellogenesis assay.  Examination of Flybase reveals that this region contains thirty genes mutable to lethal, visible, or female sterile phenotypes.  One gene stands out as a candidate for the repressor, namely, lozenge.  This is a complex locus that is mutable to female sterility (Green and Green, 1949, 1956), and it is named for a reduced-eye, smoothened-eye, mutant phenotypes.  Interestingly, certain ovo-mutant alleles are called “lozenge-like” in recognition of a similar eye defect (Oliver et al., 1987; Mevel-Ninio et al., 1989; Garfinkel et al., 1992).  The lz gene codes for a transcription factor (Dag et al., 1996). Region 6 (polytene bands 9C4; 9D1-2):  The cytological assignment of this region is based on the overlap of three deficiencies:  Df(1)N110, Df(1)H133, and Df(1)v L11.  Together, they mark a trans-acting repressor of ovo promoter activity.  According to  Pauli et al. (1995), only two of these three deficiencies behaved as if they exposed an E(ovoD) locus, while the third had no effect.  In combination with positive results from other deficiencies, Pauli et al. positioned the E(ovoD) locus at cytological region 9E-F.  Thus, it is again possible that the repressor-of-ovo we identified is distinct from a nearby E(ovoD) locus, and is among the half-dozen loci identified by Flybase as mapping into this interval.

Region 7 (polytene bands 10A6; 10F6-7):  This region contains a trans-acting repressor of ovo promoter activity.  According to Pauli et al. (1995), the defining deficiency had no significant interaction with ovoD alleles.  Examination of Flybase reveals that this region includes the somatic X-chromosome numerator element sis-a, which also has no function in germline development (Granadino et al., 1989, 1990, 1997).  Given the extent of this region, it is not  surprising that Flybase identifies 65 genes with diverse phenotypes and biochemical roles; however no strong candidate locus that may count for the repressor-of-ovo locus is apparent.

Region 8 (polytene bands11D1-2; 11F1-2):   This region contains perhaps the strongest trans-acting repressor of ovo promoter activity in the survey: deficiency heterozygous experimentals had 2-2.5 fold more lacZ specific activity in their ovaries that the balancer carrying controls.  According to Pauli et al (1995), one of the two deficiencies defining this common region showed a statistically weak enhancement of ovoDalleles, while the other had a significant Su(ovoD) phenotype.  Likewise, Belote et al. (1985) and Scott and Baker (1986) reported that the same deficiency later shown to have Su(ovoD) activity also interacted with loci in the somatic sex determination pathway.  It is an open question how these three results relate to one another.  Among sixteen genes that map into this region are two signal transduction loci: the Mek3 gene, a serine-threonine-specific protein kinase in the MAP kinase pathway, and a beta subunit of the heterotrimeric GTP-binding protein. A solitary female-sterile, fs(1) K4, also maps roughly into this region; it is germline-dependent, and yields fragile eggs, a phenotype occasionally seen in the eggs laid by ovoD3/+ females.

Region 9 (polytene bands 12D2-12E1; 12E8):  This region contains a trans-acting repressor of ovo promoter activity.  According to Pauli et al. (1995), neither deficiency defining this common region interacted with ovoDalleles.  This region contains the yolkless gene (DiMario et al., 1987), which has been cloned and codes for a member of 35 known genes, including a cluster of tRNA genes, the male-germline-specific Stellate genes, and several lethal and female-sterile genes.

Region 10 (polytene bands 13F1; 14B1):  This region contains a trans-acting repressor of ovo promoter activity.  Again, no significant interaction with ovoD allel4es was observed by Pauli et al. (1995).  Podry, Katzen and others have extensively mutagenized this region due to its containing shibiri (the Drosophila homolog of dynamin), c-myb, another Gb subunit, and the homeodomain protein extradenticle.  Their work revealed a total of twenty lethal genes, ten apparent visibles, and over a half-dozen unassigned open reading frames.

