Germline mutations in the tumor suppressor gene, BRCA1, predispose individuals to breast and ovarian cancers. Using a combination of affinity- and conventional chromatographic techniques, we have isolated a predominant form of a multiprotein BRCA1-containing complex from human cells displaying chromatin-remodeling activity. Mass spectrometric sequencing of components of this complex indicated that BRCA1 is associated with a SWI/SNF-related complex. We show that BRCA1 can directly interact with the BRG1 subunit of the SWI/SNF complex. Moreover, p53-mediated stimulation of transcription by BRCA1 was completely abrogated by either a dominant-negative mutant of BRG1 or the cancer-causing deletion in exon 11 of BRCA1. These findings reveal a direct function for BRCA1 in transcriptional control through modulation of chromatin structure.

BRAF35, a structural DNA-binding protein, initially was identified as a component of a large BRCA2-containing complex. Biochemical analysis revealed the presence of a smaller core-BRAF35 complex devoid of BRCA2. Here we report the isolation of a six-subunit core-BRAF35 complex with the capacity to deacetylate histones, termed the BRAF-histone deacetylase complex (BHC), from human cells. BHC contains polypeptides reminiscent of the chromatinremodeling complexes SWI͞SNF and NuRD (nucleosome remodeling and deacetylating). Similar to NuRD, BHC contains an Mi2-like subunit, BHC80, and a PHD zinc-finger subunit as well as histone deacetylases 1͞2 and an MTA-like subunit, the transcriptional corepressor CoREST. We show that BHC mediates repression of neuron-specific genes through the cis-regulatory element known as the repressor element 1 or neural restrictive silencer (RE1͞NRS).Chromatin-immunoprecipitation experiments demonstrate the recruitment of BHC by the neuronal repressor REST. Expression of BRAF35 containing a single point mutation in the HMG domain of the protein abrogated REST-mediated transcriptional repression. These results demonstrate a role for core-BRAF35-containing complex in the regulation of neuron-specific genes through modulation of the chromatin structure.T he genome of eukaryotes is packaged into chromatin, the fundamental unit of which is the nucleosome. The higher order chromatin structure is formed by arrangement of nucleosomes into an array. Such higher order chromatin structure presents a barrier to cellular processes such as transcription, DNA replication, and DNA repair. Therefore, controlling accessibility to the nucleosomal DNA provides an important regulatory point in these processes (1).Recent genetic and biochemical studies have culminated in the discovery of a host of multisubunit complexes that, in an ATP-dependent manner, are able to alter the structure of the nucleosome. The first of such multiprotein complexes, the SWI͞SNF complex, was discovered initially through genetic studies in yeast, and its catalytic subunit, SWI2͞SNF2, was identified as the DNA-dependent ATPase (2-4). A complex similar to that of the SWI͞SNF complex was identified recently in yeast (RSC), which unlike the SWI͞SNF complex is essential for growth (5). Complexes homologous in polypeptide composition and biochemical activity to that of SWI͞SNF have been identified in other organisms (6-10). More recently, a number of groups reported the isolation and characterization of a complex termed NuRD (nucleosome remodeling and deacetylating, also NURD and NRD), that not only contains a DNA-dependent ATPase subunit but also histone deacetylase (HDAC) 1͞2 (11-13).In addition to such chromatin-remodeling complexes a number of transcriptional regulatory complexes have been identified that contain histone acetylation or deacetylation activities. It was shown previously that the hyperacetylated chromatin correlates with active genes, whereas the repressed genes exhibit a pattern of hypoacetylation (14,15). This contention ...

