Histone Crosstalk Research Articles

Histone lysine methyltransferases MpDot1 and MpSet9 are involved in the production of lovastatin and MonAzPs by histone crosstalk modification

Increasing data suggested that histone methylation modification plays an important role in regulating biosynthesis of secondary metabolites (SMs). Monascus spp. have been applied to produce hypolipidemic drug lovastatin (also called monacolin K, MK) and edible Monascus-type azaphilone pigments (MonAzPs). However, little is known about how histone methylation regulates MK and MonAzPs. In this study, we constructed H3K9 methyltransferase deletion strain ΔMpDot1 and H4K20 methyltransferase deletion strain ΔMpSet9 using Monascus pilosus MS-1 as the parent. The result showed that deletion of MpDot1 reduced the production of MK and MonAzPs, and deletion of MpSet9 increased MonAzPs production. Real-time quantitative PCR (RT-qPCR) showed inactivation of mpdot1 and mpset9 disturbed the expression of genes responsible for the biosynthesis of MK and MonAzPs. Western blot suggested that deletion of MpDot1 reduced H3K79me and H4K16ac, and deletion of MpSet9 decreased H4K20me3 and increased H4pan acetylation. Chromatin immunoprecipitation coupled with quantitative PCR (ChIP-qPCR) showed ΔMpDot1 strain and ΔMpSet9 strain reduced the enrichment of H3K79me2 and H4K20me3 in the promoter regions of key genes for MK and MonAzPs biosynthesis, respectively. These results suggested that MpDot1 and MpSet9 affected the synthesis of SMs by regulating gene transcription and histone crosstalk, providing alternative approach for regulation of lovastatin and MonAzPs.

The COMPASS Complex Regulates Fungal Development and Virulence through Histone Crosstalk in the Fungal Pathogen Cryptococcus neoformans.

The Complex of Proteins Associated with Set1 (COMPASS) methylates lysine K4 on histone H3 (H3K4) and is conserved from yeast to humans. Its subunits and regulatory roles in the meningitis-causing fungal pathogen Cryptococcus neoformans remain unknown. Here we identified the core subunits of the COMPASS complex in C. neoformans and C. deneoformans and confirmed their conserved roles in H3K4 methylation. Through AlphaFold modeling, we found that Set1, Bre2, Swd1, and Swd3 form the catalytic core of the COMPASS complex and regulate the cryptococcal yeast-to-hypha transition, thermal tolerance, and virulence. The COMPASS complex-mediated histone H3K4 methylation requires H2B mono-ubiquitination by Rad6/Bre1 and the Paf1 complex in order to activate the expression of genes specific for the yeast-to-hypha transition in C. deneoformans. Taken together, our findings demonstrate that putative COMPASS subunits function as a unified complex, contributing to cryptococcal development and virulence.

Acidotic and hypoxic tumor microenvironment induces changes to histone acetylation and methylation in oral squamous cell carcinoma.

Hypoxia and acidosis are ubiquitous hallmarks of the tumor microenvironment (TME), and in most solid cancers they have been linked to rewired cancer cell metabolism. These TME stresses are linked to changes in histone post-translational modifications (PTMs) such as methylation and acetylation, which lead to tumorigenesis and drug resistance. Hypoxic and acidotic TME cause changes in histone PTMs by impacting the activities of histone-modifying enzymes. These alterations are yet to be extensively explored in oral squamous cell carcinoma (OSCC), one of the most prevalent cancers in developing countries. Hypoxic, acidotic, and hypoxia with acidotic TME affecting histone acetylation and methylation in the CAL27 OSCC cell line was studied using LC-MS-based proteomics. The study identified several well-known histone marks, in the context of their functionality in gene regulation, such as H2AK9Ac, H3K36me3, and H4K16Ac. The results provide insights into the histone acetylation and methylation associated with hypoxic and acidotic TME, causing changes in their level in a position-dependent manner in the OSCC cell line. Hypoxia and acidosis, separately and in combination, cause differential impacts on histone methylation and acetylation in OSCC. The work will helpuncovertumor cell adaptation to these stress stimuli in connection with histone crosstalk events.

