Active Histone Modifications Research Articles

Robust Identification of Developmentally Active Endothelial Enhancers in Zebrafish Using FANS-Assisted ATAC-Seq

Identification of tissue-specific and developmentally active enhancers provides insights into mechanisms that control gene expression during embryogenesis. However, robust detection of these regulatory elements remains challenging, especially in vertebrate genomes. Here, we apply fluorescent-activated nuclei sorting (FANS) followed by Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) to identify developmentally active endothelial enhancers in the zebrafish genome. ATAC-seq of nuclei from Tg(fli1a:egfp)y1 transgenic embryos revealed expected patterns of nucleosomal positioning at transcriptional start sites throughout the genome and association with active histone modifications. Comparison of ATAC-seq from GFP-positive and -negative nuclei identified more than 5,000 open elements specific to endothelial cells. These elements flanked genes functionally important for vascular development and that displayed endothelial-specific gene expression. Importantly, a majority of tested elements drove endothelial gene expression in zebrafish embryos. Thus, FANS-assisted ATAC-seq using transgenic zebrafish embryos provides a robust approach for genome-wide identification of active tissue-specific enhancer elements.

Abstract 3578: Methyl2Acetyl: predicting epigenetic and transcriptomic activity from DNA methylation

Abstract DNA methylation and histone modifications are interconnected and interdependent epigenetic mechanisms that regulate chromatin accessibility in cells. They establish cell’s developmental identity and modulate individual response to endogenous developmental stimuli and environmental changes. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a popular assay to study genomewide histone modification profiles. However, ChIP-seq assays require relatively large amounts of starting materials (ranging from ~105 cells using a low cell number protocol to 107 cells in a standard protocol), which remains as a major obstacle for precious biological samples including primary tumor specimens. On the other hand, tagmentation-based whole genome bisulfite sequencing (TWGBS) protocol enables unbiased assessment of the genomewide methylation pattern using ~3,000 cells. Therefore, a quantitative relationship between DNA methylation and epigenetic/transcriptomic activity is in great demand. In the current study, we developed Methyl2Acetyl, a machine learning framework to infer promoter epigenetic activities (e.g. H3K27Ac enrichment) using TWGBS data as input. The proposed model utilizes random forest learners, which allow automatic extraction of complex interactions among DNA methylation features surrounding transcription start sites. We applied Methyl2Acetyl to a set of pediatric solid tumor samples with high quality H3K27Ac ChIPseq data to evaluate its performance. The model accurately predicted promoter H3K27Ac enrichment in independent test samples (Pearson correlation against measured enrichment: 0.81 ± 0.02). To further test the robustness of our model, we trained a Methyl2Acetyl model on solid tumors and applied it to a set of 10 pediatric leukemia samples. Despite dramatic global methylome differences between solid tumors and leukemia, Methyl2Acetyl predicted promoter H3K27Ac enrichment showed strong concordance with target gene expression level in the same sample (Pearson correlation: 0.75 ± 0.02, classification AUC: 0.89 ± 0.01). Collectively, our data suggested that DNA methylation is predictive of promoter active histone modification enrichment and gene expression activity. Epigenetically active promoters can be imputed from TWGBS data in samples with limiting starting materials. Citation Format: Xiang Chen. Methyl2Acetyl: predicting epigenetic and transcriptomic activity from DNA methylation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3578. doi:10.1158/1538-7445.AM2017-3578

Epigenetic modifications of the VH region after DJH recombination in Pro-B cells.

The variable region of murine immunoglobulin heavy chain (Igh) is assembled by sequential DH -JH and VH -DJH recombination. The accessibility of the Igh locus determines the order of rearrangement. Because of the large number of VH genes and the lack of a suitable model, the epigenetic modifications of VH genes after DJH recombination have not previously been characterized. Here, we employed two v-Abl pro-B cell lines, in which the Igh locus is in germline and DJH -recombined configurations, respectively. The DJH junction displays the characteristics of a recombination centre, such as high levels of activation-associated histone modifications and recombination-activating gene protein (RAG) binding in DJH -rearranged pro-B cells, which extend the recombination centre model proposed for the germline Igh locus. The different domains of the VH region have distinct epigenetic characteristics after DJH recombination. Distal VH genes have higher levels of active histone modifications, germline transcription and Pax5 binding, and good quality recombination signal sequences. Proximal VH genes are relatively close to the DJH recombination centre, which partially compensates for the low levels of the above active epigenetic modifications. DJH recombination centre might serve as a cis-acting element to regulate the accessibility of the VH region. Furthermore, we demonstrate that RAG weakly binds to functional VH genes, which is the first detailed assessment of RAG dynamic binding to VH genes. We provide a way for VH -DJH recombination in which the VH gene is brought into close proximity with the DJH recombination centre for RAG binding by a Pax5-dependent chromosomal compaction event, and held in this position for subsequent cleavage and VH -DJH joining.

