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

Shao Y, Li Y, Zhang J, Liu D, Liu F, Zhao Y, Shen T, Li F - Nucleic Acids Res. (2010)

Bottom Line: 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.These results may contribute to understanding the molecular mechanisms of CAIX regulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Key Laboratory of Cell Biology, Ministry of Public Health, and Key Laboratory of Medical Cell Biology, Ministry of Education, China Medical University, Shenyang 110001, China.

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.

Show MeSH

Related in: MedlinePlus

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.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2875037&req=5

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.

Mentions: The MORC proteins are predicted to have the function of regulating transcription (23), but no MORC2 target genes have been identified. To search for hitherto unidentified MORC2 target genes, we carried out stable transfection experiment. SGC-7901 cells, which had low endogenous MORC2 expression (Figure 1A), were stably transfected with pcDNA3.1 or pcDNA3.1/MORC2 plasmid. Then we performed a DNA microarray hybridization experiment using RNA from the two stable-transfected cell lines and found a lot of MORC2 target genes, most of them were down-regulated. The genes were involved in a variety of biological functions including ion transport, lipid metabolism, inflammatory response, response to hypoxia, etc. CAIX is one of the genes which are down-regulated markedly. In order to examine whether MORC2 expression is relative to the expression of CAIX in different colorectal and gastric cancer cell lines, we carried out northern and western blot analysis. Total mRNA was isolated and equal amounts of mRNA were subjected to northern blot analysis. The endogenous mRNA level of MORC2 was high in CloneA, LS174T and BGC-823 cells, in which CAIX mRNA level was obviously low. Whereas in SGC-7901 cells, which did not express detectable level of MORC2 mRNA, the endogenous CAIX mRNA level was distinctly high (Figure 1A). We performed western blot analysis using the same cell lines as used in the northern blot analysis. The result showed that the protein levels of MORC2 and CAIX in these cells were similar to their mRNA expression levels (Figure 1B). Then we confirmed the MORC2-dependent decrease of the CAIX mRNA and protein levels by northern and western blot analysis, respectively (Figure 1C and D). These data suggested that MORC2 down-regulated the expression of CAIX. In order to know whether other MORC family members have the same repressive effect on CAIX expression, we carried out western blot analysis and found that ectopic expressed MORC1 also down-regulated CAIX expression. But MORC3 did not down-regulate the expression of CAIX obviously.Figure 1.


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

Shao Y, Li Y, Zhang J, Liu D, Liu F, Zhao Y, Shen T, Li F - Nucleic Acids Res. (2010)

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.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2875037&req=5

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.
Mentions: The MORC proteins are predicted to have the function of regulating transcription (23), but no MORC2 target genes have been identified. To search for hitherto unidentified MORC2 target genes, we carried out stable transfection experiment. SGC-7901 cells, which had low endogenous MORC2 expression (Figure 1A), were stably transfected with pcDNA3.1 or pcDNA3.1/MORC2 plasmid. Then we performed a DNA microarray hybridization experiment using RNA from the two stable-transfected cell lines and found a lot of MORC2 target genes, most of them were down-regulated. The genes were involved in a variety of biological functions including ion transport, lipid metabolism, inflammatory response, response to hypoxia, etc. CAIX is one of the genes which are down-regulated markedly. In order to examine whether MORC2 expression is relative to the expression of CAIX in different colorectal and gastric cancer cell lines, we carried out northern and western blot analysis. Total mRNA was isolated and equal amounts of mRNA were subjected to northern blot analysis. The endogenous mRNA level of MORC2 was high in CloneA, LS174T and BGC-823 cells, in which CAIX mRNA level was obviously low. Whereas in SGC-7901 cells, which did not express detectable level of MORC2 mRNA, the endogenous CAIX mRNA level was distinctly high (Figure 1A). We performed western blot analysis using the same cell lines as used in the northern blot analysis. The result showed that the protein levels of MORC2 and CAIX in these cells were similar to their mRNA expression levels (Figure 1B). Then we confirmed the MORC2-dependent decrease of the CAIX mRNA and protein levels by northern and western blot analysis, respectively (Figure 1C and D). These data suggested that MORC2 down-regulated the expression of CAIX. In order to know whether other MORC family members have the same repressive effect on CAIX expression, we carried out western blot analysis and found that ectopic expressed MORC1 also down-regulated CAIX expression. But MORC3 did not down-regulate the expression of CAIX obviously.Figure 1.

Bottom Line: 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.These results may contribute to understanding the molecular mechanisms of CAIX regulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Key Laboratory of Cell Biology, Ministry of Public Health, and Key Laboratory of Medical Cell Biology, Ministry of Education, China Medical University, Shenyang 110001, China.

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.

Show MeSH
Related in: MedlinePlus