Region 11 (polytene bands 14B8; 14C1):  This region contains a trans-acting activator of ovo promoter activity.  According to Pauli et al., (1995), the defining deficiency had no significant interaction with ovoD alleles.  This region is surprisingly dense genetically, as it apparently contains over forty genes.  Several behavioral genes coding for neuronal functions map here, including nonA, paralytic, and easily shocked.  The nonA gene codes for an RNA-binding protein, and is mutable to a variety of phenotypes including recessive lethality, male-courtship-strong abnormalities, and defective vision.  The location of para (a sodium channel) is particularly intriguing since parats mutations fail to complement certain napts alleles, and nap genetically overlaps the dosage compensation function maleless.  Mutations in maleless are unique among the known dosage compensation loci in having a mutant phenotype in germline clones, and they are said to suppress the female-germline-lethality of ovo null mutations.  The easily shocked locus codes for ethanolmine kinase, and mutations at this locus also interact with mle.

Region 12 (polytene bands 15F9-16A1; 16A7):  This region contains a trans-acting activator of ovo promoter activity.  According to Pauli et al. (1995), the defining deficiency had no significant interaction with ovoDalleles.  Examination of Flybase reveals that this region contains at least a dozen female-sterile loci, a dozen lethal loci (including the Bar homeodomain protein gene). There is an ambiguity in compared mean of activities.  According to the negative autoregulation mechanism, there suppose to be a linear decrease pattern correlated to increase in copy of ovo.  However, the pattern of the gene dose was reaching plato, when three copies of ovo were present in the genome. Yet, this also shows that there is a protection mechanism that counts the number of ovo versus number of X chromosome exists.  Therefore, the sex determination mechanism turns off the extra ovo in the system immediately. 

Consequently, the system prohibits more wrong information to be processed according to its default setting where if the X:A ratio equals to one the outcome is going to be prepared as female, if not turn off the mechanism towards male-like, sterile mode, or death at the embryonic stage.  This discontinuity in the linear correlation may be due to position effect of P[w+ ovo+].  Future Directions and Concluding Remarks The results of this study suggest that the ovo germline promoters are regulated by a large set of upstream factors.  Nearly a dozen of these maps to the X-chromosome, some to region that are well characterized genetically.  Further deficiency mapping experiments, and assessment of the phenotypes of single-P insertion lines with female-sterile or perhaps lethal phenotypes, would be required to identify the relevant genes.  Some regions contain candidate loci that have been cloned (e.g. lozenge); in this example, either in vitro DNA-binding experiments using Lz protein and the ovo promoter region, or computational assessment of the likelihood that the ovo promoter contains binding sites for Lz can be done. Another potential upstream factor not assessed in these experiments is the ecdysone regulatory hierarchy.  The steroid ecdysone is the endocrine hormone that controls molting and metamorphosis in arthropods.  It is an allosteric effector for a heterodimeric receptor of the steroid-receptor superfamily.  The ovaries of adult females manufacture their own ecdysone, and the gene for the rate-limiting steroidogenic enzyme transcribed beginning in Stage 7-8 egg chambers.  This stage immediately precedes the onset of the highest level of ovo transcription (Mevel-Ninio et al., 1991; Garfinkel et al., 1994).  Mutations in the E74 and E75 genes, when made homozygous in germline clones, cause arrest of oogenesis at Stage 7-8, as if egg chambers are unable to respond to endogenous ecdysone and continue differentiation.  Both E74 and E75 code for transcription factors that are induced as immediate-early primary responses to added ecdysone both in-vivo and in tissue culture assays.  Thus, it is reasonable to suggest that one or both of these proteins will bind to the ovo germline promoter in an in vivo effect on expression of the ovo::lacZ reporter using the methods established in this dissertation.  

Acknowledgement:  This work had been comppleted in the laboratory of Dr. Mark Garfinkel at Illinois Institute of Technology.   Dr. Demet Sag initiated the project with her own  ideas, was fully supported by Turkish National Merit Fellowship, and  earned NATO Advanced Science institute  Grant on Genome Structure and Functional Genomics, Elba Island, Italy, accepted to work with Dr. Mevel Ninio, based on the proposal submitted by Demet Sag on Molecular Mechanism of  ovo, through EMBO long term scholarship in France.

