Histone Modification Profiles Research Articles

Abstract 225: Chronic Alcohol Consumption Alters the Epigenetic Fingerprint of Cardiac Cell Types

The cardiac influence of chronic ethanol (EtOH) consumption in humans is dictated by the frequency of ethanol ingestion. Whereas moderate consumption (i.e. 1-2 alcoholic drinks/day) imparts a cardiac benefit to patients by reducing adverse cardiovascular event (ACE) occurrences, consumption below or beyond these moderate levels significantly increases the incidence of ACEs. Despite these observations, little is known regarding the functional impact of chronic EtOH consumption on post-myocardial infarct repair or the cellular mechanisms involved in this process. Thus, we investigated the post-AMI functional consequences of chronic ethanol consumption in mice. Mice received chronic ethanol via the Lieber-DeCarli paradigm (i.e. 0%, 1% (moderate) or 5% (high) ethanol v/v) in an isocaloric fashion for 8 weeks. After 8 weeks, mice underwent a 60-minute ischemic/reperfusion injury and the subsequent assessment of their cardiac function for 4 weeks post-AMI. As early as two weeks post-AMI, mice fed the 1% EtOH displayed modest yet significant improvements in ejection fraction and systolic ventricular volumes as compared to control mice. Conversely, the 5% EtOH diet group displayed diminished ejection fraction and increases in both systolic chamber volume and infarct size. To explore the cellular basis of these observed functional changes, primary cardiac cell types (fibroblasts and endothelial cells) treated chronically (5 days) with ethanol in vitro (i.e. 0%, 0.1% (moderate) or 0.5% (high) ethanol v/v) displayed robust changes in their epigenetic histone-modification profiles indicating a high likelihood for changes in cell specific gene expression. In addition, conditioned media from ethanol treated primary cardiac fibroblasts (PCFBs) altered the tube forming capacity of endothelial cells in a matrigel tube-formation assay in a dose pattern akin to what was observed in vivo. This data suggests that chronic ethanol directly invokes epigenetic changes in PCFBs that can modify their contribution to cardiac repair processes following AMI. Lastly, we are actively investigating whether ethanol-induced changes to endothelial cell epigenetic patterns alter the cells responses in the face of an ischemic insult.

Genome-wide analysis of histone modifications in latently HIV-1 infected T cells.

Objectives:The transcriptional silencing of HIV type 1 (HIV-1) provirus in latently infected cells is a major hurdle on the pathway to HIV-1 elimination. The epigenetic mechanisms established by histone modifications may affect the transcriptional silencing of HIV-1 and viral latency. A systematic epigenome profiling could be applicable to develop new epigenetic diagnostic markers for detecting HIV-1 latency.Design:The HIV-1 latency cell lines (NCHA1, NCHA2 and ACH2] were compared with CD4+ T-cell line (A3.01).Methods:The histone modification profiles obtained from chromatin immunoprecipiation followed by sequencing (ChIP-Seq) for histone H3K4me3 and H3K9ac were systematically examined and differential gene expression patterns along with levels of histone modifications were used for network analysis.Results:The HIV-1 latency gave rise to downregulation of histone H3K4me3 and H3K9ac levels in 387 and 493 regions and upregulation in 451 and 962 sites, respectively. By network analysis, five gene clusters were associated with downregulated histone modifications and six gene clusters came up with upregulated histone modifications. Integration of gene expression with epigenetic information revealed that the cell cycle regulatory genes such as CDKN1A (p21) and cyclin D2 (CCND2) identified by differentially modified histones might play an important role in maintaining the HIV-1 latency.Conclusion:The transcriptional regulation by epigenetic memory should play a key role in the evolution and maintenance of HIV-1 latency accompanied by modulation of signalling molecules in the host cells.

Epigenomic analysis of primary human T cells reveals enhancers associated with TH2 memory cell differentiation and asthma susceptibility.

