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.
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