背景:组蛋白甲基转移酶缺失,小,或同源盘1-like(ASH1L)由su(var)3-9,zeste的增强剂组成,三thorax(SET)域,pleckstrin同源域(PHD)域,中间(MID)域,和溴相邻同源(BAH)结构域。已知ASH1L的SET结构域介导H3K36二甲基化(H3K36me2)修饰。然而,PHD-BAH结构域的特定功能在很大程度上仍未被探索。本研究旨在探讨ASH1L中PHD-BAH结构域的生物学功能。
方法:我们采用了一系列技术,包括原核融合蛋白表达纯化系统,下拉分析,等温滴定量热法(ITC)聚合酶链反应(PCR),和定点诱变,集群定期间隔短回文重复(CRISPR-Cas9)基因编辑,细胞培养实验,westernblot,细胞增殖试验,和细胞凋亡试验。
结果:ASH1L中的PHD-BAH结构域相对于H3K4单甲基化(H3K4me1)和H3K4三甲基化(H3K4me3)肽优先结合H3K4me2肽。值得注意的是,PHD-BAH结构域内的W2603A突变可在体外破坏与H3K4me2的相互作用.与野生型胆管癌(CHOL)细胞相比,ASH1L中PHD-BAH结构域的缺失导致CHOL细胞凋亡增加和细胞增殖减少(P<0.001)。此外,W2603A突变影响了蛋白酶体20S亚基β(PSMB)家族基因集的调控。
结论:W2603A突变对于PHD-BAH结构域与H3K4me2肽之间的相互作用至关重要。ASH1L通过调节PSMB家族基因集的表达,通过其PHD-BAH结构域调节CHOL细胞的存活和增殖。
BACKGROUND: Histone methyltransferase absent, small, or homeotic discs1-like (
ASH1L) is composed of su(var)3-9, enhancer of zeste, trithorax (SET) domain, pleckstrin homology domain (PHD) domain, middle (MID) domain, and bromo adjacent homology (BAH) domain. The SET domain of
ASH1L is known to mediate mediate H3K36 dimethylation (H3K36me2) modification. However, the specific functions of the PHD-BAH domain remain largely unexplored. This study aimed to explore the biological function of the PHD-BAH domain in
ASH1L.
METHODS: We employed a range of techniques, including a prokaryotic fusion protein expression purification system, pull-down assay, Isothermal Titration Calorimetry (ITC), polymerase chain reaction (PCR), and sitedirected mutagenesis, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR-Cas9) gene editing, cell culture experiment, western blot, cell proliferation assay, and cell apoptosis test.
RESULTS: The PHD-BAH domain in
ASH1L preferentially binds to the H3K4me2 peptide over H3K4 monomethylation (H3K4me1) and H3K4 trimethylation (H3K4me3) peptide. Notably, the W2603A mutation within the PHD-BAH domain could disrupt the interaction with H3K4me2 in vitro. Compared with wild-type Cholangiocarcinoma (CHOL) cells, deletion of the PHD-BAH domain in
ASH1L led to increased CHOL cell apoptosis and reduced cell proliferation (P < 0.001). Additionally, the W2603A mutation affected the regulation of the proteasome 20S subunit beta (PSMB) family gene set.
CONCLUSIONS: W2603A mutation was crucial for the interaction between the PHD-BAH domain and the H3K4me2 peptide. ASH1L regulated CHOL cell survival and proliferation through its PHD-BAH domain by modulating the expression of the PSMB family gene set.