Locus-specific ChIP combined with NGS analysis reveals genomic regulatory regions that physically interact with the Pax5 promoter in a chicken B cell line.

Chromosomal interactions regulate genome functions, such as transcription, via dynamic chromosomal organization in the nucleus. In this study, we attempted to identify genomic regions that physically bind to the promoter region of the Pax5 gene, which encodes a master regulator for B cell lineage commitment, in a chicken B cell line, DT40, with the goal of obtaining mechanistic insight into transcriptional regulation through chromosomal interaction. We found that the Pax5 promoter bound to multiple genomic regions using locus-specific chromatin immunoprecipitation (locus-specific ChIP), a method for locus-specific isolation of target genomic regions, in combination with next-generation sequencing (NGS). Comparing chromosomal interactions in wild-type DT40 with those in a macrophage-like counterpart, we found that some of the identified chromosomal interactions were organized in a B cell-specific manner. In addition, deletion of a B cell-specific interacting genomic region in chromosome 11, which was marked by active enhancer histone modifications, resulted in moderate but significant down-regulation of Pax5 transcription. Together, these results suggested that Pax5 transcription in DT40 is regulated by B cell-specific inter-chromosomal interactions. Moreover, these analyses showed that locus-specific ChIP combined with NGS analysis is useful for non-biased identification of functional genomic regions that physically interact with a locus of interest.

Reciprocal regulation of the Il9 locus by counteracting activities of transcription factors IRF1 and IRF4

The T helper 9 (Th9) cell transcriptional network is formed by an equilibrium of signals induced by cytokines and antigen presentation. Here we show that, within this network, two interferon regulatory factors (IRF), IRF1 and IRF4, display opposing effects on Th9 differentiation. IRF4 dose-dependently promotes, whereas IRF1 inhibits, IL-9 production. Likewise, IRF1 inhibits IL-9 production by human Th9 cells. IRF1 counteracts IRF4-driven Il9 promoter activity, and IRF1 and IRF4 have opposing function on activating histone modifications, thus modulating RNA polymerase II recruitment. IRF1 occupancy correlates with decreased IRF4 abundance, suggesting an IRF1-IRF4-binding competition at the Il9 locus. Furthermore, IRF1 shapes Th9 cells with an interferon/Th1 gene signature. Consistently, IRF1 restricts the IL-9-dependent pathogenicity of Th9 cells in a mouse model of allergic asthma. Thus our study reveals that the molecular ratio between IRF4 and IRF1 balances Th9 fate, thus providing new possibilities for manipulation of Th9 differentiation.

Epigenetic regulator CXXC5 recruits DNA demethylase Tet2 to regulate TLR7/9-elicited IFN response in pDCs.

TLR7/9 signals are capable of mounting massive interferon (IFN) response in plasmacytoid dendritic cells (pDCs) immediately after viral infection, yet the involvement of epigenetic regulation in this process has not been documented. Here, we report that zinc finger CXXC family epigenetic regulator CXXC5 is highly expressed in pDCs, where it plays a crucial role in TLR7/9- and virus-induced IFN response. Notably, genetic ablation of CXXC5 resulted in aberrant methylation of the CpG-containing island (CGI) within the Irf7 gene and impaired IRF7 expression in steady-state pDCs. Mechanistically, CXXC5 is responsible for the recruitment of DNA demethylase Tet2 to maintain the hypomethylation of a subset of CGIs, a process coincident with active histone modifications and constitutive transcription of these CGI-containing genes. Consequently, CXXC5-deficient mice had compromised early IFN response and became highly vulnerable to infection by herpes simplex virus and vesicular stomatitis virus. Together, our results identify CXXC5 as a novel epigenetic regulator for pDC-mediated antiviral response.

H3K4 demethylase KDM5B regulates global dynamics of transcription elongation and alternative splicing in embryonic stem cells.

