DBRWD3 作為 Cul4-DDB1 E3泛素接合酶的受質受體在表觀遺傳調控上的功能

Abstract

Epigenetic regulation of gene transcription provides dynamic expression profile during the developmental process. The transcriptional activities of genes rely on its chromatin structure and the corresponding histone modifications. The histone modifications are reversible and could be add or removed by histone modifiers. Despite of histone modifiers with enzymatic activities that directly regulate histone modifications, there are indirected histone modifiers without enzymatic activity that could interpret the histone modification codes and trigger the subsequent functional readout by recruiting secondary histone modifiers. The indirect histone modifiers are characterized by its histone modifications-interacting domains such as bromodomain or chromodomain for recognition of histone codes. The dBRWD3 (bromodomain and WD40 repeat domain containing 3), which is associated with human X-link mental retardation, is characterized by its N-terminal WD40 repeat and C-terminal double bromodomains. The BRWD3 is evolutionally conserved from Drosophila to mammalian, in which there are three homologue called BRWD3, PHIP (pleckstrin homology domain interacting protein) and BRWD1, while there is only one orthologue (dBRWD3) in Drosophila. In addition to the putative function of BRWD3 bromodomain in recognition of histone modifications, BRWD3 is reported to be a substrate receptor in Cul4-based E3 ubiquitin ligase complex, which possesses diverse functions in epigenetic regulation, implying a role of BRWD3 as indirect histone modifier. Here, we found that dBRWD3 plays a role in promoting the structure of pericentric heterochromatin in Drosophila PEV assay. In addition, dBRWD3 is required for maintaining the level of repressive histone modification, dimethylation of lysine 9 on histone 3. Furthermore, the bromodomains of dBRWD3 are capable of interacting with histone proteins by chromatin immunoprecipitation experiment, although the specific recognition pattern of histone modifications has not yet been identified. Moreover, we have confirmed the association of BRWD3 with DDB1 (damage-specific DNA binding protein 1), which is an adaptor protein in Cul4-based E3 ubiquitin ligase by immunoprecipitation experiment and mapped the critical motif for its interaction by deletion mutation assay. The data above suggest the role of BRWD3 in recognition of primary histone modification and maintaining the secondary modification (H3K9me2) for heterochromatin formation. In future, we are going to identify the specific histone code recognized by BRWD3 via performing in vitro binding assay with histone peptide array. In addition, we will perform rescue experiments of decreased level of H3K9me2 in BRWD3 loss-of-function with mutant BRWD3 that could not be associated with Cul4-based E3 ubiquitin ligase to test if BRWD3 maintains the level of H3K9me2 in a Cul4- and DDB1-dependent manner.