The Epigenetic Regulation of Cytokine Inducible Mammalian Transcription by the 26S Proteasome

It is evident that components of the 26S proteasome function beyond protein degradation in the regulation of transcription. Studies in yeast implicate the 26S proteasome, specifically the 19S cap, in the epigenetic regulation of transcription. Saccharomyces cerevisiae 19S ATPases remodel chromatin by facilitating histone acetylation and methylation. However, it is unclear if the 19S ATPases play similar roles in mammalian cells. We previously found that the 19S ATPase Sug1 positively regulates transcription of the critical inflammatory gene MHC-II and that the MHC-II promoter fails to efficiently bind transcription factors upon Sug1 knockdown. MHC-II transcription is regulated by the critical coactivator CIITA. We now find that Sug1 is crucial for regulating histone H3 acetylation at the cytokine inducible MHC-II and CIITA promoters. Histone H3 acetylation is dramatically decreased upon Sug1 knockdown with a preferential loss occurring at lysine 18. Research in yeast indicates that the ortholog of Sug1, Rpt6, acts as a mediator between the activating modifications of histone H2B ubiquitination and H3 methylation. Therefore, we characterized the role the 19S proteasome plays in regulating additional activating modifications. As with acetylation, Sug1 is necessary for proper histone H3K4 and H3R17 methylation at cytokine inducible promoters. In the absence of Sug1, histone H3K4me3 and H3R17me2 are substantially inhibited. Our observation that the loss of Sug1 has no significant effect on H3K36me3 implies that Sug1’s regulation of histone modifications is localized to promoter regions as H3K4me3 but not H3K36me3 is clustered around gene promoters. Here we show that multiple H3K4 histone methyltransferase subunits bind constitutively to the inducible MHC-II and CIITA promoters and that over-expressing one subunit significantly enhances promoter activity. Furthermore, we identified a critical subunit of the H3K4 methyltransferase complex that binds multiple histone modifying enzymes, but fails to bind the CIITA promoter in the absence of Sug1, implicating Sug1 in recruiting multi-enzyme complexes responsible for initiating transcription. Finally, Sug1 knockdown maintains gene silencing as elevated levels of H3K27 trimethylation are observed upon Sug1 knockdown. Together these studies strongly implicate the 19S proteasome in mediating the initial reorganization events to relax the repressive chromatin structure surrounding inducible genes

cis-regulatory circuits regulating NEK6 kinase overexpression in transformed B cells Are super-enhancer independent

Alterations in distal regulatory elements that control gene expression underlie many diseases, including cancer. Epigenomic analyses of normal and diseased cells have produced correlative predictions for connections between dysregulated enhancers and target genes involved in pathogenesis. However, with few exceptions, these predicted cis-regulatory circuits remain untested. Here, we dissect cis-regulatory circuits that lead to overexpression of NEK6, a mitosis-associated kinase, in human B cell lymphoma. We find that only a minor subset of predicted enhancers is required for NEK6 expression. Indeed, an annotated super-enhancer is dispensable for NEK6 overexpression and for maintaining the architecture of a B cell-specific regulatory hub. A CTCF cluster serves as a chromatin and architectural boundary to block communication of the NEK6 regulatory hub with neighboring genes. Our findings emphasize that validation of predicted cis-regulatory circuits and super-enhancers is needed to prioritize transcriptional control elements as therapeutic targets

Roles for common MLL/COMPASS subunits and the 19S proteasome in regulating CIITA pIV and MHC class II gene expression and promoter methylation

