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 sequence-specific 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 genome-wide 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.

FIGURE 1.

HIC1 overexpression in U2OS osteosarcoma cells induces a proliferation arrest. A, expression of HIC1 and GFP in infected U2OS cells. U2OS cells were infected with Ad-FLAG-HIC1 (left panel) or Ad-GFP (right panel), and 24 h later the expression of HIC1 and GFP was analyzed. The HIC1 protein is detected by immunofluorescence with the anti-FLAG M2 monoclonal antibody in a punctate nuclear pattern. B, expression of HIC1 proteins in Ad-FLAG-HIC1-infected U2OS cells. Equal amounts of total protein extracts were subjected to Western blotting (WB) using the anti-FLAG monoclonal antibody. C, overexpression of HIC1 through adenoviral infection induces a growth arrest in U2OS cells. U2OS osteosarcoma cells were seeded in 60-mm plates and infected with Ad-FLAG-HIC1 or Ad-GFP at a multiplicity of infection of 100 to allow infection of at least 90% of the cells. At the indicated times, cell counts were made on five microscopic fields, and each point is representative of the average number of noninfected control cells, Ad-FLAG-HIC1, and Ad-GFP U2OS-infected cells.

FIGURE 2.

CXCR7 is down-regulated in Ad-FLAG-HIC1-infected U2OS cells, whereas SIRT1 is activated. A, expression levels of CXCR7 and of SIRT1. Total RNAs from U2OS cells (HIC1 null) infected with Ad-FLAG-HIC1 and Ad-GFP were prepared at the indicated times after infection (from 8 to 26 h), and Affymetrix HG U133A chips were used to measure the gene expression. Expression values were normalized to Ad-GFP-infected control cells at the same time points. The Percent of control corresponds to the ratio between the expression levels of CXCR7 and SIRT1 measured in Ad-GFP and in Ad-FLAG-HIC1-infected cells at each time point. B, confirmation of the microarray results for CXCR7 and SIRT1 by quantitative RT-PCR. qRT-PCR analyses were performed using total RNAs isolated from U2OS cells infected (time course point 16 h) with Ad-GFP (gray boxes) or with Ad-FLAG-HIC1 (black boxes) for CXCR7 (left column) and SIRT1 (right column). Values were normalized to GAPDH or actin as indicated.

FIGURE 3.

Inactivation of endogenous HIC1 in normal WI38 fibroblasts up-regulates CXCR7 and SIRT1 expression. A, WI38 cells were infected with the lentiviruses expressing a control shRNA (SHC002) and two shRNAs (SH1763 and SH1982) targeting HIC1 (Mission shRNAs; Sigma) as described previously (19). Total RNAs were extracted, and expression levels of HIC1 mRNA were assessed with real time quantitative PCR as described (9, 19). B and C, similarly, the expression levels of CXCR7 and SIRT1 were assessed using specific oligonucleotides (see supplemental Table S2). Values were normalized to actin.

FIGURE 4.