SESAME-catalyzed H3T11 phosphorylation inhibits Dot1-catalyzed H3K79me3 to regulate autophagy and telomere silencing

The glycolytic enzyme, pyruvate kinase Pyk1 maintains telomere heterochromatin by phosphorylating histone H3T11 (H3pT11), which promotes SIR (silent information regulator) complex binding at telomeres and prevents autophagy-mediated Sir2 degradation. However, the exact mechanism of action for H3pT11 is poorly understood. Here, we report that H3pT11 directly inhibits Dot1-catalyzed H3K79 tri-methylation (H3K79me3) and uncover how this histone crosstalk regulates autophagy and telomere silencing. Mechanistically, Pyk1-catalyzed H3pT11 directly reduces the binding of Dot1 to chromatin and inhibits Dot1-catalyzed H3K79me3, which leads to transcriptional repression of autophagy genes and reduced autophagy. Despite the antagonism between H3pT11 and H3K79me3, they work together to promote the binding of SIR complex at telomeres to maintain telomere silencing. Furthermore, we identify Reb1 as a telomere-associated factor that recruits Pyk1-containing SESAME (Serine-responsive SAM-containing Metabolic Enzyme) complex to telomere regions to phosphorylate H3T11 and prevent the invasion of H3K79me3 from euchromatin into heterochromatin to maintain telomere silencing. Together, these results uncover a histone crosstalk and provide insights into dynamic regulation of silent heterochromatin and autophagy in response to cell metabolism.

Cis- and trans-regulation by histone H4 basic patch R17/R19 in metazoan development.

The histone H4 basic patch is critical for chromatin structure and regulation of the chromatin machinery. However, the biological roles of these positively charged residues and the mechanisms by which they regulate gene expression remain unclear. In this study, we used histone mutagenesis to investigate the physiological function and downstream regulatory genes of H4 residues R17 and R19 in Drosophila. We found all histone mutations including R17A/E/H and R19A/E/H (R17 and R19 of H4 are substituted by A, E and H respectively) result in a range of growth defects and abnormalities in chromosomal high-order structures, whereas R17E mutation is embryonic lethal. RNA-seq demonstrates that downregulated genes in both R17A and R19A show significant overlap and are enriched in development-related pathways. In addition, Western and cytological analyses showed that the R17A mutation resulted in a significant reduction in H4K16 acetylation and male offspring, implying that the R17 may be involved in male dosage compensation mechanisms. R19 mutation on the other hand strongly affect Gpp (Dot1 homologue in flies)-mediated H3K79 methylation, possibly through histone crosstalk. Together these results provide insights into the differential impacts of positive charges of H4 basic patch R17/R19 on regulation of gene transcription during developmental processes.

H2B Lys34 Ubiquitination Induces Nucleosome Distortion to Stimulate Dot1L Activity.

Ubiquitination-dependent histone crosstalk plays critical roles in chromatin-associated processes and is highly associated with human diseases. Mechanism studies of the crosstalk have been of the central focus. Here our study on the crosstalk between H2BK34ub and Dot1L-catalyzed H3K79me suggests a novel mechanism of ubiquitination-induced nucleosome distortion to stimulate the activity of an enzyme. We determined the cryo-electron microscopy structures of Dot1L-H2BK34ub nucleosome complex and the H2BK34ub nucleosome alone. The structures reveal that H2BK34ub induces an almost identical orientation and binding pattern of Dot1L on nucleosome as H2BK120ub, which positions Dot1L for the productive conformation through direct ubiquitin-enzyme contacts. However, H2BK34-anchored ubiquitin does not directly interact with Dot1L as occurs in the case of H2BK120ub, but rather induces DNA and histone distortion around the modified site. Our findings establish the structural framework for understanding the H2BK34ub-H3K79me trans-crosstalk and highlight the diversity of mechanisms for histone ubiquitination to activate chromatin-modifying enzymes.

