High mobility group nucleosome binding (HMGN) proteins belong to the superfamily of high mobility group (HMG) proteins. HMGN1 and HMGN2 are ubiquitously expressed in all vertebrates, and are most highly expressed in embryonic tissue. Moreover, HMGN1 and HMGN2 were found to be highly expressed in neural stem/progenitor cells in the mouse brain. Here, mouse embryonal carcinoma cells (P19 EC) were used as a model system to study the role of HMGN proteins in pluripotent stem cells. Previously, experiments using short interfering RNA (siRNA) technology to knockdown HMGN1 and HMGN2 have suggested that HMGN proteins are important for the expression of key pluripotent genes, Oct4, Sox2 and Nanog, in P19 EC cells (Mohan, 2012). The aim of this thesis was to develop a lentiviral system for the long term knockdown of Hmgn2, in order to investigate more fully the role of this protein in stem cell pluripotency and differentiation. Constitutive and inducible lentiviral shRNAmir systems were tested and optimized, and a constitutive system was chosen for further work. HMGN2 knockdown in undifferentiated P19 EC cells resulted in the down-regulation of Oct4 protein levels. ChIP assays showed that HMGN2 binding over the Oct4 gene was absent in HMGN2 knockdown cells. Furthermore, binding of HMGN1 at this locus was increased in the absence of HMGN2. Consistent with the reduction in Oct4 expression, levels of the active histone modification, H3K4me3, were also decreased at the Oct4 gene. These results support a role for HMGN2 in the regulation of Oct4 expression in P19 cells, and imply that HMGN2 may be important for maintaining stem cell pluripotency.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:739205 |
| Date | January 2016 |
| Creators | Rehbini, Ohoud Mohammedsabri M. |
| Publisher | University of Glasgow |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://theses.gla.ac.uk/7190/ |
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