Transcriptional Regulation of the Evi-1 Gene

Fordham, Jeremy Leigh (1996) Transcriptional Regulation of the Evi-1 Gene. PhD thesis, University of Glasgow.

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Evi-l shows a temporally and spatially restricted pattern of expression in murine embryonic development and is expressed predominantly in the kidney, lung and developing oocytes of adult mice. The positions of DNAse 1 hypersensitive sites (DHS) within an 18kb region of the 5' Evi-1 locus have been examined to identify putative Evi- 1 gene regulatory regions, in murine kidney and spleen tissues. This analysis identified two DHS sites designated DHS I and DHS II. DHS I is located approximately 2kb upstream of the transcription initiation sites whereas DHS II maps over exon I. The transcriptional activity of the Evi-1 promoter has been investigated by inserting the 5' region of the gene into a luciferase construct and the activity examined by transient transfection of cells which express low levels of Evi-1. A substantial induction of luciferase activity is observed with a 5kb fragment of the 5' Evi-1 locus containing exon I, intron I and exon II which includes DHS I and DHS II. Subsequent deletion mutagenesis has identfied two regions within DHS II, located between -338 to -284 and -284 to -254, which upon removal result in a substantial reduction of promoter activity. One of these, located between -338 to -284, binds several proteins when examined by footprinting and electrophoretic mobility shift assays. Interestingly, the most abundant factor, designated EvBP1, has been shown to bind a 14bp imperfect palindromic sequence, tttccctggggaaa, which is absolutely conserved in the human Evi-1 promoter sequence. This sequence contains homology with putative binding sites for, AP3, AP2 and C/EBP. However, competition studies in EMSA with consensus binding site oligonucleotides failed to identify the components of EvBP1. EvBP1 might be a novel ubiquitous transcription factor which is required for regulation of the Evi-1 promoter. Furthermore, EMSA analysis of the second deleted region between -284 to -254 has identified a CCAAT binding protein, possibly CPI, which may also be important in basal promoter activity. Functional assays have failed to identify either promoter or enhancer activity for DHS I. Since there are no known appropriate high Evi-1 expressing cell lines we have established Evi-1 expressing kidney cultures as a system to examine tissue specific expression. This allowed the identification of a 3kb region necessary for higher activity in the cultures. This activity might correlate with a DHS site which is part of DHS II complex.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Chris Bartholomew
Keywords: Genetics
Date of Award: 1996
Depositing User: Enlighten Team
Unique ID: glathesis:1996-75560
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 19:27
Last Modified: 19 Nov 2019 19:27

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