Epigenetic regulation of chromatin plays a critical role in controlling embryonic stem (ES) cell self-renewal and pluripotency. However, the roles of histone demethylases and activating histone modifications such as trimethylated histone 3 lysine 4 (H3K4me3) in transcriptional events such as RNA polymerase II (RNAPII) elongation and alternative splicing are largely unknown. In this study, we show that KDM5B, which demethylates H3K4me3, plays an integral role in regulating RNAPII occupancy, transcriptional initiation and elongation, and alternative splicing events in ES cells. Depletion of KDM5B leads to altered RNAPII promoter occupancy, and decreased RNAPII initiation and elongation rates at active genes and at genes marked with broad H3K4me3 domains. Moreover, our results demonstrate that spreading of H3K4me3 from promoter to gene body regions, which is mediated by depletion of KDM5B, modulates RNAPII elongation rates and RNA splicing in ES cells. We further show that KDM5B is enriched nearby alternatively spliced exons, and depletion of KDM5B leads to altered levels of H3K4 methylation in alternatively spliced exon regions, which is accompanied by differential expression of these alternatively splice exons. Altogether, our data indicate an epigenetic role for KDM5B in regulating RNAPII elongation and alternative splicing, which may support the diverse mRNA repertoire in ES cells.

The HDAC inhibitor valproate induces a bivalent status of the CD20 promoter in CLL patients suggesting distinct epigenetic regulation of CD20 expression in CLL in vivo.

Treatment with anti-CD20 antibodies is only moderately efficient in chronic lymphocytic leukemia (CLL), a feature which has been explained by the inherently low CD20 expression in CLL. It has been shown that CD20 is epigenetically regulated and that histone deacetylase inhibitors (HDACis) can increase CD20 expression in vitro in CLL. To assess whether HDACis can upregulate CD20 also in vivo in CLL, the HDACi valproate was given to three del13q/NOTCH1wt CLL patients and CD20 levels were analysed (the PREVAIL study). Valproate treatment resulted in expected global activating histone modifications suggesting HDAC inhibitory effects. However, although valproate induced expression of CD20 mRNA and protein in the del13q/NOTCH1wt I83-E95 CLL cell line, no such effects were observed in the patients studied. In contrast to the cell line, in patients valproate treatment resulted in transient recruitment of the transcriptional repressor EZH2 to the CD20 promoter, correlating to an increase of the repressive histone mark H3K27me3. This suggests that valproate-mediated induction of CD20 may be hampered by EZH2 mediated H3K27me3 in vivo in CLL. Moreover, valproate treatment resulted in induction of EZH2 and global H3K27me3 in patient cells, suggesting transcriptionally repressive effects of valproate in CLL. Our results suggest new in vivo mechanisms of HDACis which may have implications on the design of future clinical trials in B-cell malignancies.

190 - New Development in Childhood MDS

Gonadotrope cell identity genes emerge in a stepwise process during mouse pituitary development. Cga, encoding for the α-subunit of TSH, LH, and FSH, is initially detected at E11.5 followed by Gnrhr and steroidogenic factor Sf1 at E13.5, specifying cells engaged in a gonadotrope cell fate. Lhb and Fshb appear at E16.5 and 17.5, respectively, typifying differentiated gonadotrope cells. Using the αT1-1, αT3-1 and LβT2 cell lines recapitulating these stages of gonadotrope differentiation, DNA methylation at Gnrhr and Sf1 was investigated. Regulatory regions were found hypermethylated in progenitor αT1-1 cells and hypomethylated in differentiated LβT2 cells. Abundance of RNA polymerase II together with active histone modifications including H3K4me1, H3K4me3, and H3K27ac were strictly correlated with DNA hypomethylation. Analyses of epigenomic modifications and chromatin accessibility were further extended to Isl1, Lhx3, Gata2, and Pitx2, highlighting alternative usages of specific regulatory gene domains in progenitor αT1-1, immature αT3-1, and mature LβT2 gonadotrope cells.

Chronic treatment of non-small-cell lung cancer cells with gefitinib leads to an epigenetic loss of epithelial properties associated with reductions in microRNA-155 and -200c.

