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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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Related in: MedlinePlus

Schematic showing the experimental design of the study to identify the Mta1 regulated genes with/without the effect of P53.RNA was extracted from all the samples Wild Type (WT), Mta1 knock out (Mta1-KO), Mta1 Re-expression in the Mta1 knock out MEFS (Mta1-KO/Mta1), P53 knock out (P53-KO), Mta1 over expression (OE) in the P53 knock out MEFs ( P53-KO/Mta1). cDNA was prepared, processed and hybridized onto the Affymetrix Mouse Exon 1.0 ST arrays followed by the data analysis. Samples were compared to identify the differentially regulated genes and in turn the genes regulated by Mta1 in p53 dependent/independent manner and irrespective of P53 status. Candidate genes were selected; these genes were validated using RT-qPCR assays in MEFs followed by RT-qPCR assays in MCF-7 human breast cancer cell line with the human homologs of the candidate mouse genes.
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pone-0017135-g001: Schematic showing the experimental design of the study to identify the Mta1 regulated genes with/without the effect of P53.RNA was extracted from all the samples Wild Type (WT), Mta1 knock out (Mta1-KO), Mta1 Re-expression in the Mta1 knock out MEFS (Mta1-KO/Mta1), P53 knock out (P53-KO), Mta1 over expression (OE) in the P53 knock out MEFs ( P53-KO/Mta1). cDNA was prepared, processed and hybridized onto the Affymetrix Mouse Exon 1.0 ST arrays followed by the data analysis. Samples were compared to identify the differentially regulated genes and in turn the genes regulated by Mta1 in p53 dependent/independent manner and irrespective of P53 status. Candidate genes were selected; these genes were validated using RT-qPCR assays in MEFs followed by RT-qPCR assays in MCF-7 human breast cancer cell line with the human homologs of the candidate mouse genes.

Mentions: The aim of the study is to identify the genes that are regulated by Mta1 in P53 dependent and independent manners. The detailed schematic of the strategy followed to identify the genes is shown in Figure 1. Murine Embryonic Fibroblasts (MEFs) from wild type, Mta1 knockout [35] and P53 knockout mice embryos [36] were isolated and cultured to obtain five different types of samples, each in triplicates, for the identification of the genes that are regulated by Mta1 with/without the P53 background. The sample sets are as follows: 1) Mta1-Wildtype (WT), 2) Mta1-knockout (Mta1-KO) [23], 3) Mta1 transfected into the Mta1-knock out MEFs (Mta1-KO/Mta1), 4) P53-Knock (P53-KO) and 5) Mta1 over expressed in the P53-Knockout MEFs (P53-KO/Mta1) [30]. The protein levels of Mta1 in all the five samples are compared using Western blot probed with Mta1 antibody (Figure S1). Total RNA was isolated from the MEFs, cDNA was prepared, processed and hybridized onto Affymetrix Mouse Exon 1.0 ST arrays. The gene expression data from all the samples were obtained, quality control steps were performed and the data was analyzed using GeneSpring GX 10.0.2 (Agilent Technologies).


Gene profiling of MTA1 identifies novel gene targets and functions.

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

Schematic showing the experimental design of the study to identify the Mta1 regulated genes with/without the effect of P53.RNA was extracted from all the samples Wild Type (WT), Mta1 knock out (Mta1-KO), Mta1 Re-expression in the Mta1 knock out MEFS (Mta1-KO/Mta1), P53 knock out (P53-KO), Mta1 over expression (OE) in the P53 knock out MEFs ( P53-KO/Mta1). cDNA was prepared, processed and hybridized onto the Affymetrix Mouse Exon 1.0 ST arrays followed by the data analysis. Samples were compared to identify the differentially regulated genes and in turn the genes regulated by Mta1 in p53 dependent/independent manner and irrespective of P53 status. Candidate genes were selected; these genes were validated using RT-qPCR assays in MEFs followed by RT-qPCR assays in MCF-7 human breast cancer cell line with the human homologs of the candidate mouse genes.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3045407&req=5

pone-0017135-g001: Schematic showing the experimental design of the study to identify the Mta1 regulated genes with/without the effect of P53.RNA was extracted from all the samples Wild Type (WT), Mta1 knock out (Mta1-KO), Mta1 Re-expression in the Mta1 knock out MEFS (Mta1-KO/Mta1), P53 knock out (P53-KO), Mta1 over expression (OE) in the P53 knock out MEFs ( P53-KO/Mta1). cDNA was prepared, processed and hybridized onto the Affymetrix Mouse Exon 1.0 ST arrays followed by the data analysis. Samples were compared to identify the differentially regulated genes and in turn the genes regulated by Mta1 in p53 dependent/independent manner and irrespective of P53 status. Candidate genes were selected; these genes were validated using RT-qPCR assays in MEFs followed by RT-qPCR assays in MCF-7 human breast cancer cell line with the human homologs of the candidate mouse genes.
Mentions: The aim of the study is to identify the genes that are regulated by Mta1 in P53 dependent and independent manners. The detailed schematic of the strategy followed to identify the genes is shown in Figure 1. Murine Embryonic Fibroblasts (MEFs) from wild type, Mta1 knockout [35] and P53 knockout mice embryos [36] were isolated and cultured to obtain five different types of samples, each in triplicates, for the identification of the genes that are regulated by Mta1 with/without the P53 background. The sample sets are as follows: 1) Mta1-Wildtype (WT), 2) Mta1-knockout (Mta1-KO) [23], 3) Mta1 transfected into the Mta1-knock out MEFs (Mta1-KO/Mta1), 4) P53-Knock (P53-KO) and 5) Mta1 over expressed in the P53-Knockout MEFs (P53-KO/Mta1) [30]. The protein levels of Mta1 in all the five samples are compared using Western blot probed with Mta1 antibody (Figure S1). Total RNA was isolated from the MEFs, cDNA was prepared, processed and hybridized onto Affymetrix Mouse Exon 1.0 ST arrays. The gene expression data from all the samples were obtained, quality control steps were performed and the data was analyzed using GeneSpring GX 10.0.2 (Agilent Technologies).

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