Showing papers in "Epigenetics & Chromatin in 2014"

Chromatin accessibility: a window into the genome

Abstract: Transcriptional activation throughout the eukaryotic lineage has been tightly linked with disruption of nucleosome organization at promoters, enhancers, silencers, insulators and locus control regions due to transcription factor binding. Regulatory DNA thus coincides with open or accessible genomic sites of remodeled chromatin. Current chromatin accessibility assays are used to separate the genome by enzymatic or chemical means and isolate either the accessible or protected locations. The isolated DNA is then quantified using a next-generation sequencing platform. Wide application of these assays has recently focused on the identification of the instrumental epigenetic changes responsible for differential gene expression, cell proliferation, functional diversification and disease development. Here we discuss the limitations and advantages of current genome-wide chromatin accessibility assays with especial attention on experimental precautions and sequence data analysis. We conclude with our perspective on future improvements necessary for moving the field of chromatin profiling forward.

Three-dimensional super-resolution microscopy of the inactive X chromosome territory reveals a collapse of its active nuclear compartment harboring distinct Xist RNA foci

Abstract: A Xist RNA decorated Barr body is the structural hallmark of the compacted inactive X territory in female mammals. Using super-resolution three-dimensional structured illumination microscopy (3D-SIM) and quantitative image analysis, we compared its ultrastructure with active chromosome territories (CTs) in human and mouse somatic cells, and explored the spatio-temporal process of Barr body formation at onset of inactivation in early differentiating mouse embryonic stem cells (ESCs). We demonstrate that all CTs are composed of structurally linked chromatin domain clusters (CDCs). In active CTs the periphery of CDCs harbors low-density chromatin enriched with transcriptionally competent markers, called the perichromatin region (PR). The PR borders on a contiguous channel system, the interchromatin compartment (IC), which starts at nuclear pores and pervades CTs. We propose that the PR and macromolecular complexes in IC channels together form the transcriptionally permissive active nuclear compartment (ANC). The Barr body differs from active CTs by a partially collapsed ANC with CDCs coming significantly closer together, although a rudimentary IC channel system connected to nuclear pores is maintained. Distinct Xist RNA foci, closely adjacent to the nuclear matrix scaffold attachment factor-A (SAF-A) localize throughout Xi along the rudimentary ANC. In early differentiating ESCs initial Xist RNA spreading precedes Barr body formation, which occurs concurrent with the subsequent exclusion of RNA polymerase II (RNAP II). Induction of a transgenic autosomal Xist RNA in a male ESC triggers the formation of an ‘autosomal Barr body’ with less compacted chromatin and incomplete RNAP II exclusion. 3D-SIM provides experimental evidence for profound differences between the functional architecture of transcriptionally active CTs and the Barr body. Basic structural features of CT organization such as CDCs and IC channels are however still recognized, arguing against a uniform compaction of the Barr body at the nucleosome level. The localization of distinct Xist RNA foci at boundaries of the rudimentary ANC may be considered as snap-shots of a dynamic interaction with silenced genes. Enrichment of SAF-A within Xi territories and its close spatial association with Xist RNA suggests their cooperative function for structural organization of Xi.

Epigenetic marking of sperm by post-translational modification of histones and protamines

Abstract: Background The concept that individual traits can be acquired and transmitted by the germline through epigenetic mechanisms has gained recognition in the past years. However, epigenetic marks in sperm have not been are not well identified.

Targeted Chromatin Capture (T2C): a novel high resolution high throughput method to detect genomic interactions and regulatory elements

Abstract: Background: Significant efforts have recently been put into the investigation of the spatial organization and the chromatin-interaction networks of genomes. Chromosome conformation capture (3C) technology and its derivatives are important tools used in this effort. However, many of these have limitations, such as being limited to one viewpoint, expensive with moderate to low resolution, and/or requiring a large sequencing effort. Techniques like Hi-C provide a genome-wide analysis. However, it requires massive sequencing effort with considerable costs. Here we describe a new technique termed Targeted Chromatin Capture (T2C), to interrogate large selected regions of the genome. T2C provides an unbiased view of the spatial organization of selected loci at superior resolution (single restriction fragment resolution, from 2 to 6 kbp) at much lower costs than Hi-C due to the lower sequencing effort. Results: We applied T2C on well-known model regions, the mouse β-globin locus and the human H19/IGF2 locus. In both cases we identified all known chromatin interactions. Furthermore, we compared the human H19/IGF2 locus data obtained from different chromatin conformation capturing methods with T2C data. We observed the same compartmentalization of the locus, but at a much higher resolution (single restriction fragments vs. the common 40 kbp bins) and higher coverage. Moreover, we compared the β-globin locus in two different biological samples (mouse primary erythroid cells and mouse fetal brain), where it is either actively transcribed or not, to identify possible transcriptional dependent interactions. We identified the known interactions in the β-globin locus and the same topological domains in both mouse primary erythroid cells and in mouse fetal brain with the latter having fewer interactions probably due to the inactivity of the locus. Furthermore, we show that interactions due to the important chromatin proteins, Ldb1 and Ctcf, in both tissues can be analyzed easily to reveal their role on transcriptional interactions and genome folding. Conclusions: T2C is an efficient, easy, and affordable with high (restriction fragment) resolution tool to address both genome compartmentalization and chromatin-interaction networks for specific genomic regions at high resolution for both clinical and non-clinical research.

