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Posts Tagged ‘histone modification’


Histone Turnover

Larry H Bernstein, MD, FCAP, Curator

LPBI

 

GEN News Highlights   Nov 9, 2015   Cells May Rest, but Not Histones

http://www.genengnews.com/gen-news-highlights/cells-may-rest-but-not-histones/81251953/

 

http://www.genengnews.com/Media/images/GENHighlight/Nov9_2015_MRC_HiraDeletion2438081646.jpg

 

Even between rounds of cell division, during the so-called resting state, a cell stays busy, unpacking and repacking portions of the genome, accumulating epigenetic marks, and turning specific genes on and off. So, it stands to reason that all this activity would degrade histone proteins, the genome’s packing material, and that histone proteins would be in need of constant replacement. Yet histone replacement is poorly understood, at least in the “resting” cell, where the day-to-day routine unfolds between bursts of DNA replication.

To hear the hum of histone turnover without the clamor of replication, scientists at the Babraham Institute and MRC Clinical Sciences Centre listened in on developing mouse egg cells, oocytes. Developing oocytes provide a system in which the packing and unpacking of DNA is relatively easy to study. Oocytes do not divide, and so there is no DNA replication. Also, in oocytes, genomes are highly active. As they mature and ready themselves for fertilization, oocytes turn genes on and off throughout the genome, which simultaneously undergoes epigenetic modification.

The scientists found that by deleting the gene for a histone replacement protein, they could dampen the hum of histone turnover. Moreover, using single-cell analysis, the scientists evaluated how interfering with histone turnover affected egg cell development, DNA integrity, and the accumulation of DNA methylation.

The results of this work appeared November 5 in the journal Molecular Cell, in an article entitled, “Continuous Histone Replacement by Hira Is Essential for Normal Transcriptional Regulation and De Novo DNA Methylation during Mouse Oogenesis.” This article describes how the scientists deleted the H3.3 chaperone Hira in developing mouse oocytes, and how they assessed the importance of continuous H3.3/H4 deposition in sustaining chromatin dynamics.

 

Continuous Histone Replacement by Hira Is Essential for Normal Transcriptional Regulation and De Novo DNA Methylation during Mouse Oogenesis

Nashun et al., 2015, Molecular Cell 60, 1–15      Buhe Nashun, Peter W.S. Hill, Sebastien A. Smallwood, Gopuraja Dharmalingam, Rachel Amouroux, Stephen J. Clark, et al.
DOI: http://dx.doi.org/10.1016/j.molcel.2015.10.010
Highlights

Histone H3/H4 replacement is continuous and mediated by Hira during mouse oogenesis

Loss of Hira results in chromatin abnormalities and extensive oocyte loss

Hira depletion reduces histone load, which prevents normal transcriptional regulation

Hira-mediated histone replacement is required for normal 5mC deposition in oocytes

The integrity of chromatin, which provides a dynamic template for all DNA-related processes in eukaryotes, is maintained through replication-dependent and -independent assembly pathways. To address the role of histone deposition in the absence of DNA replication, we deleted the H3.3 chaperone Hira in developing mouse oocytes. We show that chromatin of non-replicative developing oocytes is dynamic and that lack of continuous H3.3/H4 deposition alters chromatin structure, resulting in increased DNase I sensitivity, the accumulation of DNA damage, and a severe fertility phenotype. On the molecular level, abnormal chromatin structure leads to a dramatic decrease in the dynamic range of gene expression, the appearance of spurious transcripts, and inefficient de novo DNA methylation. Our study thus unequivocally shows the importance of continuous histone replacement and chromatin homeostasis for transcriptional regulation and normal developmental progression in a non-replicative system in vivo.

Figure thumbnail fx1

http://www.cell.com/cms/attachment/2040068218/2053594851/fx1.jpg

Nashun et al., Continuous Histone Replacement by Hira Is Essential for Normal Transcriptional Regulation and De Novo DNA Methylation during Mouse Oogenesis, Molecular Cell (2015), http://dx.doi.org/10.1016/j.molcel.2015.10.010

 

DNA in all eukaryotic organisms is bound by nucleosomes, forming a physiological chromatin context in which all molecular processes involving DNA operate. The integrity of the chromatin template is constantly compromised by fundamental biological processes, such as DNA replication, repair, and transcription, following which the normal chromatin structure is restored with the help of histone chaperone proteins (Gurard-Levin et al., 2014; Ransom et al., 2010). Advances in recent years have shed light on some of the molecular players involved in chromatin assembly and maintenance, separating (on the molecular level) DNA replication-dependent and -independent pathways (Burgess and Zhang, 2013). Histone proteins themselves are central to these processes: the expression and incorporation of canonical histones is tightly coupled to DNA replication; in contrast, histone variants can be incorporated into chromatin independent of the cell cycle (Maze et al., 2014).

While it has become increasingly clear that the activity of histone chaperone proteins is of critical importance during DNA replication and repair (Adam et al., 2013; Hoek and Stillman, 2003; Polo et al., 2006; Ransom et al., 2010), studies that have attempted to dissect the importance of histone replacement in the interphase nucleus have revealed only a limited contribution of histone chaperones and/or variants to transcriptional regulation (Banaszynski et al., 2013; Goldberg et al., 2010; Ho¨ dl and Basler, 2009; Sakai et al., 2009). These studies, however, have been complicated by the use of proliferative cell systems that could (at least partially) restore chromatin integrity through replication-coupled chromatin assembly (Banaszynski et al., 2013; Ho¨ dl and Basler, 2009; Sakai et al., 2009; Wyrick et al., 1999). Thus, the extent to which basic physiological processes, such as transcription, are dependent on or regulated by histone replacement remains unclear.

To overcome the limitations of these previous studies, we took advantage of the unique system presented by mammalian oogenesis. Over an extended time span and in the absence of DNA replication, postnatal mammalian oocytes execute the oogenesis-specific developmental program, involving widespread transcriptional changes and de novo DNA methylation, ultimately acquiring the competencies required for fertilization and embryogenesis (De La Fuente, 2006; Li and Albertini, 2013; Tomizawa et al., 2012).

 

Figure 1. Incorporation of H3.3 in Developing Oocytes Is Driven by Hira (A) Schematic illustration shows developmental stages and global transcriptional activity during oogenesis with indicated onset of Gdf9-Cre+ and Zp3-Cre+ expression (adapted from De La Fuente, 2006; Lan et al., 2004; Li and Albertini, 2013; and Tomizawa et al., 2012). (B) Growing oocytes (postnatal day [P]14) were subjected to mRNA microinjection of Flag-tagged H3.1, H3.2, H3.3, or H2A.X. Incorporation of histone variants was visualized by anti-Flag antibody staining.

 

Hira Is Responsible for H3.3/H4 Turnover in Postnatal Oocytes

Hira Depletion Results in a Severe Ovarian Phenotype, Associated with Extensive Oocyte Death and the Failure to Support Zygotic Reprogramming or Embryonic Development

Hira-Depleted Oocytes Show Increased DNA Accessibility and Accumulation of DNA Damage

Continuing Histone Replacement Mediated by Hira Is Required to Maintain the Full Dynamic Range of Gene Expression

 

Figure 2. Hira Is Essential for Progression through Oogenesis and Acquisition of Developmental Competence (A) Number of ovulated oocytes recovered per female after hormonal stimulation. Numbers of females scored are indicated in the columns. Error bars indicate SEM. ***p < 0.001; statistical analysis was carried out using two-tailed unpaired Student’s t test. (B) Ovaries of Hiraf/f, Hiraf/f Gdf9-Cre+ , and Hiraf/f Zp3-Cre+ females were collected from mice born on the same date and ovarian images were taken in a single picture. (C) Representative images show H&E staining of 3-week (top) and 6-week (bottom) ovarian sections (follicles indicated by arrows). (D) Maternal Hira depletion results in defects in chromosome condensation and segregation. Representative bright-field images of MII oocytes recovered from two Hiraf/f and two Hiraf/f Gdf9-Cre+ mice at 3 weeks of age are shown. Arrows (left) indicate oocytes with defective asymmetric division normally associated with first polar body extrusion (left). The oocytes were fixed and stained with DAPI (right). Arrows and arrowheads (right) indicate chromosome bridges and lagging chromosomes, respectively. Quantification of normal and abnormal MII oocytes is shown (bottom right). (E) Early developmental arrest of parthenogenetic embryos with maternal Hira depletion. Representative images show embryos (left); quantification of developmental progression is shown (right). Oocytes lacking Hira do not progress beyond the two-cell stage. Embryo numbers are indicated above each column. Error bars indicate SEM. See also Figures S2, S3, and S4

