All ETDs from UAB

Advisory Committee Chair

Michael Brenner

Advisory Committee Members

Farah Lubin

Scott Wilson

Steven L Carroll

Thomas M Ryan

Document Type

Dissertation

Date of Award

2012

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

Glial fibrillary acidic protein (GFAP) is an astrocytic intermediate filament protein whose levels are increased in response to CNS injuries. Aim one of my thesis is to find the regulatory elements of the GFAP promoter responsible for its expression properties using a transgenic approach, since previous studies showed inconsistent results between in vitro and in vivo approaches. Two contiguous DNA segments previously implicated in transcriptional control of the GFAP gene, the B region (bp -1612 to -1489), and the C1.1 and C1.2 regions (bp -1488 to -1399), were analyzed. Block mutation of each of 4 contiguous sequences together spanning the B region decreased the level of promoter activity by at least 50%, indicating that each subregion contains the sites that cooperatively contribute to transcription. Several of the block mutations also altered the brain region pattern of expression, astrocyte specificity and/or the developmental time course. Effects on expression pattern and specificity had previously been observed for C1.1 and C1.2 block mutations. To identify the specific transcription factors involved, candidate binding sites were mutated in each of the 4 B block segments as well as in C1.1 and C1.2. Mutation of the AP-1 site unexpectedly had no effect, confirming that in vitro and in vivo regulation of GFAP transcription differ. Mutation of the SP1 site significantly reduced promoter activity, indicating that SP1 is a novel GFAP trancriptional activator. Mutations of either the NFI or NF-&kappaB site resulted in significant transgene expression in hippocampal neurons, suggesting that they act to suppress GFAP expression in neurons. My second aim was to find the epigenetic mechanisms associated with region specific expression of a transgene driven by a shortened GFAP promoter, gfaABD (bp -1757 to -1489 linked to -132 to +56). I found that histone methylation, but not DNA methylation, is correlated with the region specificity. This result suggests that epigenetic mechanisms are associated with astrocyte regional heterogeneity of gene expression. My findings contribute to understanding the diverse roles of transcription factors and epigenetic modulators for GFAP in vivo expression, and will help discovery of therapeutic targets for various CNS diseases involving altered GFAP expression.

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