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Gene profiling of MTA1 identifies novel gene targets and functions.

Ghanta KS, Li DQ, Eswaran J, Kumar R - PLoS ONE (2011)

Bottom Line: Metastasis-associated protein 1 (MTA1), a master dual co-regulatory protein is found to be an integral part of NuRD (Nucleosome Remodeling and Histone Deacetylation) complex, which has indispensable transcriptional regulatory functions via histone deacetylation and chromatin remodeling.Emerging literature establishes MTA1 to be a valid DNA-damage responsive protein with a significant role in maintaining the optimum DNA-repair activity in mammalian cells exposed to genotoxic stress.Thus, the presented data emphasizes the known functions of Mta1 and serves as a rich resource which could help us identify novel Mta1 functions.

View Article: PubMed Central - PubMed

Affiliation: McCormick Genomic and Proteomic Center, The George Washington University Medical Center, Washington, D.C., United States of America.

ABSTRACT

Background: Metastasis-associated protein 1 (MTA1), a master dual co-regulatory protein is found to be an integral part of NuRD (Nucleosome Remodeling and Histone Deacetylation) complex, which has indispensable transcriptional regulatory functions via histone deacetylation and chromatin remodeling. Emerging literature establishes MTA1 to be a valid DNA-damage responsive protein with a significant role in maintaining the optimum DNA-repair activity in mammalian cells exposed to genotoxic stress. This DNA-damage responsive function of MTA1 was reported to be a P53-dependent and independent function. Here, we investigate the influence of P53 on gene regulation function of Mta1 to identify novel gene targets and functions of Mta1.

Methods: Gene expression analysis was performed on five different mouse embryonic fibroblasts (MEFs) samples (i) the Mta1 wild type, (ii) Mta1 knock out (iii) Mta1 knock out in which Mta1 was reintroduced (iv) P53 knock out (v) P53 knock out in which Mta1 was over expressed using Affymetrix Mouse Exon 1.0 ST arrays. Further Hierarchical Clustering, Gene Ontology analysis with GO terms satisfying corrected p-value<0.1, and the Ingenuity Pathway Analysis were performed. Finally, RT-qPCR was carried out on selective candidate genes.

Significance/conclusion: This study represents a complete genome wide screen for possible target genes of a coregulator, Mta1. The comparative gene profiling of Mta1 wild type, Mta1 knockout and Mta1 re-expression in the Mta1 knockout conditions define "bona fide" Mta1 target genes. Further extensive analyses of the data highlights the influence of P53 on Mta1 gene regulation. In the presence of P53 majority of the genes regulated by Mta1 are related to inflammatory and anti-microbial responses whereas in the absence of P53 the predominant target genes are involved in cancer signaling. Thus, the presented data emphasizes the known functions of Mta1 and serves as a rich resource which could help us identify novel Mta1 functions.

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RT-qPCR validation of the microarray data showing the differential regulation of the selected genes in the MEFs.The RNA extracted from the fibroblasts of the wild type (light blue bars) mice was used as the control and the treatments were Mta1-KO (red bars) & Mta1-KO/Mta1(green bars). In the presence of P53 relative mRNA levels of the genes Aw551984, Egr2, Phf17 were compared in all the three samples. The relative mRNA levels from microarray for the same sample sets were plotted and compared with the RT-qPCR. As expected, opposite trends of expression were observed between the knock out and re-expression models. To validate the genes regulated by Mta1 in the absence of P53 relative mRNA levels were compared among the three samples wild type (light blue bars), P53-KO (dark blue bars) & P53-KO/Mta1(yellow bars) for the genes Hmmr, Klf15, Rnf144a.The relative mRNA levels from the microarray were plotted and compared with RT-qPCR. Opposite trends of expression were observed between the P53-KO and P53-KO/Mta1 treatments when compared with the wild type sample.
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pone-0017135-g007: RT-qPCR validation of the microarray data showing the differential regulation of the selected genes in the MEFs.The RNA extracted from the fibroblasts of the wild type (light blue bars) mice was used as the control and the treatments were Mta1-KO (red bars) & Mta1-KO/Mta1(green bars). In the presence of P53 relative mRNA levels of the genes Aw551984, Egr2, Phf17 were compared in all the three samples. The relative mRNA levels from microarray for the same sample sets were plotted and compared with the RT-qPCR. As expected, opposite trends of expression were observed between the knock out and re-expression models. To validate the genes regulated by Mta1 in the absence of P53 relative mRNA levels were compared among the three samples wild type (light blue bars), P53-KO (dark blue bars) & P53-KO/Mta1(yellow bars) for the genes Hmmr, Klf15, Rnf144a.The relative mRNA levels from the microarray were plotted and compared with RT-qPCR. Opposite trends of expression were observed between the P53-KO and P53-KO/Mta1 treatments when compared with the wild type sample.

