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Archive for the ‘Embryology’ Category


Embryo Stem Cells Out of Skin

Reporter: Irina Robu, PhD

Researchers at Hebrew University identified a set of genes able to transform murine skin cells into three cell types such as the embryo itself, the placenta and extraembryonic tissue i.e. umbilical cord which was published in the journal Cell Stem Cell.

Dr. Oren Ram, Institute of Life Science at Hebrew University, Prof. Tommy Kaplan, School of Computer Science and Engineering found a new combination of five genes that once inserted into skin cells, reprogram the cells into each of three early embryonic cell types. Researchers identified that the gene “Eomes” pushes the cell toward placental stem-cell identity and placental development, whereas the “Esrrb” gene arranges fetus stem cells development through the temporary procurement of an extraembryonic stem cell identity.The team used this to examine the molecular forces that oversee cell fate decisions for skin cell reprogramming and the natural process of embryonic development.

Even though this groundbreaking research could provide a path toward creating entire human embryos from human cell skin cells without need for sperm of organs, that is still a long way in the future. However, for now this work can have large implications for modeling embryonic disease and placental dysfunctions in addition to solving infertility problems by creating human embryos in a petri dish.

SOURCE
https://www.jpost.com/OMG/A-baby-from-skin-cells-Israeli-team-makes-embryo-stem-cells-out-of-skin-588531

 

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Reporter and Curator: Dr. Sudipta Saha, Ph.D.

 

Leigh syndrome is one of the hundreds of so-called mitochondrial diseases, which are caused by defects in the mitochondria that produce 90 percent of the body’s energy. These disorders are rare; about 1,000 to 4,000 babies in the United States are born with one every year. But they are devastating and can result in grave impairment of nearly any bodily system. They are largely untreatable, uniformly incurable and very difficult to screen.

 

Leigh syndrome is a terrible disease. It emerges shortly after birth and claims one major organ after another. Movement becomes difficult, and then impossible. A tracheotomy and feeding tube are often necessary by toddlerhood, and as the disease progresses, lungs frequently have to be suctioned manually. Most children with the condition die by the age of 5 or 6.

 

Scientists have devised a procedure called mitochondrial replacement therapy (M.R.T.) that involves transplanting the nucleus of an affected egg (mitochondrial diseases are passed down from the mother’s side) into an unaffected one whose nucleus has been removed. The procedure is sometimes called “three-parent in vitro fertilization”. Mitochondria contain a minuscule amount of DNA, any resulting embryo would have mitochondrial DNA from the donor egg and nuclear DNA from each of its parents.

 

After decades of careful study in cell and animal research M.R.T. is now finally being tested in human clinical trials by doctors in Britain (no births confirmed yet officially). In the United States, however, this procedure is effectively illegal. M.R.T. does not involve altering any genetic code. Defective mitochondria are swapped out for healthy ones.

 

Mitochondrial DNA governs only a handful of basic cellular functions. It is separate from nuclear DNA, which helps determine individual traits like physical appearance, intelligence and personality. That means M.R.T. cannot be used to produce the genetically enhanced “designer babies” and thus should be allowed in humans. But, there is no way to know how safe or effective M.R.T. is until doctors and scientists test it in humans.

 

References:

 

 

https://pharmaceuticalintelligence.com/2016/10/07/the-three-parent-technique-to-avoid-mitochondrial-disease-in-embryo/

 

 

 

 

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Reporter and Curator: Dr. Sudipta Saha, Ph.D.

 

Hepatitis B virus can cause serious, long-term health problems, such as liver disease and cancer, and can spread from mother-to-child during delivery. According to the latest estimates from the World Health Organization (WHO), approximately 257 million people in 2015 were living with the virus. Countries in Asia have a high burden of hepatitis B. There is no cure, and antiviral drugs used to treat the infection usually need to be taken for life.

 

To prevent infection, WHO recommends that all newborns receive their first dose of hepatitis B vaccine within 24 hours of delivery. Infants born to hepatitis B-infected mothers are also given protective antibodies called hepatitis B immune globulin (HBIG). However, mother-to-child transmission can still occur in women with high levels of virus in their blood, as well as those with mutated versions of the virus.

 

Tenofovir disoproxil fumarate (TDF), an antiviral drug commonly prescribed to treat hepatitis B infection, does not significantly reduce mother-to-child transmission of hepatitis B virus when taken during pregnancy and after delivery, according to a phase III clinical trial in Thailand funded by the National Institutes of Health. The study tested TDF therapy in addition to the standard preventative regimen — administration of hepatitis B vaccine and protective antibodies at birth — to explore the drug’s potential effects on mother-to-child transmission rates. The results appear in the New England Journal of Medicine.

 

The present study was conducted at 17 hospitals of the Ministry of Public Health in Thailand. It screened more than 2,500 women for eligibility and enrolled 331 pregnant women with hepatitis B. The women received placebo (163) or TDF (168) at intervals from 28 weeks of pregnancy to two months after delivery. All infants received standard hepatitis B preventatives given in Thailand, which include HBIG at birth and five doses of the hepatitis B vaccine by age 6 months (which differs from the three doses given in the United States). A total of 294 infants (147 in each group) were followed through age 6 months.

 

Three infants in the placebo group had hepatitis B infection at age 6 months, compared to zero infants in the TDF treatment group. Given the unexpectedly low transmission rate in the placebo group, the researchers concluded that the addition of TDF to current recommendations did not significantly reduce mother-to-child transmission of the virus.

 

According to the study, the clinical trial had enough participants to detect statistical differences if the transmission rate in the placebo group reached at least 12 percent, a rate observed in previous studies. Though the reasons are unknown, the researchers speculate that the lower transmission rate seen in the study may relate to the number of doses of hepatitis B vaccine given to infants in Thailand, lower rates of amniocentesis and Cesarean section deliveries in this study, or the lower prevalence of mutated viruses that result in higher vaccine efficacy in Thailand compared to other countries.

 

References:

 

https://www.nih.gov/news-events/news-releases/antiviral-drug-not-beneficial-reducing-mother-child-transmission-hepatitis-b-when-added-existing-preventatives

 

https://www.ncbi.nlm.nih.gov/pubmed/29514030

 

https://www.ncbi.nlm.nih.gov/pubmed/29514035

 

https://www.ncbi.nlm.nih.gov/pubmed/25240752

 

https://www.ncbi.nlm.nih.gov/pubmed/28188612

 

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Reporter and Curator: Dr. Sudipta Saha, Ph.D.

 

Scientists at the Stanford University School of Medicine have completed the first-ever characterization of the meticulously timed immune system changes in women that occur during pregnancy. The findings were published in Science Immunology revealed that there is an immune clock of pregnancy and suggest it may help doctors predict preterm birth.

 

The timing of immune system changes follows a precise and predictable pattern in normal pregnancy. Although physicians have long known that the expectant mother’s immune system adjusts to prevent her body from rejecting the fetus, no one had investigated the full scope of these changes, nor asked if their timing was tightly controlled.

 

Nearly 10 percent of U.S. infants are born prematurely, arriving three or more weeks early, but physicians lack a reliable way to predict premature deliveries. Previous research at Stanford and other places suggested that inflammatory immune responses may help in triggering early labor. It suggested that if scientists identify an immune signature of impending preterm birth, they should be able to design a blood test to detect it.

 

The researchers used mass cytometry, a technique developed at Stanford, to simultaneously measure up to 50 properties of each immune cell in the blood samples. They counted the types of immune cells, assessed what signaling pathways were most active in each cell, and determined how the cells reacted to being stimulated with compounds that mimic infection with viruses and bacteria.

 

The researchers developed an algorithm that captures the immunological timeline during pregnancy that both validates previous findings and sheds new light on immune cell interaction during gestation. By defining this immunological chronology during normal term pregnancy, they can now begin to determine which alterations associate with pregnancy-related pathologies.

 

With an advanced statistical modeling technique, introduced for the first time in this study, the scientists then described in detail how the immune system changes throughout pregnancy. Instead of grouping the women’s blood samples by trimester for analysis, the model treated gestational age as a continuous variable, allowing the researchers to account for the exact time during pregnancy at which each sample was taken. The mathematical model also incorporated knowledge from the existing scientific literature of how immune cells behave in nonpregnant individuals to help determine which findings were most likely to be important.

