No abstract

Posttranslational modification of histones has emerged as a key regulatory signal in eukaryotic gene expression. Recent genetic and biochemical studies link H3-lysine 9 (H3-K9) methylation to HP1-mediated heterochromatin formation and gene silencing. However, the mechanisms that target and coordinate these activities to specific genes is poorly understood. Here we report that the KAP-1 corepressor for the KRAB-ZFP superfamily of transcriptional silencers binds to SETDB1, a novel SET domain protein with histone H3-K9-specific methyltransferase activity. Although acetylation and phosphorylation of the H3 N-terminal tail profoundly affect the efficiency of H3-K9 methylation by SETDB1, we found that methylation of H3-K4 does not affect SETDB1-mediated methylation of H3-K9. In vitro methylation of the N-terminal tail of histone H3 by SETDB1 is sufficient to enhance the binding of HP1 proteins, which requires both an intact chromodomain and chromoshadow domain. Indirect immunofluoresence staining of interphase nuclei localized SETDB1 predominantly in euchromatic regions that overlap with HP1 staining in nonpericentromeric regions of chromatin. Moreover, KAP-1, SETDB1, H3-MeK9, and HP1 are enriched at promoter sequences of a euchromatic gene silenced by the KRAB-KAP-1 repression system. Thus, KAP-1 is a molecular scaffold that is targeted by KRAB-ZFPs to specific loci and coordinates both histone methylation and the deposition of HP1 proteins to silence gene expression. Macromolecular protein complexes containing enzymatic activities that modify the N-terminal tails of the core histones have emerged as key regulators of gene expression in eukaryotes. The constellation of histone modifications, including acetylation, phosphorylation, ubiquination, and methylation, create both synergistic and antagonistic signals that correlate with the transcriptional activity of a gene. This emerging histone code is hypothesized to create an architecture in chromatin that is recognized by nonhistone chromosomal proteins, which then effect the dynamic transition between transcriptionally active versus transcriptionally silent chromatin domains (Jenuwein and Allis 2001). Moreover, the combinatorial nature of these histone modifications and the chromatin-associated proteins that recognize these signals may represent an epigenetic marking system responsible for setting and maintaining heritable programs of gene expression during cellular differentiation and organism development.The role of histone acetylation and phosphorylation in regulation of transcription has been extensively characterized (Cheung et al. 2000;Strahl and Allis 2000;Berger 2001). Although it is well established that arginine and lysine methylation of histones occurs in vivo, the function of these specific modifications remains to be fully described (Strahl et al. 1999). Similar to the discovery of histone acetyltransferases (HATs) and deacetylases (HDACs), the role of histone methylation in the regulation of chromatin structure and gene expression has been greatly facili...

The cellular DNA-damage response is a signaling network that is vigorously activated by cytotoxic DNA lesions, such as double-strand breaks (DSBs). The DSB response is mobilized by the nuclear protein kinase ATM, which modulates this process by phosphorylating key players in these pathways. A long-standing question in this field is whether DSB formation affects chromatin condensation. Here, we show that DSB formation is followed by ATM-dependent chromatin relaxation. ATM's effector in this pathway is the protein KRAB-associated protein (KAP-1, also known as TIF1beta, KRIP-1 or TRIM28), previously known as a corepressor of gene transcription. In response to DSB induction, KAP-1 is phosphorylated in an ATM-dependent manner on Ser 824. KAP-1 is phosphorylated exclusively at the damage sites, from which phosphorylated KAP-1 spreads rapidly throughout the chromatin. Ablation of the phosphorylation site of KAP-1 leads to loss of DSB-induced chromatin decondensation and renders the cells hypersensitive to DSB-inducing agents. Knocking down KAP-1, or mimicking a constitutive phosphorylation of this protein, leads to constitutive chromatin relaxation. These results suggest that chromatin relaxation is a fundamental pathway in the DNA-damage response and identify its primary mediators.

We have identi®ed a novel protein, BAP1, which binds to the RING ®nger domain of the Breast/Ovarian Cancer Susceptibility Gene product, BRCA1. BAP1 is a nuclearlocalized, ubiquitin carboxy-terminal hydrolase, suggesting that deubiquitinating enzymes may play a role in BRCA1 function. BAP1 binds to the wild-type BRCA1-RING ®nger, but not to germline mutants of the BRCA1-RING ®nger found in breast cancer kindreds. BAP1 and BRCA1 are temporally and spatially coexpressed during murine breast development and remodeling, and show overlapping patterns of subnuclear distribution. BAP1 resides on human chromosome 3p21.3; intragenic homozgyous rearrangements and deletions of BAP1 have been found in lung carcinoma cell lines. BAP1 enhances BRCA1-mediated inhibition of breast cancer cell growth and is the ®rst nuclearlocalized ubiquitin carboxy-terminal hydrolase to be identi®ed. BAP1 may be a new tumor suppressor gene which functions in the BRCA1 growth control pathway.

