Cis-regulatory functions of overlapping HIF-1alpha/E-box/AP-1-like sequences of CD164
1 Department of Cardiothoracic Surgery, Xiangya Second Hospital, Central South University, 139 Renmin Zhong Road, Changsha, Hunan, 410011, P.R. China
2 Cancer Research Institute, Key Laboratory of Carcinogenesis and Cancer Invasion of Ministry of Education, Key Laboratory of Carcinogenesis of Ministry of Health, Central South University, 110 Xiangya Road, Changsha, Hunan, 410078, P.R. China
3 Centre for Cancer Research and Cell Biology, School of Biomedical Sciences, Queen's University Belfast, Belfast, BT9 7BL, Northern Ireland, UK
4 School of Medicine, Shenzhen University, Shenzhen, P.R. China
5 The Li Ka Shing Institute of Health Sciences, Department of Orthopaedics & Traumatology, Faculty of Medicine, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, NT, Hong Kong
6 Stem Cell and Regeneration Program, School of Biomedical Sciences, The Chinese University of Hong Kong, Shatin, NT, Hong Kong, P.R. China
BMC Molecular Biology 2011, 12:44 doi:10.1186/1471-2199-12-44Published: 14 October 2011
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.