探討Zfp280d調控PDX1下游基因的分子機制

Unravel the Molecular Mechanism of Zfp280d Modulation of PDX1 Downstream Target Genes

張育豪(Yu-Hao Chang)

指導教授 ： 江明格

國立中正大學/理學院/生命科學系分子生物研究所/碩士(2014年)

摘要

在發育過程中有一些轉錄因子 (transcription factor)被發現在胰臟生成是很重要的，在這些因子中，PDX1在決定內胚層中原腸腔 (primitive gut)發育成胰臟的位子是很重要的。因此，在PDX1基因缺陷小鼠無法發育胰臟組織。
為了進一步研究PDX1，我們實驗室利用酵母菌雙雜交系統 (yeast two-hybrid assay)以PDX1當成餌搜尋胚胎10.5天胰臟芽cDNA基因庫 (E10.5 pancreatic bud cDNA library)。其中一個被辨認出來的基因是尚未被研究的鋅模組蛋白Zinc finger protein 280d (Zfp280d)。在小鼠Zfp280家族有三個成員，包含Zfp280b、Zfp280c與Zfp280d。Zfp280d包含九個鋅模組及一個DUF4195區域。有趣的是Zfp280d蛋白質序列與Zfp280b及Zfp280c很類似，除了他們的DUF4195區域。我們實驗室先前利用蛋白質交互作用沉澱 (pull-down assay)及共同免疫沉澱(co-immunoprecipitation)證實了Zfp280d與PDX1有交互作用。此外，我們也證實PDX1與Zfp280d共同座落 (co-localized)在細胞核內。
我也更進一步探討Zfp280d與PDX1的交互作用關係，並指出只有Zfp280d的DUF4195區域能與PDX1有交互作用。我們假設PDX1可能藉由Zfp280d的DUF4195區域去活化或抑制其下游基因的表現。此外，我在一個β細胞株NIT-1減弱Zfp280d的表現，觀察到Pax6及Insulin表現降低。這些結果指出Zfp280d可能在維持成熟β細胞的功能扮演著重要的角色。

關鍵字

轉分化 ； 胰臟 ； Zfp280d ； PDX1

並列摘要

During development, several transcription factors are found crucial for the formation of the pancreas. Among these factors, PDX1 plays a critical role in defining the region of the primitive gut that will form the pancreas; as a result, PDX1-deficient mice fail to develop pancreatic tissue.
To further understand the action of PDX1, we have utilized the yeast two-hybrid assay to screen an E10.5 pancreatic bud cDNA library using PDX1 as a bait. One of the identified gene is zinc finger protein 280d (Zfp280d) whose function has not been described before. There are three members in mice zinc finger protein 280 (Zfp280) family, including Zfp280b, Zfp280c and Zfp280d. Zfp280d contains nine zinc finger domains and a DUF4195 domain. Interestingly, the Zfp280d protein is very similar to Zfp280b and Zfp280c, except in its DUF4195 domain. Our lab has previously proved that Zfp280d interacts with PDX1 by pull-down and co-immunoprecipitation experiments. In addition, we have also shown that PDX1 and Zfp280d are co-localized in cell nucleus.
I have further characterized the interaction between Zfp280d and PDX1 and shown that only the DUF4195 domain in Zfp280d interacts with PDX1. We hypothesize that PDX1 might bring Zfp280d through its DUF4195 domain to activate or repress the expression of its downstream target genes. In addition, I have knockdowned the expression of Zfp280d in a β-cell line, NIT-1, and observed decreased expression of Pax6 and insulin. These results suggest Zfp280d may also play an important role in maintaining mature β-cell functions.

並列關鍵字

transdifferentiation ； pancreas ； Zfp280d ； PDX1

參考文獻

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