 

The study confirmed immune features of pregnancy that were already known. Such as the scientists saw that natural killer cells and neutrophils have enhanced action during pregnancy. The researchers also uncovered several previously unappreciated features of how the immune system changes, such as the finding that activity of the STAT5 signaling pathway in CD4+T cells progressively increases throughout pregnancy on a precise schedule, ultimately reaching levels much higher than in nonpregnant individuals. The STAT5 pathway is involved in helping another group of immune cells, regulatory T cells, to differentiate. Interestingly, prior research in animals has indicated that regulatory T cells are important for maintaining pregnancy.

 

The next step will be to conduct similar research using blood samples from women who deliver their babies prematurely to see where their trajectories of immune function differ from normal.

 

This study revealed a precisely timed chronology of immune adaptations in peripheral blood over the course of a term pregnancy. This finding was enabled by high-content, single-cell mass cytometry coupled with a csEN algorithm accounting for the modular structure of the immune system and previous knowledge. The study provided the conceptual backbone and the analytical framework to examine whether disruption of this chronology is a diagnostically useful characteristic of preterm birth and other pregnancy-related pathologies.

 

References:

 

http://immunology.sciencemag.org/content/2/15/eaan2946.full

 

http://med.stanford.edu/news/all-news/2017/09/immune-system-changes-during-pregnancy-are-precisely-timed.html

 

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3078586/

 

http://www.nature.com/nm/journal/v19/n5/full/nm.3160.html?foxtrotcallback=true

 

https://www.ncbi.nlm.nih.gov/pubmed/14758358

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Reporter and Curator: Dr. Sudipta Saha, Ph.D.

 

Babies born at or before 25 weeks have quite low survival outcomes, and in the US it is the leading cause of infant mortality and morbidity. Just a few weeks of extra ‘growing time’ can be the difference between severe health problems and a relatively healthy baby.

 

Researchers from The Children’s Hospital of Philadelphia (USA) Research Institute have shown it’s possible to nurture and protect a mammal in late stages of gestation inside an artificial womb; technology which could become a lifesaver for many premature human babies in just a few years.

 

The researchers took eight lambs between 105 to 120 days gestation (the physiological equivalent of 23 to 24 weeks in humans) and placed them inside the artificial womb. The artificial womb is a sealed and sterile bag filled with an electrolyte solution which acts like amniotic fluid in the uterus. The lamb’s own heart pumps the blood through the umbilical cord into a gas exchange machine outside the bag.

 

The artificial womb worked in this study and after just four weeks the lambs’ brains and lungs had matured like normal. They had also grown wool and could wiggle, open their eyes, and swallow. Although this study is looking incredibly promising but getting the research up to scratch for human babies still requires a big leap.

 

Nevertheless, if all goes well, the researchers hope to test the device on premature humans within three to five years. Potential therapeutic applications of this invention may include treatment of fetal growth retardation related to placental insufficiency or the salvage of preterm infants threatening to deliver after fetal intervention or fetal surgery.

 

The technology may also provide the opportunity to deliver infants affected by congenital malformations of the heart, lung and diaphragm for early correction or therapy before the institution of gas ventilation. Numerous applications related to fetal pharmacologic, stem cell or gene therapy could be facilitated by removing the possibility for maternal exposure and enabling direct delivery of therapeutic agents to the isolated fetus.

 

References:

 

https://www.nature.com/articles/ncomms15112

 

 

https://www.sciencealert.com/researchers-have-successfully-grown-premature-lambs-in-an-artificial-womb

 

http://www.npr.org/sections/health-shots/2017/04/25/525044286/scientists-create-artificial-womb-that-could-help-prematurely-born-babies

 

http://www.telegraph.co.uk/science/2017/04/25/artificial-womb-promises-boost-survival-premature-babies/

 

https://www.theguardian.com/science/2017/apr/25/artificial-womb-for-premature-babies-successful-in-animal-trials-biobag

 

http://www.theblaze.com/news/2017/04/25/new-artificial-womb-technology-could-keep-babies-born-prematurely-alive-and-healthy/

 

http://www.theverge.com/2017/4/25/15421734/artificial-womb-fetus-biobag-uterus-lamb-sheep-birth-premie-preterm-infant

 

http://www.abc.net.au/news/2017-04-26/artificial-womb-could-one-day-keep-premature-babies-alive/8472960

 

https://www.theatlantic.com/health/archive/2017/04/preemies-floating-in-fluid-filled-bags/524181/

 

http://www.independent.co.uk/news/health/artificial-womb-save-premature-babies-lives-scientists-create-childrens-hospital-philadelphia-nature-a7701546.html

 

https://www.cnet.com/news/artificial-womb-births-premature-lambs-human-infants/

 

https://science.slashdot.org/story/17/04/25/2035243/an-artificial-womb-successfully-grew-baby-sheep—-and-humans-could-be-next

 

http://newatlas.com/artificial-womb-premature-babies/49207/

 

https://www.geneticliteracyproject.org/2015/06/12/artificial-wombs-the-coming-era-of-motherless-births/

 

http://news.nationalgeographic.com/2017/04/artificial-womb-lambs-premature-babies-health-science/

 

https://motherboard.vice.com/en_us/article/artificial-womb-free-births-just-got-a-lot-more-real-cambridge-embryo-reproduction

 

http://www.disclose.tv/news/The_Artificial_Womb_Is_Born_Welcome_To_The_WORLD_Of_The_MATRIX/114199

 

 

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Embryonic Stem Cell differentiation

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Plant Homeo Domain Finger Protein 8 Regulates Mesodermal and Cardiac Differentiation of Embryonic Stem Cells Through Mediating the Histone Demethylation of pmaip1

Yan Tang1, Ya-Zhen Hong1, Hua-Jun Bai1, Qiang Wu1, Charlie Degui Chen2, Jing-Yu Lang1, Kenneth R. Boheler3 and Huang-Tian Yang1,4,*

STEM CELLS: 18 APR 2016     http://dx.doi.org:/10.1002/stem.2333

Histone demethylases have emerged as key regulators of biological processes. The H3K9me2 demethylase plant homeo domain finger protein 8(PHF8), for example, is involved in neuronal differentiation, but its potential function in the differentiation of embryonic stem cells (ESCs) to cardiomyocytes is poorly understood. Here, we explored the role of PHF8 during mesodermal and cardiac lineage commitment of mouse ESCs (mESCs). Using a phf8 knockout (ph8-/Y) model, we found that deletion ofphf8 in ESCs did not affect self-renewal, proliferation or early ectodermal/endodermal differentiation, but it did promote the mesodermal lineage commitment with the enhanced cardiomyocyte differentiation. The effects were accompanied by a reduction in apoptosis through a caspase 3-independent pathway during early ESC differentiation, without significant differences between differentiating wide-type (ph8+/Y) and ph8-/Y ESCs in cell cycle progression or proliferation. Functionally, PHF8 promoted the loss of a repressive mark H3K9me2 from the transcription start site of a proapoptotic gene pmaip1 and activated its transcription. Furthermore, knockdown ofpmaip1 mimicked the phenotype of ph8-/Y by showing the decreased apoptosis during early differentiation of ESCs and promoted mesodermal and cardiac commitment, while overexpression of pmaip1 or phf8 rescued the phenotype of ph8-/Y ESCs by increasing the apoptosis and weakening the mesodermal and cardiac differentiation. These results reveal that the histone demethylase PHF8 regulates mesodermal lineage and cell fate decisions in differentiating mESCs through epigenetic control of the gene critical to programmed cell death pathways. Stem Cells2016

 

Significance Statement

Embryonic stem cells (ESCs) have the unique ability to differentiate into derivatives of all three germ layers both in vitro and in vivo. Thus, ESCs provide a unique model for the study of early embryonic development. We report here previously unrecognized effects of histone demethylase plant homeo domain finger protein 8 (PHF8) on mesodermal and early cardiac differentiation. This effect is resulted from the regulation of PHF8 on apoptosis through activating the transcription of pro-apoptotic gene pmaip1. These findings extend the knowledge in understanding of the epigenetic modification in apoptosis during ESC differentiation and of the link between apoptosis and cell lineage decision as well as cardiogenesis.