Transgenerational epigenetics in the germline cycle of Caenorhabditis elegans

Abstract: Epigenetic mechanisms create variably stable changes in gene expression through the establishment of heritable states of chromatin architecture. While many epigenetic phenomena are, by definition, heritably passed through cell division during animal and plant development, evidence suggests that ‘epigenetic states’ may also be inherited across multiple generations. Work in the nematode Caenorhabditis elegans has uncovered a number of mechanisms that participate in regulating the transgenerational passage of epigenetic states. These mechanisms include some that establish and maintain heritable epigenetic information in the form of histone modifications, as well as those that filter the epigenetic information that is stably transmitted. The information appears to influence and help guide or regulate gene activity and repression in subsequent generations. Genome surveillance mechanisms guided by small RNAs appear to be involved in identifying and directing heritable repression of genomic elements, and thus may participate in filtering information that is inappropriate for stable transmission. This review will attempt to summarize recent findings that illustrate this simple nematode to be a truly elegant resource for defining emerging biological paradigms. As the cell lineage that links generations, the germline is the carrier of both genetic and epigenetic information. Like genetic information, information in the epigenome can heritably affect gene regulation and phenotype; yet unlike genetic information, the epigenome of the germ lineage is highly modified within each generation. Despite such alterations, some epigenetic information is highly stable across generations, leading to transgenerationally stable phenotypes that are unlinked to genetic changes. Studies in the nematode C. elegans have uncovered mechanisms that contribute to transgenerational repression as well as to the expression of genes that rely on histone modifying machinery and/or non-coding RNA-based mechanisms. These studies indicate that epigenetic mechanisms operating within the germ cell cycle of this organism filter and maintain an epigenetic memory that is required for germ cell function and can also influence gene expression in somatic lineages.

DNA methylation dynamics during ex vivo differentiation and maturation of human dendritic cells

Abstract: Dendritic cells (DCs) are important mediators of innate and adaptive immune responses, but the gene networks governing their lineage differentiation and maturation are poorly understood. To gain insight into the mechanisms that promote human DC differentiation and contribute to the acquisition of their functional phenotypes, we performed genome-wide base-resolution mapping of 5-methylcytosine in purified monocytes and in monocyte-derived immature and mature DCs. DC development and maturation were associated with a great loss of DNA methylation across many regions, most of which occurs at predicted enhancers and binding sites for known transcription factors affiliated with DC lineage specification and response to immune stimuli. In addition, we discovered novel genes that may contribute to DC differentiation and maturation. Interestingly, many genes close to demethylated CG sites were upregulated in expression. We observed dynamic changes in the expression of TET2, DNMT1, DNMT3A and DNMT3B coupled with temporal locus-specific demethylation, providing possible mechanisms accounting for the dramatic loss in DNA methylation. Our study is the first to map DNA methylation changes during human DC differentiation and maturation in purified cell populations and will greatly enhance the understanding of DC development and maturation and aid in the development of more efficacious DC-based therapeutic strategies.

Two ways to fold the genome during the cell cycle: insights obtained with chromosome conformation capture

Abstract: Genetic and epigenetic inheritance through mitosis is critical for dividing cells to maintain their state. This process occurs in the context of large-scale re-organization of chromosome conformation during prophase leading to the formation of mitotic chromosomes, and during the reformation of the interphase nucleus during telophase and early G1. This review highlights how recent studies over the last 5 years employing chromosome conformation capture combined with classical models of chromosome organization based on decades of microscopic observations, are providing new insights into the three-dimensional organization of chromatin inside the interphase nucleus and within mitotic chromosomes. One striking observation is that interphase genome organization displays cell type-specific features that are related to cell type-specific gene expression, whereas mitotic chromosome folding appears universal and tissue invariant. This raises the question of whether or not there is a need for an epigenetic memory for genome folding. Herein, the two different folding states of mammalian genomes are reviewed and then models are discussed wherein instructions for cell type-specific genome folding are locally encoded in the linear genome and transmitted through mitosis, e.g., as open chromatin sites with or without continuous binding of transcription factors. In the next cell cycle these instructions are used to re-assemble protein complexes on regulatory elements which then drive three-dimensional folding of the genome from the bottom up through local action and self-assembly into higher order levels of cell type-specific organization. In this model, no explicit epigenetic memory for cell type-specific chromosome folding is required.

Meta-analysis of human methylomes reveals stably methylated sequences surrounding CpG islands associated with high gene expression

Abstract: DNA methylation is thought to play an important role in the regulation of mammalian gene expression, partly based on the observation that a lack of CpG island methylation in gene promoters is associated with high transcriptional activity. However, the CpG island methylation level only accounts for a fraction of the variance in gene expression, and methylation in other domains is hypothesized to play a role. We hypothesized that regions of very high stability in methylation would exist and provide biological insight into the role of methylation both within and outside CpG islands. We set out to identify highly stable regions in the human methylome, based on the subset of CpGs assayed with an Illumina Infinium 450 K array. Using 1,737 samples from 30 publically available studies, we identified 15,224 CpGs that are ‘ultrastable’ in their state across tissues and developmental stages (974 always methylated; 14,250 always unmethylated). Further analysis of ultrastable CpGs led us to identify a novel subset of CpG islands, ‘ravines’, which exhibit a markedly consistent pattern of low methylation with highly methylated flanking shores and shelves. We distinguish ravines from other CpG islands characterized by a broader flanking region of low methylation. Interestingly, ravines are associated with higher gene expression compared to typical unmethylated CpG islands, and are more often found near housekeeping genes. The identification of ultrastable sites in the human methylome led us to identify a subclass of CpG islands characterized by a very stable pattern of methylation encompassing the island and flanking regions, established early in development and maintained through differentiation. This pattern is associated with particularly high levels of gene expression, providing new evidence that methylation beyond the CpG island could play a role in gene expression.

Telomere shortening and telomere position effect in mild ring 17 syndrome.