A characteristic feature of asthma is the aberrant accumulation, differentiation or function of memory CD4(+) T cells that produce type 2 cytokines (TH2 cells). By mapping genome-wide histone modification profiles for subsets of T cells isolated from peripheral blood of healthy and asthmatic individuals, we identified enhancers with known and potential roles in the normal differentiation of human TH1 cells and TH2 cells. We discovered disease-specific enhancers in T cells that differ between healthy and asthmatic individuals. Enhancers that gained the histone H3 Lys4 dimethyl (H3K4me2) mark during TH2 cell development showed the highest enrichment for asthma-associated single nucleotide polymorphisms (SNPs), which supported a pathogenic role for TH2 cells in asthma. In silico analysis of cell-specific enhancers revealed transcription factors, microRNAs and genes potentially linked to human TH2 cell differentiation. Our results establish the feasibility and utility of enhancer profiling in well-defined populations of specialized cell types involved in disease pathogenesis.

Epigenetic histone modifications in a clinically relevant rat model of chronic ethanol-binge-mediated liver injury.

Ethanol binge augments liver injury after chronic ethanol consumption in humans, but the mechanism behind the enhanced liver injury by ethanol binge is not known. In this study we used a clinically relevant rat model in which liver injury is amplified by binge after chronic ethanol treatment and investigated the importance of histone modifications. Eight-week-old Sprague-Dawley rats were fed ethanol in a liquid diet for 4weeks. Control rats were fed an isocaloric liquid diet. This was followed by three binge administrations of ethanol (intragastric 5g/kg body weight, 12h apart). In the control, ethanol was replaced by water. Four hours after the last binge administration, liver samples were analyzed for histone modifications and parameters of liver injury. Chronic ethanol administration alone caused an increase in histone H3 ser10 and ser28 (H3S10 or S28) phosphorylation, and binge ethanol reduced their levels. Levels of dually modified phosphoacetylated histone H3 (H3AcK9/PS10) increased after acute binge ethanol and remained same after chronic ethanol binge. In contrast, histone H3 lysine-9 acetylation (H3AcK9) was not increased after chronic ethanol but increased significantly after acute binge and chronic ethanol binge. Increase in histone acetylation was accompanied by increased phospho-ERK1/2 in the nuclear extracts. Increased acetylation after chronic ethanol binge was also accompanied by increased protein levels of GCN5 histone acetyl transferase and a modest increase in HDAC3 in the nucleus. Histone lysine-9 dimethylation was significantly increased after chronic ethanol binge. Chronic ethanol binge also resulted in a decrease in the SAM:SAH ratio with a relative decrease of SAM levels and a corresponding increase in SAH levels. Ethanol binge after chronic ethanol altered the profile of site-specific histone modifications and may underlie the mechanism of augmented liver injury by chronic-ethanol-binge-treated rats.

Aberrant DNA Methylation in ES Cells

Both mouse and human embryonic stem cells can be differentiated in vitro to produce a variety of somatic cell types. Using a new developmental tracing approach, we show that these cells are subject to massive aberrant CpG island de novo methylation that is exacerbated by differentiation in vitro. Bioinformatics analysis indicates that there are two distinct forms of abnormal de novo methylation, global as opposed to targeted, and in each case the resulting pattern is determined by molecular rules correlated with local pre-existing histone modification profiles. Since much of the abnormal methylation generated in vitro appears to be stably maintained, this modification may inhibit normal differentiation and could predispose to cancer if cells are used for replacement therapy. Excess CpG island methylation is also observed in normal placenta, suggesting that this process may be governed by an inherent program.

From human monocytes to genome-wide binding sites--a protocol for small amounts of blood: monocyte isolation/ChIP-protocol/library amplification/genome wide computational data analysis.

Chromatin immunoprecipitation in combination with a genome-wide analysis via high-throughput sequencing is the state of the art method to gain genome-wide representation of histone modification or transcription factor binding profiles. However, chromatin immunoprecipitation analysis in the context of human experimental samples is limited, especially in the case of blood cells. The typically extremely low yields of precipitated DNA are usually not compatible with library amplification for next generation sequencing. We developed a highly reproducible protocol to present a guideline from the first step of isolating monocytes from a blood sample to analyse the distribution of histone modifications in a genome-wide manner. Conclusion: The protocol describes the whole work flow from isolating monocytes from human blood samples followed by a high-sensitivity and small-scale chromatin immunoprecipitation assay with guidance for generating libraries compatible with next generation sequencing from small amounts of immunoprecipitated DNA.

