Transcriptional Regulation of the Macrophage Inflammatory Protein 1-Alpha/Stem Cell Inhibitor Gene

Grove, Matthew P. C (1993) Transcriptional Regulation of the Macrophage Inflammatory Protein 1-Alpha/Stem Cell Inhibitor Gene. PhD thesis, University of Glasgow.

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Macrophage inflammatory protein 1alpha (MIP1alpha) protein is an inhibitor of haemopoietic stem cell proliferation and an inflammatory mediator. MIP1alpha protein is present in normal bone marrow, and is believed to be produced there by a subpopulation of macrophages. The gene encoding the MIP1alpha protein is an early-response gene as it is present at low or undetectable levels in uninduced haemopoietic cell-types, and is rapidly inducible by inflammatory stimuli in a haemopoietic cell-specific manner. The presence of high constitutive levels of MIP1alpha gene expression in fresh haemopoietic tumour cells and in the peripheral blood and/or bone marrow of patients with leukaemic and pre-leukaemic disorders shows that the gene can be disregulated, by an unknown mechanism(s), with potentially profound effects on the haemopoietic system. As gene expression is controlled ultimately at the level of transcription, the work presented in this thesis is an examination of the transcriptional regulation of the MIP1alpha gene in macrophage cell lines compared to non-MIP1alpha- expressing cell types, in order to address the questions of how the MIP1alpha gene is regulated in-vivo and by what mechanism(s) its expression can be disregulated. A positive lambdaEMBL3 genomic clone was characterized and sequenced, confirming the presence of the MIP1alpha gene. MIP1alpha gene expression was shown to be rapidly and transiently induced by lipopolysaccharide (LPS), fresh serum and interferon alpha/beta, and to be down-regulated by transforming growth factor beta and interferon-gamma in macrophages, confirming that MIP1alpha is an early-response gene in macrophages, and consistent with a role(s) for macrophage-derived MIP1alpha protein in wound-healing and inflammation. A single constitutive nuclear DNase 1 hypersensitive site, which maps to the proximal 250-350 bp of the MIP1alpha promoter, was identified in macrophage cells but was absent in cells which do not express basal levels of MIP1alpha mRNA. Consistent with this, the promoter sequences from +36 bp to -220 bp are sufficent to confer cell-specific and inducible transcription on a reporter gene in transfection assays. This region consists of proximal and distal transcriptional regulatory elements, both of which contribute to the cell-specific promoter activity; the proximal region (+36 bp to -160 bp) confers constitutive, LPS- and serum-responsive promoter activity, and the distal region (-160 bp to -220 bp) confers constitutive and serum-responsive promoter activity. In-vitro DNA-binding studies revealed five major nucleoprotein binding sites in the proximal promoter, which bind C/EBP-, c-Rel and c-Ets family members. Cell-specific differences in DNA binding by a c-Rel family protein-containing complex correlates with the cell-specificty of endogenous MIP1alpha gene expression and the chromosomal conformation of the promoter. Cell-specific differences in DNA binding by C/EBP-, c-Rel- and c-Ets family members correlates with the cell-specificity of reporter gene expression conferred by the MIP1alpha promoter in transfection assays. Changes in promoter binding by members of the C/EBP- and c-Rel families correlate with the observed LPS-stimulated transcriptional up-regulation of the endogenous MIP1alpha gene measured by nuclear run-on analysis-, and serum- and LPS-stimulated up-regulation of proximal MIP1alpha promoter constructs in transfection assays-, in macrophages. Functional transfection studies of wild-type and point-mutated MIP1alpha promoter constructs, together with in-vitro promoter binding studies and sequence comparisons show that AP-1 can bind to the distal regulatory element, and suggest that it has a negative-regulatory activity, but that its DNA binding in unstimulated- and LPS-stimulated macrophages is occluded by positive-regulatory factors binding to adjacent promoter sites. Functional transfection studies in macrophages, of point-mutated MIP1alpha promoters, show that the positive-regulatory activity of the distal promoter element is dependent on the integrity of the distal-most C/EBP family binding site of the proximal MIP1alpha promoter element; this is supported by in-vitro DNA binding studies which suggest that non-AP-1 transcription factor binding to the distal regulatory element is co-operative. A testable model for the observed cell-specific and inducible transcriptional activity of the MIP1alpha gene is proposed, based on the in-vitro and functional data presented in this study. This model suggests mechanisms by which: 1) the kinetics of induction of the MIP1alpha gene in macrophages by different agents is different; 2) TGFbeta, IFNgamma and prostaglandins negatively-regulate MIP1alpha gene transcriptional activity in macrophages; 3) cultured macrophage cell-lines may be a good model for inflammatory macrophages but not for tissue-macrophages; 4) tissue macrophages may produce a constitutively low level of MIP1alpha protein; 5) the MIP1alpha gene is disregulated in haemopoietic fresh tumour cells; 6) the MIP1alpha gene may be inducible in certain non-haemopoietic cell-types.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Mark Plumb
Keywords: Molecular biology
Date of Award: 1993
Depositing User: Enlighten Team
Unique ID: glathesis:1993-75586
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Dec 2019 09:15
Last Modified: 19 Dec 2019 09:15

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