<p>Abstract</p> <p>Background</p> <p>Studies indicate that the 19S proteasome contributes to chromatin reorganization, independent of the role the proteasome plays in protein degradation. We have previously shown that components of the 19S proteasome are crucial for regulating inducible histone activation events in mammalian cells. The 19S ATPase Sug1 binds to histone-remodeling enzymes, and in the absence of Sug1, a subset of activating epigenetic modifications including histone H3 acetylation, H3 lysine 4 trimethylation and H3 arginine 17 dimethylation are inhibited at cytokine-inducible major histocompatibilty complex (MHC)-II and class II transactivator (CIITA) promoters, implicating Sug1 in events required to initiate mammalian transcription.</p> <p>Results</p> <p>Our previous studies indicate that H3 lysine 4 trimethylation at cytokine-inducible MHC-II and CIITA promoters is dependent on proteolytic-independent functions of 19S ATPases. In this report, we show that multiple common subunits of the mixed lineage leukemia (MLL)/complex of proteins associated with Set I (COMPASS) complexes bind to the inducible MHC-II and CIITA promoters; that overexpressing a single common MLL/COMPASS subunit significantly enhances promoter activity and MHC-II <it>HLA-DRA </it>expression; and that these common subunits are important for H3 lysine 4 trimethylation at MHC-II and CIITA promoters. In addition, we show that H3 lysine 27 trimethylation, which is inversely correlated with H3 lysine 4 trimethylation, is significantly elevated in the presence of diminished 19S ATPase Sug1.</p> <p>Conclusion</p> <p>Taken together, these experiments suggest that the 19S proteasome plays a crucial role in the initial reorganization of events enabling the relaxation of the repressive chromatin structure surrounding inducible promoters.</p

Enhancer Sequence Variants and Transcription-Factor Deregulation Synergize to Construct Pathogenic Regulatory Circuits in B-Cell Lymphoma

SummaryMost B-cell lymphomas arise in the germinal center (GC), where humoral immune responses evolve from potentially oncogenic cycles of mutation, proliferation, and clonal selection. Although lymphoma gene expression diverges significantly from GC B cells, underlying mechanisms that alter the activities of corresponding regulatory elements (REs) remain elusive. Here we define the complete pathogenic circuitry of human follicular lymphoma (FL), which activates or decommissions REs from normal GC B cells and commandeers enhancers from other lineages. Moreover, independent sets of transcription factors, whose expression was deregulated in FL, targeted commandeered versus decommissioned REs. Our approach revealed two distinct subtypes of low-grade FL, whose pathogenic circuitries resembled GC B or activated B cells. FL-altered enhancers also were enriched for sequence variants, including somatic mutations, which disrupt transcription-factor binding and expression of circuit-linked genes. Thus, the pathogenic regulatory circuitry of FL reveals distinct genetic and epigenetic etiologies for GC B-cell transformation

Loss of synergistic transcriptional feedback loops drives diverse B-cell cancers

BACKGROUND: The most common B-cell cancers, chronic lymphocytic leukemia/lymphoma (CLL), follicular and diffuse large B-cell (FL, DLBCL) lymphomas, have distinct clinical courses, yet overlapping cell-of-origin . Dynamic changes to the epigenome are essential regulators of B-cell differentiation. Therefore, we reasoned that these distinct cancers may be driven by shared mechanisms of disruption in transcriptional circuitry. METHODS: We compared purified malignant B-cells from 52 patients with normal B-cell subsets (germinal center centrocytes and centroblasts, naïve and memory B-cells) from 36 donor tonsils using \u3e325 high-resolution molecular profiling assays for histone modifications, open chromatin (ChIP-, FAIRE-seq), transcriptome (RNA-seq), transcription factor (TF) binding, and genome copy number (microarrays). FINDINGS: From the resulting data, we identified gains in active chromatin in enhancers/super-enhancers that likely promote unchecked B-cell receptor signaling, including one we validated near the immunoglobulin superfamily receptors FCMR and PIGR. More striking and pervasive was the profound loss of key B-cell identity TFs, tumor suppressors and their super-enhancers, including EBF1, OCT2(POU2F2), and RUNX3. Using a novel approach to identify transcriptional feedback, we showed that these core transcriptional circuitries are self-regulating. Their selective gain and loss form a complex, iterative, and interactive process that likely curbs B-cell maturation and spurs proliferation. INTERPRETATION: Our study is the first to map the transcriptional circuitry of the most common blood cancers. We demonstrate that a critical subset of B-cell TFs and their cognate enhancers form self-regulatory transcriptional feedback loops whose disruption is a shared mechanism underlying these diverse subtypes of B-cell lymphoma. FUNDING: National Institute of Health, Siteman Cancer Center, Barnes-Jewish Hospital Foundation, Doris Duke Foundation