 

Embryonic stem cells (ESCs) have the unique ability to differentiate into derivatives of all three germ layers both in vitro and in vivo. Due to this plasticity, mechanisms controlling cell autonomous and regulatory events critical to in vivo mammalian development have benefitted from the in vitro study of differentiating ESCs [1, 2]. Early embryogenesis and cavity formation as well as early ESC differentiation, for example, are accompanied by a reduction in proliferation and increased apoptosis [3-5]. Withdrawal of leukemia inhibitory factor (LIF) from mouse embryonic stem cells (mESCs) cultivated in vitro causes approximately 20%-30% of the cells to die by spontaneous (constitutive) apoptosis [4, 5]. This occurs secondary to the induction of cleaved caspase 3 [3] and apoptosis-inducing factor (AIF)-complex proteins [6]. Blockade of spontaneous apoptosis in vitro by a p38 mitogen-activated protein kinase (MAPK) inhibitor alters the differentiation markers and increases the abundance of both antiapoptotic proteins (Bcl-2, Bcl-XL) and Ca2+-binding proteins [4, 7]. In addition, Ca2+ released from type 3 inositol 1, 4, 5-trisphosphate receptors (IP3R3) negatively regulates this apoptotic response, which in turn modulates the mesodermal lineage commitment of early differentiating mESCs [5]. These findings explain, in part, how apoptosis contributes to specific lineage commitment during early development. However, in contrast to the relatively advanced knowledge of signaling pathways [8], little is known about the contribution of epigenetic regulators, especially, histone lysine demethylases (KDMs), in the regulation of apoptosis during ESC differentiation and how the affected programmed cell death by KDMs contributes to the lineage commitment.

Epigenetic regulators and dynamic histone modifications by KDMs are emerging as important players in ESC fate decisions [9]. Histone modifications coordinate transient changes in gene transcription and help maintaining differential patterns of gene expression during differentiation [10-13]. The molecular and biological functions of many KDMs, however, remain enigmatic during ESC differentiation. PHF8, an X-linked gene encoding an evolutionarily conserved histone demethylase harboring an N-terminal plant homeo domain (PHD) and an active jumonji-C domain (JmjC), is able to catalyze demethylation from histones [14, 15]. It is actively recruited to and enriched in the promoters of transcriptionally active genes [14], and it functions to maintain the active state of rRNA through the removal of the repressive H3K9me2 methylation mark at the active rRNA promoters. Mutation of PHF8 is associated with X-linked mental retardation with cleft lip/cleft palate in human [16-18]. Knockdown of phf8 in mouse embryonic carcinoma P19 cells impairs neuronal differentiation [19] and leads to brain defects in zebrafish by directly regulating the expression of the homeo domain transcription factor MSX1/MSXB [20]. However, the precise function of PHF8 in the regulation of lineage differentiation derived from other germ layers remains to be identified.

Here, we report previously unrecognized effects of the PHF8 histone demethylase on germ layer commitment and differentiation of mESCs. The results are based on an assessment of early steps of differentiation to mesodermal lineages and cardiomyocytes using phf8 knockout (phf8-/Y) and wild-type (phf8+/Y) mESCs. The data show that PHF8 regulates gene transcription of a proapoptotic gene pmaip1 (also named Noxa) [21]. Activation or repression of pmaip1 controlled by PHF8 ultimately determines mESC lineage commitment through the regulation of caspase 3-independent apoptosis during mesodermal and cardiac differentiation. Our data reveal that PHF8-mediated the demethylation of histone proteins coordinates ESC lineage commitment through the regulation of apoptosis in early differentiating ESCs.

 

Deletion of phf8 Promotes Mesodermal and Cardiac Lineage Commitment

The PHF8 protein was detectable in undifferentiated ESCs, but its abundance significantly increased within one day of LIF withdrawal. Then it gradually decreased to a level at day 5 lower than that observed in the undifferentiated ESCs (Fig. 1A).    http://onlinelibrary.wiley.com/store/10.1002/stem.2333/asset/image_t/stem2333-fig-0001-t.gif

 

Figure 1. Plant homeo domain finger protein 8 (PHF8) regulates the mesodermal and early cardiac differentiation of mouse embryonic stem cells (mESCs). (A): Western blot analysis of PHF8 expression in undifferentiated and differentiating ESCs. n = 3. (B): quantitative RT-PCR (qRT-PCR) analysis of pluripotency markers nanog, rex1, sox2, and oct4. n = 8. (C): qRT-PCR analysis of gene expression of pluripotency marker oct4; early mesodermal markers brachyury (T), gsc, eomes, and mesp1; cardiovascular progenitor markers flk-1 and nrp1; and the cardiac transcription factors hand1, tbx5, and mef2c during ESC differentiation. n = 5. (D): qRT-PCR analysis of the early ectodermal markersnestin and fgf5 during ESC differentiation. n = 3. (E): qRT-PCR analysis of early endodermal markers afp, foxa2, sox17, and gata4 during ESC differentiation. n = 3. Data are presented as mean ± SEM *, p < .05; **, p < .01; ***, p < .001 compared with the corresponding phf8+/Y value.

To determine the significance of phf8 gene expression on ESC fate decision, we knocked out the X-chromosome-encoded phf8 gene in one allele of male SCR012 ESCs by deletion of exons 7 and 8 through Cre-mediated recombination (Supporting Information Fig. S1A). Gene inactivation was confirmed by the lack of Phf8 mRNA and PHF8 protein expression in these targeted ESCs (Supporting Information Fig. S1B). Transcripts for pluripotency marker genes nanog, rex1 (zfp42), sox2, and oct4 (pou5f1) were not significantly different between phf8+/Y and phf8-/Y ESCs (Fig. 1B). No significant difference was observed in cell morphology (Supporting Information Fig. S1C) of undifferentiated phf8+/Y and phf8-/Y ESCs or in alkaline phosphatase activity (Supporting Information Fig. S1D). Immunofluorescence staining confirmed that the expression of pluripotency marker SOX2 and SSEA-1 did not differ between the phf8+/Y and phf8-/Y ESCs (Supporting Information Fig. S1E). These results indicate that phf8 may be dispensable for normal growth and maintenance of mESCs.

We then analyzed the role of PHF8 in the mesodermal and cardiac lineage commitment. By microarray analysis of differentiating phf8+/Y and phf8-/Y cells from days 0, 1, to 3.5, we found a significant decrease in transcripts for pluripotency markers, accompanied by a significant increase in transcripts for ectoderm, mesoderm and endoderm, while in phf8-/Y cells some transcripts for mesodermal and cardiac lineage commitment were significantly enhanced compared with those in phf8+/Y cells (Supporting Information Fig. S2A). These differentiation-dependent changes in transcript abundance were confirmed by qRT-PCR for early mesodermal markers brachyury (T) [28], goosecoid (gsc),eomes[29], and mesp1[30], cardiovascular progenitor marker flk-1[31, 32] and neuropilin 1 (nrp1) [33]. Early cardiac transcription factors, including myocyte enhancer factor 2C (mef2c) [34], hand1[35], and tbx5[36, 37] were also up-regulated in phf8-/Y cells at differentiation day 5, while no difference in the expression levels of pluripotent markersoct4 (Fig. 1C), rex1, and nanog (Supporting Information Fig. S2B) were detected between phf8+/Y and phf8-/Y cells at the time points examined.

Because mESCs can differentiate into all three germ layers, we also examined whether phf8 affected ectodermal and endodermal differentiation. qRT-PCR analysis did not show any significantly difference in the expression of early ectodermal markers nestin and fgf5 between the phf8+/Y and phf8-/Y cells (Fig. 1D). Moreover, in the induced early ectodermal differentiation system [23], the expression of ectodermal markers nestin, fgf5, and pax6 were comparable between the phf8+/Y and phf8-/Y cells (Supporting Information Fig. S3A). Besides, the expression of endodermal markers afp, foxa2, sox17, and gata4 were not significantly different between the phf8+/Y and phf8-/Y cells (Fig.1E). Consistently, the expression of endodermal markers foxa2, sox17, and gata4 were comparable during induced endodermal differentiation [24] between the phf8+/Y andphf8-/Y cells (Supporting Information Fig. S3B). Thus, phf8 appears not to affect early ectodermal and endodermal differentiation.

