BRCA1 interacts with components of the histone deacetylase complex.
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Germ-line mutations in the BRCA1 tumor-suppressor gene are associated with an increased susceptibility to breast and ovarian cancer. BRCA1 contains a carboxyl-terminal domain (BRCT) that is shared with several other proteins involved in maintaining genome integrity. In an effort to understand the function of BRCA1, we sought to isolate proteins that interact with the BRCT domain. Purified BRCT polypeptide was used as a probe to screen a human placenta cDNA expression library by Far Western analysis. Here we report that BRCA1 interacts in vivo and in vitro with the Rb-binding proteins, RbAp46 and RbAp48, as well as with Rb. Moreover, the BRCT domain associates with the histone deacetylases HDAC1 and HDAC2. These results demonstrate that BRCA1 interacts with components of the histone deacetylase complex, and therefore may explain the involvement of BRCA1 in multiple processes such as transcription, DNA repair, and recombination. (+info)
Analysis of the NuRD subunits reveals a histone deacetylase core complex and a connection with DNA methylation.
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ATP-dependent nucleosome remodeling and core histone acetylation and deacetylation represent mechanisms to alter nucleosome structure. NuRD is a multisubunit complex containing nucleosome remodeling and histone deacetylase activities. The histone deacetylases HDAC1 and HDAC2 and the histone binding proteins RbAp48 and RbAp46 form a core complex shared between NuRD and Sin3-histone deacetylase complexes. The histone deacetylase activity of the core complex is severely compromised. A novel polypeptide highly related to the metastasis-associated protein 1, MTA2, and the methyl-CpG-binding domain-containing protein, MBD3, were found to be subunits of the NuRD complex. MTA2 modulates the enzymatic activity of the histone deacetylase core complex. MBD3 mediates the association of MTA2 with the core histone deacetylase complex. MBD3 does not directly bind methylated DNA but is highly related to MBD2, a polypeptide that binds to methylated DNA and has been reported to possess demethylase activity. MBD2 interacts with the NuRD complex and directs the complex to methylated DNA. NuRD may provide a means of gene silencing by DNA methylation. (+info)
Functional analysis of the SIN3-histone deacetylase RPD3-RbAp48-histone H4 connection in the Xenopus oocyte.
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We investigated the protein associations and enzymatic requirements for the Xenopus histone deacetylase catalytic subunit RPD3 to direct transcriptional repression in Xenopus oocytes. Endogenous Xenopus RPD3 is present in nuclear and cytoplasmic pools, whereas RbAp48 and SIN3 are predominantly nuclear. We cloned Xenopus RbAp48 and SIN3 and show that expression of RPD3, but not RbAp48 or SIN3, leads to an increase in nuclear and cytoplasmic histone deacetylase activity and transcriptional repression of the TRbetaA promoter. This repression requires deacetylase activity and nuclear import of RPD3 mediated by a carboxy-terminal nuclear localization signal. Exogenous RPD3 is not incorporated into previously described oocyte deacetylase and ATPase complexes but cofractionates with a component of the endogenous RbAp48 in the oocyte nucleus. We show that RPD3 associates with RbAp48 through N- and C-terminal contacts and that RbAp48 also interacts with SIN3. Xenopus RbAp48 selectively binds to the segment of the N-terminal tail immediately proximal to the histone fold domain of histone H4 in vivo. Exogenous RPD3 may be targeted to histones through interaction with endogenous RbAp48 to direct transcriptional repression of the Xenopus TRbetaA promoter in the oocyte nucleus. However, the exogenous RPD3 deacetylase functions to repress transcription in the absence of a requirement for association with SIN3 or other targeted corepressors. (+info)
SWItched-on mobility.
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Recent studies have shown that two nucleosome-remodeling complexes, NURF and CHRAC, open chromatin for transcription and replication by using their common catalytic subunit, the nucleosomal ATPase ISWI, to increase the mobility of nucleosomes relative to DNA sequence. (+info)
RbAp48 belongs to the histone deacetylase complex that associates with the retinoblastoma protein.