Figure 3. Hira Deletion Leads to Increased DNA Accessibility and Accumulation of DNA Damage (A) Hira deletion leads to reduced histone load in the Hiraf/f Gdf9-Cre+ GV oocytes. The graph shows the quantification of the IF (pan histone antibody staining) signal normalized to DNA content (DAPI). (B) Structural alteration of chromatin in Hiraf/f Gdf9-Cre+ GV oocytes. Extensive chromatin de-condensation was observed in more than half of the examined Hiraf/f Gdf9-Cre+ GV oocytes. DNA was stained with DAPI and pseudocolored in gray. (C) Lack of Hira leads to increased DNase I accessibility. Schematic illustration shows in vivo DNase I TUNEL assay (left); see also the Supplemental Experimental Procedures. The fraction of oocytes with positive TUNEL signal is indicated in brackets. (D) Lack of Hira leads to the accumulation of DNA damage. The g-H2A.X staining of growing oocytes (P16, left) and GV oocytes (right) is shown. (E) Gene set enrichment analysis (GSEA) compares genes upregulated after DNA damage (Kyng_DNA_Damage_Up gene set) and the ranked list of gene expression changes in Hiraf/f Gdf9-Cre+ relative to Hiraf/f MII oocytes. NES, normalized enrichment score; FDR, false discovery rate (both were calculated in the GSEA program). DNA was stained with DAPI (in blue). Scale bar, 10 mm.

 

Hira-Mediated H3.3/H4 Deposition Is Essential for Transcriptional Transitions Associated with the Oocyte Developmental Program

Hira Depletion Leads to Aberrant Transcription from Regions Not Normally Transcribed within the Genome

Continuing Histone Replacement Is Essential for Efficient De Novo Methylation during Oogenesis

 

Figure 4. Continuous Histone Replacement Mediated by Hira Is Required to Maintain the Full Dynamic Range of Gene Expression (A) Detection of newly synthesized RNA by EU incorporation in growing (P14) and GV (P20) Hiraf/f and Hiraf/f Gdf9-Cre+ oocytes is shown. Scale bar, 10 mm. (B) Comparison between obtained FPKM values and absolute copy number per cell for ERCC spike-in RNA in Hiraf/f and Hiraf/f Gdf9-Cre+ MII oocytes revealed no significant difference. (C) Principal component analysis of scRNA-seq data derived from Hiraf/f and Hiraf/f Gdf9-Cre+ MII oocytes is shown. (D and E) Selected gene ontology (GO) terms significantly enriched for among differentially upregulated (D, edgeR, FDR < 0.1) and differentially downregulated (E, edgeR, FDR < 0.1) genes in Hiraf/f Gdf9-Cre+ MII oocytes are shown. x axis represents Benjamini-Hochberg adjusted p value. (F) Number of annotated genes detected, as computed by HTSeq program, is shown. (G) Relative proportion of genes distributed among ten equally sized expression level bins. All genes detected in our RNA-seq experiment were divided into ten equal bins based on their expression levels in Hiraf/f oocytes. The gene number in each bin was counted for Hiraf/f or Hiraf/f Gdf9-Cre+ , and the percentage was calculated relative to all genes detected in a given sample. (H) Boxplot shows gene expression levels of differentially upregulated (edgeR, FDR < 0.1), downregulated (edgeR, FDR < 0.1), and all annotated genes in Hiraf/f Gdf9-Cre+ MII oocytes. (I) Boxplots show distribution of gene expression fold change for each gene expression level quintile (based on Hiraf/f gene expression levels). (J) Variance of gene expression within a given Hiraf/f or Hiraf/f Gdf9-Cre+ sample. In all cases, error bars indicate SEM. Statistical analysis was carried out using two-tailed unpaired Student’s t test (F and G), Kruskal-Wallis with Dunn’s post hoc test (H) or F test (J); ns, non-significant; *p < 0.05, **p < 0.01, and ***p < 0.001. See also Figure S6.

Figure 5. Hira Is Essential for Transcriptional Transitions Associated with the Oocyte Developmental Program and Is Required for Repression of Aberrant Transcription (A) GSEA comparing genes of selected expression clusters during oogenesis (Figure S5B) and the ranked list of gene expression changes in Hiraf/f Gdf9-Cre+ MII oocytes relative to Hiraf/f oocytes. NES and FDR both were calculated in the GSEA program. For each cluster, each dot represents mean-normalized gene expression for consecutive stages of oocyte development. (B) Numbers of total transfrags and total annotated transfrags in Hiraf/f and Hiraf/f Gdf9-Cre+ MII oocytes (as computed by CuffCompare program) are shown. (C) Numbers of total annotated transfrags, unannotated entirely intronic transfrags, and unannotated entirely intergenic transfrags in Hiraf/f and Hiraf/f Gdf9-Cre+ MII oocytes (as computed by CuffCompare program) are shown. In all cases, error bars indicate SEM. Statistical analysis was carried out using two-tailed Student’s t test (*p < 0.05). See also Figures S5 and S6.

Figure 6. Continuous Histone Replacement Is Required for Efficient De Novo Methylation during Oogenesis (A) Reduced total 5mC was measured by LC-MS in GV (Hiraf/f and Hiraf/f Gdf9-Cre+ ) and MII (Hiraf/f and Hiraf/f Zp3-Cre+ ) oocytes. (B) Global levels of DNA methylation quantified by scBS-seq in the CpG (CG) and non-CpG (CHH/G) contexts are shown. (C) Example shows CpG methylation quantified over 3-kb sliding windows (1.5-kb steps) for published GV datasets (Shirane et al., 2013; WGBS, top), Hiraf/f, and Hiraf/f Gdf9-Cre+ (scBS-seq). (D) Distribution shows the 3-kb genomic windows in the indicated bins of DNA methylation in Hiraf/f oocytes (top, horizontal, percentage indicating the proportion of methylation bins) and their corresponding DNA methylation values in Hiraf/f Gdf9-Cre+ oocytes (bottom, vertical columns). (E) Pie chart distribution shows the 3-kb genomic windows presenting statistically significant (chi-square test, p < 0.01) changes in Hiraf/f Gdf9-Cre+ versus Hiraf/f (percentage indicates the proportion of each segment). (F) Effect of Hira deletion on DNA methylation is global and independent of the genomic context. DNA methylation at CpGs (five reads coverage) was determined and averaged for each genomic context. (G) CpG islands (CGIs) methylated in Hiraf/f oocytes are globally hypomethylated in Hiraf/f Gdf9-Cre+ oocytes. CGI methylation was defined for each genotype, and only CGIs hypermethylated in Hiraf/f (>80%) are displayed, for both Hiraf/f (blue) and Hiraf/f Gdf9-Cre+ (red), and ordered on the x axis based on their genomic location. (H) Effects on DNA methylation in Hiraf/f Gdf9-Cre+ oocytes are more pronounced at highly expressed genes. DNA methylation was quantified for genes binned into expression percentile based on the scRNA-seq data (boxplot with plus signs representing mean values and horizontal bars representing median values). (I) Comparison between DNA methylation and gene expression differences in Hiraf/f and Hiraf/f Gdf9-Cre+ oocytes. See also Figure S7.

 

To address the biological significance of continuous histone replacement in a physiological context, we have generated a genetic deletion of the histone chaperone Hira in the early stages of mouse oogenesis. Developing mouse oocytes represent a unique experimental system as postnatal oocytes undergo developmental transitions, including major transcriptional changes and widespread de novo DNA methylation, in the absence of DNA replication. Our results show that the chromatin of developing oocytes is highly dynamic, with histone turnover being observed also in the transcriptionally inert GV-stage oocytes (Figures 1C and 4A). We demonstrate that constant histone replacement is necessary for the maintenance of normal chromatin homeostasis in vivo. Depletion of Hira during early oocyte development leads to a severe reduction of histone load, compromised developmental progression, and progressive oocyte loss (Figures 2, 3A, and S2G). We note that, albeit pronounced, our observed phenotype is milder in comparison to the recently reported oocyte death in H3.3 knockout mice (Tang et al., 2015). We attribute this difference to the presence of an alternative ATRX/DAXX H3.3 chaperone complex, which, although present in our system, cannot functionally replace Hira-driven H3.3 deposition (Figures S2A–S2C).

Our experiments document that, while lacking the normal ability to incorporate H3.3 and H4, Hira-deleted growing oocytes remain transcriptionally active, which results in chromatin with severely reduced histone content, increased DNase I sensitivity, and signs of DNA damage (Figures 3 and S2B). Reminiscent of the phenotype associated with Hira depletion in yeast (Blackwell et al., 2004; Greenall et al., 2006) and H3.3 depletion in mice (Bush et al., 2013; Lin et al., 2013) and Drosophila (Sakai et al., 2009), the compromised chromatin structure leads to chromosome segregation defects and aberrant first polar body extrusion (Figure 2D). However, the observed chromosome segregation defects in the Hira-depleted oocytes are not linked with aberrant CenpA incorporation, as previously suggested in somatic cells (Figure S2C; Bush et al., 2013).

