Transcription Factor Binding to, and Regulation of, the HNP-1 defensin Gene Promoter

Schembri-Wismayer, Pierre (1999) Transcription Factor Binding to, and Regulation of, the HNP-1 defensin Gene Promoter. PhD thesis, University of Glasgow.

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The defensins are small antibiotic peptides produced during granulocyte differentiation and stored in the azurophilic granules of the mature granulocyte. Defensin is expressed during a brief window in differentiation with mRNA expression peaking at the promyelocyte and early myelocyte stages. Using the promyelocytic leukaemia cell line NB4, nuclear protein binding to the gene sequences immediately upstream of the main transcription start site was studied. Changes in such binding were correlated with mRNA abundance during granulocyte (high defensin) and monocyte (no defensin) NB4 differentiation. Protein-binding sites, signified by changes in DNAse digestion, were recognised at the ets site at positions -59 and -155, the overlapping c/ebp-myb site at position -120/-105 and at numerous c/ebp-aml sites along the first 240 bp of the upstream sequence. Binding to most of the sites, with the exception of the -59 ets site, was seen to be considerably greater (by footprinting studies) with granulocytic extract as opposed to undifferentiated NB4 extract. The presence of a DNase 1 hypersensitive site at position -59 (which overlaps the ets site) seems to be essential for defensin expression. The presence of this site appeared to be correlated with baseline expression since it is absent in footprints seen with the extract of monocytic NB4 cells (which do not express defensin). This site (termed footprint alpha) was shown to bind GABPalpha, the c/ebp-myb site (within footprint beta) was shown to bind C/EBPepsilon whilst the -155 site (in footprint gamma) was shown to bind PU.1 by means of electrophoretic mobility shift assays (EMSAs). Different transcription factors were also shown to compete for binding to particular footprinted sites by means of competition EMSAs. By inserting mutations into the binding sites for these various factors, the ets site at -59 and the c/ebp and myb sites at -120/-105 were shown to be very important for defensin promoter activity. For maximal activity in undifferentiated NB4 cells, both these sites were required, but in differentiated cells maximum promoter activity was obtained with a minimal promoter, -67/+15, which did not include any myb sites. Co-transfection studies showed that C/EBP and GABPalpha could up-regulate defensin expression in NB4 cells. GABPbeta did not co-operate with GABPalpha in undifferentiated cells, but synergised with it in differentiated NB4 cells. In heterologous HeLa cells, defensin promoter activity was stimulated synergistically by C/EBPe with Myb. It was also strongly transactivated by GABPalpha but such transactivation was unexpectedly inhibited by co-expression of GABPbeta, CBFalpha/beta, PU.1 or CHOP-10 were found to co-operate with GABPalphabeta to stimulate transactivation. The pattern of transactivation obtained with GABP factors differed from the classical synergism seen between GABPalpha and GABPbeta. These differences may be due to both the different reporter systems being used, and to the defensin promoter in particular, which appears to bind GABPalpha alone, much more strongly than other promoters such as the neutrophil elastase promoter. The results obtained have been used to create models of possible protein interactions on the defensin promoter. Using known patterns of expression of transcription factors during myeloid differentiation, a model is presented describing probable factor interactions responsible for initial up-regulation and later down regulation during differentiation.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: George Birnie
Keywords: Genetics
Date of Award: 1999
Depositing User: Enlighten Team
Unique ID: glathesis:1999-74907
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 27 Sep 2019 15:25
Last Modified: 27 Sep 2019 15:25

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