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

dBRWD3 functions as a substrate receptor of Cul4-DDB1 E3 ubiquitin ligase in epigenetic regulation

蔡宗翰(Tsung-Han Tsai)

指導教授 ： 吳君泰

國立臺灣大學/醫學院/分子醫學研究所/碩士(2012年)

https://doi.org/10.6342/NTU.2012.02010

摘要

在發育的過程中，表觀遺傳調控對於基因的表現上提供了。基因表現的活性決定於其所處位置上組蛋白特定官能基的修飾以及其染色質的結構。組蛋白上的修飾是可逆的，由一群稱作histone modifier 的蛋白負責加上或去除修飾。除了一部份具有酵素功能的histone modifier能夠直接調控組蛋白上修飾之外，有另一群不具有酵素能力的indirect histone modifier能夠解讀組蛋白上修飾組成的代碼，並且藉由吸引其他histone modifier到特定位置上來引發後續反應。indirect histone modifier的特徵為具有和組蛋白上修飾作用的蛋白結構如bromodomain或chromodomain來辨認特定的組蛋白修飾代碼。 BRWD3 (bromodomain and WD40 repeat domain containing 3) 為一個與人類X染色體相關的智能發展遲緩 (X-link mental retardation) 相關的疾病基因，其特徵為靠近胺基端的WD40 repeat以及靠近羧基端的兩個連續bromodomain。BRWD3在演化上從人類至果蠅皆具有高度保留性，在人類中有三個BRWD3同源基因分別稱為BRWD3、PHIP (pleckstrin homology domain interacting protein) 以及BRWD1，而在果蠅中BRWD3 (dBRWD3)並不具有其他同源基因。除了具有設想為辨認組蛋白修飾的bromodomain之外，BRWD3也被報導過為可能的受質受體在由Cul4所組成的E3泛素連接酶複合體中。Cul4組成的E3泛素連接酶複合體被報導與對於基因表觀遺傳調控上扮演多種調控角色，暗示著BRWD3可能扮演著indirect histone modifier的角色。在我們的研究中利用果蠅的PEV實驗發現到dBRWD3作用在促進形成環繞中節的異染色質的結構。另外，dBRWD3對於維持抑制性組蛋白修飾 (dimethylation of lysine 9 on histone 3)也扮演重要角色。再者，在染色質免疫沈澱的實驗中驗證了dBRWD3的bromodomain具有與組蛋白作用的能力，雖然目前我們還無法確定其專一性辨認的特定組蛋白修飾。除此之外，我們利用免疫沈澱實驗證實了dBRWD3以及Cul4組成的E3泛素連接酶複合體中扮演adaptor protein角色的 DDB1 (damage-specific DNA binding protein 1) 之間的相互作用，並且找出在dBRWD3上對於其相互作用重要的作用區域。由上述的結果可推測dBRWD3的角色在辨認染色質上已存在的特定組蛋白修飾並且維持H3K9me2的量來形成異染色質。未來我們將會利用 histone peptide array進行活體外結合試驗來找出dBRWD3辨認的特定組蛋白修飾。另一方面，我們將會利用無法與DDB1作用的突變dBRWD3對於BRWD3失去功能所造成的H3K9me2量下降來進行rescue experiment看dBRWD3對於H3K9me2的調控是否需要Cul4以及DDB1的參與。

關鍵字

BRWD3 ； heterochromatin ； Cul4 ； DDB1

並列摘要

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.

並列關鍵字

BRWD3 ； heterochromatin ； Cul4 ； DDB1

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