1996 Fiscal Year Final Research Report Summary
Transcriptional repressive function of the DNA structure-specific recognition protein Orpheus/SSRP1
| Project/Area Number |
07680747
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Molecular biology
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| Research Institution | Keio University School of Medicine |
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Principal Investigator |
IMAI Shin-Ichiro Keio University, School of Medicine Department of Microbiology, Instructor, 医学部, 助手 (20255433)
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| Project Period (FY) |
1995 – 1996
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| Keywords | DNA structure / SSRP1 / transcriptional repression / HMG box / cellular senescence / immortalization / Oct-1 |
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| Research Abstract |
A transcription factor Orpheus negatively regulates the collagenase gene, which is one of the genes whose expression is drastically changed in cellular senescence and immortalization, by interacting with an AT-rich cis-element of ISE2 in the upstream of the gene. As a candidate gene for Orpheus, we isolated a cDNA clone that encoded the structure-specific recognition protein 1 (SSRP1) by Southwestern screening. SSRP1 carries a high mobility group (HMG)-box domain homologous to HMG1 and 2. SSRP1 was constitutively expressed at protein and mRNA levels during the processes of cellular senescence and immortalization. However, the overexpression of SSRP1 mediated repression of the collagenase transcription preferentially in young preimmortalized and immortalized cells. This repressive activity of SSRP1 was not detected when the Orpheus binding site was mutated. Because the band of Orpheus was not supershifted by a SSRP1-specific polyclonal antibody, SSRP1 did not seem to be included in the
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Orpheus binding activity. By chemical footprint analysis, a weak footprint was detected on the sequence 5' adjacent to the Orpheus binding site. When this sequence was mutated, the trascriptional activity of the collagenase upstream region was enhanced about four-fold. We examined whether the corresponding factor was SSRP1 or not, by Southwestern blotting and gel shift assay with a bacterially produced SSRP1. We detected the ISE2-specific binding activity of SSRP1 by Southwestern blotting, not by gel shift assay. Although the precise reason for these results remains unclear, it may be possible that SSRP1 has an intramolecular domain which inhibits its DNA binding activity, or that SSRP1 can bind to DNA only by interacting with other regulatory molecules. Recently, we demonstrated that Orpheus was identical to a member of the POU domain family, Oct-1. Thus, SSRP1 mediates transcriptional repression in cellular senescence- and immortalization-dependent manner, possibly by interacting with Oct-1. Less
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