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FIGURES and TABLES:

Figure 1: Sex determination of D. melanogaster (1998)

Figure 2: Somatic-Germline Interactions. (1998)

Figure 3: Molecular Structure of the ovo locus

Figure 4: In vivo Biochemical_genetic Assay for Regulators

Figure 5: ovo-LacZ Reporter Construction. (1998)

Figure 6 : Establishing Stocks From Duplication Carrying Lines.

Figure 7: Control Assay for b-galactosidase Assay. (1998).

Table 1: List of Stocks for X-chromosome Screening (1998)

Table 2: Stocks Made in This Study for X-Chromosome Screening

Table 3: LacZ Specific Activities Obtained by Screening X-Chromosome with ovo3U21

Table 4: LacZ Specific Activities Obtained by Screening X-Chromosome with ovo4B8 (Results)

Table 5: Deficiency Lines Affecting the ovo Gene Activity (X-chromosome screening result)

 

Previously Posted:  

ovo Female Germline Specific Drosophila melanogaster Gene has two auto-regulation mechanism: negative and positive

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What is the Future for Genomics in Clinical Medicine?


What is the Future for Genomics in Clinical Medicine?

Author and Curator: Larry H Bernstein, MD, FCAP

 

Introduction

This is the last in a series of articles looking at the past and future of the genome revolution.  It is a revolution indeed that has had a beginning with the first phase discovery leading to the Watson-Crick model, the second phase leading to the completion of the Human Genome Project, a third phase in elaboration of ENCODE.  But we are entering a fourth phase, not so designated, except that it leads to designing a path to the patient clinical experience.
What is most remarkable on this journey, which has little to show in treatment results at this time, is that the boundary between metabolism and genomics is breaking down.  The reality is that we are a magnificent “magical” experience in evolutionary time, functioning in a bioenvironment, put rogether like a truly complex machine, and with interacting parts.  What are those parts – organelles, a genetic message that may be constrained and it may be modified based on chemical structure, feedback, crosstalk, and signaling pathways.  This brings in diet as a source of essential nutrients, exercise as a method for delay of structural loss (not in excess), stress oxidation, repair mechanisms, and an entirely unexpected impact of this knowledge on pharmacotherapy.  I illustrate this with some very new observations.

Gutenberg Redone

The first is a recent talk on how genomic medicine has constructed a novel version of the “printing press”, that led us out of the dark ages.

Topol_splash_image

In our series The Creative Destruction of Medicine, I’m trying to get into critical aspects of how we can Schumpeter or reboot the future of healthcare by leveraging the big innovations that are occurring in the digital world, including digital medicine.

We have this big thing about evidence-based medicine and, of course, the sanctimonious randomized, placebo-controlled clinical trial. Well, that’s great if one can do that, but often we’re talking about needing thousands, if not tens of thousands, of patients for these types of clinical trials. And things are changing so fast with respect to medicine and, for example, genomically guided interventions that it’s going to become increasingly difficult to justify these very large clinical trials.

For example, there was a drug trial for melanoma and the mutation of BRAF, which is the gene that is found in about 60% of people with malignant melanoma. When that trial was done, there was a placebo control, and there was a big ethical charge asking whether it is justifiable to have a body count. This was a matched drug for the biology underpinning metastatic melanoma, which is essentially a fatal condition within 1 year, and researchers were giving some individuals a placebo.

The next observation is a progression of what he have already learned. The genome has a role is cellular regulation that we could not have dreamed of 25 years ago, or less. The role is far more than just the translation of a message from DNA to RNA to construction of proteins, lipoproteins, cellular and organelle structures, and more than a regulation of glycosidic and glycolytic pathways, and under the influence of endocrine and apocrine interactions. Despite what we have learned, the strength of inter-molecular interactions, strong and weak chemical bonds, essential for 3-D folding, we know little about the importance of trace metals that have key roles in catalysis and because of their orbital structures, are essential for organic-inorganic interplay. This will not be coming soon because we know almost nothing about the intracellular, interstitial, and intrvesicular distributions and how they affect the metabolic – truly metabolic events.

I shall however, use some new information that gives real cause for joy.