Ubiquitination of Histone H2B by Proteasome Subunit RPT6 Controls Histone Methylation Chromatin Dynamics During Memory Formation

BackgroundPosttranslational histone modifications play a critical role in the regulation of gene transcription underlying synaptic plasticity and memory formation. One such epigenetic change is histone ubiquitination, a process that is mediated by the ubiquitin–proteasome system in a manner similar to that by which proteins are normally targeted for degradation. However, histone ubiquitination mechanisms are poorly understood in the brain and in learning. In this article, we describe a new role for the ubiquitin–proteasome system in histone crosstalk, showing that learning-induced monoubiquitination of histone H2B (H2Bubi) is required for increases in the transcriptionally active H3 lysine 4 trimethylation (H3K4me3) mark at learning-related genes in the hippocampus. MethodsUsing a series of molecular, biochemical, electrophysiological, and behavioral experiments, we interrogated the effects of short interfering RNA–mediated knockdown and CRISPR (clustered regularly interspaced short palindromic repeats)-mediated upregulation of ubiquitin ligases, deubiquitinating enzymes and histone methyltransferases in the rat dorsal hippocampus during memory consolidation. ResultsWe show that H2Bubi recruits H3K4me3 through a process that is dependent on the 19S proteasome subunit RPT6 and that a loss of H2Bubi in the hippocampus prevents learning-induced increases in H3K4me3, gene transcription, synaptic plasticity, and memory formation. Furthermore, we show that CRISPR–dCas9-mediated increases in H2Bubi promote H3K4me3 and memory formation under weak training conditions and that promoting histone methylation does not rescue memory impairments resulting from loss of H2Bubi. ConclusionsThese results suggest that H2B ubiquitination regulates histone crosstalk in learning by way of nonproteolytic proteasome function, demonstrating a novel mechanism by which histone modifications are coordinated in response to learning.

Histone sumoylation promotes Set3 histone-deacetylase complex-mediated transcriptional regulation.

Histones are substrates of the SUMO (small ubiquitin-like modifier) conjugation pathway. Several reports suggest histone sumoylation affects transcription negatively, but paradoxically, our genome-wide analysis shows the modification concentrated at many active genes. We find that trans-tail regulation of histone-H2B ubiquitylation and H3K4 di-methylation potentiates subsequent histone sumoylation. Consistent with the known control of the Set3 histone deacetylase complex (HDAC) by H3K4 di-methylation, histone sumoylation directly recruits the Set3 complex to both protein-coding and noncoding RNA (ncRNA) genes via a SUMO-interacting motif in the HDAC Cpr1 subunit. The altered gene expression profile caused by reducing histone sumoylation matches well to the profile in cells lacking Set3. Histone H2B sumoylation and the Set3 HDAC coordinately suppress cryptic ncRNA transcription initiation internal to mRNA genes. Our results reveal an elaborate co-transcriptional histone crosstalk pathway involving the consecutive ubiquitylation, methylation, sumoylation and deacetylation of histones, which maintains transcriptional fidelity by suppressing spurious transcription.

Histone acetyltransferase Gcn5 regulates gene expression by promoting the transcription of histone methyltransferase SET1

