07-550 Sigma-AldrichAnti-SUV39H1 Antibody

Correct chromosome segregation requires a unique chromatin environment at centromeres and in their vicinity. Here, we address how the deposition of canonical H2A and H2A.Z histone variants is controlled at pericentric heterochromatin (PHC). Whereas in euchromatin newly synthesized H2A and H2A.Z are deposited throughout the cell cycle, we reveal two discrete waves of deposition at PHC - during mid to late S phase in a replication-dependent manner for H2A and during G1 phase for H2A.Z. This G1 cell cycle restriction is lost when heterochromatin features are altered, leading to the accumulation of H2A.Z at the domain. Interestingly, compromising PHC integrity also impacts upon neighboring centric chromatin, increasing the amount of centromeric CENP-A without changing the timing of its deposition. We conclude that the higher-order chromatin structure at the pericentric domain influences dynamics at the nucleosomal level within centromeric chromatin. The two different modes of rearrangement of the PHC during the cell cycle provide distinct opportunities to replenish one or the other H2A variant, highlighting PHC integrity as a potential signal to regulate the deposition timing and stoichiometry of histone variants at the centromere.

Survival time-associated plant homeodomain (PHD) finger protein in Ovarian Cancer 1 (SPOC1, also known as PHF13) is known to modulate chromatin structure and is essential for testicular stem-cell differentiation. Here we show that SPOC1 is recruited to DNA double-strand breaks (DSBs) in an ATM-dependent manner. Moreover, SPOC1 localizes at endogenous repair foci, including OPT domains and accumulates at large DSB repair foci characteristic for delayed repair at heterochromatic sites. SPOC1 depletion enhances the kinetics of ionizing radiation-induced foci (IRIF) formation after γ-irradiation (γ-IR), non-homologous end-joining (NHEJ) repair activity, and cellular radioresistance, but impairs homologous recombination (HR) repair. Conversely, SPOC1 overexpression delays IRIF formation and γH2AX expansion, reduces NHEJ repair activity and enhances cellular radiosensitivity. SPOC1 mediates dose-dependent changes in chromatin association of DNA compaction factors KAP-1, HP1-α and H3K9 methyltransferases (KMT) GLP, G9A and SETDB1. In addition, SPOC1 interacts with KAP-1 and H3K9 KMTs, inhibits KAP-1 phosphorylation and enhances H3K9 trimethylation. These findings provide the first evidence for a function of SPOC1 in DNA damage response (DDR) and repair. SPOC1 acts as a modulator of repair kinetics and choice of pathways. This involves its dose-dependent effects on DNA damage sensors, repair mediators and key regulators of chromatin structure.

Epigenetic silencing of tumour suppressor genes is an important mechanism involved in cell transformation and tumour progression. The Set and RING-finger-associated domain-containing protein UHRF1 might be an important link between different epigenetic pathways. Here, we report that UHRF1 is frequently overexpressed in human prostate tumours and has an important role in prostate cancer pathogenesis and progression. Analysis of human prostate cancer samples by microarrays and immunohistochemistry showed increased expression of UHRF1 in about half of the cases. Moreover, UHRF1 expression was associated with reduced overall survival after prostatectomy in patients with organ-confined prostate tumours (P<0.0001). UHRF1 expression was negatively correlated with several tumour suppressor genes and positively with the histone methyltransferase (HMT) EZH2 both in prostate tumours and cell lines. UHRF1 knockdown reduced proliferation, clonogenic capability and anchorage-independent growth of prostate cancer cells. Depletion of UHRF1 resulted in reactivation of several tumour suppressor genes. Gene reactivation upon UHRF1 depletion was associated with changes in histone H3K9 methylation, acetylation and DNA methylation, and impaired binding of the H3K9 HMT Suv39H1 to the promoter of silenced genes. Co-immunoprecipitation experiments showed direct interaction between UHRF1 and Suv39H1. Our data support the notion that UHRF1, along with Suv39H1 and DNA methyltransferases, contributes to epigenetic gene silencing in prostate tumours. This could represent a parallel and convergent pathway to the H3K27 methylation catalyzed by EZH2 to synergistically promote inactivation of tumour suppressor genes. Deregulated expression of UHRF1 is involved in the prostate cancer pathogenesis and might represent a useful marker to distinguish indolent cancer from those at high risk of lethal progression.Oncogene advance online publication, 13 February 2012; doi:10.1038/onc.2011.641.