Non-replicative Hira-depleted oocytes uniquely reveal the importance of histone replacement on transcriptional regulation in the absence of replication-coupled chromatin assembly. Although transcription can continue from histone-depleted chromatin, our study shows that the lack of histone replacement has a major impact on the dynamic range of gene expression. In the context of histone-depleted chromatin, genes can neither be efficiently silenced nor effectively activated. Additionally, the lack of normal histone occupancy leads to increased spurious transcription from otherwise not-transcribed regions of the genome, suggesting an evolutionarily conserved role for the Hira histone chaperone complex (Anderson et al., 2009).

Our results additionally reveal an unexpected connection among continuous H3.3 replacement, transcription, and de novo DNA methylation in developing oocytes. Following Hira depletion, more accessible chromatin with reduced histone load leads to significantly reduced DNA methylation (Figures 6A–6G). We note that, although in other systems DNAmethylation changes result in pronounced transcriptional outcomes (Yang et al., 2014), reduced DNA methylation is not likely to contribute to the observed transcriptional changes in Hira-depleted oocytes, as Dnmt3l knockout oocytes lacking DNA methylation do not display any transcriptional phenotype (Kobayashi et al., 2012).

Our data suggest that the observed methylation phenotype cannot be attributed to downregulation of the Dnmt3a/3l complex, and, although we cannot exclude locus-specific effects on Dnmt3a/3l recruitment, the observed reduced DNA methylation is likely due to altered enzymatic activity of Dnmt3a in the absence of normal levels of chromatin-bound H3 (Figures 1C, 1E, and S2B). In this context, the N-terminal part of H3 has been shown in vitro to be required for allosteric activation of Dnmt3a catalytic activity (Guo et al., 2015). Our study thus provides the first support for this effect in an in vivo setting. The observed effect is more pronounced in highly expressed genes (Figure S7I), in agreement with the expected role of transcription in inducing nucleosome depletion in Hira-depleted oocytes. Furthermore, CpG island methylation is greatly reduced in Hira-depleted oocytes (Figure 6G) and methylation loss is more pronounced at regions with high CpG density (Figure S7K). As the Dnmt3a enzyme has been shown to operate in a non-processive manner (Dhayalan et al., 2010), stimulatory effect of the unmodified (H3K4me0) H3 tail might be necessary to ensure a high level of methylation at these regions.

Figure 7. Role of Hira in Transcriptional Regulation and De Novo DNA Methylation during Oogenesis During oogenesis, normal chromatin structure and nucleosome density is maintained by Hira-mediated continuous H3.3/H4 replacement. This is required for fine-tuned transcriptional regulation and efficient de novo DNA methylation.

Supplemental Information includes Supplemental Experimental Procedures, seven figures, and four tables and can be found with this article online at http://dx.doi.org/10.1016/j.molcel.2015.10.010.

 

Essentially, the scientists found that disturbing H3.3/H4 deposition alters chromatin structure, resulting in increased DNase I sensitivity, the accumulation of DNA damage, and a severe fertility phenotype.

“On the molecular level, abnormal chromatin structure leads to a dramatic decrease in the dynamic range of gene expression, the appearance of spurious transcripts, and inefficient de novo DNA methylation,” wrote the authors. “Our study thus unequivocally shows the importance of continuous histone replacement and chromatin homeostasis for transcriptional regulation and normal developmental progression in a non-replicative system in vivo.”

“Oocytes lacking the Hira histone chaperone showed severe developmental defects which often led to cell death,” said Gavin Kelsey, Ph.D., one of the authors of the Molecular Cell paper and a research group leader in the Babraham Institute’s epigenetics program. “The whole system is disrupted, eggs accumulate DNA damage and the altered chromatin means that genes cannot be efficiently silenced or activated. But we also uncovered an intricate relationship between the different epigenetic systems operating in the oocyte, where failure to ensure normal histone levels severely compromised deposition of methylation on the underlying DNA.”

The research addresses the importance of histone turnover in maintaining genomic fidelity and adds to our understanding about the mechanisms in place to protect the integrity of the genome as it is remodeled and reshaped. Studying this in the context of the developing oocytes provides new insights into our dynamic genome, unclouded by the complications of DNA replication, and also reveals how important maintaining chromatin dynamics is to the integrity of our gametes.

 

 

 

 

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Larry H Bernstein, MD, FCAP, Reporter and Curator

http://pharmaceyticalinnovation.com/7/10/2014/A new relationship identified in preterm stress and development of autism or schizophrenia/

 

This is a fascinating study.  It is of considerable interest because it deals with several items that need to be addressed with respect to neurodevelopmental disruptive disorders.  It leaves open some aspects that are known, but not subject to investigation in the experiments.  Then there is also no reporting of some associations that are known at the time of deveopment of these disorders – autism spectrum, and schizophrenia.  Of course, I don’t know how it would be possible to also look at prediction of a possible relationship to later development of mood disorders.

  1. The placenta functions as an endocrine organ in the conversion of androsteinedione to testosterone during pregnancy, which is delivered to the fetus.
  2. The conversion is by a known enzymatic pathway – and there is a sex difference in the depression of testosterone in males, females not affected.
  3. There is a greater susceptibility of males to autism and schizophrenia than of females, which I as reader, had not known, but if this is true, it would lend some credence to a biological advantage to protect the females of animal species, and might raise some interest into what relationship it has to protecting multitasking for females.
  4. It is well known that the twin studies that have been carried out determined that in identical twins, there is discordance as a rule.  Those studies are old, and they did not examine whether the other identical twin might be anywhere on the autism spectrum disorder (not then termed “spectrum”.
  5. However, there is a clear effect of stress on “gene expression”, and in this case we are looking at enzymation suppression at the placental level affecting trascriptional activity in the male fetus.  The same genetic signature exists in the male genetic profile, so we are not looking at a clear somatic mutation in this study.
  6. There is also much less specific an association with the MTHFR gene mutation at either one or two loci. This would have to be looked at as a possible separate post translational somatic mutation.
  7. Whether there is another component expressed later in the function of the zinc metalloproteinase under stress in the affected subject is worth considering, but can’t be commented on with respect to the study.

Penn Team Links Placental Marker of Prenatal Stress to Neurodevelopmental Problems 

By Ilene Schneider          July 8, 2014

When a woman experiences a stressful event early in pregnancy, the risk that her child will develop autism spectrum disorders or schizophrenia increases. The way in which maternal stress is transmitted to the brain of the developing fetus, leading to these problems in neurodevelopment, is poorly understood.

New findings by University of Pennsylvania School of Veterinary Medicine scientists suggest that an enzyme found in the placenta is likely playing an important role. This enzyme, O-linked-N-acetylglucosamine transferase, or OGT, translates maternal stress into a reprogramming signal for the brain before birth. The study was supported by the National Institute of Mental Health.

“By manipulating this one gene, we were able to recapitulate many aspects of early prenatal stress,” said Tracy L. Bale, senior author on the paper and a professor in the Department of Animal Biology at Penn Vet. “OGT seems to be serving a role as the ‘canary in the coal mine,’ offering a readout of mom’s stress to change the baby’s developing brain. Bale, who also holds an appointment in the Department of Psychiatry, co-authored tha paper with postdoctoral researcher Christopher L. Howerton, for PNAS.

OGT is known to play a role in gene expression through chromatin remodeling, a process that makes some genes more or less available to be converted into proteins. In a study published last year in PNAS, Bale’s lab found that placentas from male mice pups had lower levels of OGT than those from female pups, and placentas from mothers that had been exposed to stress early in gestation had lower overall levels of OGT than placentas from the mothers’ unstressed counterparts.

“People think that the placenta only serves to promote blood flow between a mom and her baby, but that’s really not all it’s doing,” Bale said. “It’s a very dynamic endocrine tissue and it’s sex-specific, and we’ve shown that tampering with it can dramatically affect a baby’s developing brain.”

To elucidate how reduced levels of OGT might be transmitting signals through the placenta to a fetus, Bale and Howerton bred mice that partially or fully lacked OGT in the placenta. They then compared these transgenic mice to animals that had been subjected to mild stressors during early gestation, such as predator odor, unfamiliar objects or unusual noises, during the first week of their pregnancies.

The researchers performed a genome-wide search for genes that were affected by the altered levels of OGT and were also affected by exposure to early prenatal stress using a specific activational histone mark and found a broad swath of common gene expression patterns.