BackgroundThe EGFR tyrosine kinase inhibitor gefitinib is used in therapy for non-small-cell lung cancer (NSCLC). However, its application is limited by resistance-accelerated disease progression, which is accompanied by the epithelial-to-mesenchymal transition (EMT). In the present study, we performed multiple expression analyses of microRNAs (miRNAs) and quantified the expression of several related EMT players in gefitinib-resistant NSCLC cells.Methods and resultsTo establish gefitinib-resistant NSCLC cells, gefitinib-sensitive HCC827 cells, which exhibit an in-frame deletion [E746-A750] in EGFR exon 19, were exposed to gefitinib for at least 1.5 months. Next, to profile “gefitinib-resistant HCC827 (HCC827GR)” cells, which have a secondary T790M mutation in EGFR exon 20, a miRNA array analysis was performed in HCC827 and HCC827GR cells. The greatest differences were seen in the levels of miR-155 and miR-200c, which essentially disappeared in HCC827GR cells. In addition to these reductions, the levels of smad2 and zeb1, which are both key players in EMT and targets for miR-155 and miR-200c, respectively, were dramatically increased in HCC827GR cells. In HCC827GR cells, the expression of epithelial-cadherin (E-cadherin) was greatly reduced with repressive histone modifications, whereas vimentin, which is expressed in mesenchymal cells, was dramatically increased with active histone modifications. In another gefitinib-resistant NSCLC cell line (H1975 cells), similar to the findings in HCC827GR cells, both miR-155 and miR-200c were absent, and the EMT was induced along with epigenetic modifications. Interestingly, the inhibition of both miR-155 and miR-200c in HCC827 cells without gefitinib induced significant increases in smad2 and zeb1 along with a dramatic decrease in E-cadherin and a slight increase in vimentin. Furthermore, although the inhibition of these miRNAs in HCC827 cells decreased gefitinib sensitivity, this dual-inhibition in HCC827 cells without gefitinib did not produce a secondary T790M mutation in EGFR exon 20.Conclusion and implicationsThese results suggest that chronic treatment of NSCLC cells with gefitinib changes the expression of miRNAs, including dramatic reductions in miR-155 and miR-200c along with an EGFR mutation. Furthermore, this depletion of miR-155 and miR-200c may be associated with the EMT along with histone modifications, and may contribute to the decrease in the sensitivity to gefitinib independent of a secondary EGFR mutation.

Promoter Methylation Pattern Controls Corticotropin Releasing Hormone Gene Activity in Human Trophoblasts.

Placental CRH production increases with advancing pregnancy in women and its course predicts gestational length. We hypothesized that CRH gene expression in the placenta is epigenetically controlled setting gestational trajectories characteristic of normal and pathological pregnancies. Here we determined histone modification and DNA methylation levels and DNA methylation patterns at the CRH promoter in primary trophoblast cultures by chromatin immunoprecipitation combined with clonal bisulfite sequencing and identified the transcriptionally active epialleles that associate with particular histone modifications and transcription factors during syncytialisation and cAMP-stimulation. CRH gene expression increased during syncytial differentiation and cAMP stimulation, which was associated with increased activating and decreased repressive histone modification levels at the promoter. DNA methylation levels remained unchanged. The nine CpGs of the CRH proximal promoter were partially and allele-independently methylated displaying many (>100) epialleles. RNA-polymerase-II (Pol-II) bound only to three particular epialleles in cAMP-stimulated cells, while phospho-cAMP response element-binding protein (pCREB) bound to only one epiallele, which was different from those selected by Pol-II. Binding of TATA-binding protein increased during syncytial differentiation preferentially at epialleles compatible with Pol-II and pCREB binding. Histone-3 acetylation was detected only at epialleles targeted by Pol-II and pCREB, while gene activating histone-4 acetylation and histone-3-lysine-4 trimethylation occurred at CRH epialleles not associated with Pol-II or pCREB. The suppressive histone-3-lysine-27 trimethyl and–lysine-9 trimethyl modifications showed little or no epiallele preference. The epiallele selectivity of activating histone modifications and transcription factor binding demonstrates the epigenetic and functional diversity of the CRH gene in trophoblasts, which is controlled predominantly by the patterns, not the overall extent, of promoter methylation. We propose that conditions impacting on epiallele distribution influence the number of transcriptionally active CRH gene copies in the trophoblast cell population determining the gestational trajectory of placental CRH production in normal and pathological pregnancies.