The increased mesodermal and cardiac marker expressions were associated with a significant increase in the total number of cardiac progenitors and cardiomyocytes in differentiating phf8-/Y cells. By flow cytometry analysis, marked increases in the population of FLK-1 positive (FLK-1+) cells were detected in phf8-/Y cells at differentiation day 3 and day 4 (Fig. 2A). Consistently, the percentage of contracting EBs was higher in phf8-/Y cells than in phf8+/Y cells (Fig. 2B). The transcripts for progenitor marker nrp1, early cardiac transcription factor tbx5, and cardiac specific genes tnnt2, myh6, myl2, and gja1 were higher in phf8-/Y EBs than those in phf8+/Y ones (Fig. 2C). The areas of immunostained EBs positive for the cardiac cytoskeletal and myofilamental proteins α-actinin and TNNT2 were also greater in phf8-/Y than in phf8+/Y EBs (Fig. 2D). Flow cytometry analysis of MYH6+ (Fig. 2E) and TNNT2+ (Fig. 2F) cells at differentiation day 9 further confirmed the increase of cardiomyocytes in phf8-/Y cells. Taken together, these data indicate that the phf8 deletion facilitates the differentiation of mesodermal and cardiac linage commitment.

Figure 2. phf8 deletion promotes cardiac differentiation of mouse embryonic stem cells (mESCs). (A): Left, representative flow cytometry plots showing FLK-1 expression at differentiation day 3 (n = 6), day 4 and day 5 (n = 3 each). Right, the quantification of flow cytometry data. (B): Differentiation profile of cardiomyocytes during embryoid bodies (EB) outgrowth. n = 6. (C): qRT-PCR analysis of ESCs for the expression of cardiac markers at differentiation day 14. n = 3. (D): Immunofluorescence analysis of TNNT2 and α-actinin in day 14 EBs. Scale bars = 400 μm. (E) Flow cytometry analysis of MYH6 positive cells and (F) TNNT2 positive cells in day 9 EBs. n = 3 each. Data are presented as mean ± SEM *, p < .05; **, p < .01; ***, p < .001 compared with the corresponding phf8+/Y value.   http://onlinelibrary.wiley.com/store/10.1002/stem.2333/asset/image_t/stem2333-fig-0002-t.gif

PHF8 Inactivation Increases Cell Viability but not Proliferation of the Differentiating ESCs

Differentiation of both phf8+/Y and phf8-/Y ESCs via EB formation produced normal round shaped EBs but, by day 3, phf8-/Y EBs were larger than those generated from phf8+/YESCs, and the size differences were visibly obvious at differentiation days 5 and 7 (Fig. 3A). Although no significant differences in cell viability could be demonstrated between undifferentiated phf8+/Y and phf8-/Y ESCs (Fig. 3B), the viability of phf8-/Y cells was significantly greater than that in phf8+/Y cells at differentiation days 3 to 7 (Fig. 3C). However, no significant change in BrdU staining was detected by flow cytometry between phf8+/Y and phf8-/Y ESCs at differentiation days 0, 3, or 5 (Fig. 3D). Moreover, no significant difference in the cell cycle distribution between the differentiating Phf8+/Y and Phf8-/Y ESCs was detected, although the percentage of cells in S phase gradually decreased while those in G1 phase increased upon differentiation (Fig. 3E). Knockout of phf8 thus increases cell numbers in the early differentiating ESCs through the improvement of cell viability without changes in cell proliferation or cell cycle progression.

Figure 3. phf8 deletion increases cell viability in differentiating mouse embryonic stem cells (mESCs) without affecting cell proliferation. (A): Left, phase-contrast images of embryoid bodies (EB) morphology during EB formation from ESCs. Scale bar = 200 μm. Right, the diameter of EB formed from ESCs. (B): Cell viability of undifferentiated and (C): differentiating ESCs analyzed by MTT assay for seven consecutive days. n = 3. (D): Flow cytometry analysis of BrdU positive proportion of undifferentiated (n = 4) and differentiating ESCs at day 3 and day 5.n = 5 each. (E): Flow cytometry analysis of cell cycle distribution by propidium iodide (PI) staining at differentiation day 3 (n = 6) and day 5 (n = 3). Data are presented as mean ± SEM *, p < .05; ***, p < .001 compared with the corresponding phf8+/Y value.   http://onlinelibrary.wiley.com/store/10.1002/stem.2333/asset/image_t/stem2333-fig-0003-t.gif

PHF8 Regulates Apoptosis During the Early Stage of Cardiac Lineage Commitment

We then examined whether cell death might account for the differences in the cell viability observed between the differentiating phf8+/Y and phf8-/Y ESCs. In undifferentiated ESCs, no significant difference was demonstrated with Annexin V (an early apoptosis marker) staining, TUNEL assay, total DNA fragmentation or caspase 3 protein cleavage between phf8+/Y and phf8-/Y cells (Fig. 4A–4C, 4E). In contrast, Annexin V staining (Fig. 4A) and TUNEL assay (Fig. 4B) showed significant decreases in the number of apoptotic cells in phf8-/Y ESCs at differentiation days 3 and 5 compared with those in phf8+/Y cells. Genomic DNA fragmentation with a pattern typical for apoptosis was detected in phf8+/Y cells at differentiation days 3 and 5, but it was reduced in phf8-/Y cells at the same time points (Fig. 4C). Moreover, approximately 35% of Annexin V+ cells were present in FLK-1+/phf8+/Y cells at differentiation day 4, whereas only 9% of the cells were Annexin V+ in FLK-1+/phf8-/Y cells (Fig. 4D). The ratio of TUNEL+ to either NESTIN+ (ectoderm) or SOX17+ (endoderm) cells did not differ between the phf8+/Y and phf8-/Y cells (Supporting Information Fig. S3C, S3D). In addition, phf8+/Y ESCs at differentiation days 3 and 5 increased the caspase 3 cleavage (Fig. 4E, upper and lower left panels) and the ratio of cleaved caspase 3 to total caspase 3 protein (Fig. 4E, lower right panel). Unexpectedly, the ratio of cleaved caspase 3 to total caspase 3 in phf8-/Y ESCs did not significantly differ from that observed in phf8+/Y ones. Consistently, a significant enhancement of the downstream target PARP1 cleavage [38, 39] was observed at differentiation days 3 and 5, but it was comparable between the phf8+/Y andphf8-/Y cells (Fig. 4F). These data suggest that the cell death associated with phf8 function does not operate through the conventional caspase 3-mediated apoptosis.

Figure 4. Plant homeo domain finger protein 8 (PHF8) regulates apoptosis during the early mouse embryonic stem cells (mESC) differentiation. (A): Left, representative flow cytometry plots showing Annexin V (x-axis), and PI (y-axis) staining in ECSs at differentiation day 0 (n = 4), day 3 (n = 3) and day 5 (n = 7). Right, the quantification of flow cytometry data. (B): Flow cytometry detection of apoptotic responses of TUNEL positive cells at differentiation day 0 (n = 3), day 3 (n = 4), and day 5 (n = 4). (C): DNA laddering analysis at differentiation days 0, 3, and 5. n = 6 each. (D): Cells double stained with FLK-1 and Annexin V were analyzed by flow cytometry at differentiation day 4. n = 3. (E): Western blot analysis of caspase 3 during the mESC differentiation. β-actin was used as a loading control. n = 4. (F): Western blot analysis of PARP1 expression during the differentiation. β-actin was used as a loading control. n = 4. Data are presented as mean ± SEM *, p < .05; ***, p < .001 compared with the corresponding phf8+/Yvalue; #, p < .05; ##, p < .01 compared with the corresponding d0 value.  http://onlinelibrary.wiley.com/store/10.1002/stem.2333/asset/image_t/stem2333-fig-0004-t.gif

pmaip1 is a Direct Target Gene of PHF8 in the Early Differentiating ESCs

To understand how PHF8 might regulate apoptosis during early ESC differentiation, we compared the profiles of apoptosis-related gene transcripts in undifferentiated and early differentiating phf8+/Y and phf8-/Y ESCs using gene expression microarrays. Among the apoptosis-related genes, the transcript to pmaip1, a proapoptotic Bcl-2 family member crucial in fine-turning the cell death decision [21, 40-42], was markedly increased during early differentiation of phf8+/Y cells but it was reduced in phf8-/Y cells at differentiation days 1 and 3.5 (Fig. 5A). These expression patterns were confirmed by qRT-PCR during cardiac differentiation (Fig. 5B), and the results were consistent with the apoptotic pattern observed during the early ESC differentiation (Fig. 4A–4C). In addition, qRT-PCR analysis showed that the expression of pmaip1 did not show any significant difference between the phf8+/Y and phf8-/Y cells during the induced ectodermal (Supporting Information Fig. S3E) or endodermal (Supporting Information Fig. S3F) differentiation.

