TRIM28在脂肪分化過程表觀遺傳調控Dlk1表現量

TRIM28 epigenetically regulates Dlk1 expression in adipogenesis

林潔如(Chieh-Ju Lin)

指導教授 ： 張瀞仁

國立臺灣大學/生命科學院/生化科學研究所/碩士(2018年)

https://doi.org/10.6342/NTU201801277

摘要

TRIM28在基因轉錄上扮演輔抑制物，可以做為支架與帶有KRAB domain相關的鋅指蛋白ZFP相互作用，並招募異染色質蛋白1(HP1)、組蛋白相關甲基化酶、組蛋白去乙醯基化酶和DNA甲基化酶等複合物共同使基因沉默。並且TRIM28是一個在發育、分化過程中以及參與印記基因表現相當重要的調節者。我們證明了敲低TRIM28會抑制3T3L1 脂肪前驅細胞正常的脂肪分化過程。但其中的關聯尚未明瞭，因此想更進一步了解TRIM28介導的脂肪分化過程其中的分子機制，而欲透過RNA-seq的結果找出TRIM28 的作用目標基因。Dlk1又稱pref-1，當在脂肪分化零天與兩天的3T3L1 脂肪前驅細胞中敲低TRIM28會導致Dlk1表現量上升。而Dlk1他是一個印記基因，並且在脂肪分化過程中作為抑制者，隨後我們也更進一步證明了在原本已經敲低TRIM28再同時敲低Dlk1可以回復脂肪細胞的分化。另外透過了螢光素酶報告基因的實驗證明了TRIM28對於Dlk1 啟動子有抑制活性的作用。另外，Dlk1的基因表現是由基因組上的甲基化差異區域所調控。我們也透過種亞硫酸鹽DNA甲基化實驗去分析Dlk1這段印記基因上DNA甲基化的情形，比較Trim28敲低的細胞跟控制組，結果顯示在Dlk1 啟動子上三個基因組甲基化差異區上DNA甲基化的程度並無明顯差異。更進一步透過染色質免疫沉澱實驗分析了在Dlk1 啟動子與各個基因組甲基化差異區上相關Trim28結合和組蛋白修飾的情況，證明了TRIM28會結合在Dlk1啟動子和IG-DMR。敲低Trim28造成在Dlk1啟動子上H3K4me3結合程度上升與H3K27me3結合程度下降與最終Dlk1基因轉錄活化吻合。最後我們透過螢光素酶報告基因分析顯示Trim28可能會藉由Zfp57蛋白的招募而更加抑制Dlk1啟動子的活性。綜合以上結果，我們的研究證明Trim28可以藉由下調Dlk1的表現使前驅脂肪細胞正常分化。

關鍵字

TRIM28 ； Dlk1 ； 表觀遺傳學 ； 脂肪分化

並列摘要

Trim28 is a scaffold corepressor, interacting with Krüppel-associated box-zinc finger proteins (KRAB-ZFPs) and recruiting heterochromatin protein 1 (HP1), histone methyltransferases, histone deacetylases and DNA methyltransferases for gene silencing. It is a crucial regulator in development, differentiation and imprinted gene expression. We demonstrated that knockdown of Trim28 in 3T3-L1 preadipocyte inhibited adipogenesis. To understand the molecular mechanism of Trim28-mediated adipogenesis, the RNA-seq was performed to find out the possible Trim28-targeted genes. Dlk1 (delta-like homolog 1), also named pref-1(preadipocyte factor-1) was increased in Trim28 knockdown 3T3-L1 cells before induction to differentiation and induced to differentiation for 2 days. Dlk1 is an imprinted gene and known as an inhibitor of adipogenesis. We have confirmed that further knockdown of Dlk1 in Trim28 knockdown 3T3-L1 can rescue cell differentiation. The luciferase reporter assay showed that Trim28 inhibited Dlk1 promoter-driven luciferase activity in a dose-dependent manner. It was known that the paternal imprinted Dlk1 expression was regulated by DMRs (differentially methylated regions). The bisulfite DNA methylation analysis indicated that there are no differences of DNA methylation pattern on Dlk1 promoter and three associated DMRs between Trim28 knockdown and control cells during adipogenesis. The chromatin-immunoprecipitation (ChIP) analysis displayed that TRIM28 targeted to the Dlk1 promoter and IG-DMR (Intergenic differentially methylated region). Moreover, knockdown of TRIM28 can increase H4K4me3 and decrease H3K27me3 on Dlk1 promoter, resulting in Dlk1 gene activation. Finally, by using luciferase reporter assay, Trim28 inhibited Dlk1 promoter-driven luciferase activity via ZFP57. Our findings provide evidence to prove that TRIM28 can downregulate Dlk1 for adipogenesis in 3T3-L1 cells.

並列關鍵字

TRIM28 ； Dlk1 ； epigenetic ； adipogenesis

參考文獻

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