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Nuclear Factor I in Malignant Glioma
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- Author / Creator
- Brun, Miranda
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Malignant glioma (MG), comprised of grade III (anaplastic astrocytoma) and grade IV (glioblastoma) astrocytoma, is the most common adult brain tumour, with an incidence of ~4 in 100 000 people. Despite aggressive treatment with surgery, radiation, and chemotherapy, survival remains dismal, with median survival of 2-3 years for grade III, and 14.6 months for grade IV astrocytomas. MGs are hypothesized to arise from cells in the glial cell lineage based on expression of glial genes including glial fibrillary acidic protein (GFAP) and brain fatty acid-binding protein (B-FABP, FABP7). In MG, B-FABP expression correlates with poor prognosis, and increased migratory activity in MG cell lines. During development, B-FABP is expressed in a type of neural stem cells called radial glial cells. Following the onset of gliogenesis, expression of GFAP is activated in these cells. The Nuclear factor I (NFI) family of transcription factors, consisting of four family members (NFIA, B, C, X), are expressed in the developing brain, and are necessary for normal glial cell differentiation, including expression of GFAP. NFI binding sites are present in the promoters of both B-FABP and GFAP, and NFI family members are expressed in MG cells. We examined the role of NFI family members in regulating expression of B-FABP and GFAP in MG cell lines. We show that NFIs bind to the promoters of the B-FABP and GFAP genes and regulate their expression, with all four NFIs contributing to their regulation. Of note, we show that NFI-dependent regulation is promoter and promoter-context dependent. We also observe compensation between NFI family members, which suggests cross-talk between NFIs. Given the vital role of NFI in gliogenesis, and expression of NFI in MG cells, we sought to identify additional NFI target genes in MG. Using chromatin immunoprecipitation (ChIP)-on-chip, we identified 403 putative NFI target genes, including the Notch effector HEY1. HEY1 promotes maintenance of neural progenitor cells during development, and its expression correlates with decreased survival in MG. Here, we show that NFI binds to NFI binding sites in the HEY1 promoter, and represses expression of HEY1 in MG cells. NFI is differentially phosphorylated in MG cell lines, and hypophosphorylated NFI correlates with expression of B-FABP and GFAP in these cells. Previously, a phosphatase activity was identified in cells with hypophosphorylated NFI that was absent in cells with hyperphosphorylated NFI. We show that the phosphatase calcineurin regulates NFI dephosphorylation and activation in MG cells. Furthermore, we identify a cleaved, activated form of calcineurin that localizes to the nucleus and is specifically found in MG cells with hypophosphorylated NFI. Immunohistochemical analysis of grade IV astrocytoma tumour tissues reveals the presence of calcineurin in the nucleus of cells found in areas of infiltration/migration. Taken together, our findings demonstrate an important role for NFI in regulation of genes involved in glial cell differentiation in MG cells, and reveal a novel calcineurin-NFI regulatory axis that further regulates NFI-dependent promoter activity in these cells.
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- Graduation date
- Fall 2015
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- Type of Item
- Thesis
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- Degree
- Doctor of Philosophy
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- License
- This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.