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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.

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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.
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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.

Mentions: In order to elucidate whether the decrease in CAIX mRNA is dependent on MORC2 as a regulator of transcription, we examined the regulation of the CAIX promoter. As a reporter, we used the −173 to +31 fragment of the CAIX promoter fused to the luciferase reporter gene. This region has been shown to be sufficient for the transcriptional induction of CAIX gene in MaTu and HeLa cells (17). MGC-803 cells were transfected with the pGL3-E-CP reporter construct and increasing amounts of MORC2 expression vector. The results showed that MORC2 down-regulated CAIX promoter activity in a dose-dependent manner (Figure 2A). To test the expression of MORC2 after the transfection, we carried out western blot analysis, at the same time, CAIX mRNA and protein levels were also examined. As can be seen in Figure 2B and C, with increasing amounts of MORC2 expression plasmid transfected, the mRNA and protein levels of CAIX were decreased. We also performed RT-PCR, real-time PCR and western blot analysis in SGC-7901 cells. The mRNA and protein levels of CAIX were reduced with the ectopic MORC2 expression level enhancing (Figure 2D and E). In sum, these results show that MORC2 is able to down-regulate the expression of CAIX in a dose-dependent manner.Figure 2.


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 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.
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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.
Mentions: In order to elucidate whether the decrease in CAIX mRNA is dependent on MORC2 as a regulator of transcription, we examined the regulation of the CAIX promoter. As a reporter, we used the −173 to +31 fragment of the CAIX promoter fused to the luciferase reporter gene. This region has been shown to be sufficient for the transcriptional induction of CAIX gene in MaTu and HeLa cells (17). MGC-803 cells were transfected with the pGL3-E-CP reporter construct and increasing amounts of MORC2 expression vector. The results showed that MORC2 down-regulated CAIX promoter activity in a dose-dependent manner (Figure 2A). To test the expression of MORC2 after the transfection, we carried out western blot analysis, at the same time, CAIX mRNA and protein levels were also examined. As can be seen in Figure 2B and C, with increasing amounts of MORC2 expression plasmid transfected, the mRNA and protein levels of CAIX were decreased. We also performed RT-PCR, real-time PCR and western blot analysis in SGC-7901 cells. The mRNA and protein levels of CAIX were reduced with the ectopic MORC2 expression level enhancing (Figure 2D and E). In sum, these results show that MORC2 is able to down-regulate the expression of CAIX in a dose-dependent manner.Figure 2.

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