İnsan nöral kök hücrelerinde NFI transkripsiyon faktörlerinn, NFIB hedef genlerinin ve NFIB ekspresyonunu düzenleyen miRNA'ların karakterizasyonu

The transcription factors of the nuclear factor one (NFI) family play an important role in regulation of gene expression during nervous system development. NFI family has four members (NFIA, NFIB, NFIC and NFIX) that are highly homologous in their N-terminal DNA binding domains, but vary in their C terminal transcription activation/repression domains. NFIs can induce or repress transcription of different genes in different cell types in a context dependent manner. Each NFI member has multiple alternative splicing isoforms; however, functions of these isoforms of NFIs have not been well described. NFI proteins recognize consensus sequence TTGGC(N5)GCCAA and bind double strand DNA as homo- or heterodimers. In addition, the post-translational modifications (glycosylation or phosphorylation) of NFI proteins can potentially affect or modulate their function. NFIs are expressed in an overlapping but distinct expression pattern in the developing embryo suggesting a role in regulation of development. All NFIs except for NFIC are expressed in the embryonic central nervous system while expression is restricted to stem cell niches in the adult brain. Deletion of NFIA, NFIB and NFIX leads to distinct phenotypes involving different brain regions such as neocortex, hippocampus, and hindbrain, altering different developmental processes like axonal outgrowth and guidance, differentiation, neurogenesis or gliogenesis. Previously, we have found that while all three neural NFI proteins are expressed in the precerebellar systems of the hindbrain, only the absence of NFIB lead to a delay in the development of precerebellar nuclei. To understand how NFIB affected neurogenesis in the precerebellar system, potential NFIB target genes were identified in precerebellar neuroprogenitors by mRNA expression profiling. Here, we set out to study regulation of downstream targets of NFIB as well as upstream regulators of NFIB in the human neural stem cell culture system derived from H9 human embryonic stem cell line. To this end, we first characterized the expression of NFI family members and three target genes (CDO, COL2A1 and FGF19) selected according to their expression patterns and known functions. Expression of NFIB, NFIC, NFIX, CDO and COL2A1 was significantly reduced while NFIA and FGF19 were up-regulated during neuronal differentiation. NFIB-4, NFIB-3 and/or NFIB-1 isoform mRNA expression was detected in this cell line. We found that NFIB-3 and NFIB-1 protein expression also decreased in differentiated hNSCs. To identify novel upstream regulators of NFIB, we first determined miRNA binding sites on the NFIB 3'UTR and looked for those miRNAs that are upregulated in hNSCs during neural differentiation. We analyzed expression of miR-153, miR-30a, miR-124a, miR-92b, miR-130a and miR-363 and found miR-153, miR-124a and miR-30a expression increased while miR-363 decreased in differentiating hNSCs. Upregulated miRNAs were selected for further study. 3'UTR fragments carrying binding sites of these miRNAs were fused to a reporter gene and screened for inhibition by miRNA mimics. Preliminary data indicate that miR-153 and miR-124a can directly repress translation of NFIB by interacting with 3'UTR_D (6418-6425) and 3'UTR_B (1842-1848) regions on the NFIB. Site directed mutagenesis of miR-153 and miR-124a binding sites appear to render these sites resistant to miRNA mimics, underlining the specificity of this repression.