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The retinoblastoma susceptibility gene product, the Rb protein, is a key regulator of mammalian cell proliferation. One of the major targets of Rb is the S phase inducing E2F transcription factor. Once bound to E2F, Rb represses the expression of E2F-regulated genes. Transcriptional repression by Rb is believed to be crucial for the proper control of cell growth. Recently, we and others showed that Rb represses transcription through the recruitment of a histone deacetylase. Interestingly, we show here that the Rb-associated histone deacetylase complex could deacetylate polynucleosomal substrates, indicating that other proteins could be present within this complex. The Rb-associated protein RbAp48 belongs to many histone deacetylase complexes. We show here that the histone deacetylase HDAC1 is able to mediate the formation of a ternary complex containing Rb and RbAp48. Moreover, less deacetylase activity was found associated with Rb in cell extracts depleted for RbAp48 containing complexes, demonstrating that Rb, histone deacetylase, and RbAp48 are physically associated in live cells. Taken together, these data indicate that RbAp48 is a component of the histone deacetylase complex recruited by Rb. Finally, we found that E2F1 and RbAp48 are physically associated in the presence of Rb and HDAC1, suggesting that RbAp48 could be involved in transcriptional repression of E2F-responsive genes. (+info)
Histone binding protein RbAp48 interacts with a complex of CREB binding protein and phosphorylated CREB.
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A CREB-CREB binding protein (CBP) complex was used as bait to screen a mouse embryo cDNA library in yeast. One of the strongest interactions identified the histone binding protein RbAp48. RbAp48 also interacted weakly with CBP alone but did not interact with phosphorylated or nonphosphorylated CREB. CBP (or its homologue p300) from HeLa cell nuclear extracts coimmunoprecipitated with RbAp48 and its homologue RbAp46 and bound to a glutathione S-transferase-RbAp48 fusion protein. This interaction was stimulated by the addition of phosphorylated CREB and allowed the association of core histones and mononucleosomes in an acetylation-dependent manner. RbAp48 lowered the K(m) of CBP histone acetylase activity and facilitated p300-mediated in vitro transcription of a chromatinized template in the presence of acetylcoenzyme A. These data indicate that the association of phosphorylated CREB with CBP promotes the binding of RbAp48 and its homologue RbAp46, allowing the formation of a complex that facilitates histone acetylation during transcriptional activation. (+info)
The histone deacetylase HDAC3 targets RbAp48 to the retinoblastoma protein.
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The product of the retinoblastoma susceptibility gene, the Rb protein, functions partly through transcriptional repression of E2F-regulated genes. Repression by Rb is mediated, at least in part, by a histone deacetylase complex, whose enzymatic activity relies on HDAC1, HDAC2 or HDAC3. Recently, we have shown that the Rb-associated histone deacetylase complex contains RbAp48 protein, which interacts with HDAC1 and HDAC2. RbAp48 could favour the deacetylation of histones since it binds directly to histone H4. In agreement with that, we show that transcriptional repression of E2F activity requires the presence of RbAp48. HDAC3 was thought not to interact with RbAp48. However, we found that it shared with HDAC1 the ability to favour the recruitment of RbAp48 to Rb. This latter effect was unlikely to be due to activation of Rb function, since HDAC3 did not increase Rb-E2F1 interaction. Rather, we found, surprisingly, that HDAC3 could physically interact with RbAp48 both in vitro and in living cells. Taken together, our data suggest a model in which Rb mediates the recruitment to E2F-regulating promoters of a repressive complex containing either HDAC1, HDAC2 or HDAC3 and the histone-binding protein RbAp48. (+info)
Interaction between the Drosophila CAF-1 and ASF1 chromatin assembly factors.
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The assembly of newly synthesized DNA into chromatin is essential for normal growth, development, and differentiation. To gain a better understanding of the assembly of chromatin during DNA synthesis, we identified, cloned, and characterized the 180- and 105-kDa polypeptides of Drosophila chromatin assembly factor 1 (dCAF-1). The purified recombinant p180+p105+p55 dCAF-1 complex is active for DNA replication-coupled chromatin assembly. Furthermore, we have established that the putative 75-kDa polypeptide of dCAF-1 is a C-terminally truncated form of p105 that does not coexist in dCAF-1 complexes containing the p105 subunit. The analysis of native and recombinant dCAF-1 revealed an interaction between dCAF-1 and the Drosophila anti-silencing function 1 (dASF1) component of replication-coupling assembly factor (RCAF). The binding of dASF1 to dCAF-1 is mediated through the p105 subunit of dCAF-1. Consistent with the interaction between dCAF-1 p105 and dASF1 in vitro, we observed that dASF1 and dCAF-1 p105 colocalized in vivo in Drosophila polytene chromosomes. This interaction between dCAF-1 and dASF1 may be a key component of the functional synergy observed between RCAF and dCAF-1 during the assembly of newly synthesized DNA into chromatin. (+info)