Mentions: Based upon our analysis and our laboratory interest, some of the genes that were regulated by Mta1 with/without P53 back ground were selected for the validation using the RT-qPCR assays. Validations were first performed in the MEFs followed by the MCF7 human breast cancer cell line. The results showing relative mRNA levels, for the selected genes, are presented in Figure 7. The controls are compared with the treatments and if the trend of expression (up or down regulation) in qPCR is in agreement with the microarray gene expression, then the respective homologous genes were selected for further validation in the human breast cancer cell line (MCF7). Aw551984 gene is the homolog of the human gene VWA5A, also known as BCSC-1. Monaco et al (1997) characterized and proposed that this gene could be a tumor suppressor [39], [40]. We found that both Aw551984/VWA5A were negatively regulated by Mta1 in MEFs and human breast cancer cells. In the case of VWA5A, the expression trend is similar in MEFs and MCF7 cells but the difference in expression levels between the WT and MTA1 knockdown in the MCF7 cell line is marginal. Up regulation of this gene was observed in Mta1-KO and MTA1-siRNA knock down samples when compared to the wild type MEFs and MCF7cells respectively. Another candidate, early growth response protein 2 [encoded by Egr2] and its human homolog was found to follow similar trend in MEFs and MCF7 cell line with significant difference in expression levels when compared between wild type and Mta1-KO. Finally, Phf17, a known apoptosis promoter which may act as a renal tumor suppressor [41] is found to be down regulated in the Mta1 knock out MEFs. As expected the expression pattern of all these candidates is in agreement with the above described microarray data. Together, the expression of these representative genes highlights the possible interplay between the tumor suppressors (Aw551984, Egr2), apoptotic protein (Phf17) and oncogene (Mta1). The bar charts (7A, 7B, 7C) in Figure 7 show the expression levels of these three genes from the RT-qPCR assay and the Affymetrix microarray data.


Gene profiling of MTA1 identifies novel gene targets and functions.

Ghanta KS, Li DQ, Eswaran J, Kumar R - PLoS ONE (2011)

RT-qPCR validation of the microarray data showing the differential regulation of the selected genes in the MEFs.The RNA extracted from the fibroblasts of the wild type (light blue bars) mice was used as the control and the treatments were Mta1-KO (red bars) & Mta1-KO/Mta1(green bars). In the presence of P53 relative mRNA levels of the genes Aw551984, Egr2, Phf17 were compared in all the three samples. The relative mRNA levels from microarray for the same sample sets were plotted and compared with the RT-qPCR. As expected, opposite trends of expression were observed between the knock out and re-expression models. To validate the genes regulated by Mta1 in the absence of P53 relative mRNA levels were compared among the three samples wild type (light blue bars), P53-KO (dark blue bars) & P53-KO/Mta1(yellow bars) for the genes Hmmr, Klf15, Rnf144a.The relative mRNA levels from the microarray were plotted and compared with RT-qPCR. Opposite trends of expression were observed between the P53-KO and P53-KO/Mta1 treatments when compared with the wild type sample.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3045407&req=5

