SiDe Li

Molecular Interplay of the Noncoding RNA ANRIL and Methylated Histone H3 Lysine 27 by Polycomb CBX7 in Transcriptional Silencing of INK4a

Expression of the INK4b/ARF/INK4a tumor suppressor locus in normal and cancerous cell growth is controlled by methylation of histone H3 at lysine 27 (H3K27me) as directed by the Polycomb group proteins. The antisense noncoding RNA ANRIL of the INK4b/ARF/INK4a locus is also important for expression of the protein-coding genes in cis, but its mechanism has remained elusive. Here we report that chromobox 7 (CBX7) within the polycomb repressive complex 1 binds to ANRIL, and both CBX7 and ANRIL are found at elevated levels in prostate cancer tissues. In concert with H3K27me recognition, binding to RNA contributes to CBX7 function, and disruption of either interaction impacts the ability of CBX7 to repress the INK4b/ARF/INK4a locus and control senescence. Structure-guided analysis reveals the molecular interplay between noncoding RNA and H3K27me as mediated by the conserved chromodomain. Our study suggests a mechanism by which noncoding RNA participates directly in epigenetic transcriptional repression.

Histone demethylase JMJD3 contributes to epigenetic control of INK4a/ARF by oncogenic RAS

The INK4a/ARF tumor suppressor locus, a key executor of cellular senescence, is regulated by members of the Polycomb group (PcG) of transcriptional repressors. Here we show that signaling from oncogenic RAS overrides PcG-mediated repression of INK4a by activating the H3K27 demethylase JMJD3 and down-regulating the methyltransferase EZH2. In human fibroblasts, JMJD3 activates INK4a, but not ARF, and causes p16(INK4a)-dependent arrest. In mouse embryo fibroblasts, Jmjd3 activates both Ink4a and Arf and elicits a p53-dependent arrest, echoing the effects of RAS in this system. Our findings directly implicate JMJD3 in the regulation of INK4a/ARF during oncogene-induced senescence and suggest that JMJD3 has the capacity to act as a tumor suppressor.

Mechanism and regulation of acetylated histone binding by the tandem PHD finger of DPF3b

Histone lysine acetylation and methylation have an important role during gene transcription in a chromatin context. Knowledge concerning the types of protein modules that can interact with acetyl-lysine has so far been limited to bromodomains. Recently, a tandem plant homeodomain (PHD) finger (PHD1–PHD2, or PHD12) of human DPF3b, which functions in association with the BAF chromatin remodelling complex to initiate gene transcription during heart and muscle development, was reported to bind histones H3 and H4 in an acetylation-sensitive manner, making it the first alternative to bromodomains for acetyl-lysine binding. Here we report the structural mechanism of acetylated histone binding by the double PHD fingers of DPF3b. Our three-dimensional solution structures and biochemical analysis of DPF3b highlight the molecular basis of the integrated tandem PHD finger, which acts as one functional unit in the sequence-specific recognition of lysine-14-acetylated histone H3 (H3K14ac). Whereas the interaction with H3 is promoted by acetylation at lysine 14, it is inhibited by methylation at lysine 4, and these opposing influences are important during transcriptional activation of the mouse DPF3b target genes Pitx2 and Jmjd1c. Binding of this tandem protein module to chromatin can thus be regulated by different histone modifications during the initiation of gene transcription.

Transcriptional repression of the cystic fibrosis transmembrane conductance regulator gene, mediated by CCAAT displacement protein/cut homolog, is associated with histone deacetylation.

