TRIM28又稱作KAP1或是TIF1β,是一個在細胞中大量表現并參與在細胞分化以及生長等多種重要生理反應的一個蛋白質。它通過其N端的RBCC domains (RING domain, 兩個 B-box zinc fingers and a coiled coil domain)來和帶有KRAB domain 的鋅指蛋白結合,並且通過C端的PHD-Bromo domains 把輔抑制物複合體帶過來,並且通過和HP1結合來誘導異染色質產生。TRIM28的這種輔抑制能力是經由轉譯後修飾來調控的,因而我們認為如果有TRIM28賴氨酸304換成穀氨醯胺來模擬乙醯化的細胞,我們應該可以看到TRIM28和鋅指蛋白的這種結合會受到影響。在K562細胞中,用CRISPR-Cas9技術製造了一個敲除了TRIM28的細胞株,並且也製造了敲入了一個在賴氨酸304的位置換成穀氨醯胺的TRIM28來模擬賴氨酸的乙醯化的細胞株。我們通過即時聚合酶鏈式反應和RNA定序來觀察穀氨醯胺304突變株的基因的表達發生了怎樣的變化。我們在榖氨醯胺304突變和TRIM28敲除突變中觀察到長非編碼RNA H19,胚胎期表現的ε球蛋白和胎兒時期表現的γ球蛋白的表現量上升;成年期表現的β球蛋白,球蛋白抑制物SOX6的表現量下降。我們也觀測到巨核細胞分化的標記物——整合素β3 和其他一些跟細胞分化有關的基因表現量上升了,這暗示賴氨酸304的乙醯化可能會促進細胞的分化。有趣的是,TRIM28的重要結合蛋白質MAGEC2的信使RNA的量在穀氨醯胺304突變株和TRIM28敲除細胞株中也下降了,具體機制目前不明。我們自己純化出了一個anti-TRIM28-N抗體用來做免疫沉澱的實驗,並把抗體抓到的蛋白質送去做質譜分析,看看正常TRIM28和突變的TRIM28各自抓到的蛋白質會有哪些不同。我們找到了一個叫做GID/CTLH複合物的蛋白質,這個蛋白質只能在正常的TRIM28的質譜資料中找到。我們進一步利用免疫沉澱法證實了這兩個蛋白質真的會相互結合而不是無選擇性抓到的雜質,這是這兩個蛋白質第一次被報導會有相互作用。綜合來看,TRIM28在賴氨酸304的乙醯化可能通過影響到那些會跟TRIM28相互作用的蛋白質來影響到那些被TRIM28調節的基因的表現。
Tripartite motif-containing 28 (TRIM28), also known as KRAB (Krüppel-associated box repression) domain associated protein 1 (KAP1) or transcriptional intermediary factor 1 beta (TIF1β), is a ubiquitously expressed protein that plays a role in many important physiological phenomena such as cellular differentiation and proliferation. It associates with KRAB-Zinc finger proteins (ZNFs) via N-terminal RBCC domains (RING domain, two B-box zinc fingers and a coiled coil domain), recruiting corepressor complexes by C-terminal PHD-Bromo domains and forms heterochromatin through interacting with HP1. The corepressor activity of TRIM28 was regulated by post-translational modifications. We have demonstrated that acetylation-mimic TRIM28-K304Q mutant weakens the interaction with KRAB-ZNFs. The TRIM28-K304Q knock-in K562 mutant and TRIM28 homozygous knock-out K562 cells have been created by CRISPR-Cas9 gene editing. In this study, we performed real-time PCR and RNA-seq to delineate gene expression in TRIM28 K304Q K562 cells. We found that lncRNA H19, embryo epsilon globin and fetal gamma globin genes were up-regulated, while adult beta globin, and a globin gene repressor SOX6 were down-regulation in the TRIM28 K304Q mutant. The expression levels of megakaryocyte marker integrin beta3 and some of differentiation-related genes were increased in TRIM28 K304Q cells, indicating constant K304 acetylation might promote cell differentiation. Interestingly, the mRNA expression of MAGEC2, a binding partner of TRIM28 for E3 ligase activity regulation, was down-regulated in K304Q and homozygous knock-out cells through unclear mechanism. Rabbit anti-TRIM28-N (N-terminal) antibody was used for isolation of TRIM28 and TRIM28-K304Q and their associated proteins precipitate TRIM28 from K562 cells by immunoprecipitation followed by mass spectrometry LC/MS/MS analysis to investigate the change in TRIM28 association protein. GID/CTLH E3 ligase complexes could only be detected in wild-type K562 while compared with the TRIM28-K304Q mutant. The protein-protein interactions of them have been further confirmed by immunoprecipitation analysis. Taken together, our results suggest that the TRIM28-K304 acetylation may modulate TRIM28-mediated gene expression via regulation of its interaction with KRAB-ZNFs.