Wang Sen, Zhen Lei, Liu Zhu, Ai Qing, Ji Ying, Du Gang, Wang Yitao, Bu Youquan
Department of Biochemistry and Molecular Biology, Chongqing Medical University, Chongqing 400016, China; Molecular Medicine and Cancer Research Center, Chongqing Medical University, Chongqing 400016, China.
Department of Biochemistry and Molecular Biology, Chongqing Medical University, Chongqing 400016, China; Molecular Medicine and Cancer Research Center, Chongqing Medical University, Chongqing 400016, China.
Biochem Biophys Res Commun. 2015 May 15;460(4):1008-14. doi: 10.1016/j.bbrc.2015.03.142. Epub 2015 Apr 2.
Hyaluronan (HA) is a key component of the vertebrate extracellular matrix that is synthesized at the plasma membrane by the hyaluronan synthases including HAS1, HAS2 and HAS3. The expression and regulation of HAS1-3 are implicated in numerous physiological and pathological processes. The promoters of human HAS1 and HAS2 genes have been identified previously whereas HAS3 promoter remains unclear. In the present study, we have for the first time identified and characterized the human HAS3 gene promoter region. 5' RACE assay revealed two novel transcriptional variants of HAS3 gene with distinct transcription start sites. Progressive deletion analysis of the 5'-flanking region of HAS3 gene demonstrated that HAS3 proximal promoter is mainly restricted to a 450-bp region (i.e. -761 to -305 bp upstream of the major HAS3 transcription start site), whereas its core promoter is located to a minimal 129-bp region (i.e. -433 to -305 bp upstream of the major HAS3 transcription start site). Transcriptional factor binding analysis indicated that HAS3 gene promoter lacks of canonical TATA box, but contains classical GC box as well as other putative binding sites for transcriptional factors such as C/EBP and NFκB. In addition, site-directed mutagenesis assay demonstrated that the proximal Sp1 binding site is essential for the robust proximal promoter activity of HAS3 gene whereas the core MTE (core promoter motif ten elements) motif is required for the basic core promoter activity of HAS3 gene. Our present study should facilitate further studies on the mechanism regulating the expression of this important gene.
透明质酸（HA）是脊椎动物细胞外基质的关键成分，由包括HAS1、HAS2和HAS3在内的透明质酸合酶在质膜上合成。HAS1 - 3的表达和调控涉及众多生理和病理过程。人类HAS1和HAS2基因的启动子此前已被鉴定，而HAS3启动子仍不清楚。在本研究中，我们首次鉴定并表征了人类HAS3基因启动子区域。5' RACE分析揭示了HAS3基因的两种新型转录变体，其转录起始位点不同。对HAS3基因5'侧翼区域的逐步缺失分析表明，HAS3近端启动子主要局限于一个450 bp的区域（即主要HAS3转录起始位点上游 - 761至 - 305 bp），而其核心启动子位于一个最小的129 bp区域（即主要HAS3转录起始位点上游 - 433至 - 305 bp）。转录因子结合分析表明，HAS3基因启动子缺乏典型的TATA盒，但含有经典的GC盒以及其他转录因子如C/EBP和NFκB的假定结合位点。此外，定点诱变分析表明，近端Sp1结合位点对于HAS3基因强大的近端启动子活性至关重要，而核心MTE（核心启动子基序十元件）基序对于HAS3基因的基本核心启动子活性是必需的。我们目前的研究应有助于进一步研究该重要基因表达的调控机制。