Human cystic fibrosis transmembrane conductance regulator gene (CFTR) transcription is tightly regulated by nucleotide sequences upstream of the initiator sequences. Our studies of human CFTR transcription focus on identifying transcription factors bound to an inverted CCAAT consensus or “Y-box element.” The human homeodomain CCAAT displacement protein/cut homolog (CDP/cut) can bind to the Y-box element through a cut repeat and homeobox. Analysis of stably transfected cell lines with wild-type and mutant humanCFTR-directed reporter genes demonstrates that human histone acetyltransferase GCN5 and transcription factor ATF-1 can potentiate CFTR transcription through the Y-box element. We have found 1) that human CDP/cut acts as a repressor ofCFTR transcription through the Y-box element by competing for the sites of transactivators hGCN5 and ATF-1; 2) that the ability of CDP/cut to repress activities of hGCN5 and ATF-1 activity is contingent on the amount of CDP/cut expression; 3) that histone acetylation may have a role in the regulation of gene transcription by altering the accessibility of the CFTRY-box for sequence-specific transcription factors; 4) that trichostatin A, an inhibitor of histone deacetylase activity, activates transcription of CFTR through the Y-box element; 5) that the inhibition of histone deacetylase activity leads to an alteration of local chromatin structure requiring an intact Y-box sequence inCFTR; 6) that immunocomplexes of CDP/cutpossess an associated histone deacetylase activity; 7) that the carboxyl region of CDP/cut, responsible for the transcriptional repressor function, interacts with the histone deacetylase, HDAC1. We propose that CFTR transcription may be regulated through interactions with factors directing the modification of chromatin and requires the conservation of the inverted CCAAT (Y-box) element of the CFTR promoter.

p53-induced growth arrest is regulated by the mitochondrial SirT3 deacetylase.

A hallmark of p53 function is to regulate a transcriptional program in response to extracellular and intracellular stress that directs cell cycle arrest, apoptosis, and cellular senescence. Independent of the role of p53 in the nucleus, some of the anti-proliferative functions of p53 reside within the mitochondria [1]. p53 can arrest cell growth in response to mitochondrial p53 in an EJ bladder carcinoma cell environment that is naïve of p53 function until induced to express p53 [2]. TP53 can independently partition with endogenous nuclear and mitochondrial proteins consistent with the ability of p53 to enact senescence. In order to address the role of p53 in navigating cellular senescence through the mitochondria, we identified SirT3 to rescue EJ/p53 cells from induced p53-mediated growth arrest. Human SirT3 function appears coupled with p53 early during the initiation of p53 expression in the mitochondria by biochemical and cellular localization analysis. Our evidence suggests that SirT3 partially abrogates p53 activity to enact growth arrest and senescence. Additionally, we identified the chaperone protein BAG-2 in averting SirT3 targeting of p53 -mediated senescence. These studies identify a complex relationship between p53, SirT3, and chaperoning factor BAG-2 that may link the salvaging and quality assurance of the p53 protein for control of cellular fate independent of transcriptional activity.

Lysine methyltransferase G9a methylates the transcription factor MyoD and regulates skeletal muscle differentiation

Skeletal muscle cells have served as a paradigm for understanding mechanisms leading to cellular differentiation. The proliferation and differentiation of muscle precursor cells require the concerted activity of myogenic regulatory factors including MyoD. In addition, chromatin modifiers mediate dynamic modifications of histone tails that are vital to reprogramming cells toward terminal differentiation. Here, we provide evidence for a unique dimension to epigenetic regulation of skeletal myogenesis. We demonstrate that the lysine methyltransferase G9a is dynamically expressed in myoblasts and impedes differentiation in a methyltransferase activity-dependent manner. In addition to mediating histone H3 lysine-9 di-methylation (H3K9me2) on MyoD target promoters, endogenous G9a interacts with MyoD in precursor cells and directly methylates it at lysine 104 (K104) to constrain its transcriptional activity. Mutation of K104 renders MyoD refractory to inhibition by G9a and enhances its myogenic activity. Interestingly, MyoD methylation is critical for G9a-mediated inhibition of myogenesis. These findings provide evidence of an unanticipated role for methyltransferases in cellular differentiation states by direct posttranslational modification of a transcription factor.

Structure and Hemimethylated CpG Binding of the SRA Domain from Human UHRF1.