Many chromatin modifying factors regulate gene expression in an as-yet-unknown indirect manner. Revealing the molecular basis for this indirect gene regulation will help understand their precise roles in gene regulation and associated biological processes. Here, we studied histone modifying enzymes that indirectly regulate gene expression by modulating the expression of histone methyltransferase, Set1. Through unbiased screening of the histone H3/H4 mutant library, we identified 13 histone substitution mutations with reduced levels of Set1 and H3K4 trimethylation (H3K4me3) and 2 mutations with increased levels of Set1 and H3K4me3, which concentrate at 3 structure clusters. Among these substitutions, the H3K14A mutant substantially reduces SET1 transcription and H3K4me3. H3K14 is acetylated by histone acetyltransferase Gcn5 at SET1 promoter, which then promotes SET1 transcription to maintain normal H3K4me3 levels. In contrast, the histone deacetylase Rpd3 deacetylates H3K14 to repress SET1 transcription and hence reduce H3K4me3 levels, establishing a dynamic crosstalk between H3K14ac and H3K4me3. By promoting the transcription of SET1 and maintaining H3K4me3 levels, Gcn5 regulates the transcription of a subset gene in an indirect manner. Collectively, we propose a model wherein Gcn5 promotes the expression of chromatin modifiers to regulate histone crosstalk and gene transcription.

Symmetric dimethylation on histone H4R3 associates with histone deacetylation to maintain properly polarized cell growth

Yeast Hsl7 is recognized as a homolog of human arginine methyltransferase 5 (PRMT5) and shows type II PRMT activity by forming symmetric dimethylarginine residues on histones. Previously, we reported that Hsl7 is responsible for in vivo symmetric dimethylation on histone H4 arginine 3 (H4R3me2s) in a transcriptionally repressed state, possibly in association with histone deacetylation by Rpd3. Here, we investigated the function of Hsl7 during cell cycle progression. We found that the accumulation of Hsl7-mediated H4R3me2s is maintained by the histone deacetylase Rpd3 during transcriptional repression and that the low level of H4R3me2s is required for proper asymmetric cell growth during cell division. Our results suggest that the hypoacetylated state of histones is connected to the function of Hsl7 in regulating properly polarized cell growth during cell division and provide new insight into the epigenetic modifications that are important for cell cycle morphogenesis checkpoint control based on the repressive histone crosstalk between symmetric arginine methylation of H4 and histone deacetylation.

A Novel Histone Crosstalk Pathway Important for Regulation of UV-Induced DNA Damage Repair in Saccharomyces cerevisiae.

Histone post-translational modifications play vital roles in a variety of nuclear processes, including DNA repair. It has been previously shown that histone H3K79 methylation is important for the cellular response to DNA damage caused by ultraviolet (UV) radiation, with evidence that specific methylation states play distinct roles in UV repair. Here, we report that H3K79 methylation is reduced in response to UV exposure in Saccharomyces cerevisiae This reduction is specific to the dimethylated state, as trimethylation levels are minimally altered by UV exposure. Inhibition of this reduction has a deleterious effect on UV-induced sister chromatid exchange, suggesting that H3K79 dimethylation levels play a regulatory role in UV repair. Further evidence implicates an additional role for H3K79 dimethylation levels in error-free translesion synthesis, but not in UV-induced G1/S checkpoint activation or double-stranded break repair. Additionally, we find that H3K79 dimethylation levels are influenced by acetylatable lysines on the histone H4 N-terminal tail, which are hyperacetylated in response to UV exposure. Preclusion of H4 acetylation prevents UV-induced reduction of H3K79 dimethylation, and similarly has a negative effect on UV-induced sister chromatid exchange. These results point to the existence of a novel histone crosstalk pathway that is important for the regulation of UV-induced DNA damage repair.

Dual modified antiphospho (Ser10)-acetyl (Lys14)-histone H3 predominantly mark the pericentromeric chromatin during mitosis in monokinetic plants.