They chose to focus on one particular differentially regulated gene called Hsd17b3, which encodes an enzyme that converts androstenedione, a steroid hormone, to testosterone. The researchers found this gene to be particularly interesting in part because neurodevelopmental disorders such as autism and schizophrenia have strong gender biases, where they either predominantly affect males or present earlier in males.

Placentas associated with male mice pups born to stressed mothers had reduced levels of the enzyme Hsd17b3, and, as a result, had higher levels of androstenedione and lower levels of testosterone than normal mice.

“This could mean that, with early prenatal stress, males have less masculinization,” Bale said. “This is important because autism tends to be thought of as the brain in a hypermasculinized state, and schizophrenia is thought of as a hypomasculinized state. It makes sense that there is something about this process of testosterone synthesis that is being disrupted.”

Furthermore, the mice born to mothers with disrupted OGT looked like the offspring of stressed mothers in other ways. Although they were born at a normal weight, their growth slowed at weaning. Their body weight as adults was 10 to 20 percent lower than control mice.

Because of the key role that that the hypothalamus plays in controlling growth and many other critical survival functions, the Penn Vet researchers then screened the mouse genome for genes with differential expression in the hypothalamus, comparing normal mice, mice with reduced OGT and mice born to stressed mothers.

They identified several gene sets related to the structure and function of mitochrondria, the powerhouses of cells that are responsible for producing energy. And indeed, when compared by an enzymatic assay that examines mitochondria biogenesis, both the mice born to stressed mothers and mice born to mothers with reduced OGT had dramatically reduced mitochondrial function in their hypothalamus compared to normal mice. These studies were done in collaboration with Narayan Avadhani’s lab at Penn Vet. Such reduced function could explain why the growth patterns of mice appeared similar until weaning, at which point energy demands go up.

“If you have a really bad furnace you might be okay if temperatures are mild,” Bale said. “But, if it’s very cold, it can’t meet demand. It could be the same for these mice. If you’re in a litter close to your siblings and mom, you don’t need to produce a lot of heat, but once you wean you have an extra demand for producing heat. They’re just not keeping up.”

Bale points out that mitochondrial dysfunction in the brain has been reported in both schizophrenia and autism patients. In future work, Bale hopes to identify a suite of maternal plasma stress biomarkers that could signal an increased risk of neurodevelopmental disease for the baby.

“With that kind of a signature, we’d have a way to detect at-risk pregnancies and think about ways to intervene much earlier than waiting to look at the term placenta,” she said.

 

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Reporter: Ritu Saxena, Ph.D.

Diabetes currently affects more than 336 million people worldwide, with healthcare costs by diabetes and its complications of up to $612 million per day in the US alone.  The islets of Langerhans, miniature endocrine organs within the pancreas, are essential regulators of blood glucose homeostasis and play a key role in the pathogenesis of diabetes.  Islets of Langerhans are composed of several types of endocrine cells.  The α- and β-cells are the most abundant and also the most important in that they secrete hormones (glucagon and insulin, respectively) crucial for glucose homeostasis (Bosco D, et al, Diabetes, May 2010;59(5):1202-10).

Diabetes is a ‘bihormonal’ disease, involving both insulin deficiency and excess glucagon.  For decades, insulin deficiency was considered to be the sole reason for diabetes; however, recent studies emphasize excess glucagon as an important part of diabetes etiology.  Thus, insulin-secreting β cells and glucagon-secreting α cells maintain physiological blood glucose levels, and their malfunction drives diabetes development.  Increasing the number of insulin-producing β cells while decreasing the number of glucagon-producing α cells, either in vitro in donor pancreatic islets before transplantation into type 1 diabetics or in vivo in type 2 diabetics, is a promising therapeutic avenue.  A huge leap has been taken in this direction by the researchers at the University of Pennsylvania (Philadelphia, PA) in collaboration with Oregon Health and Science University (Portland, OR), USA by demonstrating that α to β cell reprogramming could be promoted by manipulating the histone methylation signature of human pancreatic islets.  In fact, the treatment of cultured pancreatic islets with a histone methyltransferase inhibitor leads to colocalization of both glucagon and insulin and glucagon and insulin promoter factor 1 (PDX1) in human islets and colocalization of both glucagon and insulin in mouse islets.  The research findings were published in the Journal of Clinical Investigation.

Study design: First step was to study and analyze the epigenetic and transcriptional landscape of human pancreatic human pancreatic α, β, and exocrine cells using ChIP and RNA sequencing.  Study design for determination of the transcriptome and differential histone marks included the dispersion and FACS to of human islets to obtain cell populations highly enriched for α, β, and exocrine (duct and acinar) cells.  Then, chromatin was prepared for ChIP analysis using antibodies for histone modifications, H3K4me3 (represents gene activation) and H3K27me3 (represents gene repression).  RNA-Sequencing analysis was then performed to determine mRNA and lncRNA.  Sample purity was confirmed using qRT-PCR of insulin and glucagon expression levels of the individual α and β cell population revealing high sample purity.

Results:

  • Long noncoding transcripts: Long noncoding RNA molecules have been implicated as important developmental regulators, cell lineage allocators, and contributors to disease development.  The authors discovered 12 cell–specific and 5 α cell–specific noncoding (lnc) transcripts, indicative of the valuable research resource represented from transcriptome data.  Recently discovered lncRNA molecules in islets are regulated during development and dysregulated in type 2 diabetic islets.
  • Monovalent histone modification landscapes shared among three cell types:  Monovalent H3K4me3-enriched regions, indicative of gene activation, were identified and compared in α, β, and exocrine cells.  Strikingly, the vast majority of monovalently H3K4me3-marked genes were shared among the 3 pancreatic cell lineages (83%–95%), reflecting both their related function in protein secretion and common embryonic descent. Similarly, a high degree of overlap was observed in H3K27me3 modification patterns in all the three cell types (73%–83%).
  • Bivalent histone modifications (H3K4me3 and H3K27me3) were high in α cells: Bernstein colleagues observed bivalent marks to be common in undifferentiated cells, such as ES cells and pluripotent progenitor cells, and in most cases, one of the histone modification marks was lost during differentiation, accompanying lineage specification (Bernstein BE, et al, Cell, 21 Apr 2006; 125(2):315-26).  α cells exhibited many more genes bivalently marked, followed by β cells and exocrine cells.  Bivalent state was remarkably similar to that of hESC, suggesting a more plastic epigenomic state for α cells.
  • Monovalent histone modifications were high in β cells: Thousands of the genes that were in bivalent state in α cells were in a monovalent state, carrying only the activating or repressing mark.
  • Inhibition of histone methyltransferases led to partial cell-fate conversion: Adenosine dialdehye (Adox), a drug that interferes with histone methylation and decreases H3K27me3, when administered in human islet tissue, led to decrease of H3K27me3 enrichment at the 3 gene loci that are originally expressed bivalently in α cells and monovalently in β cells:  MAFA, PDX1 and ARX.  Adox resulted in the occasional cooccurrence of glucagon and insulin granules within the same islet cell, which was not observed in untreated islets.  Thus, inhibition of histone methyltransferases leads to partial endocrine cell-fate conversion.

Conclusion:  α cells have been reprogrammed into β cell fate in various mouse models.  The reason, as proposed by the authors, might be the presence of more bivalently marked genes that confers a more plastic epigenomic state of the cells that probably drives them to the β cell fate.  Therefore, using epigenomic information of different cell types in pancreatic islets and harnessing it for subsequent manipulation of their epigenetic signature could be utilized to reprogram cells and hence provide a path for diabetes therapy.

Source: Bramswig NC, et al, Epigenomic plasticity enables human pancreatic α to β cell reprogramming. J Clin Invest, 22 Feb 2013. pii: 66514.

Related reading on Pharmaceutical Intelligence:

Junk DNA codes for valuable miRNAs: non-coding DNA controls Diabetes

Therapeutic Targets for Diabetes and Related Metabolic Disorders

Reprogramming cell fate

CRACKING THE CODE OF HUMAN LIFE: Recent Advances in Genomic Analysis and Disease – Part IIC

2013 Genomics: The Era Beyond the Sequencing of the Human Genome: Francis Collins, Craig Venter, Eric Lander, et al.

Genome-Wide Detection of Single-Nucleotide and Copy-Number Variation of a Single Human Cell

SNAP: Predict Effect of Non-synonymous Polymorphisms: How well Genome Interpretation Tools could Translate to the Clinic

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Differentiation Therapy – Epigenetics Tackles Solid Tumors

Author-Writer: Stephen J. Williams, Ph.D.