Abstract: Background Ring chromosome 17 syndrome is a rare disease that arises from the breakage and reunion of the short and long arms of chromosome 17. Usually this abnormality results in deletion of genetic material, which explains the clinical features of the syndrome. Moreover, similar phenotypic features have been observed in cases with complete or partial loss of the telomeric repeats and conservation of the euchromatic regions. We studied two different cases of ring 17 syndrome, firstly, to clarify, by analyzing gene expression analysis using real-time qPCR, the role of the telomere absence in relationship with the clinical symptoms, and secondly, to look for a new model of the mechanism of ring chromosome transmission in a rare case of familial mosaicism, through cytomolecular and quantitative fluorescence in-situ hybridization (Q-FISH) investigations.

Assessing cellular efficacy of bromodomain inhibitors using fluorescence recovery after photobleaching.

Abstract: Background: Acetylation of lysine residues in histone tails plays an important role in the regulation of gene transcription. Bromdomains are the readers of acetylated histone marks, and, consequently, bromodomaincontaining proteins have a variety of chromatin-related functions. Moreover, they are increasingly being recognised as important mediators of a wide range of diseases. The first potent and selective bromodomain inhibitors are beginning to be described, but the diverse or unknown functions of bromodomain-containing proteins present challenges to systematically demonstrating cellular efficacy and selectivity for these inhibitors. Here we assess the viability of fluorescence recovery after photobleaching (FRAP) assays as a target agnostic method for the direct visualisation of an on-target effect of bromodomain inhibitors in living cells. Results: Mutation of a conserved asparagine crucial for binding to acetylated lysines in the bromodomains of BRD3, BRD4 and TRIM24 all resulted in reduction of FRAP recovery times, indicating loss of or significantly reduced binding to acetylated chromatin, as did the addition of known inhibitors. Significant differences between wild type and bromodomain mutants for ATAD2, BAZ2A, BRD1, BRD7, GCN5L2, SMARCA2 and ZMYND11 required the addition of the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) to amplify the binding contribution of the bromodomain. Under these conditions, known inhibitors decreased FRAP recovery times back to mutant control levels. Mutation of the bromodomain did not alter FRAP recovery times for full-length CREBBP, even in the presence of SAHA, indicating that other domains are primarily responsible for anchoring CREBBP to chromatin. However, FRAP assays with multimerised CREBBP bromodomains resulted in a good assay to assess the efficacy of bromodomain inhibitors to this target. The bromodomain and extraterminal protein inhibitor PFI-1 was inactive against other bromodomain targets, demonstrating the specificity of the method. Conclusions: Viable FRAP assays were established for 11 representative bromodomain-containing proteins that broadly cover the bromodomain phylogenetic tree. Addition of SAHA can overcome weak binding to chromatin, and the use of tandem bromodomain constructs can eliminate masking effects of other chromatin binding domains. Together, these results demonstrate that FRAP assays offer a potentially pan-bromodomain method for generating cell-based assays, allowing the testing of compounds with respect to cell permeability, on-target efficacy and selectivity.

The Mbd1-Atf7ip-Setdb1 pathway contributes to the maintenance of X chromosome inactivation

Abstract: X chromosome inactivation (XCI) is a developmental program of heterochromatin formation that initiates during early female mammalian embryonic development and is maintained through a lifetime of cell divisions in somatic cells. Despite identification of the crucial long non-coding RNA Xist and involvement of specific chromatin modifiers in the establishment and maintenance of the heterochromatin of the inactive X chromosome (Xi), interference with known pathways only partially reactivates the Xi once silencing has been established. Here, we studied ATF7IP (MCAF1), a protein previously characterized to coordinate DNA methylation and histone H3K9 methylation through interactions with the methyl-DNA binding protein MBD1 and the histone H3K9 methyltransferase SETDB1, as a candidate maintenance factor of the Xi. We found that siRNA-mediated knockdown of Atf7ip in mouse embryonic fibroblasts (MEFs) induces the activation of silenced reporter genes on the Xi in a low number of cells. Additional inhibition of two pathways known to contribute to Xi maintenance, DNA methylation and Xist RNA coating of the X chromosome, strongly increased the number of cells expressing Xi-linked genes upon Atf7ip knockdown. Despite its functional importance in Xi maintenance, ATF7IP does not accumulate on the Xi in MEFs or differentiating mouse embryonic stem cells. However, we found that depletion of two known repressive biochemical interactors of ATF7IP, MBD1 and SETDB1, but not of other unrelated H3K9 methyltransferases, also induces the activation of an Xi-linked reporter in MEFs. Together, these data indicate that Atf7ip acts in a synergistic fashion with DNA methylation and Xist RNA to maintain the silent state of the Xi in somatic cells, and that Mbd1 and Setdb1, similar to Atf7ip, play a functional role in Xi silencing. We therefore propose that ATF7IP links DNA methylation on the Xi to SETDB1-mediated H3K9 trimethylation via its interaction with MBD1, and that this function is a crucial feature of the stable silencing of the Xi in female mammalian cells.

A global assessment of cancer genomic alterations in epigenetic mechanisms

Abstract: The notion that epigenetic mechanisms may be central to cancer initiation and progression is supported by recent next-generation sequencing efforts revealing that genes involved in chromatin-mediated signaling are recurrently mutated in cancer patients. Here, we analyze mutational and transcriptional profiles from TCGA and the ICGC across a collection 441 chromatin factors and histones. Chromatin factors essential for rapid replication are frequently overexpressed, and those that maintain genome stability frequently mutated. We identify novel mutation hotspots such as K36M in histone H3.1, and uncover a general trend in which transcriptional profiles and somatic mutations in tumor samples favor increased transcriptionally repressive histone methylation, and defective chromatin remodeling. This unbiased approach confirms previously published data, uncovers novel cancer-associated aberrations targeting epigenetic mechanisms, and justifies continued monitoring of chromatin-related alterations as a class, as more cancer types and distinct cancer stages are represented in cancer genomics data repositories.

Single-base resolution of mouse offspring brain methylome reveals epigenome modifications caused by gestational folic acid.

