| The human DHRS4 gene cluster consists of DHRS4, DHRS4L2 and DHRS4L1. DHRS4, the fourth member of the dehydrogenase/reductase (SDR) family, is a gene encoding NADP(H)-dependent retinol dehydrogenase/reductase, that is a crucial enzyme in the synthesis of retinoic acid, thus correlated with the signal transduction, proliferation and differentiation of cells. In our previous study, we cloned the alternative spliced RNAs of DHRS4 and DHRS4L2, and identified exon a1 both as a novel exon contained in the RNA variants of DHRS4L2 and as an alternative transcription start site for DHRS4L2. DHRS4L1, due to an information update of GenBank in December of 2008, was renamed from the gene LOC728635, located downstream of DHRS4 and DHRS4L2 and similar to the peroxisomal short-chain alcohol dehydrogenase, but little is known about its structure, expression and function. Since DHRS4, DHRS4L2 and DHRS4L1 constitute the gene cluster as a whole, we are interested in the questions about what being the differences among them in gene structure, whether there are any common characters for them in transcription, and which factors possible participating in the transcriptional regulation for the gene cluster.To investigate the transcription and transcriptional regulation of the human DHRS4 gene cluster, in present study: (1) We firstly cloned the RNA variants of the newly named gene DHRS4L1 through RT-PCR and RACE PCR, and found that DHRS4L1 initiated its transcription from two transcriptional starts sites, exon a2 and exon 1. Exon a2 matches with the intergene sequence, approximate 29 kb upstream of DHRS4L1, and is homologous to exon a1 located upstream of DHRS4L2. (2) Through immunoprecipitation and mass spectrometry, a splicing RNA variant, designated NRDR A2, of human DHRS4 gene was identified. Green fluorescent protein-reporter assay showed that NRDR A2 protein mainly localized to the nuclei. The subcelluar localization of NRDR A2 was different with NRDR, which was previously reported localizing in peroxisomes. (3) Based on the results of our previous studies and the data from GenBank, we found that exon 1 was absent in all the spliced variants of DHRS4L2 and DHRS4L1 initiated from exon a1 and exon a2 respectively. Analysis of the sequence upward the exon 1 indicates that exon 1 lacks a canonical splice site of AG at its 5’ends. We proposed that alternative transcriptional start sites were accompanied by alternative splicing and a possible reason for the removal of exon 1 from the transcripts of DHRS4L2 and DHRS4L1. (4) Naturally occurring antisense gene C14orf167 locates in the minus strand of DHRS4 gene, belonging to the head-to-head bidirectional transcription. For the homologous identity of DHRS4 gene cluster and the relationship between C14orf167 and DHRS4, we predicted that antisense transcripts might regulate the transcription initiation and posttranscriptional process of DHRS4, DHRS4L2 and DHRS4L1 in a cis- and trans-acting manner. (5) In human hepatocarcinoma cell line HepG2 and human hepatocyte cell line HL-7702, the real-time RT-PCR results revealed that mRNA express level of DHRS4, DHRS4L2 and DHRS4L1 were upregulated after the antisense transcripts were silenced by RNAi. (6) The results of mythylation specific PCR revealed that the CG islands of DHRS4 and DHRS4L1 in HepG2 and HL-7702 were hypomethylated, and that of DHRS4L2 was hypermethylated. Knocking down the antisense transcripts resulted in the hypomethylation of DHRS4L2 CG islands.Summarily, through analyzing the gene structures and transcriptional behaviors of the DHRS4 gene cluster, we proposed a possible mechanism involving in the alternative transcriptional start sites regulating the posttranscriptional splicing of the initial exon of DHRS4L2 and DHRS4L1, and verified that antisense RNA might regulate the DNA methylation and mRNA level of DHRS4 gene cluster. The present results might provide clues for further study about the function of DHRS4 gene cluster and the mechanism of antisense RNA regulating the the transcription of plus genes. |
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