Genetic and epigenetic events within a cell which promote a block in normal development or differentiation coupled with unregulated proliferation are hallmarks of neoplastic transformation.  Differentiation therapy is a chemotherapeutic strategy directed at re-activating endogenous cellular differentiation programs in a tumor cell therefore driving the cancerous cell to a state closer resembling the normal or preneoplastic cell and therefore incurring loss of the tumorigenic phenotype.

This post will deal with:

  • Agents such as histone deacetylase inhibitors (HDACi), retinoids, and PPARϒ agonists which have been shown to reactivate terminal differentiation programs in solid tumors
  • Clinical trials in solid tumors
  • Issues regarding the use of differentiation therapy in solid tumors

This post is a follow-up post to Histone Deacetylase Inhibitors Induce Epithelial-to-Mesenchymal Transition in Prostate Cancer Cells

To put the need for alternate chemotherapeutic strategies in perspective, one is referred to the National Cancer Statistics from http://www.cancer.gov show that 33% of cancer patients, treated with standard cytolytic chemotherapy, will still die within five years (i.e. one in three will die within 5 years).  However the addition of the differentiation agent retinoic acid to standard chemotherapy regimen for treatment of acute promyelocytic leukemia (APML) had improved 5 year survival rates from a range of 50-80% up to near 90% complete remission rates while 75% become disease free, an astonishing success story.  For a review of APML please be referred to http://en.wikipedia.org/wiki/Acute_promyelocytic_leukemia.  Briefly, APML is predominantly a result of the chromosomal translocation producing a fusion gene between the promyelocytic leukemia (PML) and RARα receptor genes.  The PML-RARα fusion protein recruits transcriptional repressors, histone deacetylases (HDACs), and DNA methyltransferases.  Treatment with pharmacologic doses of retinoic acid dissociates the PML-RARα from HDACs and results in degradation of PML-RARα, eventually resulting in the differentiation of the myeloid cells in APML.

Dr. Igor Matushansky of Columbia University believes such differentiation therapy could be useful in soft tissue sarcomas, due to the existence of a connective tissue (mesenchymal) stem cell,  in vitro methods which can differentiate these cells into mature tissues, and, from a gene clustering analysis his group had performed, correlation of expression signatures of each liposarcoma subtype throughout the adipocytic differentiation spectrum, including early differentiated to more mature differentiated cells(1).   A parallel study by Riester and colleagues had been able to classify breast tumors and liposarcomas along a phylogenetic tree showing solid tumors can be reclassified based on cell of origin via expression patterns(2).  In addition, other solid tumors, such as ovarian cancer are easily classified, based both on pathologic, histologic, and expression analysis into well and poorly differentiated tumors, correlating differentiation status with prognosis.

Compound Classes which have potential in

differentiation therapy for solid tumors

A. Histone Deacetylase Inhibitors (HDACi)

In eukaryotes, epigenetic post-translational modification of histones is critical for regulation of chromatin structure and gene expression.  Histone deacetylation leads to chromatin compaction and is associated with transcriptional repression of tumor suppressors, cell growth and differentiation.  Therefore, HDACi are promising anti-tumor agents as they may affect the cell cycle, inhibit proliferation, stimulate differentiation and induce apoptotic cell death (3). In a review by Kniptein and Gore, entinostat was found to be a well-tolerated HDACi that demonstrates promising therapeutic potential in both solid and hematologic malignancies(4). The path to the discovery of suberoylanilide hydroxamic acid (SAHA, vorinostat) began over three decades ago with our studies designed to understand why dimethylsulfoxide causes terminal differentiation of the virus-transformed cells, murine erythroleukemia cells. SAHA can cause growth arrest and death of a broad variety of transformed cells both in vitro and in vivo at concentrations that have little or no toxic effects on normal cells (for references see (5). In fact, treatment of MCF-7 breast carcinoma cells with SAHA resulted in morphologic changes resembling epithelial mammary differentiation(6).

HDAC inhibitors

Figure.  Structures of some HDACi used in clinical trials for cancer (see section below)

hdacwithsaha

Figure.  HDAC with SAHA

B. Retinoids

Vitamin A and retinoids play significant roles in basic physiological processes such as vision, reproduction, growth, development, hematopoiesis and immunity (7). Retinoids are the natural derivatives and synthetic analogs of vitamin A. They have been shown to prevent mammary carcinogenesis in rodents (8), to inhibit the growth of human cancer cells in vitro  (9,10) and be effective chemopreventive and chemotherapeutic agents in a variety of human epithelial and hematopoietic tumors (11-14).

Retinoids cannot be synthesized de novo by higher animals and consequently must be consumed in the diet. The two sources of retinoids are animal products that contain retinol and retinyl esters, and plant-derived carotenoids (provitamin A). b-carotene is the most potent vitamin A precursor and has been shown to be an active inhibitor of both tumor initiation and promotion (15).

A major function of retinol, relevant to cancer, is its function as an antioxidant. The antioxidant properties of vitamin A have been shown both in vitro and in vivo (16,17). Retinol deficiency causes oxidative damage to liver mitochondria in rats that can be reversed by vitamin A supplementation (18). A caveat to this is in vitro and in vivo evidence of chronic hypervitaminosis A inducing oxidative DNA damage, as well (19-21). Therefore, it is evident that maintaining the vitamin A concentration within a physiological range is critical to normal cell function because either a deficiency or an excess of vitamin A induces oxidative stress (22). Retinoic acids (RA) (all-trans, 9-cis and 13-cis) are the major biologically active retinoids and exert their effects by regulation of gene expression by binding two families of ligand-activated nuclear retinoid receptors (23). Retinoic acid receptors (RARs) and retinoid X receptors (RXRs) regulate the transcription of a large number of target genes that contain retinoic acid response elements (RAREs) in their promoters. Many of these genes are involved in cancer (13,24) and differentiation (24-26).

Several lines of evidence suggest involvement of defects in retinol signaling in cancer, from the observation that a vitamin A-deficient (VAD) diet leads to an increase in the number of spontaneous and chemically induced tumors in animals (27-29) to the observation that RA itself can induce  differentiation and inhibit the growth of many tumor cells (30-32), as well as the identification that components of the RA signaling pathway are absent in cancer cells (33). Vitamin A and its metabolites have been proposed to have a dual effect in cancer prevention, as antioxidants (16,17,19,34) and differentiating agents (35-37). as it is well accepted that retinoid signaling is integral in maintaining the differentiated state of many cell types (13,38). Additionally, current rationale for chemoprevention with retinoids is based, in part, on the hypothesis that some tumors, may arise due to loss of normal somatic differentiation during tissue repair.

C. PPARϒ Agonists

Peroxisome proliferator-activated receptor ϒ (PPARϒ) is a member of the steroid hormone receptor superfamily that responds to changes in lipid and glucose homeostasis but has increasing roles in differentiation and tumorigenesis. The first PPAR (PPARα) was discovered during the search of a molecular target for a group of agents then referred to as peroxisome proliferators, as they increased peroxisomal numbers in rodent liver tissue, apart from improving insulin sensitivity.  One of the first agents, developed in the early 80’s for treatment of hyperlipidemia and hperlipoproteinemia, was clofibrate.  All PPAR subtypes heterodimerize with the retinoid-x-receptor (RXR) and, upon binding of ATRA, activate target genes.

PPARϒ agonists have shown potential as a therapeutic in a variety of cancer types including bladder cancer (39), colon cancer(40),  breast cancer(41), prostate cancer(42).  There are numerous studies showing that PPARϒ agonists have anti-tumorigenic activity via anti-proliferative, pro-differentiation and anti-angiogenic mechanisms of action(43). For example, Papi et al. observed that agonists for the retinoid X receptor (6-OH-11-O-hydroxyphenanthrene), retinoic acid receptor (all-trans retinoic acid (RA)) and peroxisome proliferator-activated receptor (PPAR)-γ (pioglitazone (PGZ)), reduce the survival of MS generated from breast cancer tissues and MCF7 cells, but not from normal mammary gland or MCF10 cells(44) with concomitant upregulation of differentiation markers.

A great website for further information on PPAR is Dr. Jack Vanden Heuvel, Professor of Toxicology at Penn State University at http://ppar.cas.psu.edu/general_information.html.

D. Trabectedin

Trabectedin (ecteinascidin-743 (ET-743); Yondelis) is derived from the Caribbean tunicate Ecteinascidia turbinacta has antitumor activity by binding to the DNA minor groove thus disrupting binding of transcription factors and inhibiting DNA synthesis.  However, it has also been shown, in myxoid liposarcoma (MLS) cells, to cause dissociation of transcription factor TLS-CHOP from promoter sequences resulting in downregulation of target genes such as CHOP, PTX3 and FN1 and induces an adipogenic differentiation program by enhancing activation of CAAT/enhancer binding protein (C/EBP) family of genes.  In MLS, TLS-CHOP sequesters C/EBPβ resulting in block of differentiation programs while trabectedin disrupts this association freeing up C/EBPβ to act as transcriptional activator of genes related to differentiation.

