TRIM28在脂肪前驅細胞分化過程中之調控

TRIM28又稱作KAP1或是TIF1β，它是一個調控轉錄因子，會和帶有KRAB domain的鋅指蛋白結合並抑制基因的表現。TRIM28的N端包含了Ring finger、B boxes以及coiled-coil domain，這三個domain稱作RBCC domain，此domain會形成三聚體並與KRAB domain結合，而C端的PHD以及Bromo domain則會和組蛋白去乙醯酶以及甲基轉移酶等複合物結合。TRIM28在發育及分化的過程中是個重要的調控因子。而我們想了解TRIM28在脂肪分化的過程當中所扮演的角色及功能為何。首先，利用shRNA (short hairpin RNA)將3T3-L1脂肪前驅細胞當中的TRIM28進行knockdown，藉由觀察細胞分化型態以及分析基因的表現，結果發現3T3-L1脂肪前驅細胞無法正常分化成脂肪細胞。進一步透過RNA定序分析，結果發現3T3-L1的細胞週期以及PPARγ 訊息路徑受到了knockdown TRIM28影響。另外，我們發現調控脂肪分化的抑制因子Dlk1可能會受到TRIM28的調控。TRIM28的胺基酸上有許多的修飾已經被確定，並且被證明其在基因調控上的功能為何，但詳細的機制仍然不清楚。位於473的絲胺酸之磷酸化在脂肪分化的過程中已被我們偵測到，並建立模擬磷酸化的突變S473E以及不磷酸化的突變S473A；同時，我們也想探究位於305的離胺酸之乙醯化，於是建立了模擬乙醯化的突變K305Q以及不乙醯化的突變K305R，並進行其生化及功能分析。透過電泳遲滯法證明K305Q的突變會降低與Gal4 DBD-KRAB之間的交互作用。我們也利用共免疫沉澱法證明K305Q和S473E這兩個突變會降低與zinc finger 30 (Zfp30)之間的交互作用。另外，將S473A、S473E、K305Q以及K305R這四個突變分別過表現在3T3-L1脂肪前驅細胞當中，試圖探究TRIM28之磷酸化及乙醯化在脂肪分化過程中的功能，並進一步了解他們之間與Zfp30之間的關係，希望能夠找到調控脂肪分化相關的目標基因。

關鍵字

TRIM28 ；乙醯化；脂肪前驅細胞；脂肪分化；鋅指蛋白

並列摘要

The tripartite motif-containing protein 28 (TRIM28), also known as KRAB-associated protein 1 (KAP1) or transcriptional intermediary factor 1-beta (TIF1β), is a transcription corepressor of KRAB-ZFPs (Krüppel-associated box containing zinc finger proteins). TRIM28 contains N-terminal RBCC domain for trimer formation and interacting with KRAB domain, and C-terminal PHD and Bromo domain for interacting with histone deacetylase and methyltransferase complexes. TRIM28 is a crucial regulator in development and differentiation. We would like to investigate its function and regulation in adipogenesis. Knockdown of TRIM28 by transducing lentivirus-carrying shRNAs impairs the differentiation of 3T3-L1 preadipocytes, demonstrated by morphological observation and gene expression analysis. RNA-seq analysis was performed in TRIM28 knockdown 3T3-L1 cells to identify TRIM28-regulated genes. Cell cycle regulators and PPARγ signaling pathway were significantly affected by knockdown of TRIM28. One of adipogenesis repressor, Dlk1, might be downregulated by TRIM28 during adipogenesis. Several amino acid residues of TRIM28 for phosphorylation, sumoylation and acetylation have been identified and play functions in gene regulation. The profiling of serine 473 phosphorylation was monitored during adipogenesis. To demonstrate the function of acetylation at lysine 305 which located in RBCC domain, the acetyl-mimetic K305Q and non-acetyl K305R mutants were generated for biochemical and functional analysis. The electrophoretic mobility shift assay (EMSA) showed that the K305Q mutant decreases its interaction with DNA-bound Gal4 DBD-KRAB. Co-immunoprecipitation also confirmed that K305Q or S473E mutant interacts weakly with a KRAB-ZFP, Zfp30. 3T3-L1 cells ectopically expressed K305Q or K305R mutant was generating to explore the function of TRIM28 acetylation in adipogenesis. We suggest that in response to differentiation signals, TRIM28 might be acetylated and phosphorylated to reduce interaction with KRAB-ZFPs like Zfp30, leading to activation of targeted genes.

並列關鍵字

TRIM28 ； acetylation ； 3T3-L1 ； adipogenesis ； ZFPs

參考文獻

1. Friedman, J.R., et al., KAP-1, a novel corepressor for the highly conserved KRAB repression domain. Genes Dev, 1996. 10(16): p. 2067-78.

2. Peng, H., et al., Reconstitution of the KRAB-KAP-1 repressor complex: a model system for defining the molecular anatomy of RING-B box-coiled-coil domain-mediated protein-protein interactions. J Mol Biol, 2000. 295(5): p. 1139-62.

3. Ryan, R.F., et al., KAP-1 corepressor protein interacts and colocalizes with heterochromatic and euchromatic HP1 proteins: a potential role for Kruppel-associated box-zinc finger proteins in heterochromatin-mediated gene silencing. Mol Cell Biol, 1999. 19(6): p. 4366-78.

4. Quenneville, S., et al., The KRAB-ZFP/KAP1 system contributes to the early embryonic establishment of site-specific DNA methylation patterns maintained during development. Cell Rep, 2012. 2(4): p. 766-73.

5. Cammas, F., et al., Mice lacking the transcriptional corepressor TIF1beta are defective in early postimplantation development. Development, 2000. 127(13): p. 2955-63.

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TRIM28在脂肪前驅細胞分化過程中之調控

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