Epigenetic regulatory posttranslational histone modification marks not only function individually but also capable to act in combination as a unique pattern. A total of 16 plant species belonging to 11 genera of eight families (five dicots and three monocots) including land plants, epiphytes (orchids) and the holokinetic taxa (Drosera spp.) were analysed for chromosomal distribution of dual modified antiphospho (Ser10)-acetyl (K14)-histone H3 (H3S10phK14ac) to understand the combinatorial chromatin dynamics during mitotic cell division in plants. The anti-H3S10phK14ac evidently mark the pericentromeric chromatin on mitotic chromosomes of the plants excluding the holokinetic Drosera species, which revealed the immunolabelling of whole chromosomes all along the arms. The dual modified immunosignals were absent during early stages of mitosis, appeared intensively at metaphase and remained visible until late-anaphase/telophase however, labelled the whole chromosomes during meiotic metaphase I. Colocalization of anti-H3S10phK14ac with an onion's CENH3 antibody on mitotic chromosomes of Allium revealed the chromosomal location of anti-H3S10phK14ac in the region between signals for CENH3 detection. Overall analysis suggests that the unique localization of combinatorial histone modification mark at pericentromeric chromatin might have attributed through 'phospho-acetyl' cross talk that ultimately facilitate the sister chromatid cohesion at pericentromeres following condensation events in mitotic chromosomes. Here, we propose that dual modified H3S10phK14ac histone may serve as an additional cytogenetic landmark to identify pericentromeric chromatin during mitosis in plants. The plausible role of histone cross talk and future perspectives of combinatorial histone modification marks in plant cytogenetics with special reference to chromatin dynamics have been discussed.

The RING finger domain E3 ubiquitin ligases BRCA1 and the RNF20/RNF40 complex in global loss of the chromatin mark histone H2B monoubiquitination (H2Bub1) in cell line models and primary high-grade serous ovarian cancer.

Enzymatic factors driving cancer-associated chromatin remodelling are of increasing interest as the role of the cancer epigenome in gene expression and DNA repair processes becomes elucidated. Monoubiquitination of histone H2B at lysine 120 (H2Bub1) is a central histone modification that functions in histone cross-talk, transcriptional elongation, DNA repair, maintaining centromeric chromatin and replication-dependent histone mRNA 3'-end processing, as well as being required for the differentiation of stem cells. The loss of global H2Bub1 is seen in a number of aggressive malignancies and has been linked to tumour progression and/or a poorer prognosis in some cancers. Here, we analyse a large cohort of high-grade serous ovarian cancers (HGSOC) and show loss of global H2Bub1 in 77% (313 of 407) of tumours. Loss of H2Bub1 was seen at all stages (I-IV) of HGSOC, indicating it is a relatively early epigenomic event in this aggressive malignancy. Manipulation of key H2Bub1 E3 ubiquitin ligases, RNF20, RNF40 and BRCA1, in ovarian cancer cell line models modulated H2Bub1 levels, indicative of the role of these RING finger ligases in monoubiquitination of H2Bub1 in vitro. However, in primary HGSOC, loss of RNF20 protein expression was identified in just 6% of tumours (26 of 424) and did not correlate with global H2Bub1 loss. Similarly, germline mutation of BRCA1 did not show a correlation with the global H2Bub1 loss. We conclude that the regulation of tumour-associated H2Bub1 levels is complex. Aberrant expression of alternative histone-associated 'writer' or 'eraser' enzymes are likely responsible for the global loss of H2Bub1 seen in HGSOC.

Linker histones in hormonal gene regulation

In the present review, we summarize advances in our knowledge on the role of the histone H1 family of proteins in breast cancer cells, focusing on their response to progestins. Histone H1 plays a dual role in gene regulation by hormones, both as a structural component of chromatin and as a dynamic modulator of transcription. It contributes to hormonal regulation of the MMTV promoter by stabilizing a homogeneous nucleosome positioning, which reduces basal transcription whereas at the same time promoting progesterone receptor binding and nucleosome remodeling. These combined effects enhance hormone dependent gene transcription, which eventually requires H1 phosphorylation and displacement. Various isoforms of histone H1 have specific functions in differentiated breast cancer cells and compact nucleosomal arrays to different extents in vitro. Genome-wide studies show that histone H1 has a key role in chromatin dynamics of hormone regulated genes. A complex sequence of enzymatic events, including phosphorylation by CDK2, PARylation by PARP1 and the ATP-dependent activity of NURF, are required for H1 displacement and gene de-repression, as a prerequisite for further nucleosome remodeling. Similarly, during hormone-dependent gene repression a dedicated enzymatic mechanism controls H1 deposition at promoters by a complex containing HP1γ, LSD1 and BRG1, the ATPase of the BAF complex. Thus, a broader vision of the histone code should include histone H1, as the linker histone variants actively participate in the regulation of the chromatin structure. How modifications of the core histones tails affect H1 modifications and vice versa is one of the many questions that remains to be addressed to provide a more comprehensive view of the histone cross-talk mechanisms. This article is part of a Special Issue entitled: Histone H1, edited by Dr. Albert Jordan.