Ongoing Cancer Clinical Trials with HDAC Inhibitors

The following is a listing of some clinical trials using histone deacetylase inhibitors in combination with approved chemotherapeutics in various tumors.  This data was taken from the New Medicine Oncology Knowledge Base ( at http://www.nmok.net).

hdactrial1 hdactrial2

Issues and Future of Differentiation-based Therapy

In the review by Filemon Dela Cruz and Igor Matushansky(1), the authors suggest that, like days of old of cytotoxic monotherapy, differentiation therapy would not evolve as a simplistic one-size-fits –all but mirror an extremely complicated process.  Therefore they suggest three theoretical mechanisms in which differentiation therapy may occur:

  1. Cancer directed differentiation: differentiation pathways are activated without correcting the underlying oncogenic mechanisms which produced the initial differentiation block
  2. Cancer reverted differentiation: correction of the underlying oncogenic mechanism results in restoration of endogenous differentiation pathways
  3. Cancer diverted differentiation: cancer cell is redirected to an earlier stage of differentiation

Finally the authors suggest that “the potential for reversion of the malignant cancer phenotype to a more benign, or at the very least a lower grade of biological aggressiveness, may serve as a critical clinical and biologic transition of a uniformly fatal cancer into one more amenable to management or to treatment using conventional therapeutic approaches.”

References:

1.            Cruz, F. D., and Matushansky, I. (2012) Oncotarget 3, 559-567

2.            Riester, M., Stephan-Otto Attolini, C., Downey, R. J., Singer, S., and Michor, F. (2010) PLoS computational biology 6, e1000777

3.            Seidel, C., Schnekenburger, M., Dicato, M., and Diederich, M. (2012) Genes & nutrition 7, 357-367

4.            Knipstein, J., and Gore, L. (2011) Expert opinion on investigational drugs 20, 1455-1467

5.            Marks, P. A. (2007) Oncogene 26, 1351-1356

6.            Munster, P. N., Troso-Sandoval, T., Rosen, N., Rifkind, R., Marks, P. A., and Richon, V. M. (2001) Cancer research 61, 8492-8497

7.            Napoli, J. L. (1999) Biochim Biophys Acta 1440, 139-162

8.            Moon, R., Metha, R., and Rao, K. (1994) Retinoids and cancer in experimental animals. in The Retinoids: Biology, Chemistry, and Medicine (Sporn, M., Roberts, A., and Goodman, D. eds.), 2 Ed., Raven Press, New York. pp 573-596

9.            De Luca, L. M. (1991) Faseb J 5, 2924-2933

10.          Gudas, L. J. (1992) Cell Growth Differ 3, 655-662

11.          Degos, L., and Parkinson, D. (1995) Retinoids in Oncology, Springer-Verlag, Berlin

12.          Lotan, R. (1996) Faseb J 10, 1031-1039

13.          Zhang, D., Holmes, W. F., Wu, S., Soprano, D. R., and Soprano, K. J. (2000) J Cell Physiol 185, 1-20

14.          Fontana, J. A., and Rishi, A. K. (2002) Leukemia 16, 463-472

15.          Suda, D., Schwartz, J., and Shklar, G. (1986) Carcinogenesis 7, 711-715

16.          Ciaccio, M., Valenza, M., Tesoriere, L., Bongiorno, A., Albiero, R., and Livrea, M. A. (1993) Arch Biochem Biophys 302, 103-108

17.          Palacios, A., Piergiacomi, V. A., and Catala, A. (1996) Mol Cell Biochem 154, 77-82

18.          Barber, T., Borras, E., Torres, L., Garcia, C., Cabezuelo, F., Lloret, A., Pallardo, F. V., and Vina, J. R. (2000) Free Radic Biol Med 29, 1-7

19.          Borras, E., Zaragoza, R., Morante, M., Garcia, C., Gimeno, A., Lopez-Rodas, G., Barber, T., Miralles, V. J., Vina, J. R., and Torres, L. (2003) Eur J Biochem 270, 1493-1501

20.          Omenn, G. S., Goodman, G. E., Thornquist, M. D., Balmes, J., Cullen, M. R., Glass, A., Keogh, J. P., Meyskens, F. L., Jr., Valanis, B., Williams, J. H., Jr., Barnhart, S., Cherniack, M. G., Brodkin, C. A., and Hammar, S. (1996) J Natl Cancer Inst 88, 1550-1559

21.          Murata, M., and Kawanishi, S. (2000) J Biol Chem 275, 2003-2008

22.          Schwartz, J. L. (1996) J Nutr 126, 1221S-1227S

23.          Chambon, P. (1996) Faseb J 10, 940-954

24.          Freemantle, S. J., Kerley, J. S., Olsen, S. L., Gross, R. H., and Spinella, M. J. (2002) Oncogene 21, 2880-2889

25.          Collins, S. J., Robertson, K. A., and Mueller, L. (1990) Mol Cell Biol 10, 2154-2163

26.          Grunt, T. W., Somay, C., Oeller, H., Dittrich, E., and Dittrich, C. (1992) J Cell Sci 103 ( Pt 2), 501-509

27.          Lasnitzki, I. (1955) Br J Cancer 9, 434-441

28.          Moore, T. (1965) Proc Nutr Soc 24, 129-135

29.          Saffiotti, U., Montesano, R., Sellakumar, A. R., and Borg, S. A. (1967) Cancer 20, 857-864

30.          Strickland, S., and Mahdavi, V. (1978) Cell 15, 393-403

31.          Breitman, T. R., Selonick, S. E., and Collins, S. J. (1980) Proc Natl Acad Sci U S A 77, 2936-2940

32.          Breitman, T. R., Collins, S. J., and Keene, B. R. (1981) Blood 57, 1000-1004

33.          Niles, R. M. (2000) Nutrition 16, 573-576

34.          Monagham, B., and Schmitt, F. (1932) J Biol Chem 96, 387-395

35.          Miller, W. H., Jr. (1998) Cancer 83, 1471-1482

36.          Miyauchi, J. (1999) Leuk Lymphoma 33, 267-280

37.          Reynolds, C. P. (2000) Curr Oncol Rep 2, 511-518

38.          Ortiz, M. A., Bayon, Y., Lopez-Hernandez, F. J., and Piedrafita, F. J. (2002) Drug Resist Updat 5, 162-175

39.          Mansure, J. J., Nassim, R., and Kassouf, W. (2009) Cancer biology & therapy 8, 6-15

40.          Osawa, E., Nakajima, A., Wada, K., Ishimine, S., Fujisawa, N., Kawamori, T., Matsuhashi, N., Kadowaki, T., Ochiai, M., Sekihara, H., and Nakagama, H. (2003) Gastroenterology 124, 361-367

41.          Stoll, B. A. (2002) Eur J Cancer Prev 11, 319-325

42.          Smith, M. R., and Kantoff, P. W. (2002) Investigational new drugs 20, 195-200

43.          Rumi, M. A., Ishihara, S., Kazumori, H., Kadowaki, Y., and Kinoshita, Y. (2004) Current medicinal chemistry. Anti-cancer agents 4, 465-477

44.          Papi, A., Guarnieri, T., Storci, G., Santini, D., Ceccarelli, C., Taffurelli, M., De Carolis, S., Avenia, N., Sanguinetti, A., Sidoni, A., Orlandi, M., and Bonafe, M. (2012) Cell death and differentiation 19, 1208-1219

Other research papers on Cancer and Cancer Therapeutics were published on this Scientific Web site as follows:

Histone Deacetylase Inhibitors Induce Epithelial-to-Mesenchymal Transition in Prostate Cancer Cells

PIK3CA mutation in Colorectal Cancer may serve as a Predictive Molecular Biomarker for adjuvant Aspirin therapy

Nanotechnology Tackles Brain Cancer

Response to Multiple Cancer Drugs through Regulation of TGF-β Receptor Signaling: a MED12 Control

Personalized medicine-based cure for cancer might not be far away

GSK for Personalized Medicine using Cancer Drugs needs Alacris systems biology model to determine the in silico effect of the inhibitor in its “virtual clinical trial”

Lung Cancer (NSCLC), drug administration and nanotechnology

Non-small Cell Lung Cancer drugs – where does the Future lie?

Cancer Innovations from across the Web

arrayMap: Genomic Feature Mining of Cancer Entities of Copy Number Abnormalities (CNAs) Data

How mobile elements in “Junk” DNA promote cancer. Part 1: Transposon-mediated tumorigenesis.

