Involvement of histone deacetylation in MORC2-mediated down-regulation of carbonic anhydrase IX

Abstract

Carbonic anhydrase IX (CAIX) plays an important role in the growth and survival of tumor cells. MORC2 is a member of the MORC protein family. The MORC proteins contain a CW-type zinc finger domain and are predicted to have the function of regulating transcription, but no MORC2 target genes have been identified. Here we performed a DNA microarray hybridization and found CAIX mRNA to be down-regulated 8-fold when MORC2 was overexpressed. This result was further confirmed by northern and western blot analysis. Our results also showed that the protected region 4 (PR4) was important for the repression function of MORC2. Moreover, MORC2 decreased the acetylation level of histone H3 at the CAIX promoter. Meanwhile, trichostatin A (TSA) had an increasing effect on CAIX promoter activity. Among the six HDACs tested, histone deacetylase 4 (HDAC4) had a much more prominent effect on CAIX repression. ChIP and ChIP Re-IP assays showed that MORC2 and HDAC4 were assembled on the same region of the CAIX promoter. Importantly, we further confirmed that both proteins are simultaneously present in the PR4-binding complex. These results may contribute to understanding the molecular mechanisms of CAIX regulation.

Figure 1.

MORC2 down-regulates the mRNA and protein levels of CAIX. (A) Northern blot analysis of MORC2 and CAIX mRNA levels in different colorectal and gastric cancer cell lines. Total RNA was isolated and 20 μg total RNA was analyzed by northern blot analysis as described in ‘Materials and methods’ section. GAPDH mRNA was used to assess the integrity of the RNA and to control for the RNA loading. (B) Western blot analysis of MORC2 and CAIX protein levels in different colorectal and gastric cancer cell lines. Cells were lysed as indicated in ‘Materials and methods’ section. Equal amounts of protein (60 μg) were separated by SDS–PAGE and blotted with anti-MORC2 or anti-CAIX antibodies. The expression of endogenous MORC2 and CAIX were detected using the ECL staining method. The expression of MORC2 and CAIX in SGC-7901 cells stable-transfected with pcDNA3.1 or pcDNA3.1/MORC2 were analyzed by northern blot (C) and western blot analysis (D). −, untransfected SGC-7901 cells. (E) Western blot analysis of MORC family members and CAIX protein levels in SGC-7901 cells transfected with pcDNA3.1, pcDNA3.1/MORC2, pcDNA3.1/MORC1 or pcDNA3.1/MORC3.

Figure 2.

MORC2 down-regulates CAIX promoter activity, mRNA and protein levels in a dose-dependent manner. (A) MGC-803 cells were transfected with pGL3-E-CP reporter construct (firefly luciferase expression vector), pRL-TK plasmid (renilla luciferase expression vector) and MORC2 expression vector as indicated. Total DNA of the plasmid was adjusted to the same amount by transfecting pcDNA3.1 empty vector. After 24 h of transfection, the cells were harvested and firefly and renilla luciferase activities were measured using Dual-Luciferase Reporter Assay System (Promega), with the results expressed as the ratio of firefly to renilla luciferase activity (Fluc/Rluc). The renilla luciferase activity was used as a control for normalizing the assay. All the results represented means ± SD of three independent experiments. *P < 0.05, **P < 0.01. (B) MGC-803 cells were transfected with the MORC2 expression vector as indicated. After 30 h of transfection, the mRNA levels of CAIX were measured by northern blot analysis. (C) The same transfection experiment as (B), after 30 h of transfection, cells were lysed and equal amounts of protein (100 μg) were separated by SDS–PAGE, the protein levels of MORC2 and CAIX were measured by western blot analysis. (D) MORC2 expression vectors were transiently transfected into SGC-7901 cells as indicated, and the mRNA level was estimated by RT–PCR and real-time PCR analysis. Values are means ± SD (n = 3). **P < 0.01. (E) SGC-7901 cells were transfected with MORC2 expression vector as indicated, after 30 h of transfection, the protein levels of MORC2 and CAIX were measured by western blot analysis.

Figure 3.

Specific knockdown of MORC2 leading to the up-regulation of CAIX. BGC-823 cells were transfected with siRNAs targeting MORC2 or non-silencing control. C, untransfected BGC-823 cells, NC, BGC-823 cells transfected with non-silencing control. (A) After 30 h of transfection, the mRNA levels of MORC2 and CAIX were measured by northern blot analysis. GAPDH mRNA was used to assess the integrity of the RNA and to control for the RNA loading. (B) After 30 h transfection, the protein levels of MORC2 and CAIX were measured by western blot analysis. (C) After 24 h of transfection, the cells were transfected with the reporter constructs pGL3-E-CP (firefly luciferase expression vector) and pRL-TK (renilla luciferase expression vector), 24 h later, the cells were lysed, firefly and renilla luciferase activities were measured using Dual-Luciferase Reporter Assay System (Promega). First we got the ratio of firefly to renilla luciferase activity (Fluc/Rluc, Rluc was used as a control for normalizing the assay) and then the results were shown as fold induction relative to that of cells transfected without siRNA. The results were the means ± SD of three individual experiments. *P < 0.05.

Figure 4.

Identification of the cis-elements involved in the suppression function of MORC2 on CAIX transcription. (A) Schematic representation of a series of 5′-deleted CAIX promoter/luciferase constructs. The transcription initiation site is indicated by a bent arrow. Numbers indicate base pairs from the transcription start site. (B) The basal activities of the corresponding constructs in BGC-823 cells. Luciferase activities were normalized to Renilla activities. The bar is mean ± SD from three independent experiments, in duplicate for each construct. (C) The comparison of the effects of MORC2 on the activities of 5′-deleted CAIX promoter/luciferase constructs in BGC-823 cells. BGC-823 cells were transfected with the indicated constructs and with (the grey bar) or without (the black bar) MORC2. Results are expressed as a percentage of the MORC2-untransfected control that is taken as 100%. *P < 0.05 compared with control.

