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 在哺乳动物细胞中，蛋白质的N末端乙酰化是由N末端乙酰基转移酶(NAT)介导的。在人和小鼠细胞中，NAT的主要形式是由NARG1亚基和催化亚基ARD1A组成，其中催化亚基是酶活性所必需的。在体细胞组织中，NARG1-ARD1A是NAT的主要形式。然而，在小鼠睾丸生殖细胞中，ARD1A亚单位在雄性减数分裂后被ARD1B取代。ARD1B在睾丸中的发现表明蛋白质N末端乙酰化在生殖细胞发育完成中的重要性。另一方面，ARD1a与癌症进展和神经元树突发育有关。ARD1A或ARD1B的上游和下游靶点尚未完全阐明。为了探讨蛋白质N末端乙酰化的生物学意义，我们重点研究了ARD1A和ARD1B基因表达的调控机制，以及通过基因敲除和基因敲除策略对其产物的生物学功能的影响。 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。与以往报道的不同，我们发现ARD1a是在人类细胞培养中主要表达的ARD1亚型。与其小鼠同源基因相似，我们发现位于人ARD1B基因启动子区域的CpG岛受到DNA甲基化的影响；而人ARD1B基因的表达受DNA甲基化的调控。我们利用siRNA技术击倒了ARD1A的表达，并鉴定了一组受ARD1A基因去除影响其表达水平的基因，这表明ARD1A介导的生物学功能并不局限于蛋白质修饰。大多数受影响的基因被定位到未确定特征的基因组位置。有趣的是，在没有ARD1A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之间的关系，以阐明ARD1A的生物学作用。同时，我们正在利用ARD1B基因敲除小鼠研究ARD1B在睾丸发育中的生物学作用。我们的初步数据表明，ARD1B表达缺失所产生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，以确定发育的敏感阶段，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.