Transcriptional Regulation of the Mouse betamajor Globin Gene in Murine Erytroleukaemia Cells

MacLeod, Kay Ferguson (1990) Transcriptional Regulation of the Mouse betamajor Globin Gene in Murine Erytroleukaemia Cells. PhD thesis, University of Glasgow.

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Abstract

There is considerable evidence that certain features of the mechanism of transcriptional regulation of the (betamajor globin gene are common to other erythroid-specific globin and non-globin genes (reviewed in chapter 1) . However, my work suggests that there are aspects of this regulation which are unique. My work on the binding of nuclear proteins to the mouse betamajor globin gene promoter has revealed a modular organisation of protein binding sites. These include two binding sites for the erythroid-specific factor, NF-E1 (at -65 and -210 bp) and binding by non-tissue-specific proteins to the CCAAT (-85 bp), CACCC (-95 bp) and Box 1 (-170 bp) motifs and to a nuclear factor 1 (NF-1) binding site (-255 bp). Mutational analysis of the mouse betamajor globin gene proximal promoter TATA, CCAAT and CACCC boxes by other researchers had shown that they are required for promoter function in erythroid and non-erythroid cells. Using site-directed mutagenesis, I have confirmed that the CACCC motif is required for promoter function when linked to a reporter gene and assayed in transient and stable transfection assay systems. Furthermore I have shown by mutagenesis that the NF-E1 site at -65 bp is necessary for promoter function. However mutation of the NF-E1, Box1 and NF-1 sites in the distal region of the promoter results in up-regulation of transcription from the promoter in uninduced MEL cells and it is proposed here that while the proximal promoter is essential for promoter function that the distal promoter has a negative regulatory function in uninduced MEL cells. This negative regulatory effect is derepressed when the MEL cells are induced to differentiate. In addition to the role of 5' promoter sequences in transcriptional regulation of the mouse betamajor globin gene, I have identified sequences 3' of the coding region of the gene which contain NF-E1 and Box 1 binding sites and which confer an up-regulatory effect on the transcription of a linked reporter gene from the mouse betamajor globin gene promoter. The up-regulatory effect of this enhancer is greater when the cells are induced to differentiate. Work with two different MEL cell lines, F4-12B2 and C88, and with two different inducing agents, DMSO and HMBA, suggests that there are differences in the responses of these cell lines to the same treatment and also that DMSO and HMBA act through different mechanisms to induce differen tiation.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Genetics
Date of Award: 1990
Depositing User: Enlighten Team
Unique ID: glathesis:1990-78031
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 30 Jan 2020 15:43
Last Modified: 30 Jan 2020 15:43
URI: http://theses.gla.ac.uk/id/eprint/78031

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