Figure 5.

Histone deacetylation is involved in the CAIX transcriptional repression. (A) The acetylation level of histone H3 at CAIX promoter (−173/+31) was decreased after the overexpression of MORC2. SGC-7901 cells were transfected with 8 μg MORC2 expression vector or pcDNA3.1 empty vector as the control. ChIP was carried out using antibody against acetylated histone H3 (α-AcH3), followed by PCR with primers amplifying the CAIX promoter region (−173/+31). (B) HEK-293 cells and BGC-823 cells were transfected with the pGL3-E-CP reporter construct, pRL-TK and MORC2 or pcDNA3.1 empty plasmid for 6 h followed by treatment with or without TSA (200 nM) for 24 h. Results are shown as fold induction relative to that of the cells transfected with pGL3-E-CP and pRL-TK, and without treatment of TSA. Values are means ± SD (n = 3). (C) BGC-823 cells were transfected with pGL3-E-CP plasmid together with HDAC constructs expressing HDAC1-6, respectively. Luciferase activities were determined and normalized to Renilla activity 24 h after transfection. Results are shown as fold induction relative to that of the cells transfected without HDAC plasmid and are the means ± SD from at least three individual experiments. *P < 0.05, **P < 0.01. (D) MORC2 and HDAC4 were transiently transfected into BGC-823 cells as indicated, and the mRNA level was estimated by RT-PCR and real-time PCR analysis. Values are means ± SD (n = 3), **P < 0.01. (E) BGC-823 cells were transfected with CAIX promoter deletion constructs used in Figure 4A and with or without HDAC4 expression plasmid as indicated. Luciferase activities were determined and normalized to Renilla activity 24 h after transfection. Results are expressed as a percentage of the MORC2-untransfected control that is taken as 100%. *P < 0.05 compared with control.

Figure 6.

ChIP and ChIP Re-IP assays for MORC2 and HDAC4 in association with the CAIX promoter. (A) ChIP assay to examine the binding of MORC2 to the CAIX promoter. SGC-7901 cells were transfected with 4 μg MORC2 expression vector or pcDNA3.1 empty vector as the control. ChIP was carried out using antibody against MORC2, followed by PCR with primers amplifying the CAIX promoter region (−173/+31), up- and down-stream regions of CAIX gene. (B) ChIP assay for detection of the recruitment of HDAC4 to the CAIX promoter. SGC-7901 cells were transfected with 4 μg pcDNA3.1 empty vector or Flag-HDAC4 expression plasmid. ChIP was carried out using antibody against Flag, followed by PCR with primers amplifying the CAIX promoter region. (C) ChIP Re-IP to examine whether MORC2 and HDAC4 were assembled on the same promoter. Soluble chromatin was prepared from SGC-7901 cells transfected with MORC2 and HDAC4 or pcDNA3.1 empty vector and divided into two aliquots. One aliquot was first immunoprecipitated with antibody against Flag (1° IP). The supernatant was collected and reimmunoprecipitated with antibody against MORC2 (Supernatant Re-IP). The other aliquot was first immunoprecipitated with antibody against MORC2 (1° IP), followed by reimmunoprecipitation with antibody against Flag. Similar reciprocal Re-IPs were also performed on complexes eluted from the 1° IPs (Bound Re-IP). (D) The same ChIP Re-IP experiment as (C) to examine whether MORC2 and HDAC2 were assembled on the same promoter. SGC-7901 cells were transfected with MORC2 and Flag-HDAC2 or pcDNA3.1 empty vector. The antibodies used were against MORC2 and Flag.

Figure 7.

MORC2 binds to HDAC4 and both of them bind to the PR4 region of the CAIX promoter. (A) For GST pull-down assay, GST or GST fusion proteins were incubated with MORC2 protein in vitro translated. Bound proteins were subjected to SDS–PAGE and western blot with anti-MORC2 antibody. (B) For the immunoprecipitation assay, cell lysates were immunoprecipitated by anti-GFP antibody, and precipitates were immunoblotted with anti-His antibody. The expression were checked using 20 µg of total cell lysate (TCL) blotted with the indicated antibodies (bottom panels). (C) EMSA was performed with nuclear extracts from BGC-823 cells and the PR4 oligonucleotide (*PR4) labeled with biotin. Lane 1 is *PR4 probe without nuclear extracts. The binding complexes were competed by 50-, 100- or 500-fold molar excess of unlabeled PR4, respectively, and supershifted by anti-MORC2 or anti-HDAC4 antibody. NS, non-specific; SS, super-shift; *PR4, PR4 probe labeled with biotin; PR4, unlabeled PR4. (D) EMSA was performed with in vitro translated MORC2 protein using a TNT quick coupled transcription-translation system (Promega) and PR4 oligonucleotide (*PR4) labeled with biotin. The binding complexes were competed by 50- or 100-fold molar excess of unlabeled PR4, respectively, and super-shifted by anti-MORC2 antibody.

Figure 8.

Proposed model showing roles of MORC2 and HDAC4 in regulation of the CAIX gene. MORC2 binds the protected region 4 in the CAIX promoter, and recruits HDAC4 that decreases the acetylation level of histone H3 at the CAIX promoter, leading to a closed chromatin structure and thus the transcriptional repression of CAIX gene.