DOI
https://doi.org/10.25772/ZN4C-VD09
Defense Date
2010
Document Type
Dissertation
Degree Name
Doctor of Philosophy
Department
Biochemistry
First Advisor
Tomasz Kordula
Abstract
This dissertation sheds light on several novel mechanistic findings in astrocyte specific gene regulation and function by the NFI-X transcription factor which can be further extrapolated to astrocyte differentiation and glial tumor invasion. First, we cloned and analyzed human NFI-X3, a novel splice variant of the nfix gene, which contains a unique transcriptional activation (TA) domain completely conserved in primates. In contrast to previously cloned NFI-X1, overexpression of NFI-X3 potently activates NFI reporters, including GFAP reporter, in astrocytes and glioma cells. The expression of NFI-X3 is dramatically upregulated during the differentiation of neural progenitors to astrocytes and precedes the expression of astrocyte markers such as GFAP and SPARCL1. Overexpression of NFI-X3 dramatically upregulates GFAP and SPARCL1 expression in glioma cells, while the knockdown of NFI-X3 diminishes the expression of both GFAP and SPARCL1 in astrocytes. Although activation of astrocyte-specific genes involves DNA demethylation and subsequent increase of histone acetylation, the TA domain of NFI-X3 activates GFAP expression by inducing alteration in the +1 nucleosome architecture that lead to the increased recruitment of RNA polymerase II. Thus, we propose that NFI-X3 is the major isoform of NFI-X regulating astrocyte specific gene expression during their differentiation, likely via nucleosomal remodeling of the astrocyte specific promoters. NFI-X knock-out animals display severe neuroanatomical defects including partial agenesis of the corpus callosum and hydrocephalus, however the target genes of NFI-X in the CNS remained elusive. Here, we show for the first time that YKL-40 is a novel target gene of NFI-X in astrocytes and controls their migration. In addition, we report that YKL-40 expression is activated during mouse brain development and also during the differentiation of neural progenitors into astrocytes in vitro. In primary astrocytes, YKL-40 expression is controlled by nuclear factor I-X (NFI-X) and signal transducer and activator of transcription 3 (STAT3), which are known to regulate gliogenesis. Indeed, knock-down of NFI-X and STAT3 significantly reduced YKL-40 expression in astrocytes, while overexpression of NFI-X3 (a splice isoform of NFI-X) dramatically induced YKL-40 expression in glioma cells. In addition, activation of STAT3 by oncostatin M induced YKL-40 expression in astrocytes. Interestingly, STAT3 activated YKL-40 expression via its binding site located in the YKL-40 proximal promoter, whereas direct NFI-X binding had not been detected. Surprisingly, NFI-X and STAT3 physically interact and this complex likely regulates YKL-40 expression in astrocytes. We further show that NFI-X controls migration and invasion of astrocytes and glioma cells, respectively, by regulating YKL-40 expression. These novel data suggest that YKL-40 is expressed by astrocytes during brain development and controls astrocyte migration. Since YKL-40 is used as a shared biomarker for ongoing inflammation and oncogenic transformation and its (high) levels correlate to the severity of disease, we have tested its expression in astrocytes and microglia (CNS macrophage) after treatment of various neuro-inflammatory cytokines. Here we report, that IL-1 and IL-6/OSM synergistically activate YKL-40 expression in astrocytes but not in microglia when added together. Furthermore, induced YKL-40 expression can be detected in the media from astrocytes but not from microglia. Since YKL-40 is a secreted molecule and is highly upregulated in CSF of multiple sclerosis patients, we have tested its role in oligodendrocyte differentiation. Preliminary observations clearly demonstrate that YKL-40 inhibits myelin basic protein (MBP) expression during the in-vitro differentiation of oligodendrocyte progenitor cells into myelin producing oligodendrocytes. Thus, we propose that YKL-40 is produced and secreted by reactive astrocytes during various CNS pathologies, and may inhibit MBP expression in MS. In summary, these studies have identified novel mechanisms in astrocyte gene regulation and functions, and provided new insights into astrocyte biology, with the implications for further understanding of the development and progression of CNS pathology.
Rights
© The Author
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
July 2010