Kadariya Yuwaraj, Nakatani Kaname, Nishioka Junji, Fujikawa Takahiko, Kruger Warren D, Nobori Tsutomu
Department of Laboratory Medicine, Mie University School of Medicine, Tsu, Mie 514-8507, Japan.
Biochem J. 2005 Apr 1;387(Pt 1):175-83. doi: 10.1042/BJ20041472.
hMTAP (human 5'-deoxy-5'-methylthioadenosine phosphorylase) is a key enzyme in the methionine salvage pathway and is frequently inactivated in human tumour cells. To understand the mechanism of the transcriptional regulation of the MTAP gene, we have cloned the 1.29 kb fragment of the hMTAP promoter and identified cis-acting regulatory sequences using a luciferase reporter gene assay. Maximal promoter activity was associated with sequences between -446 and -152, where two CCAAT elements were located. Electrophoretic mobility-shift assay reveals binding of specific complexes at both CCAAT motifs within the MTAP promoter, although more prominent bands were associated with the distal motif (-372 to -368). Supershift experiments and chromatin immunoprecipitation assays indicate that both the proximal and distal complexes bind CBF (CCAAT-binding factor; also known as nuclear factor-Y), and that the distal CCAAT motif has increased levels of CBF binding. We have mapped seven different transcriptional start sites between -135 and -58. Our results show that the hMTAP expression is regulated by a CBF and that the distal one of two CCAAT motifs plays a major role in the transcriptional activation of hMTAP gene.
人5'-脱氧-5'-甲硫腺苷磷酸化酶（hMTAP）是甲硫氨酸补救途径中的关键酶，在人类肿瘤细胞中经常失活。为了解MTAP基因转录调控的机制，我们克隆了hMTAP启动子的1.29 kb片段，并使用荧光素酶报告基因检测法鉴定了顺式作用调控序列。最大启动子活性与位于-446至-152之间的序列相关，该区域有两个CCAAT元件。电泳迁移率变动分析显示，MTAP启动子内两个CCAAT基序处均有特异性复合物结合，尽管与远端基序（-372至-368）相关的条带更为明显。超迁移实验和染色质免疫沉淀分析表明，近端和远端复合物均结合CCAAT结合因子（CBF；也称为核因子-Y），且远端CCAAT基序的CBF结合水平增加。我们在-135至-58之间定位了七个不同的转录起始位点。我们的结果表明，hMTAP的表达受CBF调控，且两个CCAAT基序中的远端基序在hMTAP基因的转录激活中起主要作用。