Abstract: Background Epigenetic modifications, such as cytosine methylation in CpG-rich regions, regulate multiple functions in mammalian development. Maternal nutrients affecting one-carbon metabolism during gestation can exert long-term effects on the health of the progeny. Using C57BL/6 J mice, we investigated whether the amount of ingested maternal folic acid (FA) during gestation impacted DNA methylation in the offspring’s cerebral hemispheres. Reduced representation bisulfite sequencing at single-base resolution was performed to analyze genome-wide DNA methylation profiles.

DNA methylation reader MECP2: cell type- and differentiation stage-specific protein distribution.

Abstract: Methyl-CpG binding protein 2 (MECP2) is a protein that specifically binds methylated DNA, thus regulating transcription and chromatin organization. Mutations in the gene have been identified as the principal cause of Rett syndrome, a severe neurological disorder. Although the role of MECP2 has been extensively studied in nervous tissues, still very little is known about its function and cell type specific distribution in other tissues. Using immunostaining on tissue cryosections, we characterized the distribution of MECP2 in 60 cell types of 16 mouse neuronal and non-neuronal tissues. We show that MECP2 is expressed at a very high level in all retinal neurons except rod photoreceptors. The onset of its expression during retina development coincides with massive synapse formation. In contrast to astroglia, retinal microglial cells lack MECP2, similar to microglia in the brain, cerebellum, and spinal cord. MECP2 is also present in almost all non-neural cell types, with the exception of intestinal epithelial cells, erythropoietic cells, and hair matrix keratinocytes. Our study demonstrates the role of MECP2 as a marker of the differentiated state in all studied cells other than oocytes and spermatogenic cells. MECP2-deficient male (Mecp2-/y) mice show no apparent defects in the morphology and development of the retina. The nuclear architecture of retinal neurons is also unaffected as the degree of chromocenter fusion and the distribution of major histone modifications do not differ between Mecp2-/y and Mecp2 wt mice. Surprisingly, the absence of MECP2 is not compensated by other methyl-CpG binding proteins. On the contrary, their mRNA levels were downregulated in Mecp2-/y mice. MECP2 is almost universally expressed in all studied cell types with few exceptions, including microglia. MECP2 deficiency does not change the nuclear architecture and epigenetic landscape of retinal cells despite the missing compensatory expression of other methyl-CpG binding proteins. Furthermore, retinal development and morphology are also preserved in Mecp2-null mice. Our study reveals the significance of MECP2 function in cell differentiation and sets the basis for future investigations in this direction.

G9a co-suppresses LINE1 elements in spermatogonia

Abstract: Background: Repression of retrotransposons is essential for genome integrity and the development of germ cells. Among retrotransposons, the establishment of CpG DNA methylation and epigenetic silencing of LINE1 (L1) elements and the intracisternal A particle (IAP) endogenous retrovirus (ERV) is dependent upon the piRNA pathway during embryonic germ cell reprogramming. Furthermore, the Piwi protein Mili, guided by piRNAs, cleaves expressed L1 transcripts to post-transcriptionally enforce L1 silencing in meiotic cells. The loss of both DNA methylation and the Mili piRNA pathway does not affect L1 silencing in the mitotic spermatogonia where histone H3 lysine 9 dimethylation (H3K9me2) is postulated to co-repress these elements. Results: Here we show that the histone H3 lysine 9 dimethyltransferase G9a co-suppresses L1 elements in spermatogonia. In the absence of both a functional piRNA pathway and L1 DNA methylation, G9a is both essential and sufficient to silence L1 elements. In contrast, H3K9me2 alone is insufficient to maintain IAP silencing in spermatogonia. The loss of all three repressive mechanisms has a major impact on spermatogonial populations inclusive of spermatogonial stem cells, with the loss of all germ cells observed in a high portion of seminiferous tubules. Conclusions: Our study identifies G9a-mediated H3K9me2 as a novel and important L1 repressive mechanism in the germ line. We also demonstrate fundamental differences in the requirements for the maintenance of L1 and IAP silencing during adult spermatogenesis, where H3K9me2 is sufficient to maintain L1 but not IAP silencing. Finally, we demonstrate that repression of retrotransposon activation in spermatogonia is important for the survival of this population and testicular homeostasis.

A mechanistic role for the chromatin modulator, NAP1L1, in pancreatic neuroendocrine neoplasm proliferation and metastases

Abstract: The chromatin remodeler NAP1L1, which is upregulated in small intestinal neuroendocrine neoplasms (NENs), has been implicated in cell cycle progression. As p57Kip2 (CDKN1C), a negative regulator of proliferation and a tumor suppressor, is controlled by members of the NAP1 family, we tested the hypothesis that NAP1L1 may have a mechanistic role in regulating pancreatic NEN proliferation through regulation of p57Kip2. NAP1L1 silencing (siRNA and shRNA/lipofectamine approach) decreased proliferation through inhibition of mechanistic (mammalian) target of rapamycin pathway proteins and their phosphorylation (p < 0.05) in the pancreatic neuroendocrine neoplasm cell line BON in vitro (p < 0.0001) and resulted in significantly smaller (p < 0.05) and lighter (p < 0.05) tumors in the orthotopic pancreatic NEN mouse model. Methylation of the p57 Kip2 promoter was decreased by NAP1L1 silencing (p < 0.05), and expression of p57Kip2 (transcript and protein) was upregulated. For methylation of the p57 Kip2 promoter, NAP1L1 bound directly to the promoter (−164 to +21, chromatin immunoprecipitation). In 43 pancreatic NEN samples (38 primaries and 5 metastasis), NAP1L1 was over-expressed in metastasis (p < 0.001), expression which was inversely correlated with p57Kip2 (p < 0.01) on mRNA and protein levels. Menin was not differentially expressed. NAP1L1 is over-expressed in pancreatic neuroendocrine neoplasm metastases and epigenetically promotes cell proliferation through regulation of p57 Kip2 promoter methylation.

