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Characterization of the human a9 integrin subunit gene: promoter analysis and transcriptional regulation

ABSTRACT: α9b1 is the most recent addition to the integrin family of membrane-bound receptors and consequently remains the one which is the least characterized. To better understand how transcription of the human gene encoding the α9 subunit is regulated at the molecular level, we cloned the α9 promoter and characterized the regulatory elements that are required to ensure its transcription. Transfection of α9 promoter/CAT recombinant plasmids in primary cultured cells and uveal melanoma cell lines demonstrated the presence of both negative and positive regulatory elements along the α9 promoter and positioned the basal α9 promoter to within 118 bp from the α9 mRNA start site. In vitro DNaseI footprinting and in vivo ChIP analyses demonstrated the binding of the transcription factors Sp1/Sp3, c-Myb and NFI to the most upstream α9 negative regulatory element. The transcription factors Sp1/Sp3 and NFI were found to bind the basal α9 promoter individually but Sp1 binding clearly predominates when both transcription factors are present in the same extract. Most of all, addition of tenascin-C (TNC), the ligand of α9β1, to the tissue culture plates prior to cell seeding increased α9 transcription whereas it simultaneously decreased expression of the α5 integrin subunit gene. This dual regulatory action of TNC on the transcription of the α9 and α5 genes suggests that both these integrins must work together to appropriately regulate cell adhesion, migration and differentiation that are hallmarks of tissue wound healing.

ORGANISM(S): Homo sapiens

PROVIDER: GSE62075 | GEO | 2014/10/06

SECONDARY ACCESSION(S): PRJNA263181

REPOSITORIES: GEO

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Project description:Î±9b1 is the most recent addition to the integrin family of membrane-bound receptors and consequently remains the one which is the least characterized. To better understand how transcription of the human gene encoding the Î±9 subunit is regulated at the molecular level, we cloned the Î±9 promoter and characterized the regulatory elements that are required to ensure its transcription. Transfection of Î±9 promoter/CAT recombinant plasmids in primary cultured cells and uveal melanoma cell lines demonstrated the presence of both negative and positive regulatory elements along the Î±9 promoter and positioned the basal Î±9 promoter to within 118 bp from the Î±9 mRNA start site. In vitro DNaseI footprinting and in vivo ChIP analyses demonstrated the binding of the transcription factors Sp1/Sp3, c-Myb and NFI to the most upstream Î±9 negative regulatory element. The transcription factors Sp1/Sp3 and NFI were found to bind the basal Î±9 promoter individually but Sp1 binding clearly predominates when both transcription factors are present in the same extract. Most of all, addition of tenascin-C (TNC), the ligand of Î±9Î²1, to the tissue culture plates prior to cell seeding increased Î±9 transcription whereas it simultaneously decreased expression of the Î±5 integrin subunit gene. This dual regulatory action of TNC on the transcription of the Î±9 and Î±5 genes suggests that both these integrins must work together to appropriately regulate cell adhesion, migration and differentiation that are hallmarks of tissue wound healing. Primary cultures of human corneal epithelial cells (HCECs; number of replicates: 8), human skin epithelial cells (HSEC; number of replicates: 4), human corneal fibroblast cells (HCFCs; number of replicates: 3), human skin fibroblast cells (HSFCs; number of replicates: 3), human uveal melanocytes (UVM; number of replicates: 3), and various humal uveal melanoma cell lines (T115, T142 and T143; number of replicates: 2-3) were analyzed by gene profiling on microarrays at low culture passages (between passage 2 to 7). HCECs were also cultured on either BSA (number of replicates: 2) or on tenascin-C (number of replicates: 2) and their transcriptome analyzed by gene profiling.

Project description:Important remodeling of the extracellular matrix is a hallmark of corneal wound healing. Besides the massive secretion of fibronectin (FN) that characterizes the early step of that process, alterations in the secretion of collagens also occurs as part of this wound healingresponse. In this study, we examined whether expression of the gene encoding the α5 subunit from the FN binding integrin α5β1 changes as corneal epithelial cells (CECs) are cultured in the presence of collagens. Responsiveness of the α5 gene toward collagen was determined by transfection of recombinant plasmids bearing the CAT reporter gene under the control of various segments from the human α5 promoter into primary cultured rabbit (RCECs) and human (HCECs) CECs cultured on BSA or on collagens. Electrophoretic mobility shift assays (EMSAs) and Western blot analyses were used to monitor both the DNA binding and expression of the transcription factors required to ensure basal transcription of the α5 gene. The influence collagens on the patterns of genes expressed by HCECs was also studied be gene profiling on microarrays. All collagen types repressed the transcriptional activity directed by the full-length α5 promoter/CAT construct in confluent CECs. A moderate increase in α5 promoter activity was observed in subconfluent RCECs grown on CIV but not on CI. These collagen-dependent regulatory influences also correlated with alterations in either the expression or the DNA binding of the transcription factors Sp1/Sp3, NFI and AP-1 that ensure basal transcription of the α5 gene. Gene profiling on microarray revealed that CI more profoundly alter the pattern of genes expressed by HCECs than what is observed with CIV. Collagens considerably suppressed expression of the α5 gene in CECs at confluence suggesting that the sustained staining for CI and CIV after corneal injury may play a role in controlling adhesion to the temporary FN matrix and further differentiation of CECs into suprabasal epithelial cells through a mechanism involving the α5β1 integrin.

Project description:Important remodeling of the extracellular matrix is a hallmark of corneal wound healing. Besides the massive secretion of fibronectin (FN) that characterizes the early step of that process, alterations in the secretion of collagens also occurs as part of this wound healingresponse. In this study, we examined whether expression of the gene encoding the M-NM-15 subunit from the FN binding integrin M-NM-15M-NM-21 changes as corneal epithelial cells (CECs) are cultured in the presence of collagens. Responsiveness of the M-NM-15 gene toward collagen was determined by transfection of recombinant plasmids bearing the CAT reporter gene under the control of various segments from the human M-NM-15 promoter into primary cultured rabbit (RCECs) and human (HCECs) CECs cultured on BSA or on collagens. Electrophoretic mobility shift assays (EMSAs) and Western blot analyses were used to monitor both the DNA binding and expression of the transcription factors required to ensure basal transcription of the M-NM-15 gene. The influence collagens on the patterns of genes expressed by HCECs was also studied be gene profiling on microarrays. All collagen types repressed the transcriptional activity directed by the full-length M-NM-15 promoter/CAT construct in confluent CECs. A moderate increase in M-NM-15 promoter activity was observed in subconfluent RCECs grown on CIV but not on CI. These collagen-dependent regulatory influences also correlated with alterations in either the expression or the DNA binding of the transcription factors Sp1/Sp3, NFI and AP-1 that ensure basal transcription of the M-NM-15 gene. Gene profiling on microarray revealed that CI more profoundly alter the pattern of genes expressed by HCECs than what is observed with CIV. Collagens considerably suppressed expression of the M-NM-15 gene in CECs at confluence suggesting that the sustained staining for CI and CIV after corneal injury may play a role in controlling adhesion to the temporary FN matrix and further differentiation of CECs into suprabasal epithelial cells through a mechanism involving the M-NM-15M-NM-21 integrin. Primary cultures of human corneal epithelial cells cultivated on BSA (number of replicates: 2), Collagen type I (number of replicates: 2) and Collagen type IV (number of replicates: 2) matrix.

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Characterization of the human a9 integrin subunit gene: promoter analysis and transcriptional regulation

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