TRIM28在前驅脂肪細胞分化過程中調控Dlk1表現之分子機制

Abstract

TRIM28也稱為TIF1β或KAP1，是分化和發育的重要調節因子。我們之前的研究表明敲低TRIM28的表現會抑制脂肪細胞分化。此外，在RNA-seq數據中我們發現了一種名為Dlk1的基因，該基因會被TRIM28下調。Dlk1亦稱為pref-1，已被報導為脂肪細胞分化的抑製劑，在脂肪細胞分化期間其mRNA表現會降低。我們證明了TRIM28在脂肪細胞分化過程中作用於Dlk1啟動子上並以表觀遺傳學方式調節Dlk1表達。然而，哪種DNA結合蛋白將TRIM28招集到Dlk1啟動子尚不清楚。根據先前的報導指出轉錄因子E2f1在脂肪細胞中可以轉錄活化Dlk1基因表達。除E2f1外，含有KRAB的Zfp57可以與TRIM28相互作用來維持H3K9甲基化，而Dlk1也是Zfp57的作用標的。在小鼠中，Zfp57和Zfp445也被報導在DLK1-DIO3基因座中共同作用以維持基因組印記。因此，我們假設在脂肪細胞分化過程中TRIM28介導的Dlk1表達可被E2f1、Zfp57和Zfp445調節。染色質免疫沉澱方法證實E2f1、Zfp57、Zfp445與Dlk1啟動子的結合。當敲除這些因子則會降低了Dlk1 mRNA表達並增強了脂肪生成。免疫共沉澱方法也證實它們與TRIM28的相互作用，且熒光素酶報導分子測定證實TRIM28可以下調這三種因子對Dlk1啟動子介導的熒光素酶報告基因的活性。此外，TRIM28作為與DNA甲基轉移酶和組蛋白去乙醯基化酶相互作用的一個支架蛋白。在3T3-L1細胞中加入組蛋白去乙醯基化酶抑製劑之後顯示Dlk1 mRNA表達有略微增加並抑制脂肪細胞分化。在脂肪前驅細胞上游的小鼠間充質C3H10T1/2細胞中，TRIM28可能在其中發揮不同的作用。在處理表觀遺傳學抑製劑後細胞分化良好但Dlk1表達沒有顯著變化，表明在C3H10T1/2細胞中這種表觀遺傳學調節不受Dlk1的影響。因此，我們的結果支持E2f1、Zfp57和Zfp445可以調節TRIM28對Dlk1 mRNA表達並影響3T3-L1脂肪前驅細胞分化。

TRIM28 (tripartite motif-containing protein 28), also known as TIF1β or KAP1, is an important regulator of differentiation and development. Our previous study has shown that knockdown of TRIM28 would inhibit adipogenesis. Furthermore, in the RNA-seq data we found a gene called Dlk1, which was downregulated by TRIM28. Dlk1, also called pref-1, has been reported as an inhibitor of adipogenesis, whose mRNA expression was decreased during adipogenesis. We demonstrated that TRIM28 targeted to Dlk1 promoter and epigenetically regulated Dlk1 expression during adipogenesis. However, which DNA-binding protein recruits TRIM28 to Dlk1 promoter is unclear. The transcription factor E2f1 was reported to transcriptionally activate Dlk1 gene expression in adipocytes. Except for E2f1, the KRAB-containing Zfp57 has known to interact with TRIM28 to maintain H3K9 methylation and Dlk1 is also a target of Zfp57. In mouse, Zfp57 and Zfp445 act together to maintain the genomic imprinting, especially in the DLK1-DIO3 locus. Therefore, we suggest that TRIM28-mediated Dlk1 expression may be modulated by E2f1, Zfp57 and Zfp445 during adipogenesis. Chromatin immunoprecipitation (ChIP) assay was used to confirm the binding of E2f1, Zfp57, Zfp445 on Dlk1 promoter. Knockdown of these factors decreased the Dlk1 mRNA expression and enhanced adipogenesis. Co-immunoprecipitation assay was performed to confirm their interaction with TRIM28 and luciferase reporter assays exhibited that TRIM28 can downregulate the activity of these three candidates on Dlk1 promoter-mediated luciferase reporter. In addition, TRIM28 serves as a corepressor scaffold interacting with DNA methylatransferases (DNMTs) and Histone deacetylases (HDACs). So, HDAC inhibitors was treated and showed that slightly increased Dlk1 mRNA expression to inhibit adipogenesis in 3T3-L1 cells. In the mouse mesenchymal C3H10T1/2 cells, which was upstream of preadipocytes, TRIM28 might play different role in it. After treating epigenetics inhibitors, cells differentiated well but the Dlk1 expression has no significantly alteration, indicating that this epigenetics regulation was not affected by Dlk1 in C3H10T1/2 cells. Taken together, our results provide the evidence to support that E2f1, Zfp57 and Zfp445 can regulate TRIM28 on Dlk1 mRNA expression in 3T3-L1 cells, which is important for adipogenesis.