The C. Elegans dosage compensation complex mediates interphase X chromosome compaction

Abstract: Background: Dosage compensation is a specialized gene regulatory mechanism which equalizes X-linked gene expression between sexes. In Caenorhabditis elegans, dosage compensation is achieved by the activity of the dosage compensation complex (DCC). The DCC localizes to both X chromosomes in hermaphrodites to downregulate gene expression by half. The DCC contains a subcomplex (condensin I DC ) similar to the evolutionarily conserved condensin complexes which play fundamental roles in chromosome dynamics during mitosis and meiosis. Therefore, mechanisms related to mitotic chromosome condensation have been long hypothesized to mediate dosage compensation. However experimental evidence was lacking. Results: Using 3D FISH microscopy to measure the volumes of X and chromosome I territories and to measure distances between individual loci, we show that hermaphrodite worms deficient in DCC proteins have enlarged interphase X chromosomes when compared to wild type. By contrast, chromosome I is unaffected. Interestingly, hermaphrodite worms depleted of condensin I or II show no phenotype. Therefore X chromosome compaction is specific to condensin I DC . In addition, we show that SET-1, SET-4, and SIR-2.1, histone modifiers whose activity is regulated by the DCC, need to be present for the compaction of the X chromosome territory. Conclusion: These results support the idea that condensin I DC , and the histone modifications regulated by the DCC, mediate interphase X chromosome compaction. Our results link condensin-mediated chromosome compaction, an activity connected to mitotic chromosome condensation, to chromosome-wide repression of gene expression in interphase.

The PEG13-DMR and brain-specific enhancers dictate imprinted expression within the 8q24 intellectual disability risk locus.

Abstract: Background: Genomic imprinting is the epigenetic marking of genes that results in parent-of-origin monoallelic expression. Most imprinted domains are associated with differentially DNA methylated regions (DMRs) that originate in the gametes, and are maintained in somatic tissues after fertilization. This allelic methylation profile is associated with a plethora of histone tail modifications that orchestrates higher order chromatin interactions. The mouse chromosome 15 imprinted cluster contains multiple brain-specific maternally expressed transcripts including Ago2, Chrac1, Trappc9 and Kcnk9 and a paternally expressed gene, Peg13. The promoter of Peg13 is methylated on the maternal allele and is the sole DMR within the locus. To determine the extent of imprinting within the human orthologous region on chromosome 8q24, a region associated with autosomal recessive intellectual disability, Birk-Barel mental retardation and dysmorphism syndrome, we have undertaken a systematic analysis of allelic expression and DNA methylation of genes mapping within an approximately 2 Mb region around TRAPPC9. Results: Utilizing allele-specific RT-PCR, bisulphite sequencing, chromatin immunoprecipitation and chromosome conformation capture (3C) we show the reciprocal expression of the novel, paternally expressed, PEG13 non-coding RNA and maternally expressed KCNK9 genes in brain, and the biallelic expression of flanking transcripts in a range of tissues. We identify a tandem-repeat region overlapping the PEG13 transcript that is methylated on the maternal allele, which binds CTCF-cohesin in chromatin immunoprecipitation experiments and possesses enhancer-blocker activity. Using 3C, we identify mutually exclusive approximately 58 and 500 kb chromatin loops in adult frontal cortex between a novel brain-specific enhancer, marked by H3K4me1 and H3K27ac, with the KCNK9 and PEG13 promoters which we propose regulates brain-specific expression. Conclusions: We have characterised the molecular mechanism responsible for reciprocal allelic expression of the PEG13 and KCNK9 transcripts. Therefore, our observations may have important implications for identifying the cause of intellectual disabilities associated with the 8q24 locus.

Predicting expression: the complementary power of histone modification and transcription factor binding data

Abstract: Transcription factors (TFs) and histone modifications (HMs) play critical roles in gene expression by regulating mRNA transcription. Modelling frameworks have been developed to integrate high-throughput omics data, with the aim of elucidating the regulatory logic that results from the interactions of DNA, TFs and HMs. These models have yielded an unexpected and poorly understood result: that TFs and HMs are statistically redundant in explaining mRNA transcript abundance at a genome-wide level. We constructed predictive models of gene expression by integrating RNA-sequencing, TF and HM chromatin immunoprecipitation sequencing and DNase I hypersensitivity data for two mammalian cell types. All models identified genome-wide statistical redundancy both within and between TFs and HMs, as previously reported. To investigate potential explanations, groups of genes were constructed for ontology-classified biological processes. Predictive models were constructed for each process to explore the distribution of statistical redundancy. We found significant variation in the predictive capacity of TFs and HMs across these processes and demonstrated the predictive power of HMs to be inversely proportional to process enrichment for housekeeping genes. It is well established that the roles played by TFs and HMs are not functionally redundant. Instead, we attribute the statistical redundancy reported in this and previous genome-wide modelling studies to the heterogeneous distribution of HMs across chromatin domains. Furthermore, we conclude that statistical redundancy between individual TFs can be readily explained by nucleosome-mediated cooperative binding. This could possibly help the cell confer regulatory robustness by rejecting signalling noise and allowing control via multiple pathways.

Genome-wide analysis of H3.3 dissociation reveals high nucleosome turnover at distal regulatory regions of embryonic stem cells

Abstract: Background The histone variant H3.3 plays a critical role in maintaining the pluripotency of embryonic stem cells (ESCs) by regulating gene expression programs important for lineage specification. H3.3 is deposited by various chaperones at regulatory sites, gene bodies, and certain heterochromatic sites such as telomeres and centromeres. Using Tet-inhibited expression of epitope-tagged H3.3 combined with ChIP-Seq we undertook genome-wide measurements of H3.3 dissociation rates across the ESC genome and examined the relationship between H3.3-nucleosome turnover and ESC-specific transcription factors, chromatin modifiers, and epigenetic marks.

