Transcriptional regulation of proopiomelanocortin gene expression by corticotropin-releasing hormone.
Item
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Title
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Transcriptional regulation of proopiomelanocortin gene expression by corticotropin-releasing hormone.
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Identifier
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AAI9521284
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identifier
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9521284
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Creator
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Jin, Wei Dong.
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Contributor
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Adviser: James L. Roberts
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Date
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1995
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Language
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English
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Publisher
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City University of New York.
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Subject
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Biology, Neuroscience | Biology, Molecular
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Abstract
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A corticotrophin releasing hormone (CRH) and cAMP-responsive region ({dollar}-{dollar}236/{dollar}-{dollar}133) in the rat POMC gene promoter previously reported to confer CRH/cAMP responsiveness to heterologous reporter constructs has been characterized. DNAse-footprint analysis revealed that multiple elements in this region were bound by nuclear proteins from the POMC expressing AtT20 cell. When these individual DNA elements were separately tested in heterologous reporter constructs for CRH induction, only one element, designated PCRH-RE (POMC CRH responsive element, {dollar}-{dollar}171/{dollar}-{dollar}160) was found to give strong CRH stimulation (5-7 fold). Gel shift analyses of the PCRH-RE with AtT20 cell nuclear extracts showed marked stimulation of retarded nucleoproteins following CRH stimulation. The activity of PCRH-RE binding protein was inhibited by divalent cations, with Cu{dollar}\sp{lcub}2+{rcub}{dollar} and Cd{dollar}\sp{lcub}2+{rcub}{dollar} being most effective, while Zn{dollar}\sp{lcub}2+{rcub}{dollar} had no effect. A 2.6 kb cDNA clone encoding a protein (PCRH-REB-1) binding to this element was isolated by Southwestern screening of an AtT20 expression library with radio-labeled PCRH-RE oligonucleotides. This clone was used to isolate several other cDNA clones to determine the sequence corresponding to the entire coding region of the protein (PCRH-REB), which proved to be identical to a recently described DNA binding protein of the replication factor C complex, mRFC140/MSW. Primer extension and Northern blot analysis revealed that the size of the full length mRNA is about 4.9 kb. PCRH-REB mRNA expression is not restricted to corticotrophs but is present in a broad tissue distribution as evaluated by RT-PCR analysis. A bacterially expressed {dollar}\beta{dollar}-galactosidase-PCRH-REB-1 fusion protein was shown to bind PCRH-RE efficiently. Furthermore, binding of the PCRH-REB-1 fusion protein to the POMC CRH-responsive element was inhibited by divalent cations with similar sensitivities to those observed using AtT20 nuclear extracts. The predicted PCRH-REB protein sequence presents several interesting motifs: one p-Loop motif (ATP binding site), nine PKA phosphorylation sites (implying a possible role in responding to the CRH induced cAMP signal), and regions of homology to proteins involved in DNA replication and repair. Results from functional studies suggested that CRH regulation of POMC gene transcription by {dollar}-{dollar}236/{dollar}-{dollar}133 region is mediated by a complex, multi-element involved mechanism.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
