Chromatin-mediated Regulation Research Articles

Epigenotype-genotype-phenotype correlations in SETD1A and SETD2 chromatin disorders.

Germline pathogenic variants in two genes encoding the lysine-specific histone methyltransferase genes SETD1A and SETD2 are associated with neurodevelopmental disorders (NDDs) characterized by developmental delay and congenital anomalies. The SETD1A and SETD2 gene products play a critical role in chromatin-mediated regulation of gene expression. Specific methylation episignatures have been detected for a range of chromatin gene-related NDDs and have impacted clinical practice by improving the interpretation of variant pathogenicity. To investigate if SETD1A and/or SETD2-related NDDs are associated with a detectable episignature, we undertook targeted genome-wide methylation profiling of > 2M CpGs using a next-generation sequencing-based assay. A comparison of methylation profiles in patients with SETD1A variants (n = 6) did not reveal evidence of a strong methylation episignature. A review of the clinical and genetic features of the SETD2 patient group revealed that, as reported previously, there were phenotypic differences between patients with truncating mutations (n = 4, Luscan-Lumish syndrome; MIM:616831) and those with missense codon 1740 variants [p.Arg1740Trp (n = 4) and p.Arg1740Gln (n = 2)]. Both SETD2 subgroups demonstrated a methylation episignature, which was characterized by hypomethylation and hypermethylation events, respectively. Within the codon 1740 subgroup, both the methylation changes and clinical phenotype were more severe in those with p.Arg1740Trp variants. We also noted that two of 10 cases with a SETD2-NDD had developed a neoplasm. These findings reveal novel epigenotype-genotype-phenotype correlations in SETD2-NDDs and predict a gain-of-function mechanism for SETD2 codon 1740 pathogenic variants.

Transitions in development - an interview with Bhavana Muralidharan.

Bhavana Muralidharan is an Assistant Professor at the Institute for Stem Cell Science and Regenerative Medicine (inStem), where her research focuses on chromatin-mediated regulation of neurodevelopment and neuropsychiatric disorders, and the development of cerebral organoids for modelling these disorders in vitro. We caught up with Bhavana over Zoom to discuss her research, the recent EMBO organoid meeting hosted at inStem and her creative performances outside of the lab.

CHARGE syndrome-associated proteins FAM172A and CHD7 influence male sex determination and differentiation through transcriptional and alternative splicing mechanisms.

To gain further insight into chromatin‐mediated regulation of mammalian sex determination, we analyzed the role of the CHARGE syndrome‐associated proteins FAM172A and CHD7. This study is based on our prior discoveries that a subset of corresponding mutant mice display complete male‐to‐female sex reversal, and that both of these proteins regulate co‐transcriptional alternative splicing in neural crest cells. Here, we report that FAM172A and CHD7 are present in the developing gonads when sex determination normally occurs in mice. The interactome of FAM172A in pre‐Sertoli cells again suggests a role at the chromatin‐spliceosome interface, like in neural crest cells. Accordingly, analysis of Fam172a‐mutant pre‐Sertoli cells revealed transcriptional and splicing dysregulation of hundreds of genes. Many of these genes are similarly affected in Chd7‐mutant pre‐Sertoli cells, including several known key regulators of sex determination and subsequent formation of testis cords. Among them, we notably identified Sry as a direct transcriptional target and WNT pathway‐associated Lef1 and Tcf7l2 as direct splicing targets. The identified molecular defects are also associated with the abnormal morphology of seminiferous tubules in mutant postnatal testes. Altogether, our results thus identify FAM172A and CHD7 as new players in the regulation of male sex determination and differentiation in mice, and further highlight the importance of chromatin‐mediated regulatory mechanisms in these processes.

LncRNA Mrhl orchestrates differentiation programs in mouse embryonic stem cells through chromatin mediated regulation.