Figure 5. pmaip1 is a direct target gene of plant homeo domain finger protein 8 (PHF8) in mouse embryonic stem cells (mESCs). (A): Microarray gene expression heat map depicting the expression of apoptosis-related genes at differentiation days 0, 1 and 3.5 in phf8+/Y and phf8-/Y ESCs. The expression values in log2 scale were calculated and presented on the heat map with red representing highly abundant transcripts and green representing poorly abundant transcripts. n = 3. (B): qRT-PCR analysis of pmaip1 during the ESC differentiation. (C): ChIP assay of PHF8 around the TSS of pmaip1 in phf8+/Y andphf8-/Y ESCs at differentiation days 0 and 3. n = 4 each. (D): Western blot analysis of H3K9me2 and H3 in phf8+/Y and phf8-/Y ESCs during the differentiation. H3 was used as a loading control. n = 9. (E): H3K9me2 staining inphf8+/Y and phf8-/Y embryoid bodies (EBs) at differentiation day 1. Scale bars = 25 μm. (F): ChIP assay of H3K9me2 around the TSS of pmaip1 in phf8+/Y andphf8-/Y ESCs at differentiation day 3. n = 4. Data are presented as mean ± SEM. *, p < .05; **, p < .01; ***, p < .001 compared with the corresponding phf8+/Y value or d0.  http://onlinelibrary.wiley.com/store/10.1002/stem.2333/asset/image_t/stem2333-fig-0005-t.gif

A direct link between the PHF8 and pmaip1 was then confirmed by ChIP analysis. We detected the endogenous binding of PHF8 at the transcription start site (TSS, from −45 bp to 104 bp) of pmaip1 in phf8+/Y ESCs and determined that binding was enhanced at differentiation day 3. The binding of PHF8 was not detectable above the IgG control levels in phf8-/Y cells (Fig. 5C). Global methylation (H3K9me2 normalized to H3) was unchanged at differentiation days 3 and 5, but it was significantly enhanced at differentiation day 1 in phf8-/Y cells (Fig. 5D). The augmentation of H3K9me2 methylation in phf8-/Y ESCs was then confirmed by immunostaining at differentiation day 1 (Fig.5E). An increase in the repressive mark of H3K9me2 was also observed at the TSS of pmaip1 in the early differentiating phf8-/Y ESCs (Fig. 5F), indicating that the PHF8 demethylase activity is actively involved in the regulation of pmaip1 gene.

Transient Knockdown of pmaip1 Decreases Apoptosis and Promotes Mesodermal and Cardiac Differentiation

To clarify the role of pmaip1 in mESC differentiation, we transfected specific siRNAs against pmaip1 (si-Pmaip1) into phf8+/Y ESCs followed by EB formation. The negative control siRNA (si-NC) did not alter pmaip1 transcript levels compared with untreated (NT) cells, while si-Pmaip1 significantly inhibited pmaip1 transcripts by 74%-76% at differentiation days 0 and 1 (Fig. 6A-a). si-Pmaip1 cells had fewer TUNEL+ cells compared with the NT and si-NC cells at differentiation day 3 in both phf8+/Y and phf8-/Y cells (Fig. 6A-b). We then examined whether the pmaip1 knockdown influences mesodermal and early cardiac differentiation. As shown in Figure 6B, the apoptosis of FLK-1+ cells was significantly decreased in si-Pmaip1 mESCs (Fig. 6B). The expression of T and gsc as well as nrp1 and flk-1 were increased in si-Pmaip1 cells compared with those in NT and si-NC cells at differentiation day 3. In addition, the expression of cardiac transcript factors mef2c and tbx5 was up-regulated at differentiation day 5, and myh6 andtnnt2 were up-regulated at differentiation day 9 (Fig. 6C). We also transfected si-Pmaip1 into phf8-/Y ESCs. The expression of pmaip1 was downregulated at differentiation day 0 and day 1 in phf8-/Y ESCs with si-Pmaip1 (Supporting Information as Fig. S4A-a), accompanied by a decrease in TUNEL+ cells compared with NT and si-NC (Fig. 6A-b), while Annexin V remained unchanged (Supporting Information Fig. S4A-b). The expression of nrp1 and flk1 did not significantly change in phf8-/Y ESCs with si-Pmaip1 at differentiation day 3, while mef2c was upregulated at differentiation day 5, and myh6 was upregulated at differentiation day 9 (Supporting Information as Fig. S4B). These results suggest that downregulation of pmaip1 in phf8-/Y ESCs may not lead to as robust of a phenotype as it did in phf8+/Y ESCs. This difference is likely due to the level ofpmaip1 during early differentiation of phf8-/Y ESCs was already decreased to a low level similar to that observed in the undifferentiated cells (Fig. 5B). Taken together, these data demonstrate that the decreased apoptosis via down-regulation of pmaip1 contributes, at least partially, to the phf8-/Y-facilitated mesodermal and cardiomyocyte commitment.

Figure 6. Plant homeo domain finger protein 8 (PHF8) regulates the mesodermal and cardiac differentiation through pmaip1. (A-a): qRT-PCR analysis of thepmaip1 expression in phf8+/Y ESCs after being transiently transfected with si-NC or si-Pmaip1. n = 4. (A-b): Apoptosis cells were quantified by flow cytometry analysis of TUNEL assay at differentiation day 3 in phf8+/Y and phf8-/Y ESCs after transient transfection with si-NC or si-Pmaip1. n = 3. (B): Cells double stained with FLK-1 and Annexin V were analyzed by flow cytometry at differentiation day 4. n = 4. (C): qRT-PCR analysis of the expression of T, gsc, flk-1, nrp1, tbx5, mef2c, myh6, and tnnt2 in phf8+/Y ESCs after transient transfection with si-NC or si-Pmaip1. n = 5. (D): Flow cytometry detection of TUNEL positive cells at differentiation day 3, Annexin V positive cells and double stained FLK-1 and Annexin V at differentiation day 4 in phf8-/Y, phf8-NC-/+, phf8-pmaip1-/+, and phf8-hPHF8-/+ mouse embryonic stem cells (mESCs). n = 4. (E): qRT-PCR analysis of the expression of T, gsc, flk-1, nrp1, tbx5, mef2c, myh6, and tnnt2 in phf8-/Y, phf8-NC-/+, phf8-pmaip1-/+, and phf8-hPHF8-/+ mESCs. n = 3. Data are presented as mean ± SEM. *, p < .05; **, p < .01; ***, p < .001 compared with the corresponding phf8+/Y or phf8-/Y value.
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Overexpression of pmaip1 or hPHF8 in phf8-/Y ESCs Increases Apoptosis and Weakens Mesodermal and Cardiac Differentiation