Cancer Genomics – Leading the Way by Cancer Genomics Program at UC Santa Cruz

Closing the gap towards real-time, imaging-guided treatment of cancer patients.

Closing the gap towards real-time, imaging-guided treatment of cancer patients.

mRNA interference with cancer expression

Search Results for ‘cancer’ on this web site

Cancer Genomics – Leading the Way by Cancer Genomics Program at UC Santa Cruz

Closing the gap towards real-time, imaging-guided treatment of cancer patients.

Lipid Profile, Saturated Fats, Raman Spectrosopy, Cancer Cytology

mRNA interference with cancer expression

Pancreatic cancer genomes: Axon guidance pathway genes – aberrations revealed

Biomarker tool development for Early Diagnosis of Pancreatic Cancer: Van Andel Institute and Emory University

Is the Warburg Effect the cause or the effect of cancer: A 21st Century View?

Crucial role of Nitric Oxide in Cancer

Targeting Glucose Deprived Network Along with Targeted Cancer Therapy Can be a Possible Method of Treatment

Read Full Post »


Histone Deacetylase Inhibitors Induce Epithelial-to-Mesenchymal Transition in Prostate Cancer Cells(1)

Authors: Dejuan Kong, Aamir Ahmad, Bin Bao, Yiwei Li, Sanjeev Banarjee, Fazlul H. Sarkar, Wayne State University School of Medicine

Reporter-Curator: Stephen J. Williams, Ph.D.

Clinically, there has not been much success in treating solid tumors with histone deacetylase inhibitors (HDACi). Histone acetylation and deacetylation play an important role in transcriptional regulation of genes and increased activity is associated with many cancers, therefore it was thought that HDAC inhibition might be fruitful as a therapy.  There have been several phase I and II clinical trials using HDACi for treatment of various malignancies, including hematological and solid malignancies(2), with most success seen in hematologic malignancies such as cutaneous T-cell lymphoma and peripheral T-cell lymphoma and little or no positive outcome with solid tumors.  Many mechanisms of resistance to HDACi in solid tumors have been described, most of which are seen with other chemotherapeutics such as increased multidrug resistance gene MDR1, increased anti-apoptotic proteins and activation of cell survival pathways(3).

A report in PLOS One by Dr. Dejuan Kong, Dr. Fazlul Sarkar, and colleagues from Wayne State University School of Medicine, demonstrate another possible mechanism of resistance to HDACi in prostate cancer, by induction of the epithelial-to-mesenchymal transition (EMT), which has been associated with the development of resistance to chemotherapies in other malignancies of epithelial origin(4,5).

EMT is an important differentiation process in embryogenesis and felt to be important in progression of cancer.  Epithelial cells will acquire a mesenchymal morphology (on plastic this looks like a cuboidal epithelial cell gaining a more flattened, elongated, tri-corner morphology; see paper Figure 1) and down-regulate epithelial markers such as cytokeratin, up-regulation of mesenchymal markers, increased migration and invasiveness in standard assays, and increased resistance to chemotherapeutics, and similarity to cancer stem cells(6-10).

ImageFigure 1. HDACis led to the induction of EMT phemotype. (A and B) PC3 cells treated with TSA and SAHA for 24 h at indicated doses.  The photomicrographs of PC3 cells treated with TSA and SAHA exhibited a fibroblastic-type phenotype, while cells treated with DMAO control displayed rounded epithelial cell morphology (original magnification, x 100). (C) Treated PC3 cells show increased mesenchymal markers vimentin and ZEB1 and F-actin reorganization.  Figure taken from Kong, D., Ahmad, A., Bao, B., Li, Y., Banerjee, S., and Sarkar, F. H. (2012) PloS one 7, e45045

In this study the authors found that treatment of prostate carcinoma cells with two different HDACis (trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA)) induced EMT phenotype mediated through up-regulation of transcription factors ZEB1, ZEB2 and Slug, increased expression of mesenchymal markers vimentin, N-cadherin and fibronectin by promoting histone 3 acetylation on gene promoters.  In addition TSA increased the stem cell markers Sox2 and Nanog with concomitant EMT morphology and increased cell motility.

Below is the abstract of this paper(1):

ABSTRACT

Clinical experience of histone deacetylase inhibitors (HDACIs) in patients with solid tumors has been disappointing; however, the molecular mechanism of treatment failure is not known. Therefore, we sought to investigate the molecular mechanism of treatment failure of HDACIs in the present study. We found that HDACIs Trichostatin A (TSA) and Suberoylanilide hydroxamic acid (SAHA) could induce epithelial-to-mesenchymal transition (EMT) phenotype in prostate cancer (PCa) cells, which was associated with changes in cellular morphology consistent with increased expression of transcription factors ZEB1, ZEB2 and Slug, and mesenchymal markers such as vimentin, N-cadherin and Fibronectin. CHIP assay showed acetylation of histone 3 on proximal promoters of selected genes, which was in part responsible for increased expression of EMT markers. Moreover, TSA treatment led to further increase in the expression of Sox2 and Nanog in PCa cells with EMT phenotype, which was associated with cancer stem-like cell (CSLC) characteristics consistent with increased cell motility. Our results suggest that HDACIs alone would lead to tumor aggressiveness, and thus strategies for reverting EMT-phenotype to mesenchymal-to-epithelial transition (MET) phenotype or the reversal of CSLC characteristics prior to the use of HDACIs would be beneficial to realize the value of HDACIs for the treatment of solid tumors especially PCa.

Highlights of the research include:

  • TSA and SAHA induce morphologic changes  in prostate carcinoma LNCaP and PC3 cells related to EMT by microscopy as well as accumulation of mesenchymal markers ZEB1, vimentin, and F-actin reorganization shown by immunofluorescence microscopy and increased expression of these markers shown by real-time PCR
  • Western blotting showed TSA treatment resulted in hyperacetyulation of histone 3 whi8le CHIP analysis revealed increased histone 3 acetylation on the promoters of vimentin, ZEB2, Slug, and MMP2
  • Western analysis revealed that HDACi not only induced EMT but increased the expression of cancer stem cell markers associated with increased motility such as Sox2 and Nanog.  Increased cell migration was measured by Transwell migration assays and increased cell motility was measured via cell detachment assays

1.            Kong, D., Ahmad, A., Bao, B., Li, Y., Banerjee, S., and Sarkar, F. H. (2012) PloS one 7, e45045

2.            Bertino, E. M., and Otterson, G. A. (2011) Expert opinion on investigational drugs 20, 1151-1158

3.            Robey, R. W., Chakraborty, A. R., Basseville, A., Luchenko, V., Bahr, J., Zhan, Z., and Bates, S. E. (2011) Molecular pharmaceutics 8, 2021-2031

4.            Wang, Z., Li, Y., Kong, D., Banerjee, S., Ahmad, A., Azmi, A. S., Ali, S., Abbruzzese, J. L., Gallick, G. E., and Sarkar, F. H. (2009) Cancer research 69, 2400-2407

5.            Wang, Z., Li, Y., Ahmad, A., Azmi, A. S., Kong, D., Banerjee, S., and Sarkar, F. H. (2010) Drug resistance updates : reviews and commentaries in antimicrobial and anticancer chemotherapy 13, 109-118

6.            Hugo, H., Ackland, M. L., Blick, T., Lawrence, M. G., Clements, J. A., Williams, E. D., and Thompson, E. W. (2007) Journal of cellular physiology 213, 374-383

7.            Thiery, J. P. (2002) Nature reviews. Cancer 2, 442-454

8.            Kong, D., Banerjee, S., Ahmad, A., Li, Y., Wang, Z., Sethi, S., and Sarkar, F. H. (2010) PloS one 5, e12445

9.            Kong, D., Li, Y., Wang, Z., and Sarkar, F. H. (2011) Cancers 3, 716-729

10.          Bao, B., Wang, Z., Ali, S., Kong, D., Li, Y., Ahmad, A., Banerjee, S., Azmi, A. S., Miele, L., and Sarkar, F. H. (2011) Cancer letters 307, 26-36

Other research papers on Cancer and Cancer Therapeutics were published on this Scientific Web site as follows:

PIK3CA mutation in Colorectal Cancer may serve as a Predictive Molecular Biomarker for adjuvant Aspirin therapy

Nanotechnology Tackles Brain Cancer

Response to Multiple Cancer Drugs through Regulation of TGF-β Receptor Signaling: a MED12 Control

Personalized medicine-based cure for cancer might not be far away

GSK for Personalized Medicine using Cancer Drugs needs Alacris systems biology model to determine the in silico effect of the inhibitor in its “virtual clinical trial”

Lung Cancer (NSCLC), drug administration and nanotechnology

Non-small Cell Lung Cancer drugs – where does the Future lie?