Histone deacetylase (HDAC) inhibitors are a promising class of anticancer agents for the treatment of solid and hematological malignancies. The precise mechanism by which HDAC inhibitors mediate their effects on tumor cell growth, differentiation, and/or apoptosis is the subject of intense research. Previously we described a family of multiprotein complexes that contain histone deacetylase 1/2 (HDAC1/2) and the histone demethylase BHC110 (LSD1). Here we show that HDAC inhibitors diminish histone H3 lysine 4 (H3K4) demethylation by BHC110 in vitro. In vivo analysis revealed an increased H3K4 methylation concomitant with inhibition of nucleosomal deacetylation by HDAC inhibitors. Reconstitution of recombinant complexes revealed a functional connection between HDAC1 and BHC110 only when nucleosomal substrates were used. Importantly, while the enzymatic activity of BHC110 is required to achieve optimal deacetylation in vitro, in vivo analysis following ectopic expression of an enzymatically dead mutant of BHC110 (K661A) confirmed the functional cross talk between the demethylase and deacetylase enzymes. Our studies not only reveal an intimate link between the histone demethylase and deacetylase enzymes but also identify histone demethylation as a secondary target of HDAC inhibitors.

Nucleosomal DNA is arranged in a higher-order structure that presents a barrier to most cellular processes involving protein DNA interactions. The cellular machinery involved in sister chromatid cohesion, the cohesin complex, also requires access to the nucleosomal DNA to perform its function in chromosome segregation. The machineries that provide this accessibility are termed chromatin remodelling factors. Here, we report the isolation of a human ISWI (SNF2h)-containing chromatin remodelling complex that encompasses components of the cohesin and NuRD complexes. We show that the hRAD21 subunit of the cohesin complex directly interacts with the ATPase subunit SNF2h. Mapping of hRAD21, SNF2h and Mi2 binding sites by chromatin immunoprecipitation experiments reveals the specific association of these three proteins with human DNA elements containing Alu sequences. We find a correlation between modification of histone tails and association of the SNF2h/cohesin complex with chromatin. Moreover, we show that the association of the cohesin complex with chromatin can be regulated by the state of DNA methylation. Finally, we present evidence pointing to a role for the ATPase activity of SNF2h in the loading of hRAD21 on chromatin.

ABSTRACT3-hydroxy-3-methylglutaryl-CoA (HMGCoA) reductase is the rate-limiting enzyme and the first committed step in the biosynthesis of cholesterol in mammals. We have determined the crystal structures of two nonproductive ternary complexes of HMG-CoA reductase, HMG-CoA͞ NAD ؉ and mevalonate͞NADH, at 2.8 Å resolution. In the structure of the Pseudomonas mevalonii apoenzyme, the last 50 residues of the C terminus (the f lap domain), including the catalytic residue His381, were not visible. The structures of the ternary complexes reported here reveal a substrate-induced closing of the f lap domain that completes the active site and aligns the catalytic histidine proximal to the thioester of HMG-CoA. The structures also present evidence that Lys267 is critically involved in catalysis and provide insights into the catalytic mechanism.The reaction catalyzed by 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA), the conversion of (S)-HMG-CoA to (R)-mevalonate, represents a major point of control for isoprenoid biogenesis (for a review, see ref. 1). Because in mammals this reaction is the first committed step in cholesterol biosynthesis, HMG-CoA reductase is a primary target enzyme for chemotherapy of hypercholesterolemias (2). The crystal structure of the HMG-CoA reductase from Pseudomonas mevalonii previously was solved at 3.0 Å resolution (3). The structure revealed a tightly bound dimer that brings together conserved residues implicated in binding and catalysis at the subunitinterface active site. Each monomer is composed of two major domains. The large domain (residues 1-108 and 220-375) binds HMG-CoA and consists of a central 24-residue ␣-helix surrounded by three roughly triangular walls. The small NAD(H) binding domain (residues 110-215) has a nonclassical dinucleotide-binding fold. This domain consists of a fourstrand antiparallel ␤-sheet with two crossover helices that lie on one side of the sheet. Connecting the third strand and the second helix in the small domain, there is a highly conserved sequence, the DAMG loop (residues 180-186), which is analogous to the G-rich loop in the classic dinucleotidebinding domain.Knowledge of the spatial location of catalytic residues is crucial for understanding the mechanism of this enzyme. However, the last 50 residues of the C terminus (377-428), which include the catalytic His381 (4, 5), were not visible in the electron density maps of the HMG-CoA reductase apoenzyme and were presumably disordered. It was proposed that these C-terminal residues form a flap domain that closes over the active site when substrates are bound (3). We have now determined the crystal structures of two nonproductive ternary complexes, HMG-CoA͞NAD ϩ and mevalonate͞NADH, at 2.8 Å resolution. The structures demonstrate that the flexible flap domain closes on binding of the substrates and positions the catalytic residue His381 close to the scissile bond of HMG-CoA, completing the active site in its catalytic conformation. MATERIALS AND METHODSCrystallization. P. mevalonii HMG-CoA reductase was crystalliz...