Genome-wide histone state profiling of fibroblasts from the opossum, Monodelphis domestica, identifies the first marsupial-specific imprinted gene

BackgroundImprinted genes have been extensively documented in eutherian mammals and found to exhibit significant interspecific variation in the suites of genes that are imprinted and in their regulation between tissues and developmental stages. Much less is known about imprinted loci in metatherian (marsupial) mammals, wherein studies have been limited to a small number of genes previously known to be imprinted in eutherians. We describe the first ab initio search for imprinted marsupial genes, in fibroblasts from the opossum, Monodelphis domestica, based on a genome-wide ChIP-seq strategy to identify promoters that are simultaneously marked by mutually exclusive, transcriptionally opposing histone modifications.ResultsWe identified a novel imprinted gene (Meis1) and two additional monoallelically expressed genes, one of which (Cstb) showed allele-specific, but non-imprinted expression. Imprinted vs. allele-specific expression could not be resolved for the third monoallelically expressed gene (Rpl17). Transcriptionally opposing histone modifications H3K4me3, H3K9Ac, and H3K9me3 were found at the promoters of all three genes, but differential DNA methylation was not detected at CpG islands at any of these promoters.ConclusionsIn generating the first genome-wide histone modification profiles for a marsupial, we identified the first gene that is imprinted in a marsupial but not in eutherian mammals. This outcome demonstrates the practicality of an ab initio discovery strategy and implicates histone modification, but not differential DNA methylation, as a conserved mechanism for marking imprinted genes in all therian mammals. Our findings suggest that marsupials use multiple epigenetic mechanisms for imprinting and support the concept that lineage-specific selective forces can produce sets of imprinted genes that differ between metatherian and eutherian lines.

Hypermethylation of the alternative AWT1 promoter in hematological malignancies is a highly specific marker for acute myeloid leukemias despite high expression levels

BackgroundWilms tumor 1 (WT1) is over-expressed in numerous cancers with respect to normal cells, and has either a tumor suppressor or oncogenic role depending on cellular context. This gene is associated with numerous alternatively spliced transcripts, which initiate from two different unique first exons within the WT1 and the alternative (A)WT1 promoter intervals. Within the hematological system, WT1 expression is restricted to CD34+/CD38- cells and is undetectable after differentiation. Detectable expression of this gene is an excellent marker for minimal residual disease in acute myeloid leukemia (AML), but the underlying epigenetic alterations are unknown.MethodsTo determine the changes in the underlying epigenetic landscape responsible for this expression, we characterized expression, DNA methylation and histone modification profiles in 28 hematological cancer cell lines and confirmed the methylation signature in 356 cytogenetically well-characterized primary hematological malignancies.ResultsDespite high expression of WT1 and AWT1 transcripts in AML-derived cell lines, we observe robust hypermethylation of the AWT1 promoter and an epigenetic switch from a permissive to repressive chromatin structure between normal cells and AML cell lines. Subsequent methylation analysis in our primary leukemia and lymphoma cohort revealed that the epigenetic signature identified in cell lines is specific to myeloid-lineage malignancies, irrespective of underlying mutational status or translocation. In addition to being a highly specific marker for AML diagnosis (positive predictive value 100%; sensitivity 86.1%; negative predictive value 89.4%), we show that AWT1 hypermethylation also discriminates patients that relapse from those achieving complete remission after hematopoietic stem cell transplantation, with similar efficiency to WT1 expression profiling.ConclusionsWe describe a methylation signature of the AWT1 promoter CpG island that is a promising marker for classifying myeloid-derived leukemias. In addition AWT1 hypermethylation is ideally suited to monitor the recurrence of disease during remission in patients undergoing allogeneic stem cell transfer.

Histone Modification Patterns and Their Responses to Environment

Histone modifications play important roles in the epigenetic regulation of gene expression. Recent genome-wide profiling of histone modifications with deep sequencing-based methods provides novel insights of crosstalk between histone modifications, leading to recent proposals of histone “language” or “web” as an alternative of “code” to appreciate combinatorial modification patterns. Environmental factor–altered histone modifications now may be addressed dynamically and systematically with the recognition and use of these interesting combinatorial patterns.