Chromatin States in Mouse Sperm Correlate with Embryonic and Adult Regulatory Landscapes

The mammalian sperm genome is thought to lack substantial information for the regulation of future expression after fertilization. Here, we show that most promoters in mouse sperm are flanked by well-positioned nucleosomes marked by active histone modifications. Analysis of these modifications suggests that many enhancers and super-enhancers functional in embryonic and adult tissues are already specified in sperm. The sperm genome is bound by CTCF and cohesin at sites that are also present in round spermatids and embryonic stem cells (ESCs). These sites mediate interactions that organize the sperm genome into domains and compartments that overlap extensively with those found in mESCs. These results suggest that sperm carry a rich source of regulatory information, encoded in part by its three-dimensional folding specified by CTCF and cohesin. This information may contribute to future expression during embryonic and adult life, suggesting mechanisms by which environmental effects on the paternal germline are transmitted transgenerationally.

Histone modifications influence skipped exons inclusion.

Alternative splicing (AS), by which individual genes can produce multiple mRNA, associates with genomic complexity, disease, and development. Histone modifications show important roles in both transcription initiation and mRNA splicing. Here, we intended to find the link between AS and histone modifications in flanking regions through analyzing publicly available data in two human cell lines, GM12878 and K562 cell lines. According to exon inclusion levels, exons were classified into three types, included skipped exons, excluded skipped exons and expressed constitutive exons. We revealed that the inclusion levels of skipped exons (SEs) were negatively correlated with the enrichment of active histone marks in SEs, indicating a role of histone modifications in AS. We also found that active histone modifications were enriched in the upstream exons of SEs, especially around 5[Formula: see text] splicing sites. We inferred that the histone modifications around the 5[Formula: see text] splicing sites in upstream exon of the SEs could help RNA Polymerase II complex to recruit the effector proteins and facilitate AS. It was indicated that nucleosome occupancy had little influence on the inclusion levels of SEs. At last, we proposed an integrated model that describe how histone modifications affected the pre-mRNA splicing.

Sex-specific chromatin landscapes in an ultra-compact chordate genome

BackgroundIn multicellular organisms, epigenome dynamics are associated with transitions in the cell cycle, development, germline specification, gametogenesis and inheritance. Evolutionarily, regulatory space has increased in complex metazoans to accommodate these functions. In tunicates, the sister lineage to vertebrates, we examine epigenome adaptations to strong secondary genome compaction, sex chromosome evolution and cell cycle modes.ResultsAcross the 70 MB Oikopleura dioica genome, we profiled 19 histone modifications, and RNA polymerase II, CTCF and p300 occupancies, to define chromatin states within two homogeneous tissues with distinct cell cycle modes: ovarian endocycling nurse nuclei and mitotically proliferating germ nuclei in testes. Nurse nuclei had active chromatin states similar to other metazoan epigenomes, with large domains of operon-associated transcription, a general lack of heterochromatin, and a possible role of Polycomb PRC2 in dosage compensation. Testis chromatin states reflected transcriptional activity linked to spermatogenesis and epigenetic marks that have been associated with establishment of transgenerational inheritance in other organisms. We also uncovered an unusual chromatin state specific to the Y-chromosome, which combined active and heterochromatic histone modifications on specific transposable elements classes, perhaps involved in regulating their activity.ConclusionsCompacted regulatory space in this tunicate genome is accompanied by reduced heterochromatin and chromatin state domain widths. Enhancers, promoters and protein-coding genes have conserved epigenomic features, with adaptations to the organization of a proportion of genes in operon units. We further identified features specific to sex chromosomes, cell cycle modes, germline identity and dosage compensation, and unusual combinations of histone PTMs with opposing consensus functions.

Rapid Recall Ability of Memory T cells is Encoded in their Epigenome

Even though T-cell receptor (TCR) stimulation together with co-stimulation is sufficient for the activation of both naïve and memory T cells, the memory cells are capable of producing lineage specific cytokines much more rapidly than the naïve cells. The mechanisms behind this rapid recall response of the memory cells are still not completely understood. Here, we performed epigenetic profiling of human resting naïve, central and effector memory T cells using ChIP-Seq and found that unlike the naïve cells, the regulatory elements of the cytokine genes in the memory T cells are marked by activating histone modifications even in the resting state. Therefore, the ability to induce expression of rapid recall genes upon activation is associated with the deposition of positive histone modifications during memory T cell differentiation. We propose a model of T cell memory, in which immunological memory state is encoded epigenetically, through poising and transcriptional memory.