Long non-coding RNAs (lncRNAs) have been well-established to act as regulators and mediators of development and cell fate specification programs. LncRNA Mrhl (meiotic recombination hotspot locus) has been shown to act in a negative feedback loop with WNT signaling to regulate male germ cell meiotic commitment. In our current study, we have addressed the role of Mrhl in development and differentiation using mouse embryonic stem cells (mESCs) as our model system of study. Mrhl is a nuclear-localized, chromatin-bound lncRNA with moderately stable expression in mESCs. Transcriptome analyses and loss-of-function phenotype studies revealed dysregulation of developmental processes, lineage-specific transcription factors and key networks along with aberrance in specification of early lineages during differentiation of mESCs. Genome-wide chromatin occupancy studies suggest regulation of chromatin architecture at key target loci through triplex formation. Our studies thus reveal a role for lncRNA Mrhl in regulating differentiation programs in mESCs in the context of appropriate cues through chromatin-mediated responses.

Positioning of nucleosomes containing \u03b3-H2AX precedes active DNA demethylation and transcription initiation

In addition to nucleosomes, chromatin contains non-histone chromatin-associated proteins, of which the high-mobility group proteins are the most abundant. Chromatin-mediated regulation of transcription involves DNA methylation and histone modifications. However, the order of events and the precise function of high-mobility group proteins during transcription initiation remain unclear. Here we show that high-mobility group AT-hook 2 protein (HMGA2) induces DNA nicks at the transcription start site, which are required by the histone chaperone FACT complex to incorporate nucleosomes containing the histone variant H2A.X. Further, phosphorylation of H2A.X at S139 (γ-H2AX) is required for repair-mediated DNA demethylation and transcription activation. The relevance of these findings is demonstrated within the context of TGFB1 signaling and idiopathic pulmonary fibrosis, suggesting therapies against this lethal disease. Our data support the concept that chromatin opening during transcriptional initiation involves intermediates with DNA breaks that subsequently require DNA repair mechanisms to ensure genome integrity.

Combinations of histone post-translational modifications.

Histones are essential proteins that package the eukaryotic genome into its physiological state of nucleosomes, chromatin, and chromosomes. Post-translational modifications (PTMs) of histones are crucial to both the dynamic and persistent regulation of the genome. Histone PTMs store and convey complex signals about the state of the genome. This is often achieved by multiple variable PTM sites, occupied or unoccupied, on the same histone molecule or nucleosome functioning in concert. These mechanisms are supported by the structures of 'readers' that transduce the signal from the presence or absence of PTMs in specific cellular contexts. We provide background on PTMs and their complexes, review the known combinatorial function of PTMs, and assess the value and limitations of common approaches to measure combinatorial PTMs. This review serves as both a reference and a path forward to investigate combinatorial PTM functions, discover new synergies, and gather additional evidence supporting that combinations of histone PTMs are the central currency of chromatin-mediated regulation of the genome.

MNase Profiling of Promoter Chromatin in Salmonella typhimurium-Stimulated GM12878 Cells Reveals Dynamic and Response-Specific Nucleosome Architecture.

The nucleosome is the primary unit of chromatin structure and commonly imputed as a regulator of nuclear events, although the exact mechanisms remain unclear. Recent studies have shown that certain nucleosomes can have different sensitivities to micrococcal nuclease (MNase) digestion, resulting in the release of populations of nucleosomes dependent on the concentration of MNase. Mapping MNase sensitivity of nucleosomes at transcription start sites genome-wide reveals an important functional nucleosome organization that correlates with gene expression levels and transcription factor binding. In order to understand nucleosome distribution and sensitivity dynamics during a robust genome response, we mapped nucleosome position and sensitivity using multiple concentrations of MNase. We used the innate immune response as a model system to understand chromatin-mediated regulation. Herein we demonstrate that stimulation of a human lymphoblastoid cell line (GM12878) with heat-killed Salmonella typhimurium (HKST) results in changes in nucleosome sensitivity to MNase. We show that the HKST response alters the sensitivity of -1 nucleosomes at highly expressed promoters. Finally, we correlate the increased sensitivity with response-specific transcription factor binding. These results indicate that nucleosome sensitivity dynamics reflect the cellular response to HKST and pave the way for further studies that will deepen our understanding of the specificity of genome response.