ChIP-less analysis of chromatin states

Abstract: Histone post-translational modifications (PTMs) are key epigenetic regulators in chromatin-based processes. Increasing evidence suggests that vast combinations of PTMs exist within chromatin histones. These complex patterns, rather than individual PTMs, are thought to define functional chromatin states. However, the ability to interrogate combinatorial histone PTM patterns at the nucleosome level has been limited by the lack of direct molecular tools. Here we demonstrate an efficient, quantitative, antibody-free, chromatin immunoprecipitation-less (ChIP-less) method for interrogating diverse epigenetic states. At the heart of the workflow are recombinant chromatin reader domains, which target distinct chromatin states with combinatorial PTM patterns. Utilizing a newly designed combinatorial histone peptide microarray, we showed that three reader domains (ATRX-ADD, ING2-PHD and AIRE-PHD) displayed greater specificity towards combinatorial PTM patterns than corresponding commercial histone antibodies. Such specific recognitions were employed to develop a chromatin reader-based affinity enrichment platform (matrix-assisted reader chromatin capture, or MARCC). We successfully applied the reader-based platform to capture unique chromatin states, which were quantitatively profiled by mass spectrometry to reveal interconnections between nucleosomal histone PTMs. Specifically, a highly enriched signature that harbored H3K4me0, H3K9me2/3, H3K79me0 and H4K20me2/3 within the same nucleosome was identified from chromatin enriched by ATRX-ADD. This newly reported PTM combination was enriched in heterochromatin, as revealed by the associated DNA. Our results suggest the broad utility of recombinant reader domains as an enrichment tool specific to combinatorial PTM patterns, which are difficult to probe directly by antibody-based approaches. The reader affinity platform is compatible with several downstream analyses to investigate the physical coexistence of nucleosomal PTM states associated with specific genomic loci. Collectively, the reader-based workflow will greatly facilitate our understanding of how distinct chromatin states and reader domains function in gene regulatory mechanisms.

Phosphorylation and arginine methylation mark histone H2A prior to deposition during Xenopus laevis development

Abstract: Stored, soluble histones in eggs are essential for early development, in particular during the maternally controlled early cell cycles in the absence of transcription. Histone post-translational modifications (PTMs) direct and regulate chromatin-templated transactions, so understanding the nature and function of pre-deposition maternal histones is essential to deciphering mechanisms of regulation of development, chromatin assembly, and transcription. Little is known about histone H2A pre-deposition modifications nor known about the transitions that occur upon the onset of zygotic control of the cell cycle and transcription at the mid-blastula transition (MBT). We isolated histones from staged Xenopus laevis oocytes, eggs, embryos, and assembled pronuclei to identify changes in histone H2A modifications prior to deposition and in chromatin. Soluble and chromatin-bound histones from eggs and embryos demonstrated distinct patterns of maternal and zygotic H2A PTMs, with significant pre-deposition quantities of S1ph and R3me1, and R3me2s. We observed the first functional distinction between H2A and H4 S1 phosphorylation, as we showed that H2A and H2A.X-F (also known as H2A.X.3) serine 1 (S1) is phosphorylated concomitant with germinal vesicle breakdown (GVBD) while H4 serine 1 phosphorylation occurs post-MBT. In egg extract H2A/H4 S1 phosphorylation is independent of the cell cycle, chromatin assembly, and DNA replication. H2AS1ph is highly enriched on blastula chromatin during repression of zygotic gene expression while H4S1ph is correlated with the beginning of maternal gene expression and the lengthening of the cell cycle, consistent with distinct biological roles for H2A and H4 S1 phosphorylation. We isolated soluble H2A and H2A.X-F from the egg and chromatin-bound in pronuclei and analyzed them by mass spectrometry analysis to quantitatively determine abundances of S1ph and R3 methylation. We show that H2A and H4 S1ph, R3me1 and R3me2s are enriched on nucleosomes containing both active and repressive histone PTMs in human A549 cells and Xenopus embryos. Significantly, we demonstrated that H2A phosphorylation and H4 arginine methylation form a new class of bona fide pre-deposition modifications in the vertebrate embryo. We show that S1ph and R3me containing chromatin domains are not correlated with H3 regulatory PTMs, suggesting a unique role for phosphorylation and arginine methylation.

Protection of CpG islands against de novo DNA methylation during oogenesis is associated with the recognition site of E2f1 and E2f2

Abstract: Background: Epigenetic reprogramming during early mammalian embryonic and germ cell development is a genome-wide process. CpG islands (CGIs), central to the regulation of mammalian gene expression, are exceptional in terms of whether, when and how they are affected by epigenetic reprogramming. Results: We investigated the DNA sequences of CGIs in the context of genome-wide data on DNA methylation and transcription during oogenesis and early embryogenesis to identify signals associated with methylation establishment and protection from de novo methylation in oocytes and associated with post-fertilisation methylation maintenance. We find no evidence for a characteristic DNA sequence motif in oocyte-methylated CGIs. Neither do we find evidence for a general role of regular CpG spacing in methylation establishment at CGIs in oocytes. In contrast, the resistance of most CGIs to de novo methylation during oogenesis is associated with the motif CGCGC, the recognition site of E2f1 and E2f2, transcription factors highly expressed specifically in oocytes. This association is independent of prominent known hypomethylation-associated factors: CGI promoter activity, H3K4me3, Cfp1 binding or R-loop formation potential. Conclusions: Our results support a DNA sequence-independent and transcription-driven model of de novo CGI methylation during oogenesis. In contrast, our results for CGIs that remain unmethylated are consistent with a model of protection from methylation involving sequence recognition by DNA-binding proteins, E2f1 and E2f2 being probable candidates.

