MBD3 and HDAC1, Two Components of the NuRD Complex, Are Localized at Aurora-A-positive Centrosomes in M Phase

Abstract

MBD3, a component of the histone deacetylase NuRD complex, contains the methyl-CpG-binding domain (MBD), yet does not possess appreciable mCpG-specific binding activity. The functional significance of MBD3 in the NuRD complex remains enigmatic, partly because of the limited availability of biochemical approaches, such as immunoprecipitation, to analyze MBD3. In this study, we stably expressed the FLAG-tagged version of MBD3 in HeLa cells. We found that MBD3-FLAG was incorporated into the NuRD complex, and the MBD3-FLAG-containing NuRD complex was efficiently immunoprecipitated by anti-FLAG antibodies. By exploiting this system, we found that MBD3 is phosphorylated in vivo in the late G 2 and early M phases. Moreover, we found that Aurora-A, a serine/threonine kinase active specifically in the late G 2 and early M phases, phosphorylates MBD3 in vitro, physically associates with MBD3 in vivo, and co-localizes with MBD3 at the centrosomes in the early M phase. Interestingly, HDAC1 is distributed at the centrosomes in a manner similar to MBD3. These results suggest the highly dynamic nature of the temporal and spatial distributions, as well as the biochemical modification, of the NuRD complex in M phase, probably through an interaction with kinases, including Aurora-A. These observations will contribute significantly to the elucidation of the yet-uncharacterized cell cyclecontrolled functions of the NuRD complex.Core histones are subjected to a variety of post-translational modifications, including acetylation and methylation (1). These modifications affect the chromatin structure and/or the accessibility of transcriptional regulatory factors to chromatin and thereby influence gene expression. Acetylation has been the most extensively investigated among them. Generally, hyperacetylated histones are correlated with the transcriptionally active state, whereas hypoacetylated histones are correlated with the silent state (reviewed in Ref. 2). The relative level of histone acetylation is determined by the equilibrium between two opposite enzymatic activities, histone acetyltransferases (HATs) 1 and histone deacetylases (HDACs). Each of the HAT and HDAC groups comprises a protein family consisting of many member proteins. These proteins do not function by themselves. Instead, they form large multiprotein complexes together with other components, such as DNA/chromatin-binding proteins and co-activator/co-repressor, as well as other rather ill-defined proteins. Among the members of the HDAC family, HDAC1, HDAC2, and HDAC3 comprise one subgroup (Class I HDACs) because of their homology to yeast HDAC Rpd3. HDAC1 and HDAC2 are present as two major complexes, namely, NuRD and SIN3 (reviewed in Ref. 3). These two HDAC complexes share some components, including HDAC1 and HDAC2, and contain specific components. NuRD is unique in that it possesses another catalytic activity, the ATP-dependent chromatin-remodeling activity performed by Mi2.The acetylation of histones is not a stationary process. Instead, it is known that the genera...

hMLH1 promoter methylation and silencing in primary endometrial cancers are associated with specific alterations in MBDs occupancy and histone modifications

“…For example, MBD2, but not MBD1 or MeCP2, was found to interact with methylated P16/Ink4A, P14/ ARF [32] and GSTP1 [33]. MBD3 is part of the NuRD complex involved in control of gene expression in early development [34]. The current study demonstrated that MeCP2, MBD1 and MBD2, but not MBD3 and MBD4, bind to methylated hMLH1 promoters.…”

Dynamic epigenetic states of ribosomal RNA promoters during the cell cycle

“…49 UBF is also phosphorylated and inactivated in mitosis. 5 MBD3 undergoes phosphorylation at G 2 /M by the cell cycle dependent kinase Aurora A, 50 however it is unknown whether this affects its ability to bind DNA. SL-1 is reactivated and remains bound in G 1 , 5 which is thought to allow the basal transcriptional levels of rRNA, while acetylation and phosphorylation by cdk4/cyclin D1 and cdk2/Cyclin E&A of UBF promotes its interaction with Pol I, 6,7,13 thus allowing for increased transcription.…”

Function and regulation of Aurora/Ipl1p kinase family in cell division

“…However, it remains to be established the physiological relevance of the TPX2 phosphorylation in bipolar spindle formation during mitosis and whether Aurora-A mediated phosphorylation alters the properties of TPX2 microtubule interaction. MBD3, a component of the histone deacetylase NuRD complex, was also found to interact with Aurora-A and colocalized with it on the centrosomes [32]. In addition, MBD3 is a substrate of Aurora-A.…”

“…Phosphorylation of histone H3 at serine 10 occurs during mitosis in diverse eukaryotes and correlates closely with mitotic and meiotic chromosome condensation. Failure in phosphorylated H3 showed abnormal chromosome Role of the chromosomal passenger complex on chromosomes and the central spindle [32]. A. INCENP targe Aurora-B to histone H3 through associating with heterocromatin protein HP-1, coordinating survivin.…”