Chromatin-Mediated Regulation of Genome Plasticity in Human Fungal Pathogens.

Human fungal pathogens, such as Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans, are a public health problem, causing millions of infections and killing almost half a million people annually. The ability of these pathogens to colonise almost every organ in the human body and cause life-threating infections relies on their capacity to adapt and thrive in diverse hostile host-niche environments. Stress-induced genome instability is a key adaptive strategy used by human fungal pathogens as it increases genetic diversity, thereby allowing selection of genotype(s) better adapted to a new environment. Heterochromatin represses gene expression and deleterious recombination and could play a key role in modulating genome stability in response to environmental changes. However, very little is known about heterochromatin structure and function in human fungal pathogens. In this review, I use our knowledge of heterochromatin structure and function in fungal model systems as a road map to review the role of heterochromatin in regulating genome plasticity in the most common human fungal pathogens: Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans.

The role of histone lysine demethylases in cancer cells' resistance to tyrosine kinase inhibitors.

Current cancer therapies are often associated with treatment failure and reduced patients’ survival due to drug resistance. There are various mechanisms involved in the acquisition of cancer drug resistance, including the selection of advantageous mutations, overexpression of transporter proteins and epigenetic alterations. In this context, epigenetic alterations refer to chromatin-mediated regulation of gene expression that results in heritable changes in the cellular phenotype. There is an ever-growing body of evidence suggesting that epigenetic mechanisms play an important role in bringing about drug resistance in cancer cells. While the relationship between chemotherapy and epigenetics has been widely discussed, emerging evidence indicates that specific epigenetic effectors are also crucial for the development of resistance to tyrosine kinase inhibitors (TKIs). One particular gene that encodes the histone lysine demethylase KDM5A is overexpressed in several cancers. In breast cancer tissues, cells with KDM5A gene amplification were found to be more resistant to erlotinib, an inhibitor of the tyrosine kinase epidermal growth factor receptor (EGFR), when compared to cells without the same amplification. KDM5A was also shown to mediate resistance to a second EGFR inhibitor called gefitinib, in EGFR-mutant lung cancer cell lines. This evidence indicates that KDM5A could activate alternative survival pathways involved in overcoming EGFR inhibition. In line with these results, another histone demethylase (i.e., KDM1A) promotes liver cancer cells’ resistance to the TKI sorafenib. Current evidence provides a suitable rationale to consider the use of specific KDMs inhibitors to sensitize cells to tyrosine kinase targeted therapies and thus, presents an opportunity to prevent the further development of drug resistance. This review discusses the involvement of histone lysine demethylases in the development of resistance to TKI and highlights the importance to develop new cancer treatment regimens to counteract this phenomenon.

Transcriptional Control of Brown and Beige Fat Development and Function.

Adipose tissue, once viewed as an inert organ of energy storage, is now appreciated to be a central node for the dynamic regulation of systemic metabolism. There are three general types of adipose tissue: white, brown, and brown-in-white or “beige” fat. All three types of adipose tissue communicate extensively with other organs in the body including skin, liver, pancreas, muscle, and brain to maintain energy homeostasis. When energy intake chronically exceeds energy expenditure, obesity and its comorbidities can develop. Thus, understanding the molecular mechanisms by which different types of adipose tissues develop and function could uncover new therapies for combating disorders of energy imbalance. In this article, we highlight recent findings on the transcriptional and chromatin-mediated regulation of brown and beige adipose tissue activity.

PARP1 restricts Epstein Barr Virus lytic reactivation by binding the BZLF1 promoter

The Epstein Barr virus (EBV) genome persists in infected host cells as a chromatinized episome and is subject to chromatin-mediated regulation. Binding of the host insulator protein CTCF to the EBV genome has an established role in maintaining viral latency type, and in other herpesviruses, loss of CTCF binding at specific regions correlates with viral reactivation. Here, we demonstrate that binding of PARP1, an important cofactor of CTCF, at the BZLF1 lytic switch promoter restricts EBV reactivation. Knockdown of PARP1 in the Akata-EBV cell line significantly increases viral copy number and lytic protein expression. Interestingly, CTCF knockdown has no effect on viral reactivation, and CTCF binding across the EBV genome is largely unchanged following reactivation. Moreover, EBV reactivation attenuates PARP activity, and Zta expression alone is sufficient to decrease PARP activity. Here we demonstrate a restrictive function of PARP1 in EBV lytic reactivation.