Histone Cross-talk Connects Protein Phosphatase 1α (PP1α) and Histone Deacetylase (HDAC) Pathways to Regulate the Functional Transition of Bromodomain-containing 4 (BRD4) for Inducible Gene Expression

Transcription elongation has been recognized as a rate-limiting step for the expression of signal-inducible genes. Through recruitment of positive transcription elongation factor P-TEFb, the bromodomain-containing protein BRD4 plays critical roles in regulating the transcription elongation of a vast array of inducible genes that are important for multiple cellular processes. The diverse biological roles of BRD4 have been proposed to rely on its functional transition between chromatin targeting and transcription regulation. The signaling pathways and the molecular mechanism for regulating this transition process, however, are largely unknown. Here, we report a novel role of phosphorylated Ser(10) of histone H3 (H3S10ph) in governing the functional transition of BRD4. We identified that the acetylated lysines 5 and 8 of nucleosomal histone H4 (H4K5ac/K8ac) is the BRD4 binding site, and the protein phosphatase PP1α and class I histone deacetylase (HDAC1/2/3) signaling pathways are essential for the stress-induced BRD4 release from chromatin. In the unstressed state, phosphorylated H3S10 prevents the deacetylation of nucleosomal H4K5ac/K8ac by HDAC1/2/3, thereby locking up the majority of BRD4 onto chromatin. Upon stress, PP1α-mediated dephosphorylation of H3S10ph allows the deacetylation of nucleosomal H4K5ac/K8ac by HDAC1/2/3, thereby leading to the release of chromatin-bound BRD4 for subsequent recruitment of P-TEFb to enhance the expression of inducible genes. Therefore, our study revealed a novel mechanism that the histone cross-talk between H3S10ph and H4K5ac/K8ac connects PP1α and HDACs to govern the functional transition of BRD4. Combined with previous studies on the regulation of P-TEFb activation, the intricate signaling network for the tight control of transcription elongation is established.

Histone H2B monoubiquitination: roles to play in human malignancy.

Ubiquitination has traditionally been viewed in the context of polyubiquitination that is essential for marking proteins for degradation via the proteasome. Recent discoveries have shed light on key cellular roles for monoubiquitination, including as a post-translational modification (PTM) of histones such as histone H2B. Monoubiquitination plays a significant role as one of the largest histone PTMs, alongside smaller, better-studied modifications such as methylation, acetylation and phosphorylation. Monoubiquitination of histone H2B at lysine 120 (H2Bub1) has been shown to have key roles in transcription, the DNA damage response and stem cell differentiation. The H2Bub1 enzymatic cascade involves E3 RING finger ubiquitin ligases, with the main E3 generally accepted to be the RNF20-RNF40 complex, and deubiquitinases including ubiquitin-specific protease 7 (USP7), USP22 and USP44. H2Bub1 has been shown to physically disrupt chromatin strands, fostering a more open chromatin structure accessible to transcription factors and DNA repair proteins. It also acts as a recruiting signal, actively attracting proteins with roles in transcription and DNA damage. H2Bub1 also appears to play central roles in histone cross-talk, influencing methylation events on histone H3, including H3K4 and H3K79. Most significantly, global levels of H2Bub1 are low to absent in advanced cancers including breast, colorectal, lung and parathyroid, marking H2Bub1 and the enzymes that regulate it as key molecules of interest as possible new therapeutic targets for the treatment of cancer. This review offers an overview of current knowledge regarding H2Bub1 and highlights links between dysregulation of H2Bub1-associated enzymes, stem cells and malignancy.