Losartan reverses permissive epigenetic changes in renal glomeruli of diabetic db/db mice

Epigenetic mechanisms such as chromatin histone H3 lysine methylation and acetylation have been implicated in diabetic vascular complications. However, histone modification profiles at pathologic genes associated with diabetic nephropathy in vivo and their regulation by the angiotensin II type 1 receptor (AT1R) are not clear. Here we tested whether treatment of type 2 diabetic db/db mice with the AT1R blocker Losartan not only ameliorates diabetic nephropathy, but also reverses epigenetic changes. As expected, the db/db mice had increased blood pressure, mesangial hypertrophy, proteinuria and glomerular expression of RAGE and PAI-1 versus control db/+ mice. This was associated with increased RNA Polymerase II recruitment and permissive histone marks as well as decreased repressive histone marks at these genes, and altered expression of relevant histone modification enzymes. Increased MCP-1 mRNA levels were not associated with such epigenetic changes, suggesting post-transcriptional regulation. Losartan attenuated key parameters of diabetic nephropathy and gene expression, and reversed some but not all the epigenetic changes in db/db mice. Losartan also attenuated increased H3K9/14Ac at RAGE, PAI-1 and MCP-1 promoters in mesangial cells cultured under diabetic conditions. Our results provide novel information about the chromatin state at key pathologic genes in vivo in diabetic nephropathy mediated in part by AT1R. Thus combination therapies targeting epigenetic regulators and AT1R could be evaluated for more effective treatment of diabetic nephropathy.

CR Cistrome: a ChIP-Seq database for chromatin regulators and histone modification linkages in human and mouse

Diversified histone modifications (HMs) are essential epigenetic features. They play important roles in fundamental biological processes including transcription, DNA repair and DNA replication. Chromatin regulators (CRs), which are indispensable in epigenetics, can mediate HMs to adjust chromatin structures and functions. With the development of ChIP-Seq technology, there is an opportunity to study CR and HM profiles at the whole-genome scale. However, no specific resource for the integration of CR ChIP-Seq data or CR-HM ChIP-Seq linkage pairs is currently available. Therefore, we constructed the CR Cistrome database, available online at http://compbio.tongji.edu.cn/cr and http://cistrome.org/cr/, to further elucidate CR functions and CR-HM linkages. Within this database, we collected all publicly available ChIP-Seq data on CRs in human and mouse and categorized the data into four cohorts: the reader, writer, eraser and remodeler cohorts, together with curated introductions and ChIP-Seq data analysis results. For the HM readers, writers and erasers, we provided further ChIP-Seq analysis data for the targeted HMs and schematized the relationships between them. We believe CR Cistrome is a valuable resource for the epigenetics community.

EPITRANS: a database that integrates epigenome and transcriptome data.

Epigenetic modifications affect gene expression and thereby govern a wide range of biological processes such as differentiation, development and tumorigenesis. Recent initiatives to define genome-wide DNA methylation and histone modification profiles by microarray and sequencing methods have led to the construction of databases. These databases are repositories for international epigenetic consortiums or provide mining results from PubMed, but do not integrate the epigenetic information with gene expression changes. In order to overcome this limitation, we constructed EPITRANS, a novel database that visualizes the relationships between gene expression and epigenetic modifications. EPITRANS uses combined analysis of epigenetic modification and gene expression to search for cell function-related epigenetic and transcriptomic alterations (Freely available on the web at http://epitrans.org ).

A DNA element regulates drug tolerance and withdrawal in Drosophila.

Drug tolerance and withdrawal are insidious responses to drugs of abuse; the first increases drug consumption while the second punishes abstention. Drosophila generate functional tolerance to benzyl alcohol sedation by increasing neural expression of the slo BK-type Ca2+ activated K+ channel gene. After drug clearance this change produces a withdrawal phenotype—increased seizure susceptibility. The drug-induced histone modification profile identified the 6b element (60 nt) as a drug responsive element. Genomic deletion of 6b produces the allele, slo Δ6b, that reacts more strongly to the drug with increased induction, a massive increase in the duration of tolerance, and an increase in the withdrawal phenotype yet does not alter other slo-dependent behaviors. The 6b element is a homeostatic regulator of BK channel gene expression and is the first cis-acting DNA element shown to specifically affect the duration of a drug action.

