探討dBRWD3在表觀遺傳調控上之角色

Abstract

真核生物發育過程中，染色質上基因之表現會精密地受到表觀遺傳調控，而這樣的調控會導致染色質狀態在異染色質及真染色質間動態的改變，藉以調控基因轉錄。過去研究顯示組蛋白變異體H3.3置入染色質，以及核小體中組蛋白之後轉錄修飾的變動，在染色質狀態之動態調控上扮演著重要的角色。在細胞中，組蛋白修飾可以透過所謂的表觀遺傳編輯酵素，例如組蛋白乙醯基轉移酶(histone acetyltransferases，HATs) 作為編寫者；或者例如組蛋白去乙醯基酶(histone deacetylase，HDACs)作為抹除者。另一方面尚有所謂的表觀遺傳讀取蛋白，可藉由其所具有之特別的domain，像是bromodomain，來讀取染色質上特定的組蛋白修飾，並且吸引其他具有酵素催化活性之蛋白來改變組蛋白之後轉譯修飾。目前有很多帶有表觀遺傳讀取domain的蛋白被研究，尤其是具有bromodomain的蛋白們，以及其在辨識乙醯基化組蛋白及影響基因轉錄的特性。而在我們實驗室我們研究一個名為dBRWD3(Drosophila bromodomain and WD40 repeat domain containing 3)的蛋白，是一個具有兩個bromodomain的蛋白。dBRWD3是人類3個BRWD家族蛋白，BRWD3、PHIP (Pleckstrin homology domain interacting protein)及BRWD1在果蠅中唯一的同源基因(homologous gene)。BRWD3在演化上自果蠅到人類都居有高度的演化保留性，並且在文獻報導中指出BRWD3為人類X染色體連結智能發展遲緩 (X-link intellectual disability)疾病之相關蛋白。我們著重於研究dBRWD3是因為其所具有的兩個表觀遺傳讀取domain，分別是位在 N-terminal 的WD40 repeat domain 以及位在 C-terminal 之兩個前後排列的bromodomains。dBRWD3也被報導指出會做為受質受體蛋白，參與在DDB1-CUL4-ROC1 所組成之 E3 泛素酶複合體中。我們亦發現dBRWD3會負向調控組蛋白H3之變異體H3.3置入於染色質，而當dBRWD3突變時會導致組蛋白修飾之表現程度受影，暗示著dBRWD3在表觀遺傳調控上具有多樣化角色。考慮到dBRWD3所具有的兩個前後排列之bromodomains，我們好奇dBRWD3是否能藉由bromodomains來讀取特殊的組蛋白修飾，進而間接改變組蛋白後轉譯修飾來動態調控染色質狀態及基因轉錄程度。 我們利用histone peptide array analysis，藉由體外試驗許多不同的組蛋白修飾來找出dBRWD3的兩個bromodomains個別之專一性辨認位點：bromodomain I會辨識H4K12ac及H4K16ac，而bromodomain II 則會辨識H2AK9ac、H2AK13ac、H3K18ac、 H3K14ac、H4K5ac以及H4K8ac。外我們也確認了在dBRWD3突變下染色質上組蛋白變異體H3.3的置入，以及轉錄相關組蛋白標記，例如H3K4me1 and H3K9me2表現程度的改變，暗示著dBRWD3在基因轉錄調控上扮演重要角。最後我們利用免疫沉澱純化出帶有Flag標記之dBRWD3，透過質譜分析來找出在表觀遺傳調控上會與之有交互作用的相關蛋白。我們好奇dBRWD3在表觀遺傳調控對基因表現調控上的角色，而我們的發現對於dBRWD3如何透過間接改變染色質狀態來調控基因轉錄提供新的線索。

In eukaryotes development, the gene expression on chromatin can be accurately managed through epigenetic regulation, and such regulation can change chromatin status from hetrochromatin to euchromatin or vice versa to turn on or turn off gene transcription. The deposition of histone variant H3.3 and changes of the post-translational modification (PTM) on histones of nucleosome have been implicated in regulating the chromatin status. In cells, histone modification can be directly regulated through epigenetic-editor enzymes, for instance, histone acetyltransferases (HATs) as the writer, or histone deacetylase (HDACs) as the eraser. On the other hand, the epigenetic-reader proteins recognize specific histone modifications on chromatin through specific domain such as bromodomain, and recruit other modifier proteins with enzymatic activity to modify the PTM of hisotnes. Lots of these epigenetic-reader domain containing proteins have been studied, especially the bromodomain containing proteins due to their recognition on acetylated histones and gene transcriptional regulation. In our lab, the protein named dBRWD3 (Drosophila bromodomain and WD40 repeat domain containing 3) is a double bromodomain-containg protein. dBRWD3 is the homologue of 3 human BRWD family proteins, the BRWD3, PHIP (pleckstrin homology domain interacting protein), and BRWD1. BRWD3 is evolutionarily conserved from Drosophila to human and has been reported as a human X-link intellectual disability-associated protein. We focused on dBRWD3 owing to its two epigenetic-reader domains, the N-terminal WD40 repeat domain and the C-terminal tandem bromodomains. dBRWD3 was reported as a substrate receptor in DDB1-CUL4-ROC1-based E3 ubiquitin ligase complexes. In our lab, we previously discovered that dBRWD3 regulates gene expression through a negative regulation of histone H3 variant H3.3 deposition on chromatin, and we uncovered that mutations in dBRWD3 change the expression levels of many histone modifications, implying a versatile role of dBRWD3 in epigenetic regulation. Considering its tandem bromodomains, we wondered whether dBRWD3 can recognize specific histone modification by its bromodomains, and indirectly edit the PTM of histone to dynamically regulate chromatin status and gene expression. We performed histone peptide array analysis, examining the binding of the two bromodomains with various histone modifications in vitro. The histone peptide array analysis revealed the specific recognitions of bromodomain I and II respectively. Bromodomain I can recognize H4K12ac and H4K16ac, and bromodomain II can recognize H2AK9ac, H2AK13ac, H3K18ac, H3K14ac, H4K5ac and H4K8ac. In addition, we also confirmed that under dBRWD3 mutation, the chromatin incorporation of histone variant H3.3 increased. The levels of transcriptional related histone markers, such as H3K4me1 and H3K9me2 changed in dBRWD3 mutant cells, implying the critical roles of dBRWD3 in gene expression. Finally, we performed immunoprecipitation to purify Flag-tagged dBRWD3 for mass spectrometry analysis in order to identify proteins associated with dBRWD3 in epigenetic regulation. We investigate the role of dBRWD3 in epigenetic regulation, and our findings provide new insights to how dBRWD3 regulates gene expression by indirectly editing chromatin status.