The focus of this proposal is the study of transcription factors involved in IFN-g- induced expression of gp91-phox (the NADPH oxidase heavy chain). Expression of gp91-phox is restricted to terminally differentiated phagocytes and is increased by interferon gamma (IFN-g). Transcription of gp91phox is part of the """"""""secondary response"""""""" to IFN-g. The gp91phox promoter contains no g-activation site consensus sequence and IFN-g stimulated gp91phox transcription requires new protein synthesis. Transcription factors involved in gl91phox gene regulation provide a link between the IFN-g """"""""early responses"""""""" and the temporally remote events of proliferation arrest and inflammatory mediation. These linking events are at present poorly characterized. Three unique enhancer elements in the gp91phox promoter addictively increase IFN-g expression. This has been demonstrated using reporter constructs of gp91phox 5' flanking region coupled with hugh expression in an RIA assay (showing a two-fold increase), and in an RNAse protection assay (showing a ten-fold increase). The three enhancer elements are recognized by a common protein complex (BID1, for binding induced during differentiation) and in vitro binding of the complex is increased by differentiation in myeloid cell lines. Each enhancer element is immediately 5' to a binding site for the repressor CDP which had been identified by Dr. Orkin's group. An enhancer element binding protein (BID1p) has been identified and cloned using an oligonucleotide sequence corresponding to one of the 5' promoter sequences to screen a commercial leukocyte lgt11 CDNA library. Genbank search demonstrated no homology between BID1p and previously described transcription factors. Several lines of evidence indicate that BID1p plays a role in regulation of gp91- phox transcription: over-expression of BID1p in myeloid cell lines disrupts in vitro CDP binding to the gp91phox repressor elements and increases the abundance of gp91phox message. Treatment of myeloid cell lines with IFNg increases the abundance of BID1p MRNA at 3 hours, suggesting the possibility that BID1p is an IFNg early response gene. This proposal therefore proposes to 1) investigate the role of BID1p in the regulation of IFN-g responsive myeloid genes. This will be done by examining the effect of overexpression of BID1p in the human PLB985 cells, and in separate studies using expression of BID1p antisense message expression to determine if BID1p expression is necessary for gp91phox transcription. The effect of IFN-g on transcription of other proteins (such as CDP) will also be examined. These studies will be extended to investigate the role of BID1p in a non-malignant hematopoiesis model using the IL3-dependent 32DC13 cells Murine BID1p will be cloned by screening a mouse spleen CDNA library with the human CDNA already in hand.
The second aim i s to investigation the regulation of BID1p binding to gp91phox enhancer elements, using an anti-BID1p antisera to be generated. This antiserum will be used to examine the effect of IFNg on BID1p protein abundance and subcellular localization, to determine whether Bid1p undergoes modifications such as phosphorylation, and an attempt to demonstrate immunologically that BID1p is present in the gel-shifted BID1 binding experiments. The effect of IFN-g on BID1p message transcription and message stability will also be examined for stability and specificity.
The third aim i s to investigate the in vivo changes of proteins binding to the gp91phox promoter, an extension of the in vitro footprinting methods that have been utilized to date for this project.
| Wang, Pen-Cheng; Vilaire, Gaston; DeGrado, William F et al. (2007) Interactions of ADP-stimulated human platelets with PEGylated polystyrene substrates prepared by surface amidation. Colloids Surf B Biointerfaces 58:225-30 |