Epigenetic Regulation of Caveolin-1 Gene Expression in Lung Fibroblasts.

Fibrotic disorders are associated with tissue accumulation of fibroblasts. We recently showed that caveolin (Cav)-1 gene suppression by a profibrotic cytokine, transforming growth factor (TGF)-β1, contributes to fibroblast proliferation and apoptosis resistance. Cav-1 has been shown to be constitutively suppressed in idiopathic pulmonary fibrosis (IPF), but mechanisms for this suppression are incompletely understood. We hypothesized that epigenetic processes contribute to Cav-1 down-regulation in IPF lung fibroblasts, and after fibrogenic stimuli. Cav-1 expression levels, DNA methylation status, and histone modifications associated with the Cav-1 promoter were examined by PCR, Western blots, pyrosequencing, or chromatin immunoprecipitation assays in IPF lung fibroblasts, normal fibroblasts after TGF-β1 stimulation, or in murine lung fibroblasts after bleomycin injury. Methylation-specific PCR demonstrated methylated and unmethylated Cav-1 DNA copies in all groups. Despite significant changes in Cav-1 expression, no changes in DNA methylation were observed in CpG islands or CpG island shores of the Cav-1 promoter by pyrosequencing of lung fibroblasts from IPF lungs, in response to TGF-β1, or after bleomycin-induced murine lung injury, when compared with respective controls. In contrast, the association of Cav-1 promoter with the active histone modification mark, H3 lysine 4 trimethylation, correlated with Cav-1 down-regulation in activated/fibrotic lung fibroblasts. Our data indicate that Cav-1 gene silencing in lung fibroblasts is actively regulated by epigenetic mechanisms that involve histone modifications, in particular H3 lysine 4 trimethylation, whereas DNA methylation does not appear to be a primary mechanism. These findings support therapeutic strategies that target histone modifications to restore Cav-1 expression in fibroblasts participating in pathogenic tissue remodeling.

Role of Histone Deacetylase-Mediated Gene Silencing in Chronic Lymphocytic Leukemia Progression

Chronic lymphocytic leukemia (CLL) follows a variable clinical course mostly dependent upon genomic factors, with a subset of patients having low risk disease and others displaying rapid progression associated with clonal evolution. Epigenetic mechanisms such as DNA promoter hypermethylation were shown to have a role in CLL evolution where the acquisition of increasingly heterogeneous DNA methylation patters occurred in conjunction with clonal evolution of genetic aberrations and was associated with disease progression. However the role of epigenetic mechanisms regulated by the histone deacetylase group of transcriptional repressors in the progression of CLL has not been well characterized.The histone deacetylases (HDACs) 1 and 2 are recruited onto gene promoters and form a complex with the histone demethylase KDM1. Once recruited, the complex mediate the removal of acetyl groups from specific lysines on histones (H3K9 and H3K14) thus triggering the demethylation of lysine 4 (H3K4me3) and the silencing of gene expression.CLL is characterized by the dysregulation of numerous coding and non coding genes, many of which have key roles in regulating the survival or progression of CLL. For instance, our group showed that the levels of HDAC1 were elevated in high risk as compared to low risk CLL or normal lymphocytes and this over-expression was responsible for the silencing of miR-106b, mR-15, miR-16, and miR-29b which affected CLL survival by modulating the expression of key anti-apoptotic proteins Bcl-2 and Mcl-1.To characterize the HDAC-repressed gene signature in high risk CLL, we conducted chromatin immunoprecipitation (ChIP) of the nuclear lysates from 3 high risk and 3 low risk CLL patients using antibodies against HDAC1, HDAC2 and KDM1 or non-specific IgG, sequenced and aligned the eluted DNA to a reference genome and determined the binding of HDAC1, HDAC2 and KDM1 at the promoters for all protein coding and microRNA genes. Preliminary results from this ChIP-seq showed a strong recruitment of HDAC1, HDAC2 and KDM1 to the promoters of several microRNA as well as protein coding genes in high risk CLL.To further corroborate these data we performed ChIP-Seq in the same 6 CLL samples to analyze the levels of H3K4me2 and H3K4me3 around gene promoters before and after 6h exposure to the HDACi panobinostat. Our goal was to demonstrate that HDAC inhibition elicited an increase in the levels of acetylation on histones and triggered the accrual of H3K4me2 at the repressed promoter, events likely to facilitate the recruitment of RNA polymerase II to this promoter. Initial analysis confirmed a robust accumulation of H3K4me2 and H3K4me3 marks at the gene promoters of representative genes that recruited HDAC1 and its co-repressors in the previous ChIP-Seq analysis in high risk CLL patients.Finally, 5 aggressive CLL samples were treated with the HDACi abexinostat for 48h and RNA before and after treatment was subjected to RNA-seq for small and large RNA to confirm that the regions of chromatin uncoiled by HDACi treatment were actively transcribed.HDAC inhibition induced the expression of a large number of miRNA genes as well as key protein coding genes, such as miR-29b, miR-210, miR-182, miR-183, miR-95, miR-940, FOXO3, EBF1 and BCL2L11. Of note, some of the predicted or validated targets of the induced miRNAs were key facilitators in the progression of CLL, such as BTK, SYK, MCL-1, BCL-2, TCL1, and ROR1. Moreover, RNA-seq showed that the expression of these protein coding genes was reduced by 2-33 folds upon HDAC inhibition.We plan to extend the RNA-seq to 5 CLL samples with indolent disease and combine all the data to identify a common signature of protein coding and miRNA genes that recruited the HDAC1 complex, accumulated activating histone modifications upon treatment with HDACi and altered gene and miRNA expression after HDAC inhibition in high risk CLL versus low risk CLL. The signature will be than validated on a large cohort of indolent and aggressive CLL patients.Our final goal is to define a signature of coding and non coding genes silenced by HDACs in high risk CLL and its role in facilitating disease progression. DisclosuresWoyach:Acerta: Research Funding; Karyopharm: Research Funding; Morphosys: Research Funding.