Purine metabolism controls innate lymphoid cell function and protects against intestinal injury

Inflammatory bowel disease (IBD) is a condition of chronic inflammatory intestinal disorder with increasing prevalence but limited effective therapies. The purine metabolic pathway is involved in various inflammatory processes including IBD. However, the mechanisms through which purine metabolism modulates IBD remain to be established. Here, we found that mucosal expression of genes involved in the purine metabolic pathway is altered in patients with active ulcerative colitis (UC), which is associated with elevated gene expression signatures of the group 3 innate lymphoid cell (ILC3)–interleukin (IL)‐22 pathway. In mice, blockade of ectonucleotidases (NTPDases), critical enzymes for purine metabolism by hydrolysis of extracellular adenosine 5′‐triphosphate (eATP) into adenosine, exacerbates dextran‐sulfate sodium‐induced intestinal injury. This exacerbation of colitis is associated with reduction of colonic IL‐22‐producing ILC3s, which afford essential protection against intestinal inflammation, and is rescued by exogenous IL‐22. Mechanistically, activation of ILC3s for IL‐22 production is reciprocally mediated by eATP and adenosine. These findings reveal that the NTPDase‐mediated balance between eATP and adenosine regulates ILC3 cell function to provide protection against intestinal injury and suggest potential therapeutic strategies for treating IBD by targeting the purine–ILC3 axis

Lineage-specific compaction of Tcrb requires a chromatin barrier to protect the function of a long-range tethering element

Gene regulation relies on dynamic changes in three-dimensional chromatin conformation, which are shaped by composite regulatory and architectural elements. However, mechanisms that govern such conformational switches within chromosomal domains remain unknown. We identify a novel mechanism by which cis-elements promote long-range interactions, inducing conformational changes critical for diversification of the TCRβ antigen receptor locus (Tcrb). Association between distal Vβ gene segments and the highly expressed DβJβ clusters, termed the recombination center (RC), is independent of enhancer function and recruitment of V(D)J recombinase. Instead, we find that tissue-specific folding of Tcrb relies on two distinct architectural elements located upstream of the RC. The first, a CTCF-containing element, directly tethers distal portions of the Vβ array to the RC. The second element is a chromatin barrier that protects the tether from hyperactive RC chromatin. When the second element is removed, active RC chromatin spreads upstream, forcing the tether to serve as a new barrier. Acquisition of barrier function by the CTCF element disrupts contacts between distal Vβ gene segments and significantly alters Tcrb repertoires. Our findings reveal a separation of function for RC-flanking regions, in which anchors for long-range recombination must be cordoned off from hyperactive RC landscapes by chromatin barriers

Multiple Histone Methyl and Acetyltransferase Complex Components Bind the HLA-DRA Gene