Reprogramming Alters Cells’ Fate

Kathy Liszewski
Gordon Conference  Report: June 21, 2012;32(11)
New and emerging strategies were showcased at Gordon Conference’s recent “Reprogramming Cell Fate” meeting. For example, cutting-edge studies described how only a handful of key transcription factors were needed to entirely reprogram cells.
M. Azim Surani, Ph.D., Marshall-Walton professor at the Gurdon Institute, University of Cambridge, U.K., is examining cellular reprogramming in a mouse model. Epiblast stem cells are derived from the early-stage embryonic stage after implantation of blastocysts, about six days into development, and retain the potential to undergo reversion to embryonic stem cells (ESCs) or to PGCs.”  They report two critical steps both of which are needed for exploring epigenetic reprogramming.  “Although there are two X chromosomes in females, the inactivation of one is necessary for cell differentiation. Only after epigenetic reprogramming of the X chromosome can pluripotency be acquired. Pluripotent stem cells can generate any fetal or adult cell type but are not capable of developing into a complete organism.”
The second read-out is the activation of Oct4, a key transcription factor involved in ESC development. The expression of Oct4 in epiSCs requires its proximal enhancer.  Dr. Surani said that their cell-based system demonstrates how a systematic analysis can be performed to analyze how other key genes contribute to the many-faceted events involved in reprogramming the germline.
Reprogramming Expressway
A number of other recent studies have shown the importance of Oct4 for self-renewal of undifferentiated ESCs. It is sufficient to induce pluripotency in neural tissues and somatic cells, among others. The expression of Oct4 must be tightly regulated to control cellular differentiation. But, Oct4 is much more than a simple regulator of pluripotency, according to Hans R. Schöler, Ph.D., professor in the department of cell and developmental biology at the Max Planck Institute for Molecular Biomedicine.
Oct4 has a critical role in committing pluripotent cells into the somatic cellular pathway. When embryonic stem cells overexpress Oct4, they undergo rapid differentiation and then lose their ability for pluripotency. Other studies have shown that Oct4 expression in somatic cells reprograms them for transformation into a particular germ cell layer and also gives rise to induced pluripotent stem cells (iPSCs) under specific culture conditions.
Oct4 is the gatekeeper into and out of the reprogramming expressway. By modifying experimental conditions, Oct4 plus additional factors can induce formation of iPSCs, epiblast stem cells, neural cells, or cardiac cells. Dr. Schöler suggests that Oct4 a potentially key factor not only for inducing iPSCs but also for transdifferention.  “Therapeutic applications might eventually focus less on pluripotency and more on multipotency, especially if one can dedifferentiate cells within the same lineage. Although fibroblasts are from a different germ layer, we recently showed that adding a cocktail of transcription factors induces mouse fibroblasts to directly acquire a neural stem cell identity.
Stem cell diagram illustrates a human fetus st...

Stem cell diagram illustrates a human fetus stem cell and possible uses on the circulatory, nervous, and immune systems. (Photo credit: Wikipedia)

English: Embryonic Stem Cells. (A) shows hESCs...

English: Embryonic Stem Cells. (A) shows hESCs. (B) shows neurons derived from hESCs. (Photo credit: Wikipedia)

Transforming growth factor beta (TGF-β) is a s...

Transforming growth factor beta (TGF-β) is a secreted protein that controls proliferation, cellular differentiation, and other functions in most cells. http://en.wikipedia.org/wiki/TGFbeta (Photo credit: Wikipedia)

Pioneer Transcription Factors

Pioneer transcription factors take the lead in facilitating cellular reprogramming and responses to environmental cues. Multicellular organisms consist of functionally distinct cellular types produced by differential activation of gene expression. They seek out and bind specific regulatory sequences in DNA. Even though DNA is coated with and condensed into a thick fiber of chromatin. The pioneer factor, discovered by Prof. KS Zaret at UPenn SOM in 1996, he says, endows the competence for gene activity, being among the first transcription factors to engage and pry open the target sites in chromatin.
FoxA factors, expressed in the foregut endoderm of the mouse,are necessary for induction of the liver program. They found that nearly one-third of the DNA sites bound by FoxA in the adult liver occur near silent genes