To further determine whether PHF8 contributes to mesoderm and cardiac cell commitment through the regulation of apoptosis via targeting pmaip1, we rescued the expression of pmaip1 and phf8 in phf8-/Y ESCs by generating pmaip1-ovexpressing phf8-/Y mESCs (phf8-pmaip1-/+ mESCs) and hPHF8-overexpressing phf8-/Y mESCs (phf8-hPHF8-/+ mESCs). The qRT-PCR analysis confirmed that the expression of hPHF8 or pmaip1 was significantly upregulated in the respective overexpressing cell lines (Supporting Information Fig. S4C). The expression of pmaip1 in undifferentiated phf8-/Y mESCs was not affected by hPHF8 overexpression. However, Pmaip1 transcripts increased by differentiation day 3 in overexpressing cells (Supporting Information Fig. S4D), indicating that PHF8 does regulate the expression of pmaip1 during differentiation. Both TUNEL and Annexin V analysis revealed significant increases of apoptosis in phf8-pmaip1-/+ and phf8-hPHF8-/+ mESCs compared with the phf8-/Y andphf8-NC-/+ mESCs at differentiation day 3 or day 4, accompanied by a higher apoptosis ratio in FLK-1+ cells (Fig. 6D). Moreover, the expression of T and gsc as well as nrp1and flk-1 were significantly decreased in phf8-pmaip1-/+ and phf8-hPHF8-/+ mESCs at differentiation day 3, followed by a down-regulation of mef2c and tbx5 at differentiation day 5, and myh6 and tnnt2 at differentiation day 9 (Fig. 6E). In addition, TUNEL analysis showed no changes in the apoptotic responses either through knockout or overexpression of phf8 compared with the corresponding wild-type cells or phf8+/Y cells during induced ectodermal differentiation (Supporting information Fig. S4E). These data are consistent with a regulatory role of phf8 on mesodermal and cardiac differentiation through targeting of pmaip1

Discussion

This is the first study to unravel a regulatory role of histone demethylase in the differentiation of ESCs through the control of apoptosis and subsequent effects on cell lineage commitment. The role of PHF8 in the regulation of ESC differentiation to the mesodermal lineage and cardiac differentiation is supported by selective changes in RNA markers for mesodermal lineages, and an increase in cardiomyocyte progenitors and cardiomyocytes (Figs. 1C, 2C). Moreover, deletion of phf8 specifically inhibits apoptosis of Flk-1+ mesodermal cells with a concomitant reduction in Annexin V+ staining (Fig. 4D) and cardiac differentiation (Fig. 2B–E), while the ratio of TUNEL+ to either NESTIN+(ectodermal cells) or SOX17+ (endodermal cells) cells does not differ between the phf8+/Y and phf8-/Y lines (Supporting Information Fig. S3C, S3D). Consistently, the proportion of early apoptotic cells (Annexin V+) in pmaip1-knockdown (Fig. 6B) is also decreased, while pmaip1-overexpression or hPHF8-overexpression in phf8-/Y cells increase the proportion of TUNEL+ and Annexin V+ cells simultaneously with a reduction in mesodermal and cardiac differentiation (Fig. 6D, 6E). These findings indicate that the PHF8 functions, at least partially, through regulation of apoptosis.

It is well known that the regulation of apoptosis is of critical importance for proper ESC differentiation and embryo development [8, 43]. ESC differentiation is regulated by apoptosis induced by MAPK activation [7] and IP3R3-regulated Ca2+ release [5]. Previously only histone 3 lysine 4 methyltransferase MLL2 had been shown to activate the antiapoptotic gene bcl2 to inhibit apoptosis during ESC differentiation [44]. The data presented, here, extends and reveals the importance of epigenetic controls in the activation of proapoptotic gene associated with ESC differentiation.

Mesodermal and cardiac differentiation have been shown to be regulated by the histone demethylase ubiquitously transcribed tetratricopeptide repeat, X chromosome (UTX)[13, 45] and jumonji domain–containing protein 3 (JMJD3) [12] through transcriptional activation of mesodermal and cardiac genes. These findings together with those presented in this paper support the critical role of histone demethylases in lineage commitment through regulatory mechanisms that control the expression of core lineage specific transcription factors and apoptotic genes. The decrease in apoptosis through deletion of phf8 can be attributed to the maintenance of repressive H3K9me2 mark on the TSS of pmaip1 after phf8 deletion, resulting in a ∼70% downregulation of pmaip1 at differentiation day 3 in the phf8-/Y cells (Fig. 5B). The pro-apoptotic gene pmaip1 is, therefore, epigenetically regulated by the histone demethylase, which subsequently affects the mesodermal and cardiac differentiation.

PMAIP1 is a Bcl2 homology domain 3 (BH3)-only protein that acts as an important mediator of apoptosis [46]. Its expression is regulated transcriptionally by various transcription factors and, when present, it acts to promote cell death in a variety of ways [21] including caspase 3 dependent [47] and independent apoptosis [48] and autophagy [40]. Here, we find that PHF8 and its regulation on the pmaip1 promote DNA fragmentation and cell death most likely through a caspase 3-independent pathway. This conclusion is based on the observation that neither the ratio of cleaved caspase 3 to total caspase 3 [49, 50] nor PARP1, a downstream target of caspase 3, is significantly affected. While this may be explained as the inhibitor of apoptosis proteins can counteract the function of caspase 3 [51, 52], the exact mechanisms we observed here need to be further explored.

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Genomics and epigenetics link to DNA structure

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Sequence and Epigenetic Factors Determine Overall DNA Structure

http://www.genengnews.com/gen-news-highlights/sequence-and-epigenetic-factors-determine-overall-dna-structure/81252592/

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Atomic-level simulations show electrostatic forces between each atom. [Alek Aksimentiev, University of Illinois at Urbana-Champaign]

 

The traditionally held hypothesis about the highly ordered organization of DNA describes the interaction of various proteins with DNA sequences to mediate the dynamic structure of the molecule. However, recent evidence has emerged that stretches of homologous DNA sequences can associate preferentially with one another, even in the absence of proteins.

Researchers at the University of Illinois Center for the Physics of Living Cells, Johns Hopkins University, and Ulsan National Institute of Science and Technology (UNIST) in South Korea found that DNA molecules interact directly with one another in ways that are dependent on the sequence of the DNA and epigenetic factors, such as methylation.

The researchers described evidence they found for sequence-dependent attractive interactions between double-stranded DNA molecules that neither involve intermolecular strand exchange nor are mediated by DNA-binding proteins.

“DNA molecules tend to repel each other in water, but in the presence of special types of cations, they can attract each other just like nuclei pulling each other by sharing electrons in between,” explained lead study author Hajin Kim, Ph.D., assistant professor of biophysics at UNIST. “Our study suggests that the attractive force strongly depends on the nucleic acid sequence and also the epigenetic modifications.”

The investigators used atomic-level supercomputer simulations to measure the forces between a pair of double-stranded DNA helices and proposed that the distribution of methyl groups on the DNA was the key to regulating this sequence-dependent attraction. To verify their findings experimentally, the scientists were able to observe a single pair of DNA molecules within nanoscale bubbles.

“Here we combine molecular dynamics simulations with single-molecule fluorescence resonance energy transfer experiments to examine the interactions between duplex DNA in the presence of spermine, a biological polycation,” the authors wrote. “We find that AT-rich DNA duplexes associate more strongly than GC-rich duplexes, regardless of the sequence homology. Methyl groups of thymine act as a steric block, relocating spermine from major grooves to interhelical regions, thereby increasing DNA–DNA attraction.”

The findings from this study were published recently in Nature Communications in an article entitled “Direct Evidence for Sequence-Dependent Attraction Between Double-Stranded DNA Controlled by Methylation.”

After conducting numerous further simulations, the research team concluded that direct DNA–DNA interactions could play a central role in how chromosomes are organized in the cell and which ones are expanded or folded up compactly, ultimately determining functions of different cell types or regulating the cell cycle.

“Biophysics is a fascinating subject that explores the fundamental principles behind a variety of biological processes and life phenomena,” Dr. Kim noted. “Our study requires cross-disciplinary efforts from physicists, biologists, chemists, and engineering scientists and we pursue the diversity of scientific disciplines within the group.”

Dr. Kim concluded by stating that “in our lab, we try to unravel the mysteries within human cells based on the principles of physics and the mechanisms of biology. In the long run, we are seeking for ways to prevent chronic illnesses and diseases associated with aging.”

 

Direct evidence for sequence-dependent attraction between double-stranded DNA controlled by methylation

Jejoong Yoo, Hajin Kim, Aleksei Aksimentiev, and Taekjip Ha
Nature Communications 7 11045 (2016)    DOI:10.1038/ncomms11045BibTex

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Although proteins mediate highly ordered DNA organization in vivo, theoretical studies suggest that homologous DNA duplexes can preferentially associate with one another even in the absence of proteins. Here we combine molecular dynamics simulations with single-molecule fluorescence resonance energy transfer experiments to examine the interactions between duplex DNA in the presence of spermine, a biological polycation. We find that AT-rich DNA duplexes associate more strongly than GC-rich duplexes, regardless of the sequence homology. Methyl groups of thymine acts as a steric block, relocating spermine from major grooves to interhelical regions, thereby increasing DNA–DNA attraction. Indeed, methylation of cytosines makes attraction between GC-rich DNA as strong as that between AT-rich DNA. Recent genome-wide chromosome organization studies showed that remote contact frequencies are higher for AT-rich and methylated DNA, suggesting that direct DNA–DNA interactions that we report here may play a role in the chromosome organization and gene regulation.