The SETD8/PR-Set7 Methyltransferase Functions as a Barrier to Prevent Senescence-Associated Metabolic Remodeling

Cellular senescence is an irreversible growth arrest that contributes to development, tumor suppression, and age-related conditions. Senescent cells show active metabolism compared with proliferating cells, but the underlying mechanisms remain unclear. Here we show that the SETD8/PR-Set7 methyltransferase, which catalyzes mono-methylation of histone H4 atlysine 20 (H4K20me1), suppresses nucleolar and mitochondrial activities to prevent cellular senescence. SETD8 protein was selectively downregulated in both oncogene-induced and replicative senescence. Inhibition of SETD8 alone was sufficientto trigger senescence. Under these states, theexpression of genes encoding ribosomal proteins(RPs) and ribosomal RNAs as well as the cyclin-dependent kinase (CDK) inhibitor p16INK4A was increased, with a corresponding reduction of H4K20me1 at each locus. As a result, the loss of SETD8 concurrently stimulated nucleolar function and retinoblastoma protein-mediated mitochondrial metabolism. In conclusion, our data demonstrate that SETD8 acts as a barrier to prevent cellular senescence through chromatin-mediated regulation of senescence-associated metabolic remodeling.

Life span extension by targeting a link between metabolism and histone acetylation in Drosophila.

Old age is associated with a progressive decline of mitochondrial function and changes in nuclear chromatin. However, little is known about how metabolic activity and epigenetic modifications change as organisms reach their midlife. Here, we assessed how cellular metabolism and protein acetylation change during early aging in Drosophila melanogaster. Contrary to common assumptions, we find that flies increase oxygen consumption and become less sensitive to histone deacetylase inhibitors as they reach midlife. Further, midlife flies show changes in the metabolome, elevated acetyl‐CoA levels, alterations in protein—notably histone—acetylation, as well as associated transcriptome changes. Based on these observations, we decreased the activity of the acetyl‐CoA‐synthesizing enzyme ATP citrate lyase (ATPCL) or the levels of the histone H4 K12‐specific acetyltransferase Chameau. We find that these targeted interventions both alleviate the observed aging‐associated changes and promote longevity. Our findings reveal a pathway that couples changes of intermediate metabolism during aging with the chromatin‐mediated regulation of transcription and changes in the activity of associated enzymes that modulate organismal life span.

Analysis of histone modifications at human ribosomal DNA in liver cancer cell.

Human liver cancer is the cancer commonly seen clinically. The transcription of ribosomal DNA (rDNA) is a critical step for cells, and epigenetic marks such as post-translational histone modifications have been involved in the regulation of rDNA transcription. But less is known about the pathogenesis of the liver cancers concerning the rDNA transcription regulation. Here we aligned the ChIP-seq data of histone modification markers and CTCF to the human genome assembly which contains a single rDNA repeat in human liver cancer cell and validated their distribution with ChIP-QPCR. Human liver cancer cell possesses a higher enrichment of H3K4me1 and H3K27me3 at ~28 kb within the intergenic spacer (IGS) of rDNA and a higher enrichment of H3K4me3 and H3K27ac upstream of TSS. Furtherly, we studied whether UBF could affect histone modification markers and CTCF at rDNA in human liver cancer cell. UBF depletion leads to a decrease of gene activation mark H3K4me3 across the rDNA promoter. And other histone modification marks and CTCF were not altered after UBF depletion. Taken together, our data showed a high resolution map of histone modification marks at rDNA in human liver cancer cell and provide novel evidence to decipher chromatin-mediated regulation of rDNA in liver cancer.

An integrative analysis of post-translational histone modifications in the marine diatom Phaeodactylum tricornutum.