Context dependency of Set1/COMPASS-mediated histone H3 Lys4 trimethylation

The stimulation of trimethylation of histone H3 Lys4 (H3K4) by H2B monoubiquitination (H2Bub) has been widely studied, with multiple mechanisms having been proposed for this form of histone cross-talk. Cps35/Swd2 within COMPASS (complex of proteins associated with Set1) is considered to bridge these different processes. However, a truncated form of Set1 (762-Set1) is reported to function in H3K4 trimethylation (H3K4me3) without interacting with Cps35/Swd2, and such cross-talk is attributed to the n-SET domain of Set1 and its interaction with the Cps40/Spp1 subunit of COMPASS. Here, we used biochemical, structural, in vivo, and chromatin immunoprecipitation (ChIP) sequencing (ChIP-seq) approaches to demonstrate that Cps40/Spp1 and the n-SET domain of Set1 are required for the stability of Set1 and not the cross-talk. Furthermore, the apparent wild-type levels of H3K4me3 in the 762-Set1 strain are due to the rogue methylase activity of this mutant, resulting in the mislocalization of H3K4me3 from the promoter-proximal regions to the gene bodies and intergenic regions. We also performed detailed screens and identified yeast strains lacking H2Bub but containing intact H2Bub enzymes that have normal levels of H3K4me3, suggesting that monoubiquitination may not directly stimulate COMPASS but rather works in the context of the PAF and Rad6/Bre1 complexes. Our study demonstrates that the monoubiquitination machinery and Cps35/Swd2 function to focus COMPASS's H3K4me3 activity at promoter-proximal regions in a context-dependent manner.

A nucleosomal approach to inferring causal relationships of histone modifications

MotivationHistone proteins are subject to various posttranslational modifications (PTMs). Elucidating their functional relationships is crucial toward understanding many biological processes. Bayesian network (BN)-based approaches have shown the advantage of revealing causal relationships, rather than simple cooccurrences, of PTMs. Previous works employing BNs to infer causal relationships of PTMs require that all confounders should be included. This assumption, however, is unavoidably violated given the fact that several modifications are often regulated by a common but unobserved factor. An existing non-parametric method can be applied to tackle the problem but the complexity and inflexibility make it impractical.ResultsWe propose a novel BN-based method to infer causal relationships of histone modifications. First, from the evidence that nucleosome organization in vivo significantly affects the activities of PTM regulators working on chromatin substrate, hidden confounders of PTMs are selectively introduced by an information-theoretic criterion. Causal relationships are then inferred from a network model of both PTMs and the derived confounders. Application on human epigenomic data shows the advantage of the proposed method, in terms of computational performance and support from literature. Requiring less strict data assumptions also makes it more practical. Interestingly, analysis of the most significant relationships suggests that the proposed method can recover biologically relevant causal effects between histone modifications, which should be important for future investigation of histone crosstalk.

Abstract A29: Loss of histone H2B monoubiquitination in ovarian cancer – new therapeutic targeting opportunities based on chromatin relaxation