Drosophila linker histone H1 coordinates STAT-dependent organization of heterochromatin and suppresses tumorigenesis caused by hyperactive JAK-STAT signaling.

Abstract: Within the nucleus of eukaryotic cells, chromatin is organized into compact, silent regions called heterochromatin and more loosely packaged regions of euchromatin where transcription is more active. Although the existence of heterochromatin has been known for many years, the cellular factors responsible for its formation have only recently been identified. Two key factors involved in heterochromatin formation in Drosophila are the H3 lysine 9 methyltransferase Su(var)3-9 and heterochromatin protein 1 (HP1). The linker histone H1 also plays a major role in heterochromatin formation in Drosophila by interacting with Su(var)3-9 and helping to recruit it to heterochromatin. Drosophila STAT (Signal transducer and activator of transcription) (STAT92E) has also been shown to be involved in the maintenance of heterochromatin, but its relationship to the H1-Su(var)3-9 heterochromatin pathway is unknown. STAT92E is also involved in tumor formation in flies. Hyperactive Janus kinase (JAK)-STAT signaling due to a mutation in Drosophila JAK (Hopscotch) causes hematopoietic tumors We show here that STAT92E is a second partner of H1 in the regulation of heterochromatin structure. H1 physically interacts with STAT92E and regulates its ectopic localization in the chromatin. Mis-localization of STAT92E due to its hyperphosphorylation or H1 depletion disrupts heterochromatin integrity. The contribution of the H1-STAT pathway to heterochromatin formation is mechanistically distinct from that of H1 and Su(var)3-9. The recruitment of STAT92E to chromatin by H1 also plays an important regulatory role in JAK-STAT induced tumors in flies. Depleting the linker histone H1 in flies carrying the oncogenic hopscotch Tum-l allele enhances tumorigenesis, and H1 overexpression suppresses tumorigenesis. Our results suggest the existence of two independent pathways for heterochromatin formation in Drosophila, one involving Su(var)3-9 and HP1 and the other involving STAT92E and HP1. The H1 linker histone directs both pathways through physical interactions with Su(var)3-9 and STAT92E, as well with HP1. The physical interaction of H1 and STAT92E confers a regulatory role on H1 in JAK-STAT signaling. H1 serves as a molecular reservoir for STAT92E in chromatin, enabling H1 to act as a tumor suppressor and oppose an oncogenic mutation in the JAK-STAT signaling pathway.

Mechanistic stochastic model of histone modification pattern formation

Abstract: Background: The activity of a single gene is influenced by the composition of the chromatin in which it is embedded. Nucleosome turnover, conformational dynamics, and covalent histone modifications each induce changes in the structure of chromatin and its affinity for regulatory proteins. The dynamics of histone modifications and the persistence of modification patterns for long periods are still largely unknown. Results: In this study, we present a stochastic mathematical model that describes the molecular mechanisms of histone modification pattern formation along a single gene, with non-phenomenological, physical parameters. We find that diffusion and recruitment properties of histone modifying enzymes together with chromatin connectivity allow for a rich repertoire of stochastic histone modification dynamics and pattern formation. We demonstrate that histone modification patterns at a single gene can be established or removed within a few minutes through diffusion and weak recruitment mechanisms of histone modification spreading. Moreover, we show that strong synergism between diffusion and weak recruitment mechanisms leads to nearly irreversible transitions in histone modification patterns providing stable patterns. In the absence of chromatin connectivity spontaneous and dynamic histone modification boundaries can be formed that are highly unstable, and spontaneous fluctuations cause them to diffuse randomly. Chromatin connectivity destabilizes this synergistic system and introduces bistability, illustrating state switching between opposing modification states of the model gene. The observed bistable long-range and localized pattern formation are critical effectors of gene expression regulation. Conclusion: This study illustrates how the cooperative interactions between regulatory proteins and the chromatin state generate complex stochastic dynamics of gene expression regulation.

Chd1 co-localizes with early transcription elongation factors independently of H3K36 methylation and releases stalled RNA polymerase II at introns

Abstract: Background Chromatin consists of ordered nucleosomal arrays that are controlled by highly conserved adenosine triphosphate (ATP)-dependent chromatin remodeling complexes. One such remodeler, chromodomain helicase DNA binding protein 1 (Chd1), is believed to play an integral role in nucleosomal organization, as the loss of Chd1 is known to disrupt chromatin. However, the specificity and basis for the functional and physical localization of Chd1 on chromatin remains largely unknown.

HP1β-dependent recruitment of UBF1 to irradiated chromatin occurs simultaneously with CPDs

Abstract: The repair of spontaneous and induced DNA lesions is a multistep process. Depending on the type of injury, damaged DNA is recognized by many proteins specifically involved in distinct DNA repair pathways. We analyzed the DNA-damage response after ultraviolet A (UVA) and γ irradiation of mouse embryonic fibroblasts and focused on upstream binding factor 1 (UBF1), a key protein in the regulation of ribosomal gene transcription. We found that UBF1, but not nucleolar proteins RPA194, TCOF, or fibrillarin, was recruited to UVA-irradiated chromatin concurrently with an increase in heterochromatin protein 1β (HP1β) level. Moreover, Forster Resonance Energy Transfer (FRET) confirmed interaction between UBF1 and HP1β that was dependent on a functional chromo shadow domain of HP1β. Thus, overexpression of HP1β with a deleted chromo shadow domain had a dominant-negative effect on UBF1 recruitment to UVA-damaged chromatin. Transcription factor UBF1 also interacted directly with DNA inside the nucleolus but no interaction of UBF1 and DNA was confirmed outside the nucleolus, where UBF1 recruitment to DNA lesions appeared simultaneously with cyclobutane pyrimidine dimers; this occurrence was cell-cycle-independent. We propose that the simultaneous presence and interaction of UBF1 and HP1β at DNA lesions is activated by the presence of cyclobutane pyrimidine dimers and mediated by the chromo shadow domain of HP1β. This might have functional significance for nucleotide excision repair.