Long Non-Coding RNAs Annotation and Their Involvement in Multiple Myeloma

Increasing amount of evidence indicates that the deregulation of non-coding elements is a common feature of cancer and therefore, its investigation may uncover new molecular oncogenic mechanisms. In multiple myeloma (MM), the altered expression of a small number of long non-coding RNAs (lncRNAs) has been associated with progression and decreased survival, suggesting that these elements may play a more important role in this disease than previously expected. Nevertheless, an extensive high-throughput analysis that characterizes the deregulation of lncRNAs in MM has not yet been performed.To characterize the transcriptome, including all genomic types of lncRNAs, of MM we performed a paired end strand-specific RNA sequencing (ssRNA-seq) in 38 purified plasma cell (PC) samples from MM patients, as well as PC samples from tonsils (TPCs, n=5) and bone marrow (BMPCs, n=3) of healthy donors as controls. Principal component analysis (PCA) demonstrated that normal PC samples from tonsil and bone marrow cluster separately, suggesting that in spite of being the same cell type, their coding and non coding transcriptomes are very different. Therefore, we selected BMPCs as the normal counterparts for comparison with BM of MM samples. PCA analysis also demonstrated that the well known heterogeneity of MM patients rely not only on the coding transcriptome but also on the lncRNA expression profile. Comparison of MM to BMPCs samples showed 70 previously annotated lncRNAs that were deregulated in MM patients, with 3 lncRNAs showing higher and 67 lower expression than normal BMPCs. Moreover, we identified 40.552 novel MM-specific lncRNAs that were present in at least 3 of the 38 patients, highlighting the magnitude of the deregulation of these non coding elements in MM.To determine the functional role of altered lncRNAs in the biology of MM plasma cells we focused on the study of LINC-MSL1 (Myeloma-Specific LncRNA 1). Analysis of the expression of this lncRNA at different stages of B-cell differentiation (Naïve, Germinal Center, Memory and PC) indicated that it is not expressed at any stage, except for a modest expression in BMPCs. Interestingly, its overexpression was detected in 40% of MM specimens when compared to normal BMPCs which was validated by qPCR in an independent cohort of MM patients. To determine whether the expression of this lncRNA is regulated by epigenetic mechanisms, we studied the DNA methylation state of this gene. DNA methylation analysis in MM demonstrated that the CpGs located upstream of LINC-MSL1 were differentially methylated in comparison with normal counterpart BMPC. These CpGs showed 70% DNA methylation in control samples, about 40% in MGUS, whereas the average of MM was about 20%, showing a remarkable hypomethylation. We validated these results by pyrosequencing, which showed a significantly lower DNA methylation at the promoter region in comparison with B cell populations from tonsil, normal BMPCs and cell lines that do not overexpress LINC-MSL1. We also have observed a gain of active chromatin states analyzed by ChiP-seq in the promoter region of LINC-MSL1 in MM patient samples. These data suggest that epigenetic mechanisms, namely the progressive hypomethylation and the gain of active histone modifications, are the cause of the overexpression of LINC-MSL1 in MM.To analyze the role of the overexpression of LINC-MSL1 in MM, we engineered two MM cell lines that show high levels of LINC-MSL1, MM.1S and MM.1R, to express shRNAs against this lncRNA. Knockdown of LINC-MSL1 by two different shRNAs resulted in a reduced proliferation of the cell lines over time. This effect was not associated with a cell cycle arrest but with a marked increased in the percentage of Annexin V-positive apoptotic cells, indicating that the overexpression of LINC-MSL1 is necessary for the survival of MM cells.All together, these data demonstrate that the alteration of lncRNAs is an important an unexplored feature that contributes to MM pathogenesis. The overexpression of LINC-MSL1 is essential for MM survival and is very specific of MM BMPCs, suggesting it could be a relevant therapeutic target. DisclosuresPaiva:Celgene: Honoraria, Research Funding; Janssen: Honoraria; Takeda: Honoraria, Research Funding; Sanofi: Consultancy, Research Funding; EngMab: Research Funding; Amgen: Honoraria; Binding Site: Research Funding. Melnick:Janssen: Research Funding.

