Department of Cardiothoracic Surgery, Xiangya Second Hospital, Central South University, Changsha, Hunan, 410011, PR China.
BMC Mol Biol. 2011 Oct 14;12:44. doi: 10.1186/1471-2199-12-44.
CD164 (also known as MGC-24v or endolyn) is a sialomucin which has been suggested to participate in regulating the proliferation, cell adhesion and differentiation of hematopoietic stem and progenitor cells. CD164 is also involved in the development of cancer. The functions of cis-regulatory elements of CD164 remain relatively unknown.
In this study, we investigated the function of cis-regulatory elements within the promoter of CD164. We fused the 5'-flanking region of CD164 to a luciferase reporter vector. The minimal promoter region was confirmed by luciferase reporter assay. Using in silico analysis, we found the presence of one HIF-1alpha (HIF-1A) motif (5_-RCGTG-3_) overlapping E-box (CACGTG) and two AP-1-like binding sites (CGCTGTCCC, GTCTGTTG), one of which is also overlapped with HIF-1alpha sequence. Dual-luciferase assay was performed to examine the transcriptional activity of AP-1 and HIF-1alpha of CD164 promoter. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was performed to measure CD164 expression. Chromatin Immunoprecipitation was used to confirm the binding of HIF-1alpha and CD164.
Co-transfection of c-jun, HIF-1alpha and minimal promoter region construct demonstrated that c-jun and HIF-1alpha bound the CD164 promoter and promoted CD164 expression. Hypoxia treatment also led to the up-regulation of CD164 expression. The mutation of overlapping sequences resulted in the reduced expression of CD164 induced by HIF-1alpha. Chromatin Immunoprecipitation demonstrated that the HIF-1alpha bound the minimal promoter region.
Determination of the optimal promoter region and transcription factors governing CD164 expression is useful in understanding CD164 functions. These results suggest that cis-regulatory elements of CD164 overlapping HIF-1alpha/E-box/AP-1-like sequences may play important regulatory roles.
CD164（也称为 MGC-24v 或 endolyn）是一种唾液酸粘蛋白，据推测它参与调节造血干细胞和祖细胞的增殖、细胞黏附和分化。CD164 也参与癌症的发展。CD164 的顺式调控元件的功能仍然相对未知。
在这项研究中，我们研究了 CD164 启动子内顺式调控元件的功能。我们将 CD164 的 5' 侧翼区与荧光素酶报告载体融合。通过荧光素酶报告基因检测证实了最小启动子区。通过计算机分析，我们发现存在一个 HIF-1alpha（HIF-1A）基序（5'-RCGTG-3_）与 E 盒（CACGTG）重叠，以及两个 AP-1 样结合位点（CGCTGTCCC，GTCTGTTG），其中一个也与 HIF-1alpha 序列重叠。双荧光素酶检测用于检测 CD164 启动子的 AP-1 和 HIF-1alpha 的转录活性。定量逆转录聚合酶链反应（qRT-PCR）用于测量 CD164 的表达。染色质免疫沉淀用于证实 HIF-1alpha 和 CD164 的结合。
c-jun、HIF-1alpha 和最小启动子区构建体的共转染表明 c-jun 和 HIF-1alpha 结合 CD164 启动子并促进 CD164 表达。缺氧处理也导致 CD164 表达上调。重叠序列的突变导致 HIF-1alpha 诱导的 CD164 表达减少。染色质免疫沉淀表明 HIF-1alpha 结合最小启动子区。
确定调控 CD164 表达的最佳启动子区和转录因子对于理解 CD164 功能很有用。这些结果表明，CD164 的顺式调控元件与 HIF-1alpha/E 盒/AP-1 样序列重叠可能发挥重要的调控作用。
