探討ZNF883在人類富潛能幹細胞中調控自我更新的功能及機制

The functions and mechanisms of ZNF883 in regulating self-renewal human pluripotent stem cells

江法萱(Fa-Hsuan Chiang)

指導教授 ： 黃筱鈞

共同指導教授 ： 呂仁(Jean Lu)

國立臺灣大學/生命科學院/分子與細胞生物學研究所/碩士(2021年)

https://doi.org/10.6342/NTU202101723

摘要

根據呂仁實驗室陳薇如博士先前的研究，發現PODXL在人類富潛能幹細胞中，可以藉由調控膽固醇細胞的代謝維持細胞自我更新的能力。鋅手指蛋白883 (Zinc finger protein 883, ZNF883) 為PODXL的下游基因。然而，ZNF883是個新穎的基因，從未在細胞中探討過它的功能。本研究揭露ZNF883富含於未分化的人類富潛能幹細胞中，但於分化細胞中會減少表現。抑制ZNF883會導致幹細胞自我更新的能力受損，且造成KLF4 和 OCT4的表現量在人類富潛能幹細胞中下降。在人類誘導性富潛能幹細胞進行逆編程的過程中，ZNF883會在後期增加表現量。另外，我建立ZNF883過度表達的系統，細胞經去氧羥四環素 (doxycycline) 處理後，ZNF883會被誘發增加表現量。ZNF883過量表達，可促進細胞自我更新。為了進一步探討ZNF883的調控路徑，我透過互補核苷酸微陣列 (cDNA microarray)及染色質免疫沉澱-測序 (ChIP-seq)，選出五個ZNF883的下游基因BAK1、 SACM1L、ARHGAP42、FNBP1L和 EIF2AK4。當抑制BAK1時，可以挽救在人類誘導性富潛能幹細胞中剔除ZNF883所造成的幹細胞自我更新能力的損傷及細胞凋亡。不論是在有無過度表現ZNF883的細胞中減少SACM1L、ARHGAP42、FNBP1L和 EIF2AK4表達，皆會造成細胞數量及鹼性磷酸酶 (alkaline phosphatase, ALP) 的活性下降。總結以上發現，ZNF883對於維持人類富潛能幹細胞中自我更新的能力是很重要的。而ZNF883如何藉由下游基因去維持幹細胞自我更新的調控機制仍尚待研究。

關鍵字

鋅指蛋白 ； 人類胚胎幹細胞 ； 人類誘導性富潛能幹細胞 ； 幹細胞自我更新 ； 細胞逆編程序

並列摘要

According to the previous research from Dr. Wei-Ju Chen of Dr. Jean Lu’s lab, that PODXL controls the cholesterol metabolism to maintain the self-renewal in human pluripotent stem cells (hPSC). Zinc finger protein 883 (ZNF883) is a downstream gene of PODXL. However, ZNF883 is a novel gene, its function in the cells has never been explored. Here, I showed that ZNF883 is enriched in the undifferentiated hPSCs, while it is decreased in the differentiated cells. Downregulation of ZNF883 leads to impair the self-renewal and decreased the expression levels of core self-renewal markers such as KLF4 and OCT4 in hPSCs. During the human-induced pluripotent stem cell (hiPSC) reprogramming, ZNF883 is highly expressed at the later time points. In addition, I generated an inducible ZNF883-overexpressed human embryonic stem cell (hESC) lines, ZNF883 is upregulated by doxycycline treatment. The upregulation promotes hESC self-renewal. To further explore the ZNF883 regulatory biological pathway, I selected the five downstream genes (BAK1, SACM1L, ARHGAP42, FNBP1L and EIF2AK4) of ZNF883 by cDNA microarray and ChIP-seq. BAK1 is upregulated and other selected genes SACM1L, ARHGAP42, FNBP1L and EIF2AK4 are downregulated upon ZNF883 knockout in the hiPSCs. Moreover, BAK1 knockdown rescues the depletion of self-renewal and apoptosis caused by ZNF883 knockout in hiPSCs. The reduction of SACM1L, ARHGAP42, FNBP1L and EIF2AK4 are decreased the relative cell numbers and alkaline phosphatase (ALP) activities in ZNF883-overexpressed or normal expressed cells. In summary, ZNF883 is important to maintain the self-renewal in hPSCs. The mechanism of how ZNF883 targets its downstream genes to regulate self-renewal remains to be studied.

並列關鍵字

Zinc-finger protein ； human embryonic stem cell ； human induced pluripotent stem cell ； self-renewal ； reprogramming

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