The molecular events that modulate chromatin structure during skeletal muscle differentiation are still poorly understood. We report in this paper that expression of the H3-K4 histone methyltransferase Set7 is increased when myoblasts differentiate into myotubes and is required for skeletal muscle development, expression of muscle contractile proteins, and myofibril assembly. Knockdown of Set7 or expression of a dominant-negative Set7 mutant impairs skeletal muscle differentiation, accompanied by a decrease in levels of histone monomethylation (H3-K4me1). Set7 directly interacts with MyoD to enhance expression of muscle differentiation genes. Expression of myocyte enhancer factor 2 and genes encoding contractile proteins is decreased in Set7 knockdown myocytes. Furthermore, we demonstrate that Set7 also activates muscle gene expression by precluding Suv39h1-mediated H3-K9 methylation on the promoters of myogenic differentiation genes. Together, our experiments define a biological function for Set7 in muscle differentiation and provide a molecular mechanism by which Set7 modulates myogenic transcription factors during muscle differentiation.

Sequence analysis of the genome of the strict intracellular pathogen Chlamydia trachomatis revealed the presence of a SET domain containing protein, proteins that primarily function as histone methyltransferases. In these studies, we demonstrated secretion of this protein via a type III secretion mechanism. During infection, the protein is translocated to the host cell nucleus and associates with chromatin. We therefore named the protein nuclear effector (NUE). Expression of NUE in mammalian cells by transfection reconstituted nuclear targeting and chromatin association. In vitro methylation assays confirmed NUE is a histone methyltransferase that targets histones H2B, H3 and H4 and itself (automethylation). Mutants deficient in automethylation demonstrated diminished activity towards histones suggesting automethylation functions to enhance enzymatic activity. Thus, NUE is secreted by Chlamydia, translocates to the host cell nucleus and has enzymatic activity towards eukaryotic substrates. This work is the first description of a bacterial effector that directly targets mammalian histones.

Core Binding Factor or CBF is a transcription factor composed of two subunits, Runx1/AML-1 and CBF beta or CBFbeta. CBF was originally described as a regulator of hematopoiesis.Here we show that CBF is involved in the control of skeletal muscle terminal differentiation. Indeed, downregulation of either Runx1 or CBFbeta protein level accelerates cell cycle exit and muscle terminal differentiation. Conversely, overexpression of CBFbeta in myoblasts slows terminal differentiation. CBF interacts directly with the master myogenic transcription factor MyoD, preferentially in proliferating myoblasts, via Runx1 subunit. In addition, we show a preferential recruitment of Runx1 protein to MyoD target genes in proliferating myoblasts. The MyoD/CBF complex contains several chromatin modifying enzymes that inhibits MyoD activity, such as HDACs, Suv39h1 and HP1beta. When overexpressed, CBFbeta induced an inhibition of activating histone modification marks concomitant with an increase in repressive modifications at MyoD target promoters.Taken together, our data show a new role for Runx1/CBFbeta in the control of the proliferation/differentiation in skeletal myoblasts.

Mechanisms of transcriptional repression are important during cell differentiation. Mammalian heterochromatin protein 1 isoforms HP1alpha, HP1beta, and HP1gamma play important roles in the regulation of chromatin structure and function. We explored the possibility of different roles for the three HP1 isoforms in an integrated system, skeletal muscle terminal differentiation. In this system, terminal differentiation is initiated by the transcription factor MyoD, whose target genes remain mainly silent until myoblasts are induced to differentiate. Here we show that HP1alpha and HP1beta isoforms, but not HP1gamma, interact with MyoD in myoblasts. This interaction is direct, as shown using recombinant proteins in vitro. A gene reporter assay revealed that HP1alpha and HP1beta, but not HP1gamma, inhibit MyoD transcriptional activity, suggesting a model in which MyoD could serve as a bridge between nucleosomes and chromatin-binding proteins such as HDACs and HP1. Chromatin immunoprecipitation assays show a preferential recruitment of HP1 proteins on MyoD target genes in proliferating myoblasts. Finally, modulation of HP1 protein level impairs MyoD target gene expression and muscle terminal differentiation. Together, our data show a nonconventional interaction between HP1 and a tissue-specific transcription factor, MyoD. In addition, they strongly suggest that HP1 isoforms play important roles during muscle terminal differentiation in an isoform-dependent manner.