ATP-dependent chromatin remodeling by the CHD family of proteins plays an important role in the regulation of gene transcription. Here we report that full-length CHD8 interacts directly with ␤-catenin and that CHD8 is also recruited specifically to the promoter regions of several ␤-catenin-responsive genes. Our results indicate that CHD8 negatively regulates ␤-catenin-targeted gene expression, since short hairpin RNA against CHD8 results in the activation of several ␤-catenin target genes. This regulation is also conserved through evolution; RNA interference against kismet, the apparent Drosophila ortholog of CHD8, results in a similar activation of ␤-catenin target genes. We also report the first demonstration of chromatin remodeling activity for a member of the CHD6-9 family of proteins, suggesting that CHD8 functions in transcription through the ATP-dependent modulation of chromatin structure.The alteration of chromatin structure provides a key regulatory step for all processes that act upon DNA (41). The factors that regulate this structure, commonly referred to as chromatin remodeling enzymes, can be grouped into two broad categories: complexes that alter chromatin structure via the covalent modification of histones (25,42,83) and complexes that use the energy of ATP hydrolysis to alter the structure or position of the nucleosome (6,47,57,67).ATP-dependent chromatin remodeling enzymes modulate the contacts between histones and DNA. In vitro, these enzymes catalyze structural changes that allow factors to access nucleosomal DNA, reposition nucleosomes on a template, transfer histone octamers to donor DNA, and replace histones with histone variants (27,43,80). In vivo, these activities are crucial for transcription, replication, repair, and recombination of the eukaryotic genome (2,21,59,65). These remodeling enzymes can be divided into numerous families based on domain architecture. One such family is the CHD (chromodomain, helicase, DNA binding) group of proteins, which are critical regulators of chromatin structure (23,26,29,49). These enzymes are characterized by tandem chromodomains N-terminal to their catalytic Snf2 helicase domain.The CHD family can be further subdivided into three subfamilies: CHD1-2, CHD3-5, and CHD6-9. While the first two subfamilies have been extensively studied, very little is known about the CHD6-9 family (29, 49). Previous studies have indicated that CHD8 may regulate the Wnt signaling pathway, since an N-terminal fragment of CHD8 was previously identified as a protein in Rattus norvegicus that binds ␤-catenin both in vivo and in vitro (61). This N-terminal fragment, termed Duplin, contains only the chromodomains and lacks the Snf2 helicase domain and C-terminal sequences. Overexpression of this N-terminal fragment results in inhibition of Tcf4-dependent transcription, and studies of Xenopus embryos demonstrated that this fragment inhibited axis formation and ␤-catenin-mediated axis duplication (61).The "canonical" Wnt signaling pathway functions by controlling the soluble pool ...

Chromatin remodeling complexes have been implicated in the disruption or reformation of nucleosomal arrays resulting in modulation of transcription, DNA replication, and DNA repair. Here we report the isolation of WCRF, a new chromatin-remodeling complex from HeLa cells. WCRF is composed of two subunits, WCRF135, the human homolog of Drosophila ISWI, and WCRF180, a protein related to the Williams syndrome transcription factor. WCRF180 is a member of a family of proteins sharing a putative heterochromatin localization domain, a PHD finger, and a bromodomain, prevalent in factors involved in regulation of chromatin structure.

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