Chromatin remodeling during in vivo neural stem cells differentiating to neurons in early Drosophila embryos

Neurons are a key component of the nervous system and differentiate from multipotent neural stem cells (NSCs). Chromatin remodeling has a critical role in the differentiation process. However, its in vivo epigenetic regulatory role remains unknown. We show here that nucleosome depletion regions (NDRs) form in both proximal promoters and distal enhancers during NSCs differentiating into neurons in the early Drosophila embryonic development. NDR formation in the regulatory regions involves nucleosome shift and eviction. Nucleosome occupancy in promoter NDRs is inversely proportional to the gene activity. Genes with promoter NDR formation during differentiation are enriched for functions related to neuron development and maturation. Active histone-modification signals (H3K4me3 and H3K9ac) in promoters are gained in neurons in two modes: de novo establishment to high levels or increase from the existing levels in NSCs. The gene sets corresponding to the two modes have different neuron-related functions. Dynamic changes of H3K27ac and H3K9ac signals in enhancers and promoters synergistically repress genes associated with neural stem or progenitor cell-related pluripotency and upregulate genes associated with neuron projection morphogenesis, neuron differentiation, and so on. Our results offer new insights into chromatin remodeling during in vivo neuron development and lay a foundation for its epigenetic regulatory mechanism study of other lineage specification.

Dynamic and Antagonistic Allele-Specific Epigenetic Modifications Controlling the Expression of Imprinted Genes in Maize Endosperm

Genomic imprinting is often associated with allele-specific epigenetic modifications. Although many reports suggested potential roles of DNA methylation and H3K27me3 in regulating genomic imprinting, the contributions of allele-specific active histone modifications to imprinting remain still unclear in plants. Here, we report the identification of 337 high-stringency allele-specific H3K4me3 and H3K36me3 peaks in maize endosperm. Paternally preferred H3K4me3 and H3K36me3 peaks mostly co-localized with paternally expressed genes (PEGs), while endosperm-specific maternally expressed genes (endo-MEGs) were associated with maternally preferred H3K4me3 and H3K36me3 peaks. A unique signature for PEGs was observed, where the active H3K4me4 and H3K36me3 as well as repressive H3K27me3 appeared together. At the gene body of con-PEGs (constitutively expressed PEG), H3K27me3 and H3K36me3 were specifically deposited on hypomethylated maternal alleles and hypermethylated paternal alleles, respectively. Around the transcription start sites of endo-MEGs, DNA methylation and H3K4me3 specifically marked paternal and maternal alleles, respectively. In addition, 35 maternally expressed non-coding RNAs exhibited the same allele-specific epigenetic features as endo-MEGs, indicating similar mechanisms for the regulation of imprinted genes and non-coding RNAs. Taken together, our results uncover the complex patterns of mutually exclusive epigenetic modifications deposited at different alleles of imprinted genes that are required for genomic imprinting in maize endosperm.