ARID1A, a chromatin remodeler, shows one of the highest mutation rates across many cancer types. Notably, ARID1A is mutated in over 50% of ovarian clear cell carcinomas, which currently has no effective therapy. To date, clinically applicable targeted cancer therapy based on ARID1A mutational status has not been described. Here we show that inhibition of the EZH2 methyltransferase acts in a synthetic lethal manner in ARID1A mutated ovarian cancer cells. ARID1A mutational status correlates with response to the EZH2 inhibitor. We identified PIK3IP1 as a direct ARID1A/EZH2 target, which is upregulated by EZH2 inhibition and contributes to the observed synthetic lethality by inhibiting PI3K/AKT signaling. Significantly, EZH2 inhibition causes regression of ARID1A mutated ovarian tumors in vivo. Together, these data demonstrate for the first time a synthetic lethality between ARID1A mutation and EZH2 inhibition. They indicate that pharmacological inhibition of EZH2 represents a novel treatment strategy for ARID1A mutated cancers.

Tandem PHD and bromodomains are often found in chromatin-associated proteins and have been shown to cooperate in gene silencing. Each domain can bind specifically modified histones: the mechanisms of cooperation between these domains are unknown. We show that the PHD domain of the KAP1 corepressor functions as an intramolecular E3 ligase for sumoylation of the adjacent bromodomain. The RING finger-like structure of the PHD domain is required for both Ubc9 binding and sumoylation and directs modification to specific lysine residues in the bromodomain. Sumoylation is required for KAP1-mediated gene silencing and functions by directly recruiting the SETDB1 histone methyltransferase and the CHD3/Mi2 component of the NuRD complex via SUMO-interacting motifs. Sumoylated KAP1 stimulates the histone methyltransferase activity of SETDB1. These data provide a mechanistic explanation for the cooperation of PHD and bromodomains in gene regulation and describe a function of the PHD domain as an intramolecular E3 SUMO ligase.

No abstract

Macromolecular complexes containing histone deacetylase and ATPase activities regulate chromatin dynamics and are vitally responsible for transcriptional gene silencing in eukaryotes. The mechanisms that target these assemblies to specific loci are not as well understood. We show that the corepressor KAP-1, via its PHD (plant homeodomain) and bromodomain, links the superfamily of Krü ppel associated box (KRAB) zinc finger proteins (ZFP) to the NuRD complex. We demonstrate that the tandem PHD finger and bromodomain of KAP-1, an arrangement often found in cofactor proteins but functionally ill-defined, form a cooperative unit that is required for transcriptional repression. The complement of transcription factors organized at a gene promoter integrates different transcriptional regulatory signals that orchestrate cellular responses such as proliferation, differentiation, and apoptosis. Regulation of RNA polymerase II involves a complex interplay between DNA-protein interactions and protein-protein interactions. Whereas the general transcription factors regulate the accurate initiation of transcription, proteins that bind gene-specific DNA elements are instrumental in either negatively or positively regulating the rate of transcription (Orphanides et al. 1996;Hampsey and Reinberg 1999). Studies aimed at understanding the mechanisms of transcriptional regulation by site-specific factors have been aided by the realization that these proteins are highly modular in architecture. In addition to sequence-specific DNA binding domains, these proteins possess an independent, functionally separable effector domain that can either activate or repress transcription. The affects of sequence-specific transcription factors in the regulation of transcription have been greatly facilitated by the identification of cofactors with which they bind and the intrinsic or associated biochemical activities of these proteins. It is clear from this strategy that these proteins possess enzymatic activities which either utilize ATP to remodel chromatin structure or covalently modify the amino-terminal tails of the core histones, creating a biochemical code that regulates gene transcription of chromatin templates (Tyler and Kadonaga 1999;Strahl and Allis 2000). Thus, sequence-specific repressor-corepressor complexes likely serve as scaffolds for the assembly of macromolecular complexes which integrate diverse transcription regulatory signals.The Krü ppel associated box (KRAB) domain is one example of an abundant amino acid sequence motif found at the N terminus of nearly one-third of all Krü ppel/ TFIIIA-type C2H2 zinc finger proteins (Bellefroid et al. 1991). This highly conserved domain displays potent,