BackgroundNucleosomes are the building blocks of chromatin where gene regulation takes place. Chromatin landscapes have been profiled for several species, providing insights into the fundamental mechanisms of chromatin-mediated transcriptional regulation of gene expression. However, knowledge is missing for several major and deep-branching eukaryotic groups, such as the Stramenopiles, which include the diatoms. Diatoms are highly diverse and ubiquitous species of phytoplankton that play a key role in global biogeochemical cycles. Dissecting chromatin-mediated regulation of genes in diatoms will help understand the ecological success of these organisms in contemporary oceans.ResultsHere, we use high resolution mass spectrometry to identify a full repertoire of post-translational modifications on histones of the marine diatom Phaeodactylum tricornutum, including eight novel modifications. We map five histone marks coupled with expression data and show that P. tricornutum displays both unique and broadly conserved chromatin features, reflecting the chimeric nature of its genome. Combinatorial analysis of histone marks and DNA methylation demonstrates the presence of an epigenetic code defining activating or repressive chromatin states. We further profile three specific histone marks under conditions of nitrate depletion and show that the histone code is dynamic and targets specific sets of genes.ConclusionsThis study is the first genome-wide characterization of the histone code from a stramenopile and a marine phytoplankton. The work represents an important initial step for understanding the evolutionary history of chromatin and how epigenetic modifications affect gene expression in response to environmental cues in marine environments.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-015-0671-8) contains supplementary material, which is available to authorized users.

The prognostic significance of bromodomain PHD-finger transcription factor in colorectal carcinoma and association with vimentin and E-cadherin.

Bromodomain PHD-finger transcription factor (BPTF) is a chromatin-mediated regulation of transcription factor, playing an important role in embryogenesis and differentiation. Epithelial-mesenchymal transition (EMT) has a pivotal role in colorectal cancer (CRC) progression, sharing the similar characteristic with BPTF. Therefore, the aim of this study was to examine the expression and clinical value of BPTF and the correlation with EMT markers in patients with CRC. Real-time PCR and Western blot were performed to evaluate the mRNA and protein expression levels of BPTF in 20 pairs of fresh-frozen CRC and non-tumor adjacent tissues (NATs). The expressions of BPTF, vimentin and E-cadherin were examined by immunohistochemical staining in 105 cases of paraffin-embedded primary CRC specimens. In addition, the clinicopathological significance and the prognostic value of BPTF, vimentin and E-cadherin expression were further determined. Then, the correlation of BPTF with vimentin and E-cadherin was also explored. BPTF mRNA and protein expression were significantly overexpressed in CRC tissues when compared with paired NATs (P < 0.01). The expression levels of BPTF and vimentin in CRC paraffin-embedded specimens were significantly higher than the expression in NATs (P < 0.01), while the expressions of E-cadherin in tumors were obviously lower than in NATs (P < 0.01). Tumors with high expression of BPTF and vimentin, as well as low expression of E-cadherin, were significantly correlated with various adverse clinicopathological factors (P < 0.05). The CRC patients with a high BPTF or vimentin expression had shorter overall survival than those with lower expression (P < 0.05). Furthermore, univariate analysis and multivariate analysis showed that high BPTF expression was an independent prognostic factor for patients with CRC. The last and more interesting Spearman rank correlation analysis and microscopic observation found that the expression of BPTF obviously correlated with the expression of EMT markers vimentin and E-cadherin. Our results strongly suggested that the high BPTF expression was significantly correlated with tumor progression and may be a potent prognostic marker of CRC. Moreover, BPTF expression was significantly associated with EMT markers vimentin and E-cadherin.

