Transcription regulation and functional studies of germ cell specific genes

N-末端乙酰转移酶的生物学功能及其基因表达调控 蛋白质N-末端乙酰化在哺乳动物细胞中由N-末端乙酰转移酶（NAT）介导。 NAT在人类和小鼠细胞中的主要形式由NARG 1亚基和催化亚基ARD 1A组成，其中催化亚基是酶活性所必需的。 在体细胞组织中，NARG 1-ARD 1A是NAT的主要形式。然而，在小鼠睾丸生殖细胞中，ARD 1A亚基在雄性减数分裂后被ARD 1B取代。 睾丸中ARD 1B的鉴定表明蛋白质N-末端乙酰化在生殖细胞发育完成中的重要性。 另一方面，ARD 1A与癌症进展和神经元树突发育有关。 ARD 1A或ARD 1B的上游和下游靶点尚未完全阐明。 为探讨蛋白质N端乙酰化的生物学意义，我们采用基因敲低和基因敲除策略，研究了ARD 1A和ARD 1B基因表达的调控机制及其产物的生物学功能。 我们以人癌细胞培养为模型系统，研究N-末端乙酰化过程的生物学意义。 与以前报道的不同，我们发现ARD 1A是在人类细胞培养中表达的主要ARD 1亚型。 与其小鼠同源基因相似，我们发现人ARD 1B基因启动子区的CpG岛存在DNA甲基化，人ARD 1B基因的表达受DNA甲基化调控。 我们通过siRNA技术敲低了ARD 1A的表达，并鉴定了一组有限的基因，其表达水平受到ARD 1A基因的去除的影响，这表明ARD 1A介导的生物学功能不仅限于蛋白质修饰。 大多数受影响的基因被定位到未表征的基因组位点。 有趣的是，在缺乏ARD 1A的情况下上调的基因包括非编码RNA和参与RNA加工和结合的基因。 我们现在正在研究这些基因和转录本的关系，以阐明ARD 1A的生物学作用。 同时，我们正在使用ARD 1B基因敲除小鼠研究ARD 1B在睾丸发育中的生物学作用。 我们的初步数据表明，ARD 1B表达的缺乏导致的生物学效应发生在雄性生殖细胞发育的后期。 进一步的研究正在进行中，以确定发育的易感阶段，并确定NAT在雄性生殖细胞中靶向的蛋白质底物。

Biological function of N-terminal acetyltransferase and its gene expression regulation Protein N-terminal acetylation is mediated in mammalian cells by N-terminal acetyltransferase (NAT). The major form of NAT in human and mouse cells is composed of the NARG1 subunit and the catalytic subunit ARD1A, in which the catalytic subunit is indispensible to enzymatic activity. In somatic tissues, the NARG1-ARD1A is the predominant form of NAT. In mouse testicular germ cells, however, the ARD1A subunit is replaced by ARD1B after male meiosis. The identification of ARD1B in the testis suggests the importance of protein N-terminal acetylation in the completion of germ cell development. On the other hand, ARD1A is implicated in cancer progression and neuronal dendritic development. The upstream and downstream targets of ARD1A or ARD1B are not completely elucidated. To investigate the biological significance of protein N-terminal acetylation, we focus on studying the regulatory mechanism of ARD1A and ARD1B gene expression, and the biological functions of their products by gene knockdown and gene knockout strategies. We used human cancer cell culture as a model system to study the biological significance of N-terminal acetylation process. Different from what was reported before, we found that ARD1A is the predominant ARD1 isoform expressed in human cell culture. Similar to its mouse gene ortholog, we discovered the CpG island located at the promoter region of human ARD1B gene is subject to DNA methylation; and the expression of human ARD1B is regulated by DNA methylation. We knocked down the expression of ARD1A by siRNA technology, and identified a limited set of genes whose expression levels are affected by the removal of ARD1A gene, suggesting that the biological function mediated by ARD1A is not limited to protein modification. The majority of the affected genes are mapped to uncharacterized genomic loci. Interestingly, the genes being upregulated in the absence of ARD1A include a non-coding RNA and genes that are involved in RNA processing and binding. We are now studying the relationship of these genes and transcripts to elucidate the biological role of ARD1A. Meanwhile, we are studying the biological role of ARD1B in testis development using ARD1B knockout mice. Our preliminary data suggest the biological effect resulting from the absence of ARD1B expression occurs late in male germ cell development. Further studies are underway to pinpoint the susceptible stage of development and to identify the protein substrates that are targeted by NAT in male germ cells.