A Nontranscriptional Role for HIF-1α as a Direct Inhibitor of DNA Replication

ME Hubbi, K Shitiz, DM Gilkes, S Rey,….GL Semenza. Johns Hopkins University SOM
Sci. Signal 2013; 6(262) 10pgs. [DOI: 10.1126/scisignal.2003417]   http:dx.doi.org/10.1126/scisignal.2003417

http://SciSignal.com/A Nontranscriptional Role for HIF-1α as a Direct Inhibitor of DNA Replication/

Many of the cellular responses to reduced O2 availability are mediated through the transcriptional activity of hypoxia-inducible factor 1 (HIF-1). We report a role for the isolated HIF-1α subunit as an inhibitor of DNA replication, and this role was independent of HIF-1β and transcriptional regulation. In response to hypoxia, HIF-1α bound to Cdc6, a protein that is essential for loading of the mini-chromosome maintenance (MCM) complex (which has DNA helicase activity) onto DNA, and promoted the interaction between Cdc6 and the MCM complex. The binding of HIF-1α to the complex decreased phosphorylation and activation of the MCM complex by the kinase Cdc7. As a result, HIF-1α inhibited firing of replication origins, decreased DNA replication, and induced cell cycle arrest in various cell types. To whom correspondence should be addressed. E-mail: gsemenza@jhmi.edu
Citation: M. E. Hubbi, Kshitiz, D. M. Gilkes, S. Rey, C. C. Wong, W. Luo, D.-H. Kim, C. V. Dang, A. Levchenko, G. L. Semenza, A Nontranscriptional Role for HIF-1α as a Direct Inhibitor of DNA Replication. Sci. Signal. 6, ra10 (2013).

Identification of a Candidate Therapeutic Autophagy-inducing Peptide

Nature 2013;494(7436).    http://nature.com/Identification_of_a_candidate_therapeutic_autophagy-inducing_peptide/   http://www.ncbi.nlm.nih.gov/pubmed/23364696
http://www.readcube.com/articles/10.1038/nature11866

Beth Levine and colleagues have constructed a cell-permeable peptide derived from part of an autophagy protein called beclin 1. This peptide is a potent inducer of autophagy in mammalian cells and in vivo in mice and was effective in the clearance of several viruses including chikungunya virus, West Nile virus and HIV-1.

Could this small autophagy-inducing peptide may be effective in the prevention and treatment of human diseases?

PR-Set7 Is a Nucleosome-Specific Methyltransferase that Modifies Lysine 20 of

Histone H4 and Is Associated with Silent Chromatin

K Nishioka, JC Rice, K Sarma, H Erdjument-Bromage, …, D Reinberg.   Molecular Cell, Vol. 9, 1201–1213, June, 2002, Copyright 2002 by Cell Press   http://www.cell.com/molecular-cell/abstract/S1097-2765(02)00548-8

http://www.sciencedirect.com/science/article/pii/S1097276502005488           http://www.ncbi.nlm.nih.gov/pubmed/12086618
http://www.cienciavida.cl/publications/b46e8d324fa4aefa771c4d6ece4d2e27_PR-Set7_Is_a_Nucleosome-Specific.pdf

We have purified a human histone H4 lysine 20methyl-transferase and cloned the encoding gene, PR/SET07. A mutation in Drosophila pr-set7 is lethal: second in-star larval death coincides with the loss of H4 lysine 20 methylation, indicating a fundamental role for PR-Set7 in development. Transcriptionally competent regions lack H4 lysine 20 methylation, but the modification coincided with condensed chromosomal regions polytene chromosomes, including chromocenter euchromatic arms. The Drosophila male X chromosome, which is hyperacetylated at H4 lysine 16, has significantly decreased levels of lysine 20 methylation compared to that of females. In vitro, methylation of lysine 20 and acetylation of lysine 16 on the H4 tail are competitive. Taken together, these results support the hypothesis that methylation of H4 lysine 20 maintains silent chromatin, in part, by precluding neighboring acetylation on the H4 tail.