Major histocompatibility complex class II (MHC-II) genes are fundamental components that contribute to adaptive immune responses. While characterization of the chromatin features at the core promoter region of these genes has been studied, the scope of histone modifications and the modifying factors responsible for activation of these genes are less well defined. Using the MHC-II gene HLA-DRA as a model, the extent and distribution of major histone modifications associated with active expression were defined in interferon-γ induced epithelial cells, B cells, and B-cell mutants for MHC-II expression. With active transcription, nucleosome density around the proximal regulatory region was diminished and histone acetylation and methylation modifications were distributed throughout the gene in distinct patterns that were dependent on the modification examined. Irrespective of the location, the majority of these modifications were dependent on the binding of either the X-box binding factor RFX or the class II transactivator (CIITA) to the proximal regulatory region. Importantly, once established, the modifications were stable through multiple cell divisions after the activating stimulus was removed, suggesting that activation of this system resulted in an epigenetic state. A dual crosslinking chromatin immunoprecipitation method was used to detect histone modifying protein components that interacted across the gene. Components of the MLL methyltransferase and GCN5 acetyltransferase complexes were identified. Some MLL complex components were found to be CIITA independent, including MLL1, ASH2L and RbBP5. Likewise, GCN5 containing acetyltransferase complex components belonging to the ATAC and STAGA complexes were also identified. These results suggest that multiple complexes are either used or are assembled as the gene is activated for expression. Together the results define and illustrate a complex network of histone modifying proteins and multisubunit complexes participating in MHC-II transcription

Arabidopsis RPT2a, 19S Proteasome Subunit, Regulates Gene Silencing via DNA Methylation

The ubiquitin/proteasome pathway plays a crucial role in many biological processes. Here we report a novel role for the Arabidopsis 19S proteasome subunit RPT2a in regulating gene activity at the transcriptional level via DNA methylation. Knockout mutation of the RPT2a gene did not alter global protein levels; however, the transcriptional activities of reporter transgenes were severely reduced compared to those in the wild type. This transcriptional gene silencing (TGS) was observed for transgenes under control of either the constitutive CaMV 35S promoter or the cold-inducible RD29A promoter. Bisulfite sequencing analysis revealed that both the transgene and endogenous RD29A promoter regions were hypermethylated at CG and non-CG contexts in the rpt2a mutant. Moreover, the TGS of transgenes driven by the CaMV 35S promoters was released by treatment with the DNA methylation inhibitor 5-aza-2′-deoxycytidine, but not by application of the inhibitor of histone deacetylase Trichostatin A. Genetic crosses with the DNA methyltransferase met1 single or drm1drm2cmt3 triple mutants also resulted in a release of CaMV 35S transgene TGS in the rpt2a mutant background. Increased methylation was also found at transposon sequences, suggesting that the 19S proteasome containing AtRPT2a negatively regulates TGS at transgenes and at specific endogenous genes through DNA methylation

The 19S proteasome subcomplex promotes the targeting of NuA4 HAT to the promoters of ribosomal protein genes to facilitate the recruitment of TFIID for transcriptional initiation in vivo

Previous studies have implicated SAGA (Spt-Ada-Gcn5-acetyltransferase) and TFIID (Transcription factor-IID)-dependent mechanisms of transcriptional activation in yeast. SAGA-dependent transcriptional activation is further regulated by the 19S proteasome subcomplex. However, the role of the 19S proteasome subcomplex in transcriptional activation of the TFIID-dependent genes has not been elucidated. Therefore, we have performed a series of chromatin immunoprecipitation, mutational and transcriptional analyses at the TFIID-dependent ribosomal protein genes such as RPS5, RPL2B and RPS11B. We find that the 19S proteasome subcomplex is recruited to the promoters of these ribosomal protein genes, and promotes the association of NuA4 (Nucleosome acetyltransferase of histone H4) co-activator, but not activator Rap1p (repressor-activator protein 1). These observations support that the 19S proteasome subcomplex enhances the targeting of co-activator at the TFIID-dependent promoter. Such an enhanced targeting of NuA4 HAT (histone acetyltransferase) promotes the recruitment of the TFIID complex for transcriptional initiation. Collectively, our data demonstrate that the 19S proteasome subcomplex enhances the targeting of NuA4 HAT to activator Rap1p at the promoters of ribosomal protein genes to facilitate the recruitment of TFIID for transcriptional stimulation, hence providing a new role of the 19S proteasome subcomplex in establishing a specific regulatory network at the TFIID-dependent promoter for productive transcriptional initiation in vivo