Abstract Post-translational modifications of histones are involved in diverse functions, including gene transcription and the DNA damage response. Targeting elements of the DNA damage response pathway is of increasing interest for the treatment of epithelial ovarian cancer (EOC). Monoubiquitination of histone H2B at lysine 120 (H2Bub1) is present at the coding region of highly expressed genes such as the gene encoding p21, CDKN1A, under conditions of DNA damage. Addition of this histone mark relies on the E3 ubiquitin ligase ring finger protein complex RNF20-RNF40 that we, and others, have previously shown interacts with members of the PAF1 complex necessary for RNA polymerase II-based transcriptional elongation. At 8.5 kDa, ubiquitin constitutes a bulky addition likely sufficient to interfere with condensed chromatin fibres, leading to a more open fibre conformation with greater accessibility for transcription factors and DNA repair proteins. H2Bub1 has been described as a master switch for mammalian gene regulation, with roles in histone cross-talk. Furthermore, H2Bub1 has recently been linked to the cellular DNA damage response, with the protein kinase ATM phosphorylating RNF20-RNF40 to induce H2Bub1 at sites of double strand breaks, leading to a more open chromatin structure for optimal repair. Global H2Bub1 loss has been reported in breast, colon, lung and parathyroid tumors1, and in some of these tumor types has been associated with advancing stage. We have undertaken immunohistochemical analyses of global H2Bub1 and total histone H2B using tissue microarrays of EOC sourced from the Australian Ovarian Cancer Study and the Gynaecological Oncology Biobank at Westmead. Expression of total histone H2B was high and consistent across all tumors, whereas over 70% of tumors showed total loss of global H2Bub1. Contrary to reports that global H2Bub1 levels decreased with advanced stage in breast cancer, we did not observe this for ovarian cancer. This may be related to the fact that cohorts of EOC are skewed towards tumors of advanced stage, as most women present with Stage III-IV disease. The mechanism of H2Bub1 loss in ovarian cancer is currently unknown, but is likely to involve genetic or epigenetic regulation of genes encoding proteins that function as components of E3 ubiquitin ligase machinery. We have previously shown that loss-of-function mutations in the tumor suppressor CDC73, a PAF1 complex member, lead to loss of H2Bub1 in parathyroid cancer1; however, we observed consistent staining of CDC73 in ovarian tumors. We are currently analysing EOCs for expression of RNF20 given that the RNF20 promoter is hypermethylated in breast cancer and TCGA-ovarian reports RNF20 missense mutations with neutral to medium predicted impact scores. This data will be presented. Given the apparent fundamental role of H2Bub1 in determining the accessibility of chromatin for molecules such as DNA repair proteins, H2Bub1 and/or its associated ubiquitin ligase machinery, may be considered as potential therapeutic targets for the treatment of EOC. 1Hahn MA, Dickson K-A, Jackson S, Clarkson A, Gill AJ and Marsh DJ. The tumor suppressor CDC73 interacts with the ring finger proteins RNF20 and RNF40 and is required for the maintenance of histone 2B monoubiquitination Human Molecular Genetics 2012; 21(3):559-568. Citation Format: Deborah J. Marsh, Anna deFazio, Adele Clarkson, Catherine Kennedy, Gregory G. Gard, Anthony J. Gill. Loss of histone H2B monoubiquitination in ovarian cancer – new therapeutic targeting opportunities based on chromatin relaxation. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A29.

Discovery of Histone Modification Crosstalk Networks by Stable Isotope Labeling of Amino Acids in Cell Culture Mass Spectrometry (SILAC MS)

In this paper we describe an approach that combines stable isotope labeling of amino acids in cells culture, high mass accuracy liquid chromatography tandem mass spectrometry and a novel data analysis approach to accurately determine relative peptide post-translational modification levels. This paper describes the application of this approach to the discovery of novel histone modification crosstalk networks in Saccharomyces cerevisiae. Yeast histone mutants were generated to mimic the presence/absence of 44 well-known modifications on core histones H2A, H2B, H3, and H4. In each mutant strain the relative change in H3 K79 methylation and H3 K56 acetylation were determined using stable isotope labeling of amino acids in cells culture. This approach showed relative changes in H3 K79 methylation and H3 K56 acetylation that are consistent with known histone crosstalk networks. More importantly, this study revealed additional histone modification sites that affect H3 K79 methylation and H3 K56 acetylation.