Next-Generation Sequencing vs. Microarrays

Shawn C. Baker, Ph.D., CSO of BlueSEQ
GEN Feb 2013
With recent advancements and a radical decline in sequencing costs, the popularity of next generation sequencing (NGS) has skyrocketed. As costs become less prohibitive and methods become simpler and more widespread, researchers are choosing NGS over microarrays for more of their genomic applications. The immense number of journal articles citing NGS technologies it looks like NGS is no longer just for the early adopters. Once thought of as cost prohibitive and technically out of reach, NGS has become a mainstream option for many laboratories, allowing researchers to generate more complete and scientifically accurate data than previously possible with microarrays.

Gene Expression

Researchers have been eager to use NGS for gene expression experiments for a detailed look at the transcriptome. Arrays suffer from fundamental ‘design bias’ —they only return results from those regions for which probes have been designed. The various RNA-Seq methods cover all aspects of the transcriptome without any a priori knowledge of it, allowing for the analysis of such things as novel transcripts, splice junctions and noncoding RNAs. Despite NGS advancements, expression arrays are still cheaper and easier when processing large numbers of samples (e.g., hundreds to thousands).
Methylation
While NGS unquestionably provides a more complete picture of the methylome, whole genome methods are still quite expensive. To reduce costs and increase throughput, some researchers are using targeted methods, which only look at a portion of the methylome. Because details of exactly how methylation impacts the genome and transcriptome are still being investigated, many researchers find a combination of NGS for discovery and microarrays for rapid profiling.

Diagnostics

They are interested in ease of use, consistent results, and regulatory approval, which microarrays offer. With NGS, there’s always the possibility of revealing something new and unexpected. Clinicians aren’t prepared for the extra information NGS offers. But the power and potential cost savings of NGS-based diagnostics is alluring, leading to their cautious adoption for certain tests such as non-invasive prenatal testing.
Cytogenetics
Perhaps the application that has made the least progress in transitioning to NGS is cytogenetics. Researchers and clinicians, who are used to using older technologies such as karyotyping, are just now starting to embrace microarrays. NGS has the potential to offer even higher resolution and a more comprehensive view of the genome, but it currently comes at a substantially higher price due to the greater sequencing depth. While dropping prices and maturing technology are causing NGS to make headway in becoming the technology of choice for a wide range of applications, the transition away from microarrays is a long and varied one. Different applications have different requirements, so researchers need to carefully weigh their options when making the choice to switch to a new technology or platform. Regardless of which technology they choose, genomic researchers have never had more options.

Sequencing Hones In on Targets

Greg Crowther, Ph.D.

GEN Feb 2013

Cliff Han, PhD, team leader at the Joint Genome Institute in the Los Alamo National Lab, was one of a number of scientists who made presentations regarding target enrichment at the “Sequencing, Finishing, and Analysis in the Future” (SFAF) conference in Santa Fe, which was co-sponsored by the Los Alamos National Laboratory and DOE Joint Genome Institute. One of the main challenges is that of target enrichment: the selective sequencing of genomic or transcriptomic regions. The polymerase chain reaction (PCR) can be considered the original target-enrichment technique and continues to be useful in contexts such as genome finishing. “One target set is the unique gaps—the gaps in the unique sequence regions. Another is to enrich the repetitive sequences…ribosomal RNA regions, which together are about 5 kb or 6 kb.” The unique-sequence gaps targeted for PCR with 40-nucleotide primers complementary to sequences adjacent to the gaps, did not yield the several-hundred-fold enrichment expected based on previously published work. “We got a maximum of 70-fold enrichment and generally in the dozens of fold of enrichment,” noted Dr. Han.