Polycomb-repressive complex 2 (PRC2)-mediated histone methylation plays an important role in aberrant cancer gene silencing and is a potential target for cancer therapy. Here we show that S-adenosylhomocysteine hydrolase inhibitor 3-Deazaneplanocin A (DZNep) induces efficient apoptotic cell death in cancer cells but not in normal cells. We found that DZNep effectively depleted cellular levels of PRC2 components EZH2, SUZ12, and EED and inhibited associated histone H3 Lys 27 methylation (but not H3 Lys 9 methylation). By integrating RNA interference (RNAi), genome-wide expression analysis, and chromatin immunoprecipitation (ChIP) studies, we have identified a prominent set of genes selectively repressed by PRC2 in breast cancer that can be reactivated by DZNep. We further demonstrate that the preferential reactivation of a set of these genes by DZNep, including a novel apoptosis affector, FBXO32, contributes to DZNep-induced apoptosis in breast cancer cells. Our results demonstrate the unique feature of DZNep as a novel chromatin remodeling compound and suggest that pharmacologic reversal of PRC2-mediated gene repression by DZNep may constitute a novel approach for cancer therapy.

Telomeres are capping structures at the ends of eukaryotic chromosomes composed of TTAGGG repeats bound to an array of specialized proteins. Telomeres are heterochromatic regions. Yeast and flies with defects in activities that modify the state of chromatin also have abnormal telomere function, but the putative role of chromatin-modifying activities in regulating telomeres in mammals is unknown. Here we report on telomere length and function in mice null with respect to both the histone methyltransferases (HMTases) Suv39h1 and Suv39h2 (called SUV39DN mice). Suv39h1 and Suv39h2 govern methylation of histone H3 Lys9 (H3-Lys9) in heterochromatic regions. We show that primary cells derived from SUV39DN mice have abnormally long telomeres relative to wild-type controls. Using chromatin immunoprecipitation (ChIP) analysis, we found that telomeres were enriched in di- and trimethylated H3-Lys9 but that telomeres of SUV39DN cells had less dimethylated and trimethylated H3-Lys9 but more monomethylated H3-Lys9. Concomitant with the decrease in H3-Lys9 methylation, telomeres in SUV39DN cells had reduced binding of the chromobox proteins Cbx1, Cbx3 and Cbx5, homologs of Drosophila melanogaster heterochromatin protein 1 (HP1). These findings indicate substantial changes in the state of telomeric heterochromatin in SUV39DN cells, which are associated with abnormal telomere elongation. Taken together, the results indicate epigenetic regulation of telomere length in mammals by Suv39h1 and Suv39h2.

In eukaryotic cells the histone methylase SUV39H1 and the methyl-lysine binding protein HP1 functionally interact to repress transcription at heterochromatic sites. Lysine 9 of histone H3 is methylated by SUV39H1 (ref. 2), creating a binding site for the chromo domain of HP1 (refs 3, 4). Here we show that SUV39H1 and HP1 are both involved in the repressive functions of the retinoblastoma (Rb) protein. Rb associates with SUV39H1 and HP1 in vivo by means of its pocket domain. SUV39H1 cooperates with Rb to repress the cyclin E promoter, and in fibroblasts that are disrupted for SUV39, the activity of the cyclin E and cyclin A2 genes are specifically elevated. Chromatin immunoprecipitations show that Rb is necessary to direct methylation of histone H3, and is necessary for binding of HP1 to the cyclin E promoter. These results indicate that the SUV39H1-HP1 complex is not only involved in heterochromatic silencing but also has a role in repression of euchromatic genes by Rb and perhaps other co-repressor proteins.