Human UHRF1 (ubiquitin-like PHD and RING finger 1) functions to maintain CpG DNA methylation patterns through DNA replication by co-localizing with the DNA methyltransferase DNMT1 at chromatin in mammals. Recent studies show that UHRF1 binds selectively to hemimethylated CpG via its conserved SRA (SET- and RING finger-associated) domain. However, the underlying molecular mechanism is not known. Here, we report a 1.95Å resolution crystal structure of the SRA domain of human UHRF1. Using NMR structure-guided mutagenesis, electrophoretic mobility shift assay, and fluorescence anisotropy analysis, we determined key amino acid residues for methyl-DNA binding that are conserved in the SRA domain.

Structure and chromosomal DNA binding of the SWIRM domain.

The evolutionarily conserved Swi3p, Rsc8p and Moira (SWIRM) domain is found in many chromosomal proteins involved in chromatin modifications or remodeling. Here we report the three-dimensional solution structure of the SWIRM domain from the human transcriptional adaptor ADA2α. The structure reveals a five-helix bundle consisting of two helix-turn-helix motifs connected by a central long helix, reminiscent of the histone fold. Using structural and biochemical analyses, we showed that the SWIRM domains of human ADA2α and SMARC2 bind to double-stranded and nucleosomal DNA, and we identified amino acid residues required for this function. We demonstrated that the ADA2α SWIRM domain is colocalized with lysine-acetylated histone H3 in the cell nucleus and that it potentiates the ACF remodeling activity by enhancing accessibility of nucleosomal linker DNA bound to histone H1. These data suggest a functional role of the SWIRM domain in chromatin remodeling.

The ZNF217 oncogene is a candidate organizer of repressive histone modifiers

The zinc finger protein 217 (ZNF217) is an important oncogene based on the high frequency of amplification and over-expression in many cancer types, but its molecular mode of gene regulation is poorly understood. We purified a complex of nuclear ZNF217-binding proteins by affinity chromatography and identified its components by mass spectrometry as Jarid1b/Plu-1, G9a, LSD1, CoREST, and CtBP1. Individual binding of these with ZNF217 was confirmed by co-immunoprecipiation (IP). Known activities of these proteins suggested a role of the ZNF217 complex in histone modification. Using in vitro assays the following activities were demonstrated: Histone H3 lysine 4 trimethyl (H3K4me3) demethylase activity, which co-fractionated with Jarid1b/Plu-1 in anion-exchange chromatography; H3K9 methylation, the known principal activity of G9a; and H3K27 methylation. The latter suggested EZH2 as another ZNF217 binding candidate, which could be confirmed by co-IP. Taken together, these findings suggest that ZNF217 assembles a distinct set of histone modifying proteins at target DNA sites that act synergistically in transcriptional repression.

Modulation of long noncoding RNAs by risk SNPs underlying genetic predispositions to prostate cancer

Long noncoding RNAs (lncRNAs) represent an attractive class of candidates to mediate cancer risk. Through integrative analysis of the lncRNA transcriptome with genomic data and SNP data from prostate cancer genome-wide association studies (GWAS), we identified 45 candidate lncRNAs associated with risk to prostate cancer. We further evaluated the mechanism underlying the top hit, PCAT1, and found that a risk-associated variant at rs7463708 increases binding of ONECUT2, a novel androgen receptor (AR)-interacting transcription factor, at a distal enhancer that loops to the PCAT1 promoter, resulting in upregulation of PCAT1 upon prolonged androgen treatment. In addition, PCAT1 interacts with AR and LSD1 and is required for their recruitment to the enhancers of GNMT and DHCR24, two androgen late-response genes implicated in prostate cancer development and progression. PCAT1 promotes prostate cancer cell proliferation and tumor growth in vitro and in vivo. These findings suggest that modulating lncRNA expression is an important mechanism for risk-associated SNPs in promoting prostate transformation.