Ubiquitous human ‘master’ origins of replication are encoded in the DNA sequence via a local enrichment in nucleosome excluding energy barriers

As the elementary building block of eukaryotic chromatin, the nucleosome is at the heart of the compromise between the necessity of compacting DNA in the cell nucleus and the required accessibility to regulatory proteins. The recent availability of genome-wide experimental maps of nucleosome positions for many different organisms and cell types has provided an unprecedented opportunity to elucidate to what extent the DNA sequence conditions the primary structure of chromatin and in turn participates in the chromatin-mediated regulation of nuclear functions, such as gene expression and DNA replication. In this study, we use in vivo and in vitro genome-wide nucleosome occupancy data together with the set of nucleosome-free regions (NFRs) predicted by a physical model of nucleosome formation based on sequence-dependent bending properties of the DNA double-helix, to investigate the role of intrinsic nucleosome occupancy in the regulation of the replication spatio-temporal programme in human. We focus our analysis on the so-called replication U/N-domains that were shown to cover about half of the human genome in the germline (skew-N domains) as well as in embryonic stem cells, somatic and HeLa cells (mean replication timing U-domains). The ‘master’ origins of replication (MaOris) that border these megabase-sized U/N-domains were found to be specified by a few hundred kb wide regions that are hyper-sensitive to DNase I cleavage, hypomethylated, and enriched in epigenetic marks involved in transcription regulation, the hallmarks of localized open chromatin structures. Here we show that replication U/N-domain borders that are conserved in all considered cell lines have an environment highly enriched in nucleosome-excluding-energy barriers, suggesting that these ubiquitous MaOris have been selected during evolution. In contrast, MaOris that are cell-type-specific are mainly regulated epigenetically and are no longer favoured by a local abundance of intrinsic NFRs encoded in the DNA sequence. At the smaller few hundred bp scale of gene promoters, CpG-rich promoters of housekeeping genes found nearby ubiquitous MaOris as well as CpG-poor promoters of tissue-specific genes found nearby cell-type-specific MaOris, both correspond to in vivo NFRs that are not coded as nucleosome-excluding-energy barriers. Whereas the former promoters are likely to correspond to high occupancy transcription factor binding regions, the latter are an illustration that gene regulation in human is typically cell-type-specific.

Big Data Approach to Identify Molecular Basis for Drug Sensitivity Phenotypes in Acute Myeloid Leukemia

Background: The use of high-throughput ¡®omic¡¯ data holds great promise to better match patients to drugs. A necessary step to realize this goal is to identify molecular markers that are predictive of clinical phenotypes such as sensitivity to drugs. Although large expression datasets from AML patients with clinical, genetic, and molecular annotation exist, due to the high-dimensionality (i.e., number of genes >> number of patients), it is an open challenge to identify robust molecular markers that are consistently associated with a phenotype across studies.Method: To resolve this challenge, we developed a novel computational method, named SPARROW, to reduce the dimensionality of expression data by identifying genes that represent important molecular events in publicly available AML expression data. In particular, it aims to identify hub regulators whose expression levels are predictive of many downstream genes¡¯ expression levels (Fig 1), by using a novel statistical technique to discriminate direct associations from complex correlations. We measured expression and in vitro drug sensitivity in 30 primary AML samples. Out of 160 drugs considered, 55 drugs exhibited activity against at least half the patient samples. We processed the drug sensitivity data by curve fitting and then extracting summary statistics, such as the activity area (AA), area under the curve (AUC), IC50, EC50, and Amax.Results: The top N hubs selected by SPARROW (with 2 methods for choosing sparsity level) are highly enriched for genes important in AML (Fig 2). The significance of enrichment was much better than 5 other widely used hub detection methods. SPARROW identifies hubs that are direct targets of perturbation of the underlying AML disease process. Hub expression is therefore especially informative of the molecular state of the disease and correspondingly the sensitivity or resistance of patient cells to drug treatment. We took the 400 top SPARROW hubs and tested for association with the summary statistics of 55 drugs. Considering the top 400 hubs increases statistical power to detect significant associations than when all 18,000 are considered (Fig 3).For each SPARROW hub that was significantly associated (FDR=0.05) with AUC for multiple drugs, we tested for enrichment of the associated drugs for a shared functional class. We present the results for four classes: 1) High expression of FLT3 was associated with increased sensitivity to the FLT3 inhibitors sunitinib, AP24534, and tandutinib (p-value: 0.01). 2) TRF2 and PRMT6 are enriched for association (p -values: 0.007, 0.034) with sensitivity to the nucleoside analogues (azacitidine, cladribine, clofarabine, fludarabine) or alkylators (melphalan, mitomycin C). The protection of human telomeres depends primarily on TRF2. PRMT6 dimethylates histone H3, which inhibits H3K4 trimethylation by MLL. 3) Notably, SMARCA4 and TRF2 are enriched for increased sensitivity (p-values: 0.015, 0.005) to daunorubicin, mitoxantrone, etoposide, and topotecan, all topoisomerase inhibitors. SMARCA4 is essential for proliferation of both normal hematopoietic leukemia stem cells. 4) For HDAC inhibitors belinostat, MS-275, panobinostat, andvorinostat, 2 hubs were significantly associated with increased sensitivity: RNF24 and BAZ2B (p-values: 6.5x10-5, 5.7x10-4). Suggestively, BAZ2B has a bromodomain, commonly involved in chromatin mediated regulation. Thus, it is intriguing that high expression level of these genes is associated with chemotherapy drug sensitivity, bringing new insight into the mechanisms that govern individual patient response to chemotherapy.Conclusion: These results demonstrate that SPARROW can reduce the dimensionality of expression data into a highly informative set of genes, which can facilitate the identification of robust molecular markers for chemotherapy drugs. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Natural variation of histone modification and its impact on gene expression in the rat genome.