PAT-ChIP coupled with laser microdissection allows the study of chromatin in selected cell populations from paraffin-embedded patient samples.

Abstract: Background: The recent introduction of pathology tissue-chromatin immunoprecipitation (PAT-ChIP), a technique allowing chromatin immunoprecipitation from formalin-fixed and paraffin-embedded (FFPE) tissues, has expanded the application potential of epigenetic studies in tissue samples. However, FFPE tissue section analysis is strongly limited by tissue heterogeneity, which hinders linking the observed epigenetic events to the corresponding cellular population. Thus, ideally, to take full advantage of PAT-ChIP approaches, procedures able to increase the purity and homogeneity of cell populations from FFPE tissues are required. Results: In this study, we tested the use of both core needle biopsies (CNBs) and laser microdissection (LMD), evaluating the compatibility of these methods with the PAT-ChIP procedure. Modifications of the original protocols were introduced in order to increase reproducibility and reduce experimental time. We first demonstrated that chromatin can be prepared and effectively immunoprecipitated starting from 0.6-mm-diameter CNBs. Subsequently, in order to assess the applicability of PAT-ChIP to LMD samples, we tested the effects of hematoxylin or eosin staining on chromatin extraction and immunoprecipitation, as well as the reproducibility of our technique when using particularly low quantities of starting material. Finally, we carried out the PAT-ChIP using chromatin extracted from either normal tissue or neoplastic lesions, the latter obtained by LMD from FFPE lung sections derived from mutant K-ras v12 transgenic mice or from human adeno- or squamous lung carcinoma samples. Well characterized histone post-translational modifications (HPTMs), such as H3K4me3, H3K27me3, H3K27Ac, and H3K9me3, were specifically immunoselected, as well as the CTCF transcription factor and RNA polymerase II (Pol II). Conclusions: Epigenetic profiling can be performed on enriched cell populations obtained from FFPE tissue sections. The improved PAT-ChIP protocol will be used for the discovery and/or validation of novel epigenetic biomarkers in FFPE human samples.

Differential expression of histone H3 genes and selective association of the variant H3.7 with a specific sequence class in Stylonychia macronuclear development.

Abstract: Regulation of chromatin structure involves deposition of selective histone variants into nucleosome arrays. Numerous histone H3 variants become differentially expressed by individual nanochromosomes in the course of macronuclear differentiation in the spirotrichous ciliate Stylonychia lemnae. Their biological relevance remains to be elucidated. We show that the differential assembly of H3 variants into chromatin is strongly correlated with the functional separation of chromatin structures in developing macronuclei during sexual reproduction in Stylonychia, thus probably determining the fate of specific sequences. Specific H3 variants approximately 15 kDa or 20 kDa in length are selectively targeted by post-translational modifications. We found that only the 15 kDa H3 variants including H3.3 and H3.5, accumulate in the early developing macronucleus, and these also occur in mature macronuclei. H3.7 is a 20 kDa variant that specifically becomes enriched in macronuclear anlagen during chromosome polytenization. H3.7, acetylated at lysine-32 (probably equivalent to lysine-36 of most H3 variants), is specifically associated with a sequence class that is retained in the mature macronucleus and therefore does not undergo developmental DNA elimination. H3.8 is another 20 kDa variant that is restricted to the micronucleus. H3.8 is selectively targeted by lysine methylation and by serine or threonine phosphorylation. Intriguingly, the expression and chromatin localization of the histone variant H3.3 was impaired during macronuclear differentiation after RNA interference knock-down of Piwi expression. Differential deposition of H3 variants into chromatin strongly correlates with the functional distinction of genomic sequence classes on the chromatin level, thus helping to determine the fate of specific DNA sequences during sexual reproduction in Stylonychia. Consequently, H3 variants are selectively targeted by post-translational modifications, possibly as a result of deviations within the recognition motifs, which allow binding of effector proteins. We propose that differential assembly of histone variants into chromatin of various nuclear types could contribute to nuclear identity, for example, during differential development of either new micronuclei or a macronuclear anlage from mitosis products of the zygote nucleus (synkaryon). The observation that the Piwi-non-coding RNA (ncRNA) pathway influences the expression and deposition of H3.3 in macronuclear anlagen indicates for the first time that selective histone variant assembly into chromatin might possibly depend on ncRNA.

Targeted gene suppression by inducing de novo DNA methylation in the gene promoter

Abstract: Targeted gene silencing is an important approach in both drug development and basic research. However, the selection of a potent suppressor has become a significant hurdle to implementing maximal gene inhibition for this approach. We attempted to construct a ‘super suppressor’ by combining the activities of two suppressors that function through distinct epigenetic mechanisms. Gene targeting vectors were constructed by fusing a GAL4 DNA-binding domain with a epigenetic suppressor, including CpG DNA methylase Sss1, histone H3 lysine 27 methylase vSET domain, and Kruppel-associated suppression box (KRAB). We found that both Sss1 and KRAB suppressors significantly inhibited the expression of luciferase and copGFP reporter genes. However, the histone H3 lysine 27 methylase vSET did not show significant suppression in this system. Constructs containing both Sss1 and KRAB showed better inhibition than either one alone. In addition, we show that KRAB suppressed gene expression by altering the histone code, but not DNA methylation in the gene promoter. Sss1, on the other hand, not only induced de novo DNA methylation and recruited Heterochromatin Protein 1 (HP1a), but also increased H3K27 and H3K9 methylation in the promoter. Epigenetic studies can provide useful data for the selection of suppressors in constructing therapeutic vectors for targeted gene silencing.