CXCR7 is down-regulated by HIC1. A, nucleotide sequence of the 5′ region of the human CXCR7 gene. The transcription start site as well as the first noncoding exon are described in GenBank^TM under accession number NM_020311; gi 114155149. The conserved gt dinucleotides of the splice donor site (sd) are indicated in boldface letters and underlined. Upstream genomic sequences were extracted from the human genome sequence (NC_000002). Potential HiREs are indicated with the 4-bp core consensus 5′-GGCA-3′ (reverse complement strand TGCC) shown in boldface and underlined (5). These sites are numbered from 1 to 11. The sites indicated by roman numerals and highlighted in light gray boxes are those conserved in the primate (human and chimpanzee) and rodent (mice and rat) genomes (see also supplemental Fig. 3 for a CLUSTAL alignment). The 5′ boundaries of the various deletions in the CXCR7 promoter obtained by PCR are indicated by arrows with the numbering relative to the +1 position of the transcription initiation site. A unique SpeI site (position −75 to −70) used to obtain the −863/+168 ΔXI construct is boxed. Primers used to amplify the CXCR7 promoter fragment in the ChIP experiment presented in Figs. 6 and 7 are indicated by an arrow. B, from top to bottom, a schematic drawing of the 5′ promoter region and the first noncoding exon of CXCR7 is shown. Numbering is relative to the transcription start site (TSS) (bent arrow, position +1). The potential HiREs (5) are shown as black ovals for those found in the primate and rodent genomes or as white ovals for those found only in the primate genomes. The small arrows indicate the position of the primers used to amplify the relevant region of CXCR7 in the ChIP and ChIP upon ChIP experiments. The left panel shows a schematic drawing of various fragments of the human CXCR7 first noncoding exon 5′ sequences subcloned in the luciferase reporter plasmid pGL3 basic. Reporter constructs were transfected in triplicate into U2OS cells and assayed for luciferase activity. Luciferase (luc) activity was normalized for transfection efficiency using a co-transfected β-galactosidase reporter. Repression of transcription of each construct by HIC1 was calculated by first dividing luciferase activity in the absence of HIC1 by the activity in the presence of HIC1 (normalized for transfection efficiency using a co-transfected β-galactosidase reporter). The value obtained for each construct was then divided by the repressive effect elicited by HIC1 on the empty pGL3 basic vector to obtain the final fold of activation. Results, expressed relative to a value of 1.0 for cells transfected with the pGL3 empty vector, are expressed as the mean of three different experiments, and error bars represent standard deviations.

FIGURE 5.

Phylogenetically conserved HiRE XI is essential for the transcriptional repression of CXCR7. A, nucleotide sequence of the 5′ region of the human CXCR7 gene showing the mutation introduced into the conserved HiRE XI site (TGC in the core motif replaced by CAT) to impede HIC1 binding (5). B, reporter constructs schematically shown in the left panel were transfected in triplicate into U2OS cells and assayed for luciferase activity. Luciferase (luc) activity is shown in the right panel (gray boxes). Repression of transcription of each construct by HIC1 was calculated exactly as detailed in legend to Fig. 4B. Results, expressed relative to a value of 1.0 for cells transfected with the pGL3 empty vector, are expressed as the mean of four different experiments, and error bars represent standard deviations. The p values are indicated (*, p values < 0.5; **, p values < 0.05). C, same experiments were performed with the −813/+168 wild-type and ΔXI constructs. The results are the mean of two independent transfections in triplicate with the p value indicated as in B.

FIGURE 6.

ChIP analyses of HIC1 on CXCR7 and SIRT1 promoters. Normal human WI38 fibroblasts (A) or transformed human SK-N-MC Ewing/PNET cells (B) that both express endogenous HIC1 proteins (see supplemental Fig. 1) were cross-linked with 1% formaldehyde. Cross-linked chromatin immunoprecipitated (IP) with polyclonal anti-HIC1 antibody (Ab) (325), with rabbit IgG, or no antibody were used in PCR amplification with primers flanking the functional HiREs identified by our luciferase assays in the CXCR7 promoter. The region previously identified in the SIRT1 promoter in WI38 cells (10) was used as positive control, whereas PCR with the 5′ promoter of GAPDH was used as an internal nonbinding control (10). An H₂O control corresponding to PCR without DNA yielded no amplified products (data not shown). Representative gels of one ChIP among three experiments are shown.

FIGURE 7.

ChIP upon ChIP assays demonstrate that HIC1 and CtBP might form a stable complex on SIRT1 and CXCR7 promoters. Normal human WI38 fibroblasts were cross-linked with 1% formaldehyde. Cross-linked chromatin was sonicated and immunoprecipitated with polyclonal anti-HIC1 antibody (325) (1st IP HIC1). The bound material was eluted, divided in two, and subjected to a second round of immunoprecipitation with anti-CtBP antibodies (2nd IP CtBP1) or with normal rabbit IgG (2nd IP IgG). PCR amplifications were performed using primers flanking the functional HiREs previously identified in SIRT1 (10) and in CXCR7 (this study). PCR with the 5′ promoter of GAPDH was used as an internal nonbinding control as described in Fig. 6. Representative gels of one ChIP upon ChIP among two experiments are shown.