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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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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.
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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.

Mentions: As co-repressors, HDACs require specific transcription factors for recruiting them to target DNA elements for regulatory functions. In this study, we showed that both of MORC2 and HDAC4 were associated with the CAIX promoter by ChIP assays (Figure 6A and B). We also described experimental results to show that MORC2 and HDAC4 were assembled on the same promoter (Figure 6C). We performed additional ChIP Re-IP experiment to prove the specificity of such a co-interaction (Figure 6D). In addition, the in vitro and in vivo interaction of MORC2 and HDAC4 was confirmed by GST-pull down and IP experiments (Figure 7A and B). Furthermore, we performed EMSA with PR4 and proved by super-shift that both proteins were simultaneously present in the PR4-binding complex (Figure 7C and D). HDAC4 is often found to form multisubunit complex with other corepressors, such as N-CoR etc, to regulate the transcription of target genes (34). Our findings indicated that MORC2 and HDAC4 were in the inhibitory complex to suppress the expression of the CAIX gene. Further studies are needed to identify other corepressors which might form a complex with HDAC4 to inhibit the transcription of the CAIX gene. Based on the previous knowledge, as well as findings from this study, we propose a hypothesized model in which MORC2 binds the PR4 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 (Figure 8).Figure 8.


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)

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

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

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.
Mentions: As co-repressors, HDACs require specific transcription factors for recruiting them to target DNA elements for regulatory functions. In this study, we showed that both of MORC2 and HDAC4 were associated with the CAIX promoter by ChIP assays (Figure 6A and B). We also described experimental results to show that MORC2 and HDAC4 were assembled on the same promoter (Figure 6C). We performed additional ChIP Re-IP experiment to prove the specificity of such a co-interaction (Figure 6D). In addition, the in vitro and in vivo interaction of MORC2 and HDAC4 was confirmed by GST-pull down and IP experiments (Figure 7A and B). Furthermore, we performed EMSA with PR4 and proved by super-shift that both proteins were simultaneously present in the PR4-binding complex (Figure 7C and D). HDAC4 is often found to form multisubunit complex with other corepressors, such as N-CoR etc, to regulate the transcription of target genes (34). Our findings indicated that MORC2 and HDAC4 were in the inhibitory complex to suppress the expression of the CAIX gene. Further studies are needed to identify other corepressors which might form a complex with HDAC4 to inhibit the transcription of the CAIX gene. Based on the previous knowledge, as well as findings from this study, we propose a hypothesized model in which MORC2 binds the PR4 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 (Figure 8).Figure 8.

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