15: An intron of IRF-8 harbors myeloid lineage specific regulatory element that is regulated by dynamic chromatin architecture

Interferon Regulatory Factor-8 (IRF-8) is an IFN-gamma responsive gene that serves as a key factor in the hierarchical differentiation of bone marrow derived cells towards monocyte/macrophage lineage. In attempt to identify the molecular mechanisms leading to this lineage restricted expression of IRF-8, we are using IRF-8 Bacterial Artificial Chromosome (BAC) reporter constructs transfected either to permissive macrophage cell line or to restrictive non-hematopoietic fibroblast cell line. We present data showing that the control of lineage specific expression of IRF-8 is confined to an intronic segment. Deletion of this intron leads to loss of lineage specific expression of BAC IRF-8 reporter. Interestingly, this IRF-8 intron exhibits evolutionary conserved regions, which are not typical to “benign” intronic sequences. To gain molecular insight to this restricted expression, we compared histone modifications and nucleosome occupancy in this intron between expression permissive and restrictive cell lines. For that purpose, we employed Chromatin Immuno-Precipitation (ChIP) and Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) analyses. The ChIP data revealed subtle differences in the overall histone modification profile (”histone code”). The FAIRE analysis pointed to a significant nucleosome density difference between permissive and restrictive cell lines. Accordingly, IRF-8 repression in a restrictive myeloid progenitor cell line was partially alleviated upon treatment with histone methyltransferase inhibitors. Altogether, our data clearly suggests that an intron of IRF-8 harbors myeloid lineage specific regulatory element that is regulated by dynamic chromatin architecture. Taken together, it is most probable that this intron serves as nucleation core for chromatin condensation in restrictive cells.

Msl2 Is a Novel Component of the Vertebrate DNA Damage Response

hMSL2 (male-specific lethal 2, human) is a RING finger protein with ubiquitin ligase activity. Although it has been shown to target histone H2B at lysine 34 and p53 at lysine 351, suggesting roles in transcription regulation and apoptosis, its function in these and other processes remains poorly defined. To further characterize this protein, we have disrupted the Msl2 gene in chicken DT40 cells. Msl2−/− cells are viable, with minor growth defects. Biochemical analysis of the chromatin in these cells revealed aberrations in the levels of several histone modifications involved in DNA damage response pathways. DNA repair assays show that both Msl2−/− chicken cells and hMSL2-depleted human cells have defects in non-homologous end joining (NHEJ) repair. DNA damage assays also demonstrate that both Msl2 and hMSL2 proteins are modified and stabilized shortly after induction of DNA damage. Moreover, hMSL2 mediates modification, presumably ubiquitylation, of a key DNA repair mediator 53BP1 at lysine 1690. Similarly, hMSL1 and hMOF (males absent on the first) are modified in the presence of hMSL2 shortly after DNA damage. These data identify a novel role for Msl2/hMSL2 in the cellular response to DNA damage. The kinetics of its stabilization suggests a function early in the NHEJ repair pathway. Moreover, Msl2 plays a role in maintaining normal histone modification profiles, which may also contribute to the DNA damage response.

Sorafenib Inhibits Epithelial-Mesenchymal Transition through an Epigenetic-Based Mechanism in Human Lung Epithelial Cells

The epithelial to mesenchymal transition (EMT) has been well recognized for many decades as an essential early step in the progression of primary tumors towards metastases. Widespread epigenetic reprogramming of DNA and histone modifications tightly regulates gene expression and cellular activity during carcinogenesis, and epigenetic therapy has been developed to design efficient strategies for cancer treatment. As the first oral agent approved for the clinical treatment of cancer, sorafenib has significant inhibitory effects on tumor growth and EMT. However, a detailed understanding of the underlying epigenetic mechanism remains elusive. In this manuscript, we performed a ChIP-seq assay to evaluate the activity of sorafenib on the genome-wide profiling of histone modifications. We demonstrate that sorafenib largely reverses the changes in histone modifications that occur during EMT in A549 alveolar epithelial cells. Sorafenib also significantly reduces the coordinated epigenetic switching of critical EMT-associated genes in accordance with their expression levels. Furthermore, we show that sorafenib potentiates histone acetylation by regulating the expression levels of histone-modifying enzymes. Collectively, these findings provide the first evidence that sorafenib inhibits the EMT process through an epigenetic mechanism, which holds enormous promise for identifying novel epigenetic candidate diagnostic markers and drug targets for the treatment of human malignancies.