Formation of a DNA double helix occurs through Watson–Crick pairing mediated by the complementary hydrogen bond patterns of the two DNA strands and base stacking. Interactions between double-stranded (ds)DNA molecules in typical experimental conditions containing mono- and divalent cations are repulsive1, but can turn attractive in the presence of high-valence cations2. Theoretical studies have identified the ion–ion correlation effect as a possible microscopic mechanism of the DNA condensation phenomena3, 4, 5. Theoretical investigations have also suggested that sequence-specific attractive forces might exist between two homologous fragments of dsDNA6, and this ‘homology recognition’ hypothesis was supported by in vitro atomic force microscopy7 and in vivo point mutation assays8. However, the systems used in these measurements were too complex to rule out other possible causes such as Watson–Crick strand exchange between partially melted DNA or protein-mediated association of DNA.

Here we present direct evidence for sequence-dependent attractive interactions between dsDNA molecules that neither involve intermolecular strand exchange nor are mediated by proteins. Further, we find that the sequence-dependent attraction is controlled not by homology—contradictory to the ‘homology recognition’ hypothesis6—but by a methylation pattern. Unlike the previous in vitro study that used monovalent (Na+) or divalent (Mg2+) cations7, we presumed that for the sequence-dependent attractive interactions to operate polyamines would have to be present. Polyamine is a biological polycation present at a millimolar concentration in most eukaryotic cells and essential for cell growth and proliferation9, 10. Polyamines are also known to condense DNA in a concentration-dependent manner2, 11. In this study, we use spermine4+(Sm4+) that contains four positively charged amine groups per molecule.

Sequence dependence of DNA–DNA forces

To characterize the molecular mechanisms of DNA–DNA attraction mediated by polyamines, we performed molecular dynamics (MD) simulations where two effectively infinite parallel dsDNA molecules, 20 base pairs (bp) each in a periodic unit cell, were restrained to maintain a prescribed inter-DNA distance; the DNA molecules were free to rotate about their axes. The two DNA molecules were submerged in 100mM aqueous solution of NaCl that also contained 20 Sm4+molecules; thus, the total charge of Sm4+, 80 e, was equal in magnitude to the total charge of DNA (2 × 2 × 20 e, two unit charges per base pair; Fig. 1a). Repeating such simulations at various inter-DNA distances and applying weighted histogram analysis12 yielded the change in the interaction free energy (ΔG) as a function of the DNA–DNA distance (Fig. 1b,c). In a broad agreement with previous experimental findings13, ΔG had a minimum, ΔGmin, at the inter-DNA distance of 25−30Å for all sequences examined, indeed showing that two duplex DNA molecules can attract each other. The free energy of inter-duplex attraction was at least an order of magnitude smaller than the Watson–Crick interaction free energy of the same length DNA duplex. A minimum of ΔG was not observed in the absence of polyamines, for example, when divalent or monovalent ions were used instead14, 15.

Figure 1: Polyamine-mediated DNA sequence recognition observed in MD simulations and smFRET experiments.
Polyamine-mediated DNA sequence recognition observed in MD simulations and smFRET experiments.

(a) Set-up of MD simulations. A pair of parallel 20-bp dsDNA duplexes is surrounded by aqueous solution (semi-transparent surface) containing 20 Sm4+ molecules (which compensates exactly the charge of DNA) and 100mM NaCl. Under periodic boundary conditions, the DNA molecules are effectively infinite. A harmonic potential (not shown) is applied to maintain the prescribed distance between the dsDNA molecules. (b,c) Interaction free energy of the two DNA helices as a function of the DNA–DNA distance for repeat-sequence DNA fragments (b) and DNA homopolymers (c). (d) Schematic of experimental design. A pair of 120-bp dsDNA labelled with a Cy3/Cy5 FRET pair was encapsulated in a ~200-nm diameter lipid vesicle; the vesicles were immobilized on a quartz slide through biotin–neutravidin binding. Sm4+ molecules added after immobilization penetrated into the porous vesicles. The fluorescence signals were measured using a total internal reflection microscope. (e) Typical fluorescence signals indicative of DNA–DNA binding. Brief jumps in the FRET signal indicate binding events. (f) The fraction of traces exhibiting binding events at different Sm4+ concentrations for AT-rich, GC-rich, AT nonhomologous and CpG-methylated DNA pairs. The sequence of the CpG-methylated DNA specifies the methylation sites (CG sequence, orange), restriction sites (BstUI, triangle) and primer region (underlined). The degree of attractive interaction for the AT nonhomologous and CpG-methylated DNA pairs was similar to that of the AT-rich pair. All measurements were done at [NaCl]=50mM and T=25°C. (g) Design of the hybrid DNA constructs: 40-bp AT-rich and 40-bp GC-rich regions were flanked by 20-bp common primers. The two labelling configurations permit distinguishing parallel from anti-parallel orientation of the DNA. (h) The fraction of traces exhibiting binding events as a function of NaCl concentration at fixed concentration of Sm4+ (1mM). The fraction is significantly higher for parallel orientation of the DNA fragments.

Unexpectedly, we found that DNA sequence has a profound impact on the strength of attractive interaction. The absolute value of ΔG at minimum relative to the value at maximum separation, |ΔGmin|, showed a clearly rank-ordered dependence on the DNA sequence: |ΔGmin| of (A)20>|ΔGmin| of (AT)10>|ΔGmin| of (GC)10>|ΔGmin| of (G)20. Two trends can be noted. First, AT-rich sequences attract each other more strongly than GC-rich sequences16. For example, |ΔGmin| of (AT)10 (1.5kcalmol−1 per turn) is about twice |ΔGmin| of (GC)10 (0.8kcalmol−1 per turn) (Fig. 1b). Second, duplexes having identical AT content but different partitioning of the nucleotides between the strands (that is, (A)20 versus (AT)10 or (G)20 versus (GC)10) exhibit statistically significant differences (~0.3kcalmol−1 per turn) in the value of |ΔGmin|.

To validate the findings of MD simulations, we performed single-molecule fluorescence resonance energy transfer (smFRET)17 experiments of vesicle-encapsulated DNA molecules. Equimolar mixture of donor- and acceptor-labelled 120-bp dsDNA molecules was encapsulated in sub-micron size, porous lipid vesicles18 so that we could observe and quantitate rare binding events between a pair of dsDNA molecules without triggering large-scale DNA condensation2. Our DNA constructs were long enough to ensure dsDNA–dsDNA binding that is stable on the timescale of an smFRET measurement, but shorter than the DNA’s persistence length (~150bp (ref. 19)) to avoid intramolecular condensation20. The vesicles were immobilized on a polymer-passivated surface, and fluorescence signals from individual vesicles containing one donor and one acceptor were selectively analysed (Fig. 1d). Binding of two dsDNA molecules brings their fluorescent labels in close proximity, increasing the FRET efficiency (Fig. 1e).

FRET signals from individual vesicles were diverse. Sporadic binding events were observed in some vesicles, while others exhibited stable binding; traces indicative of frequent conformational transitions were also observed (Supplementary Fig. 1A). Such diverse behaviours could be expected from non-specific interactions of two large biomolecules having structural degrees of freedom. No binding events were observed in the absence of Sm4+ (Supplementary Fig. 1B) or when no DNA molecules were present. To quantitatively assess the propensity of forming a bound state, we chose to use the fraction of single-molecule traces that showed any binding events within the observation time of 2min (Methods). This binding fraction for the pair of AT-rich dsDNAs (AT1, 100% AT in the middle 80-bp section of the 120-bp construct) reached a maximum at ~2mM Sm4+(Fig. 1f), which is consistent with the results of previous experimental studies2, 3. In accordance with the prediction of our MD simulations, GC-rich dsDNAs (GC1, 75% GC in the middle 80bp) showed much lower binding fraction at all Sm4+ concentrations (Fig. 1b,c). Regardless of the DNA sequence, the binding fraction reduced back to zero at high Sm4+ concentrations, likely due to the resolubilization of now positively charged DNA–Sm4+ complexes2, 3, 13.

