Expression and localization of components of the histone deacetylases multiprotein repressory complexes in the mouse preimplantation embryo

DNA methylation had been implicated in the assembly of multiprotein repressory complexes that affect chromatin architecture thereby rendering genes inactive. Proteins containing methyl binding domains (MBDs) are major components of these complexes. MBD3 is a component of the HDAC associated chromatin remodeling complex Mi2/NuRD. The addition of MBD2 to the Mi2/NuRD complex creates MeCP1, a complex that is known to inactivate methylated promoters. The undermethylated state of the mouse preimplantation embryo prompted us to investigate the known repressory complexes at this developmental stage. We found individual components of Mi2/NuRD: MBD3, Mi2, HDAC1 and HDAC2 to be expressed from a very early stage of embryo development and to localize in close proximity with each other and with constitutive heterochromatin by the blastula stage. Expression of MBD2, a component of MeCP1, starts in the blastula stage. Then it is also found to be in proximity with heterochromatin (based on DAPI staining) and with MBD3, Mi2 and HDAC1. In contrast, expression of MeCP2, an MBD containing component of a third repressory complex (MeCP2/Sin3A), is not seen in the preimplantation embryo. Our results suggest that both Mi2/NuRD and MeCP1 complexes are already present at the very early stages of embryo development, while a MeCP2 complex is added to the arsenal of repressory complexes post-implantation at a stage when DNA methylation takes place.     

Role of the Sin3-Histone Deacetylase Complex in Growth Regulation by the Candidate Tumor Suppressor p33ING1

Sin3 is an evolutionarily conserved corepressor that exists in different complexes with the histone deacetylases HDAC1 and HDAC2. Sin3-HDAC complexes are believed to deacetylate nucleosomes in the vicinity of Sin3-regulated promoters, resulting in a repressed chromatin structure. We have previously found that a human Sin3-HDAC complex includes HDAC1 and HDAC2, the histone-binding proteins RbAp46 and RbAp48, and two novel polypeptides SAP30 and SAP18. SAP30 is a specific component of Sin3 complexes since it is absent in other HDAC1/2-containing complexes such as NuRD. SAP30 mediates interactions with different polypeptides providing specificity to Sin3 complexes. We have identified p33ING1b, a negative growth regulator involved in the p53 pathway, as a SAP30-associated protein. Two distinct Sin3-p33ING1b-containing complexes were isolated, one of which associates with the subunits of the Brg1-based Swi/Snf chromatin remodeling complex. The N terminus of p33ING1b, which is divergent among a family of ING1 polypeptides, associates with the Sin3 complex through direct interaction with SAP30. The N-terminal domain of p33 is present in several uncharacterized human proteins. We show that overexpression of p33ING1b suppresses cell growth in a manner dependent on the intact Sin3-HDAC-interacting domain.     

Protein Kinase CK2 Regulates the Dimerization of Histone Deacetylase 1 (HDAC1) and HDAC2 during Mitosis

Histone deacetylase 1 (HDAC1) and HDAC2 are components of corepressor complexes that are involved in chromatin remodeling and regulation of gene expression by regulating dynamic protein acetylation. HDAC1 and -2 form homo- and heterodimers, and their activity is dependent upon dimer formation. Phosphorylation of HDAC1 and/or HDAC2 in interphase cells is required for the formation of HDAC corepressor complexes. In this study, we show that during mitosis, HDAC2 and, to a lesser extent, HDAC1 phosphorylation levels dramatically increase. When HDAC1 and -2 are displaced from the chromosome during metaphase, they dissociate from each other, but each enzyme remains in association with components of the HDAC corepressor complexes Sin3, NuRD, and CoREST as homodimers. Enzyme inhibition studies and mutational analyses demonstrated that protein kinase CK2-catalyzed phosphorylation of HDAC1 and -2 is crucial for the dissociation of these two enzymes. These results suggest that corepressor complexes, including HDAC1 or HDAC2 homodimers, might target different cellular proteins during mitosis.     

人源HDAC1/2-RbAp46/48核心复合物三维空间构象的电子显微分析

HDAC1、HDAC2和RbAp46、RbAp48是许多重要功能复合物(如NuRD、Sin3等)的核心亚基.这4个亚基在空间上相互作用,形成一个具有去乙酰化酶活性的核心复合物.但该核心复合物的三维空间构象及其对去乙酰化、染色质重塑等功能的可能影响还所知甚少.本研究中,我们包装了含4个亚基的杆状病毒,利用昆虫细胞表达、纯化了HDAC1/2-RbAp46/48核心复合物.在此基础上,利用电子显微镜单颗粒分析方法对该去乙酰化酶核心复合物的三维结构进行了初步解析.结果表明,HDAC1、HDAC2、RbAp46和RbAp48可以形成一个较为稳定均一的复合物,但该复合物中各个亚基并不是以单拷贝、等比例形式存在的.该核心复合物呈现一个非对称的鞍型结构,其背部隆起,大致形成一个三角形,两边分别有一大一小的两翼,两翼中间有个凹槽,直径大约为6 nm,推测为该核心复合物与核小体的结合位置.本研究结果为了解HDAC1/2-RbAp46/48去乙酰化酶复合物各亚基的空间结构组成、与核小体和染色质的可能相互作用以及研究去乙酰化酶活性的作用机理等提供了有益的信息.     

Multiple histone deacetylases are recruited by corepressor Sin3 and contribute to gene repression mediated by Opi1 regulator of phospholipid biosynthesis in the yeast Saccharomyces cerevisiae

Yeast genes of phospholipid biosynthesis are negatively regulated by repressor protein Opi1 when precursor molecules inositol and choline (IC) are available. Opi1-triggered gene repression is mediated by recruitment of the Sin3 corepressor complex. In this study, we systematically investigated the regulatory contribution of subunits of Sin3 complexes and identified Pho23 as important for IC-dependent gene repression. Two non-overlapping regions within Pho23 mediate its direct interaction with Sin3. Previous work has shown that Sin3 recruits the histone deacetylase (HDAC) Rpd3 to execute gene repression. While deletion of SIN3 strongly alleviates gene repression by IC, an rpd3 null mutant shows almost normal regulation. We thus hypothesized that various HDACs may contribute to Sin3-mediated repression of IC-regulated genes. Indeed, a triple mutant lacking HDACs, Rpd3, Hda1 and Hos1, could phenocopy a sin3 single mutant. We show that these proteins are able to contact Sin3 in vitro and in vivo and mapped three distinct HDAC interaction domains, designated HID1, HID2 and HID3. HID3, which is identical to the previously described structural motif PAH4 (paired amphipathic helix), can bind all HDACs tested. Chromatin immunoprecipitation studies finally confirmed that Hda1 and Hos1 are recruited to promoters of phospholipid biosynthetic genes INO1 and CHO2.