Cancer Innovations from across the Web

arrayMap: Genomic Feature Mining of Cancer Entities of Copy Number Abnormalities (CNAs) Data

How mobile elements in “Junk” DNA promote cancer. Part 1: Transposon-mediated tumorigenesis.

Cancer Genomics – Leading the Way by Cancer Genomics Program at UC Santa Cruz

Closing the gap towards real-time, imaging-guided treatment of cancer patients.

Closing the gap towards real-time, imaging-guided treatment of cancer patients.

mRNA interference with cancer expression

Search Results for ‘cancer’ on this web site

Cancer Genomics – Leading the Way by Cancer Genomics Program at UC Santa Cruz

Closing the gap towards real-time, imaging-guided treatment of cancer patients.

Lipid Profile, Saturated Fats, Raman Spectrosopy, Cancer Cytology

mRNA interference with cancer expression

Pancreatic cancer genomes: Axon guidance pathway genes – aberrations revealed

Biomarker tool development for Early Diagnosis of Pancreatic Cancer: Van Andel Institute and Emory University

Is the Warburg Effect the cause or the effect of cancer: A 21st Century View?

Crucial role of Nitric Oxide in Cancer

Targeting Glucose Deprived Network Along with Targeted Cancer Therapy Can be a Possible Method of Treatment

Read Full Post »


ENCODE data reveals important information from Genome Wide Association Studies relevant to understanding complex genetic diseases

Author: Ritu Saxena, Ph.D.

Introduction

“The depth, quality, and diversity of the ENCODE data are unprecedented” is what was stated by John Stamatoyannopoulos, professor of genomic sciences at the University of Washington and one of the many principle investigators of ENCODE project. ENCODE (Encyclopedia of DNA elements), indeed, was an ambitious project launched as a pilot in 2003 and then expanded in 2007 for the whole genome analysis and identification of all the functional elements of the human genome. The findings were striking as they challenged the definition of “gene” and ‘the central dogma of genetics (Gene-mRNA-protein). Infact, the non-coding part that constitutes about 80% of the genome or the so-called “junk DNA” was found to contain elements crucial for gene regulation. The elements, in large part, include RNA transcripts that are not transcribed into proteins but might have a regulatory role. For detailed reading, refer to the findings published in the issue of Nature, The ENCODE Project Consortium Nature 489, 57–74 (2012) An integrated encyclopedia of DNA elements in the human genome

Key features of the data, as explained in the National Human Genome Research Institute website (National Human Genome Research Institute News feature), include comprehensive mapping of:

  • Protein-coding genes — Proteins are molecules made of amino acids linked together in a specific sequence; the amino acid sequence is encoded by the sequence of DNA subunits called nucleotides that make up genes.
  • Non-coding genes — Stretches of DNA that are read by the cell as if they were genes but do not encode proteins. These appear to help regulate the activity of the genome.
  • Chromatin structure features — Complex physical structures made from a combination of DNA and binding proteins that make up the contents of the nucleus and affects genome function.
  • Histone modifications — Histones are the proteins that make up the chromatin structures that help shape and control the genome. In addition, histone proteins can be physically modified by adding chemical groups, such as a methyl molecule, that further regulates genomic activity.
  • DNA methylation — Just like histones, methyl groups can be added to DNA itself in a process called DNA methylation. Chemically attaching methyl groups to DNA physically changes the ability of enzymes to reach the DNA and thus alters the gene expression pattern in cells. Methylation helps cells “remember what they are doing” or alter levels of gene expression, and it is a crucial part of normal development and cellular differentiation in higher organisms.
  • Transcription factor binding sites — Transcription factors are proteins that bind to specific DNA sequences, controlling the flow (or transcription) of genetic information from DNA to mRNA. Mapping the binding sites can help researchers understand how genomic activity is controlled.

How could ENCODE be helpful in the study of complex human diseases?

Complex diseases and Genome wide association studies (GWAS)

Coronary artery disease, type 2 diabetes and many forms of cancer are complex human diseases that have a significant genetic component. Unlike mendelian disorders that have defined loci, the genetic component of complex disorders lies in the form of genetic variations in the genome making an individual susceptible to these complex diseases.

Researchers have performed Genome-wide association studies (GWAS) of the human genome, leading to the identification of thousands of DNA variants that could be linked with complex traits and diseases. However, identifying the variants, referred to as SNPs (Single Nucleotide Polymorphisms), that actually contribute to the disease, and understanding how they exert influence on a disease has been more of a mystery.

How would ENCODE solve the puzzle?

The puzzle lies in interpreting how the SNPs found in the genome affect a person’s susceptibility to a particular trait or disease and what is the mechanism behind it. As identified in the GWAS, most variants that are associated with the phenotype of the trait or disease lie in the non-coding region of the genome. Infact, in more than 400 studies compiled in the GWAS catalog only a small minority of the trait/disease-associated SNPs occur in protein-coding regions; the large majority (89%) are in noncoding regions. These variants fall in the gene deserts that lie far from protein-coding region, similar to those where cis-regulatory modules (CRMs) are found. CRMs such as promoters and enhancers are a group of binding sites for transcription factors, and the presence of transcription factors bound to these sites is a good indicator of the potential regulatory regions.

The integrative analysis of ENCODE data has give important insights to the results of GWAS studies. Investigators have employed ENCODE data as an initial guide to discover regulatory regions in which genetic variation is affecting a complex trait. Additionally, ENCODE study when examined the SNPs from GWAS that were associated with the phenotype of the trait, found that these regions are enriched in DNase-sensitive regions i.e, lie in the function-associated DNA region of the genome as it could be bound by transcription factors affecting the regulation of gene expression. Thus, the project demonstrates that non-coding regions must be considered when interpreting GWAS results, and it provides a strong motivation for reinterpreting previous GWAS findings.

Using ENCODE Data to Interpret GWAS Results

ENCODE and predisposition to CANCER:

C-Myc, a proto-oncogene, codes for a transcripton factor, when expressed constitutively leads to uninhibited cell proliferation resulting in cancer. It has been observed that common variants within a ~1 Mb region upstream of c-Myc gene have been associated with cancers of the colon, prostate, and breast. Several SNPs have been reported in this region, that although affect the phenotype, lie in the distal cis-region of the MYC gene. Alignment of the ENCODE data in this region with the significant variants from the GWAS also reveals that key variants are found in the transcription factor occupied DNA segments mapped by this consortium. One variant rs698327, lies within a DNase hypersensitive site that is bound by several transcription factors, enhancer-associated protein p300, and contains histone modifications relative to enhancers (high H3K4me1, low H3K4me3). ENCODE data indicates that non-coding regions in the human chromosome 8q24 loci are associated with cancer and as observed in the case of c-myc gene, similar studies on cancer-related genes could help explain predisposition to cancer.

ENCODE and fetal hemoglobin expression:

Another example of the use of ENCODE data is that of gene regulation of fetal hemoglobin. Several regions were predicted via ENCODE that were involved in the regulation of fetal hemoglobin. It was found that these predicted regions are close to the SNPs in the BLC11A gene that is associated with persistent expression of fetal hemoglobin.

Future perspective

As evident from the above examples, the ENCODE data shows that genetic variants do affect regulated expression of a target gene. Recently, several research groups in the UK performed a large-scale GWAS study to determine the genetic predisposition to fracture risk. The collaborative effort, published in a recent issue of the PLoS journal, was made to identify genetic variants associated with cortical bone thickness (CBT) and bone mineral density (BMD) with data from more than 10,000 subjects. http://www.plosgenetics.org/article/info%3Adoi%2F10.1371%2Fjournal.pgen.1002745 The study generated a wealth of data including the result – identification of SNPs in the WNT16 and its adjacent gene, FAM3C were found to be relevant to CBT and BMD. ENCODE data, in this case, could be helpful in interpreting more detailed information including determining additional SNPs, the regulatory information of the genes involved and much more. Thus, it could be concluded that ENCODE data could be immensely useful in interpreting associations between disease and DNA sequences that can vary from person to person.

Sources:

Research articles

An integrated encyclopedia of DNA elements in the human genome

A User’s Guide to the Encyclopedia of DNA Elements (ENCODE)

What does our genome encode?

Genome-wide Epigenetic Data Facilitate Understanding of Disease Susceptibility Association Studies

Genomics: ENCODE explained

ENCODE Project Writes Eulogy For Junk DNA

WNT16 Influences Bone Mineral Density, Cortical Bone Thickness, Bone Strength, and Osteoporotic Fracture Risk

 News articles

ENCODE project: In massive genome analysis new data suggests ‘gene’ redefinition

National Human Genome Research Institute News feature

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