Histone modifications are epigenetic marks that play fundamental roles in many biological processes including the control of chromatin-mediated regulation of gene expression. Little is known about interindividual variability of histone modification levels across the genome and to what extent they are influenced by genetic variation. We annotated the rat genome with histone modification maps, identified differences in histone trimethyl-lysine levels among strains, and described their underlying genetic basis at the genome-wide scale using ChIP-seq in heart and liver tissues in a panel of rat recombinant inbred and their progenitor strains. We identified extensive variation of histone methylation levels among individuals and mapped hundreds of underlying cis- and trans-acting loci throughout the genome that regulate histone methylation levels in an allele-specific manner. Interestingly, most histone methylation level variation was trans-linked and the most prominent QTL identified influenced H3K4me3 levels at 899 putative promoters throughout the genome in the heart. Cis- acting variation was enriched in binding sites of distinct transcription factors in heart and liver. The integrated analysis of DNA variation together with histone methylation and gene expression levels showed that histoneQTLs are an important predictor of gene expression and that a joint analysis significantly enhanced the prediction of gene expression traits (eQTLs). Our data suggest that genetic variation has a widespread impact on histone trimethylation marks that may help to uncover novel genotype–phenotype relationships.

Ribosomal Protein S6, a Target of Rapamycin, Is Involved in the Regulation of rRNA Genes by Possible Epigenetic Changes in Arabidopsis

The target of rapamycin (TOR) kinase pathway regulates various biological processes, including translation, synthesis of ribosomal proteins, and transcription of rRNA. The ribosomal protein S6 (RPS6) is one of the well known downstream components of the TOR pathway. Ribosomal proteins have been known to have diverse functions in regulating cellular metabolism as well as protein synthesis. So far, however, little is known about other possible role(s) of RPS6 in plants, besides being a component of the 40 S ribosomal subunit and acting as a target of TOR. Here, we report that RPS6 may have a novel function via interaction with histone deacetylase 2B (AtHD2B) that belongs to the plant-specific histone deacetylase HD2 family. RPS6 and AtHD2B were localized to the nucleolus. Co-expression of RPS6 and AtHD2B caused a change in the location of both RPS6 and AtHD2B to one or several nucleolar spots. ChIP analysis suggests that RPS6 directly interacts with the rRNA gene promoter. Protoplasts overexpressing both AtHD2B and RPS6 exhibited down-regulation of pre-18 S rRNA synthesis with a concomitant decrease in transcription of some of the ribosomal proteins, suggesting their direct role in ribosome biogenesis and plant development. This is consistent with the mutation in rps6b that results in reduction in 18 S rRNA transcription and decreased root growth. We propose that the interaction between RPS6 and AtHD2B brings about a change in the chromatin structure of rDNA and thus plays an important role in linking TOR signaling to rDNA transcription and ribosome biogenesis in plants.