Scavenger Chemokine (CXC Motif) Receptor 7 (CXCR7) Is a Direct Target Gene of HIC1 (Hypermethylated in Cancer 1)

Abstract

The tumor suppressor gene HIC1 (Hypermethylated in Cancer 1) that is epigenetically silenced in many human tumors and is essential for mammalian development encodes a sequencespecific transcriptional repressor. The few genes that have been reported to be directly regulated by HIC1 include ATOH1, FGFBP1, SIRT1, and E2F1. HIC1 is thus involved in the complex regulatory loops modulating p53-dependent and E2F1-dependent cell survival and stress responses. We performed genomewide expression profiling analyses to identify new HIC1 target genes, using HIC1-deficient U2OS human osteosarcoma cells infected with adenoviruses expressing either HIC1 or GFP as a negative control. These studies identified several putative direct target genes, including CXCR7, a G-protein-coupled receptor recently identified as a scavenger receptor for the chemokine SDF-1/CXCL12. CXCR7 is highly expressed in human breast, lung, and prostate cancers. Using quantitative reverse transcription-PCR analyses, we demonstrated that CXCR7 was repressed in U2OS cells overexpressing HIC1. Inversely, inactivation of endogenous HIC1 by RNA interference in normal human WI38 fibroblasts results in up-regulation of CXCR7 and SIRT1. In silico analyses followed by deletion studies and luciferase reporter assays identified a functional and phylogenetically conserved HIC1-responsive element in the human CXCR7 promoter. Moreover, chromatin immunoprecipitation (ChIP) and ChIP upon ChIP experiments demonstrated that endogenous HIC1 proteins are bound together with the C-terminal binding protein corepressor to the CXCR7 and SIRT1 promoters in WI38 cells. Taken together, our results implicate the tumor suppressor HIC1 in the transcriptional regulation of the chemokine receptor CXCR7, a key player in the promotion of tumorigenesis in a wide variety of cell types.HIC1 (Hypermethylated in Cancer 1) is a tumor suppressor gene that resides on the short arm of chromosome 17, a region that is frequently deleted and epigenetically silenced in human cancers (1-4). HIC1 encodes a transcriptional repressor with five Krüppel-like C 2 H 2 zinc fingers mediating DNA binding via its consensus binding site consisting of a 5Ј-(C/G)NG(C/G)-GGGCA(C/A)CC-3Ј sequence centered on a GGCA motif (2, 5). It also contains a central region that recruits CtBP 5 corepressor complexes (6) as well as an N-terminal BTB-POZ domain capable of autonomous transcriptional repression (7).HIC1 is a direct target gene of p53 transactivation through a p53-responsive element (2,8,9). A regulatory feedback loop between HIC1 and p53 has been deciphered in which HIC1 directly represses the transcription of SIRT1, which deacetylates and thereby inactivates p53 (10, 11). Therefore, inactivation of one allele of HIC1 results in the de-repression of SIRT1 causing decreased p53-mediated transactivation of the remaining HIC1 allele. In addition, SIRT1 also deacetylates HIC1 and thereby favors its SUMOylation, thus establishing optimal transcriptional repression (12). Recently, HIC1, SIRT1, and E2F1 have also been impli...

Loss of Hypermethylated in Cancer 1 (HIC1) in Breast Cancer Cells Contributes to Stress-induced Migration and Invasion through β-2 Adrenergic Receptor (ADRB2) Misregulation

“…Depending on the cell type, re-expression of HIC1 leads to proliferation arrest, differentiation, and apoptosis (1,14). Our recent results also demonstrate a role for HIC1 in the regulation of cell migration and invasion.…”

“…7 Nevertheless, we hypothesized that other target genes could also be implicated in these important biological processes. In a previous report of gene expression profiling, we published a list of genes repressed in U2OS osteosarcoma cancer cells following adenoviral infection and reexpression of HIC1 (14). Among them, we already validated the receptors coding genes CXCR7 and EphA2 as new HIC1 target genes (14,34).…”

“…U2OS cells were transfected in OptiMEM (Invitrogen) by the PEI (Euromedex) method in 12-well plates with 500 ng of DNA (14). Cells were transfected for 6 h and then were incubated in fresh complete medium.…”

“…In a previous report of gene expression profiling, we published a list of genes repressed in U2OS osteosarcoma cancer cells following adenoviral infection and reexpression of HIC1 (14). Among them, we already validated the receptors coding genes CXCR7 and EphA2 as new HIC1 target genes (14,34). Another gene present in our list, ADRB2, coding for a G-protein-coupled receptor, was repressed 5-fold in Ad-HIC1-infected cells as compared with control infected cells as early than 8 h postinfection (Fig.…”

“…To date, ten target genes play supporting roles in developmental and cell cycle control: histone deacetylase SIRT1 (10), the transcription factors ATOH1 (11), Sox9 (12), and ⌬Np73 (13), the G-protein coupled receptor CXCR7 (14), Cyclin D1 and P57KIP2 (CDKN1C) (8) and EFNA1, a cell surface ligand for Eph tyrosine kinase receptors (15).…”