“We enrich the genome, put the enriched fragments onto the Pacific Biosciences sequencer, and sequence the repeats,” continued Dr. Han. “In many parts of the sequence there will be a unique sequence anchored at one or both ends of it, and that will help us to link these scaffolds together.” This work, while promising, will remain unpublished for now, as the Joint Genome Institute has shifted its resources to other projects.
At the SFAF conference Dr. Jones focused on going beyond basic target enrichment and described new tools for more efficient NGS research. “Hybridization methods are flexible and have multiple stop-start sites, and you can capture very large sizes, but they require library prep,” said Jennifer Carter Jones, Ph.D., a genomics field applications scientist at Agilent. “With PCR-based methods, you have to design PCR primers and you’re doing multiplexed PCR, so it’s limited in the size that you can target. But the workflow is quick because there’s no library preparation; you’re just doing PCR.” She discussed Agilent’s recently acquired HaloPlex technology, a hybrid system that includes both a hybridization step and a PCR step. Because no library preparation is required, sequencing results can be obtained in about six hours, making it suitable for clinical uses. However, the hybridization step allows capture of targets of up to 5 megabases—longer than purely PCR-based methods can deliver. The Agilent talk also provided details on the applications of SureSelect, the company’s hybridization technology, to Methyl-Seq and RNA-Seq research. With this technology, 120-mer baits hybridize to targets, then are pulled down with streptavidin-coated magnetic beads.
These are selections from the SFAF conference, which is expected to be a boost to work on the microbiome, and lead to infectious disease therapeutic approaches.

Summary

We have finished a breathtaking ride through the genomic universe in several sessions.  This has been a thorough review of genomic structure and function in cellular regulation.  The items that have been discussed and can be studied in detail include:

  1.  the classical model of the DNA structure
  2. the role of ubiquitinylation in managing cellular function and in autophagy, mitophagy, macrophagy, and protein degradation
  3. the nature of the tight folding of the chromatin in the nucleus
  4. intramolecular bonds and short distance hydrophobic and hydrophilic interactions
  5. trace metals in molecular structure
  6. nuclear to membrane interactions
  7. the importance of the Human Genome Project followed by Encode
  8. the Fractal nature of chromosome structure
  9. the oligomeric formation of short sequences and single nucletide polymorphisms (SNPs)and the potential to identify drug targets
  10. Enzymatic components of gene regulation (ligase, kinases, phosphatases)
  11. Methods of computational analysis in genomics
  12. Methods of sequencing that have become more accurate and are dropping in cost
  13. Chromatin remodeling
  14. Triplex and quadruplex models not possible to construct at the time of Watson-Crick
  15. sequencing errors
  16. propagation of errors
  17. oxidative stress and its expected and unintended effects
  18. origins of cardiovascular disease
  19. starvation and effect on protein loss
  20. ribosomal damage and repair
  21. mitochondrial damage and repair
  22. miscoding and mutational changes
  23. personalized medicine
  24. Genomics to the clinics
  25. Pharmacotherapy horizons
  26. driver mutations
  27. induced pluripotential embryonic stem cell (iPSCs)
  28. The association of key targets with disease
  29. The real possibility of moving genomic information to the bedside
  30. Requirements for the next generation of electronic health record to enable item 29

Other Related articles on this Open Access Online Scientific Journal, include the following:

https://pharmaceuticalintelligence.com/2013/01/14/oogonial-stem-cells-purified-a-view-towards-the-future-of-reproductive-biology/   SSaha

https://pharmaceuticalintelligence.com/2012/10/22/blood-vessel-generating-stem-cells-discovered/ RSaxena

https://pharmaceuticalintelligence.com/2012/08/22/a-possible-light-by-stem-cell-therapy-in-painful-dark-of-osteoarthritis-kartogenin-a-small-molecule-differentiates-stem-cells-to-chondrocyte-healthy-cartilage-cells/   ASarkar and RSaxena

https://pharmaceuticalintelligence.com/2012/08/07/human-embryonic-pluripotent-stem-cells-and-healing-post-myocardial-infarction/    LHB

https://pharmaceuticalintelligence.com/2013/02/03/genome-wide-detection-of-single-nucleotide-and-copy-number-variation-of-a-single-human-cell/  SJWilliams

https://pharmaceuticalintelligence.com/2013/01/09/gene-therapy-into-healthy-heart-muscle-reprogramming-scar-tissue-in-damaged-hearts/ ALev-Ari

https://pharmaceuticalintelligence.com/2013/01/03/differentiation-therapy-epigenetics-tackles-solid-tumors/  SJWilliams

https://pharmaceuticalintelligence.com/2012/12/09/naotech-therapy-for-breast-cancer/  TBarliya

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