Because the donor and acceptor fluorophores were attached to the same sequence of DNA, it remained possible that the sequence homology between the donor-labelled DNA and the acceptor-labelled DNA was necessary for their interaction6. To test this possibility, we designed another AT-rich DNA construct AT2 by scrambling the central 80-bp section of AT1 to remove the sequence homology (Supplementary Table 1). The fraction of binding traces for this nonhomologous pair of donor-labelled AT1 and acceptor-labelled AT2 was comparable to that for the homologous AT-rich pair (donor-labelled AT1 and acceptor-labelled AT1) at all Sm4+ concentrations tested (Fig. 1f). Furthermore, this data set rules out the possibility that the higher binding fraction observed experimentally for the AT-rich constructs was caused by inter-duplex Watson–Crick base pairing of the partially melted constructs.

Next, we designed a DNA construct named ATGC, containing, in its middle section, a 40-bp AT-rich segment followed by a 40-bp GC-rich segment (Fig. 1g). By attaching the acceptor to the end of either the AT-rich or GC-rich segments, we could compare the likelihood of observing the parallel binding mode that brings the two AT-rich segments together and the anti-parallel binding mode. Measurements at 1mM Sm4+ and 25 or 50mM NaCl indicated a preference for the parallel binding mode by ~30% (Fig. 1h). Therefore, AT content can modulate DNA–DNA interactions even in a complex sequence context. Note that increasing the concentration of NaCl while keeping the concentration of Sm4+ constant enhances competition between Na+ and Sm4+ counterions, which reduces the concentration of Sm4+ near DNA and hence the frequency of dsDNA–dsDNA binding events (Supplementary Fig. 2).

Methylation determines the strength of DNA–DNA attraction

Analysis of the MD simulations revealed the molecular mechanism of the polyamine-mediated sequence-dependent attraction (Fig. 2). In the case of the AT-rich fragments, the bulky methyl group of thymine base blocks Sm4+ binding to the N7 nitrogen atom of adenine, which is the cation-binding hotspot21, 22. As a result, Sm4+ is not found in the major grooves of the AT-rich duplexes and resides mostly near the DNA backbone (Fig. 2a,d). Such relocated Sm4+ molecules bridge the two DNA duplexes better, accounting for the stronger attraction16, 23, 24, 25. In contrast, significant amount of Sm4+ is adsorbed to the major groove of the GC-rich helices that lacks cation-blocking methyl group (Fig. 2b,e).

Figure 2: Molecular mechanism of polyamine-mediated DNA sequence recognition.
Molecular mechanism of polyamine-mediated DNA sequence recognition.

(ac) Representative configurations of Sm4+ molecules at the DNA–DNA distance of 28Å for the (AT)10–(AT)10 (a), (GC)10–(GC)10 (b) and (GmC)10–(GmC)10 (c) DNA pairs. The backbone and bases of DNA are shown as ribbon and molecular bond, respectively; Sm4+ molecules are shown as molecular bonds. Spheres indicate the location of the N7 atoms and the methyl groups. (df) The average distributions of cations for the three sequence pairs featured in ac. Top: density of Sm4+ nitrogen atoms (d=28Å) averaged over the corresponding MD trajectory and the z axis. White circles (20Å in diameter) indicate the location of the DNA helices. Bottom: the average density of Sm4+ nitrogen (blue), DNA phosphate (black) and sodium (red) atoms projected onto the DNA–DNA distance axis (x axis). The plot was obtained by averaging the corresponding heat map data over y=[−10, 10] Å. See Supplementary Figs 4 and 5 for the cation distributions at d=30, 32, 34 and 36Å.

If indeed the extra methyl group in thymine, which is not found in cytosine, is responsible for stronger DNA–DNA interactions, we can predict that cytosine methylation, which occurs naturally in many eukaryotic organisms and is an essential epigenetic regulation mechanism26, would also increase the strength of DNA–DNA attraction. MD simulations showed that the GC-rich helices containing methylated cytosines (mC) lose the adsorbed Sm4+ (Fig. 2c,f) and that |ΔGmin| of (GC)10 increases on methylation of cytosines to become similar to |ΔGmin| of (AT)10 (Fig. 1b).

To experimentally assess the effect of cytosine methylation, we designed another GC-rich construct GC2 that had the same GC content as GC1 but a higher density of CpG sites (Supplementary Table 1). The CpG sites were then fully methylated using M. SssI methyltransferase (Supplementary Fig. 3; Methods). As predicted from the MD simulations, methylation of the GC-rich constructs increased the binding fraction to the level of the AT-rich constructs (Fig. 1f).

The sequence dependence of |ΔGmin| and its relation to the Sm4+ adsorption patterns can be rationalized by examining the number of Sm4+ molecules shared by the dsDNA molecules (Fig. 3a). An Sm4+ cation adsorbed to the major groove of one dsDNA is separated from the other dsDNA by at least 10Å, contributing much less to the effective DNA–DNA attractive force than a cation positioned between the helices, that is, the ‘bridging’ Sm4+ (ref. 23). An adsorbed Sm4+ also repels other Sm4+ molecules due to like-charge repulsion, lowering the concentration of bridging Sm4+. To demonstrate that the concentration of bridging Sm4+ controls the strength of DNA–DNA attraction, we computed the number of bridging Sm4+ molecules, Nspm (Fig. 3b). Indeed, the number of bridging Sm4+ molecules ranks in the same order as |ΔGmin|: Nspm of (A)20>Nspm of (AT)10Nspm of (GmC)10>Nspm of (GC)10>Nspm of (G)20. Thus, the number density of nucleotides carrying a methyl group (T and mC) is the primary determinant of the strength of attractive interaction between two dsDNA molecules. At the same time, the spatial arrangement of the methyl group carrying nucleotides can affect the interaction strength as well (Fig. 3c). The number of methyl groups and their distribution in the (AT)10 and (GmC)10 duplex DNA are identical, and so are their interaction free energies, |ΔGmin| of (AT)10Gmin| of (GmC)10. For AT-rich DNA sequences, clustering of the methyl groups repels Sm4+ from the major groove more efficiently than when the same number of methyl groups is distributed along the DNA (Fig. 3b). Hence, |ΔGmin| of (A)20>|ΔGmin| of (AT)10. For GC-rich DNA sequences, clustering of the cation-binding sites (N7 nitrogen) attracts more Sm4+ than when such sites are distributed along the DNA (Fig. 3b), hence |ΔGmin| is larger for (GC)10 than for (G)20.

Figure 3: Methylation modulates the interaction free energy of two dsDNA molecules by altering the number of bridging Sm4+.
Methylation modulates the interaction free energy of two dsDNA molecules by altering the number of bridging Sm4+.

(a) Typical spatial arrangement of Sm4+ molecules around a pair of DNA helices. The phosphates groups of DNA and the amine groups of Sm4+ are shown as red and blue spheres, respectively. ‘Bridging’ Sm4+molecules reside between the DNA helices. Orange rectangles illustrate the volume used for counting the number of bridging Sm4+ molecules. (b) The number of bridging amine groups as a function of the inter-DNA distance. The total number of Sm4+ nitrogen atoms was computed by averaging over the corresponding MD trajectory and the 10Å (x axis) by 20Å (y axis) rectangle prism volume (a) centred between the DNA molecules. (c) Schematic representation of the dependence of the interaction free energy of two DNA molecules on their nucleotide sequence. The number and spatial arrangement of nucleotides carrying a methyl group (T or mC) determine the interaction free energy of two dsDNA molecules.

Genome-wide investigations of chromosome conformations using the Hi–C technique revealed that AT-rich loci form tight clusters in human nucleus27, 28. Gene or chromosome inactivation is often accompanied by increased methylation of DNA29 and compaction of facultative heterochromatin regions30. The consistency between those phenomena and our findings suggest the possibility that the polyamine-mediated sequence-dependent DNA–DNA interaction might play a role in chromosome folding and epigenetic regulation of gene expression.

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