pone-0017135-g007: RT-qPCR validation of the microarray data showing the differential regulation of the selected genes in the MEFs.The RNA extracted from the fibroblasts of the wild type (light blue bars) mice was used as the control and the treatments were Mta1-KO (red bars) & Mta1-KO/Mta1(green bars). In the presence of P53 relative mRNA levels of the genes Aw551984, Egr2, Phf17 were compared in all the three samples. The relative mRNA levels from microarray for the same sample sets were plotted and compared with the RT-qPCR. As expected, opposite trends of expression were observed between the knock out and re-expression models. To validate the genes regulated by Mta1 in the absence of P53 relative mRNA levels were compared among the three samples wild type (light blue bars), P53-KO (dark blue bars) & P53-KO/Mta1(yellow bars) for the genes Hmmr, Klf15, Rnf144a.The relative mRNA levels from the microarray were plotted and compared with RT-qPCR. Opposite trends of expression were observed between the P53-KO and P53-KO/Mta1 treatments when compared with the wild type sample.
Mentions: Based upon our analysis and our laboratory interest, some of the genes that were regulated by Mta1 with/without P53 back ground were selected for the validation using the RT-qPCR assays. Validations were first performed in the MEFs followed by the MCF7 human breast cancer cell line. The results showing relative mRNA levels, for the selected genes, are presented in Figure 7. The controls are compared with the treatments and if the trend of expression (up or down regulation) in qPCR is in agreement with the microarray gene expression, then the respective homologous genes were selected for further validation in the human breast cancer cell line (MCF7). Aw551984 gene is the homolog of the human gene VWA5A, also known as BCSC-1. Monaco et al (1997) characterized and proposed that this gene could be a tumor suppressor [39], [40]. We found that both Aw551984/VWA5A were negatively regulated by Mta1 in MEFs and human breast cancer cells. In the case of VWA5A, the expression trend is similar in MEFs and MCF7 cells but the difference in expression levels between the WT and MTA1 knockdown in the MCF7 cell line is marginal. Up regulation of this gene was observed in Mta1-KO and MTA1-siRNA knock down samples when compared to the wild type MEFs and MCF7cells respectively. Another candidate, early growth response protein 2 [encoded by Egr2] and its human homolog was found to follow similar trend in MEFs and MCF7 cell line with significant difference in expression levels when compared between wild type and Mta1-KO. Finally, Phf17, a known apoptosis promoter which may act as a renal tumor suppressor [41] is found to be down regulated in the Mta1 knock out MEFs. As expected the expression pattern of all these candidates is in agreement with the above described microarray data. Together, the expression of these representative genes highlights the possible interplay between the tumor suppressors (Aw551984, Egr2), apoptotic protein (Phf17) and oncogene (Mta1). The bar charts (7A, 7B, 7C) in Figure 7 show the expression levels of these three genes from the RT-qPCR assay and the Affymetrix microarray data.

Bottom Line: Metastasis-associated protein 1 (MTA1), a master dual co-regulatory protein is found to be an integral part of NuRD (Nucleosome Remodeling and Histone Deacetylation) complex, which has indispensable transcriptional regulatory functions via histone deacetylation and chromatin remodeling.Emerging literature establishes MTA1 to be a valid DNA-damage responsive protein with a significant role in maintaining the optimum DNA-repair activity in mammalian cells exposed to genotoxic stress.Thus, the presented data emphasizes the known functions of Mta1 and serves as a rich resource which could help us identify novel Mta1 functions.

View Article: PubMed Central - PubMed

Affiliation: McCormick Genomic and Proteomic Center, The George Washington University Medical Center, Washington, D.C., United States of America.

ABSTRACT

Background: Metastasis-associated protein 1 (MTA1), a master dual co-regulatory protein is found to be an integral part of NuRD (Nucleosome Remodeling and Histone Deacetylation) complex, which has indispensable transcriptional regulatory functions via histone deacetylation and chromatin remodeling. Emerging literature establishes MTA1 to be a valid DNA-damage responsive protein with a significant role in maintaining the optimum DNA-repair activity in mammalian cells exposed to genotoxic stress. This DNA-damage responsive function of MTA1 was reported to be a P53-dependent and independent function. Here, we investigate the influence of P53 on gene regulation function of Mta1 to identify novel gene targets and functions of Mta1.

Methods: Gene expression analysis was performed on five different mouse embryonic fibroblasts (MEFs) samples (i) the Mta1 wild type, (ii) Mta1 knock out (iii) Mta1 knock out in which Mta1 was reintroduced (iv) P53 knock out (v) P53 knock out in which Mta1 was over expressed using Affymetrix Mouse Exon 1.0 ST arrays. Further Hierarchical Clustering, Gene Ontology analysis with GO terms satisfying corrected p-value<0.1, and the Ingenuity Pathway Analysis were performed. Finally, RT-qPCR was carried out on selective candidate genes.

Significance/conclusion: This study represents a complete genome wide screen for possible target genes of a coregulator, Mta1. The comparative gene profiling of Mta1 wild type, Mta1 knockout and Mta1 re-expression in the Mta1 knockout conditions define "bona fide" Mta1 target genes. Further extensive analyses of the data highlights the influence of P53 on Mta1 gene regulation. In the presence of P53 majority of the genes regulated by Mta1 are related to inflammatory and anti-microbial responses whereas in the absence of P53 the predominant target genes are involved in cancer signaling. Thus, the presented data emphasizes the known functions of Mta1 and serves as a rich resource which could help us identify novel Mta1 functions.

Show MeSH
Related in: MedlinePlus