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甲胎蛋白(AFP)在胎肝中高表达,出生后不久就快速下调至很低水平。有关肝脏AFP基因出生后的转录抑制机制一直是未解之谜。AFP基因的增强子、抑制区域和启动子等顺式调控元件可能参与其转录抑制的调节,但有关的关键性转录抑制因子尚不明确。我们发现了一种新型锌指蛋白ZBTB20,为研究其体内的生理功能,我们利用条件打靶技术,建立了ZBTB20的组织特异性基因敲除小鼠模型,发现肝细胞特异性ZBTB20基因敲除小鼠出生后的肝脏AFP基因一直维持近乎胎肝的高水平表达,而其肝细胞处于正常的静息状态。生化分析显示,ZBTB20具有转录抑制活性,体外可有效抑制AFP启动子的转录活性;染色质免疫沉淀和EMSA实验结果显示ZBTB20体内和体外能直接结合AFP启动子。在小鼠肝脏的发育过程中,ZBTB20基因被渐进激活,与AFP基因的表达水平呈负相关,提示激活的ZBTB20参与了AFP基因的转录抑制。上述结果表明ZBTB20是调控AFP基因表达的关键转录因子,由此我们认为肝脏ZBTB20基因出生后的表达上调及其发挥的转录抑制作用是导致AFP基因出生后快速下调的主要原因。
Alpha-fetoprotein (AFP) is highly expressed in the fetal liver and rapidly decreases to very low levels shortly after birth. The mechanism of transcriptional repression after the birth of liver AFP gene has been a mystery. AFP gene enhancers, inhibitory regions and promoters and other cis-regulatory elements may participate in the regulation of transcriptional repression, but the relevant key transcriptional repressor is not yet clear. We found a new zinc finger protein ZBTB20, in order to study its physiological function in vivo, we use conditional targeting technology to establish ZBTB20 tissue-specific knockout mouse model and found that hepatocyte-specific ZBTB20 knockout mice Postnatal liver AFP gene has been maintained near the high level of fetal liver expression, and its liver cells in normal resting state. Biochemical analysis showed that ZBTB20 had transcriptional inhibitory activity and could effectively inhibit the transcriptional activity of AFP promoter in vitro. Chromatin immunoprecipitation and EMSA showed that ZBTB20 could directly bind AFP promoter in vivo and in vitro. During the development of mouse liver, the ZBTB20 gene was gradually activated, and negatively correlated with the expression level of AFP gene, suggesting that activated ZBTB20 is involved in the transcriptional repression of AFP gene. These results indicate that ZBTB20 is a key transcription factor that regulates AFP gene expression. Therefore, we think that the up-regulation of liver ZBTB20 gene expression and its transcriptional repression are the main reasons leading to the rapid down-regulation of AFP gene after birth.