Epigenetic reprogramming: preparing the epigenome for the next generation

Epigenetic reprogramming of germ cells involves the genome-wide erasure and subsequent re-establishment of DNA methylation, along with reprogramming of histone modification profiles and the eventual incorporation of histone variants. These linked processes appear to be key for the establishment of the correct epigenetic regulation of this cell lineage. Mouse studies indicate that DNA demethylation may be initiated at E (embryonic day) 8 with rapid and substantial erasure occurring between E11.5 and E12.5. This is accompanied by a reduction in H3K9 dimethylation and an increase in H3K27 trimethylation. DNA remethylation subsequently occurs in late gestation in male germ cells and postnatally in female germ cells. This reprogramming occurs throughout the genome, with the exception of specific sequences. The conservation of this process across species remains largely undetermined, and, with recent discoveries of new DNA modifications, there is still much to be explored.

Rapid recall ability of memory T cells is associated with epigenetic gene poising (P1134)

Abstract While association of histone modifications with expressed or silent genes has been established, it remains unclear how changes in chromatin environment relate to changes in gene expression. We used ChIP-Seq to analyze the genome-wide changes in chromatin modifications during short-term activation of total human CD4+ T cells by T cell receptor (TCR) signaling. In resting and activated T cells, expressed genes were strongly associated with “active” modifications (e.g. H3K4me1/2/3, H2A.Z) and RNA Polymerase II (Pol II), while silent genes were typically associated with repressive marks. However, we found that about 20-30% of silent genes were poised - they possessed positive modifications and sometimes even Pol II at their promoters. Interestingly, majority of genes induced during T cell activation were poised in resting cells even before the TCR signaling was initiated. Similarly, genes that were silenced upon T cell activation retained active chromatin modifications even after being silenced. This suggested that poising might result from memory of past transcription. We next proceeded to compare histone modification profiles of naïve and memory T cells and discovered that ability of memory cells to produce characteristic cytokines is correlated with the presence of poising marks at the enhancers and promoters of these genes. This led us to propose a model of T cell memory in which immunological memory state is determined epigenetically - by transcriptional memory and poising.

The developmental epigenomics toolbox: ChIP-seq and MethylCap-seq profiling of early zebrafish embryos

Genome-wide profiling of DNA methylation and histone modifications answered many questions as to how the genes are regulated on a global scale and what their epigenetic makeup is. Yet, little is known about the function of these marks during early vertebrate embryogenesis. Here we provide detailed protocols for ChIP-seq and MethylCap-seq procedures applied to zebrafish (Danio rerio) embryonic material at four developmental stages. As a proof of principle, we have profiled on a global scale a number of post-translational histone modifications including H3K4me1, H3K4me3 and H3K27ac. We demonstrate that these marks are dynamic during early development and that such developmental transitions can be detected by ChIP-seq. In addition, we applied MethylCap-seq to show that developmentally-regulated DNA methylation remodeling can be detected by such a procedure. Our MethylCap-seq data concur with previous DNA methylation studies of early zebrafish development rendering this method highly suitable for the global assessment of DNA methylation in early vertebrate embryos.

RFECS: A Random-Forest Based Algorithm for Enhancer Identification from Chromatin State

Transcriptional enhancers play critical roles in regulation of gene expression, but their identification in the eukaryotic genome has been challenging. Recently, it was shown that enhancers in the mammalian genome are associated with characteristic histone modification patterns, which have been increasingly exploited for enhancer identification. However, only a limited number of cell types or chromatin marks have previously been investigated for this purpose, leaving the question unanswered whether there exists an optimal set of histone modifications for enhancer prediction in different cell types. Here, we address this issue by exploring genome-wide profiles of 24 histone modifications in two distinct human cell types, embryonic stem cells and lung fibroblasts. We developed a Random-Forest based algorithm, RFECS (Random Forest based Enhancer identification from Chromatin States) to integrate histone modification profiles for identification of enhancers, and used it to identify enhancers in a number of cell-types. We show that RFECS not only leads to more accurate and precise prediction of enhancers than previous methods, but also helps identify the most informative and robust set of three chromatin marks for enhancer prediction.