Regulation of heat shock protein 27 gene expression /
A GC-rich oligonucleotide containing an estrogen responsive
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| Format: | Thesis Book |
| Language: | English |
| Published: |
[Place of publication not identified] :
[publisher not identified] ;
1997.
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| Subjects: | |
| Online Access: | http://proxy.library.tamu.edu/login?url=http://proquest.umi.com/pqdweb?did=739887841&sid=1&Fmt=2&clientId=2945&RQT=309&VName=PQD |
| Summary: | A GC-rich oligonucleotide containing an estrogen responsive element (ERE) halfsite from the heat shock protein 27 (Hsp 27) gene promoter (- 1 05 to -84) formed a complex with the Sp I and estrogen receptor (ER) proteins. Promoter-reporter constructs containing this sequence (- 1 08 to -84 or - 1 08 to +23; Hsp-CATs and Hsp-CATI, respectively) are estrogen- responsive. Mutation of the ERE half-site did not result in loss of estrogen-responsiveness in transient transfection studies suggesting that estrogen inducibility was mediated through the Sp I -DNA motif. Gel mobility shift assays using relabeled wildtype and ERE mutant Sp 1 (N), OERE and consensus Sp I oligonucleotides showed that Sp I formed a DNA-protein complex with all three oligonucleotides and the intensities of the retarded bands were enhanced by coincubation with wild-type ER and 1 1 C-ER, which does not contain the DNA binding domain. ER mutants in which N- terminal (I 9C-ER) and C-Terminal (I 5 C-ER) regions were deleted did not enhance Sp 1 -DNA binding or hormone-induced transactivation of GC-rich promoter-reporter constructs in ER-negative NMA-MB231 cells, whereas both wild-type and I I C-ER restored inducibility. Immunoprecipitation studies confirmed that the Sp I and ER proteins interact. Treatment of MCF-7 cells with ICI 164,384 resulted in a 2-fold induction of Hsp 27 MRNA levels. Transient transfection assays utilizing Sp I -directed promoter reporter constructs in MDA-231 cells cotransfected with wild-type ER, I I C-ER, 15C-ER and 19C-ER showed that both class I and class 11 antiestrogens tamoxifen and ICI 164,384 induced transcription from GC-rich reporter constructs and this transactivation required the DNA binding domain of the ER. 2,3,7,8- Tetrachlorodibenzo-p-dioxin (TCDD) decreased estrogen-induced Hsp 27 MRNA levels in MCF-7 cells within 2 h and this response persisted up to 24 h. Analysis of the 5'-promoter region of the Hsp 27 gene identified an IDRE at the +1 start site. TCDD inhibited estrogen-induced Hsp-CATI transcription, whereas no inhibition was observed with an plasmid containing mutations in the DRE motif. Incubation of an BrdU-DRE oligonucleotide derived from the Hsp 27 promoter with TCDD treated MCF-7 nuclear extracts gave a specifically bound cross-linked 200-kDa band. The formation of this crosslinked band was inhibited by coincubation with unlabeled wild-type DRE and by preincubation with AhR antibodies (immunodepletion). |
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| Item Description: | Vita. "Major Subject: Toxicology". |
| Physical Description: | xi, 141 leaves : illustrations ; 28 cm. Issued also on microfiche from University Microfilms Inc. |
| Bibliography: | Includes bibliographical references: pages 114-139. |