Investigation of micrornas that interact with the NFIX 3′untranslated region

Nuclear factor I (NFI) transcription factor family controls many important cellular and developmental events in the embryo and the adult. This family is made up of four members in vertebrates: NFIA, NFIB, NFIC and NFIX. NFIs carry an N-terminal DNA binding-dimerization and a C-terminal transcriptional activation and repression domain which includes a proline-rich region. The N-terminal DNA binding and dimerization domain is highly conserved among NFI members, while the C-terminal domain varies. There are four conserved cysteine residues in the NFI N-terminal domain, one of which is sensitive to oxidative inactivation while the other cysteine residues are necessary for NFI DNA binding capacity. NFIs bind as homo- or heterodimers to the TTGGC(N5)GCCAA consensus sequence on duplex DNA. NFIs can regulate gene expression in a cell and tissue specific manner by various mechanisms: interaction with basal transcription factors, co-regulation with other transcription factors, binding to co-activators or co-repressors, alteration of the nucleosome structure and modification of the methylation pattern near the promoter of the target gene. NFIA, NFIB and NFIX are expressed in neural progenitors, neurons and glia of the central nervous system during embryonic development, however expression of these NFI members is restricted to stem cells in the adult brain. NFIs regulate processes such as neurogenesis and gliogenesis in different brain regions in the embryo and the survival of progenitor cells in adults. Nfia, Nfib and Nfix null mice demonstrate enlarged ventricles in the cerebral cortex, indicating misregulation of differentiation and neural progenitors. In the cerebellum, NFIs are required for neuronal migration and terminal differentiation and in cerebellar granule neurons they regulate the expression of molecules that are associated with these processes such as the extracellular signaling molecule (Wnt), cell adhesion molecules (Ephrin-B1, N-cadherin and Tag1). Nfix promotes neurogenesis by repressing of Sox9, a transcription factor involved in stem cell self-renewal in hippocampus. In Nfix null post-natal mice, progenitor cell specific markers (Pax6, Hes1, Hes5 and Sox2) are upregulated indicating expansion of progenitors while neuronal and glial differentiation is disrupted as well. In addition, Nfix also regulates neuronal migration, and one of its targets is a neurotropic factor (Gdnf) that promotes migration of progenitors in the olfactory bulb. In the spinal cord, Nfia and Nfib both induce Gfap expression, an astrocyte differentiation marker, Nfib also induces Nfix expression. Nfix, in turn, regulates astrocyte terminal differentiation by upregulation of Gfap and other astrocyte specific genes. In addition to its role in development, Nfix also involved in the regulation of stem cell quiescence and neurogenesis in the adult hippocampus. Interestingly, in cell culture, Nfix is upregulated in quiescent stem cells and is necessary and sufficient to induce quiescence. On the other hand, conditional knock-out experiments show that Nfix promotes neuroblast differentiation and represses oligodendrogenesis inhibiting expression of oligodendrocyte specific genes (Olig1, Olig2, Ptprz1 and Ntrk2) in vivo. Moreover, Nfix deletion in mice leads to fewer neurons and disrupted dendritic formation and process branching. Therefore, NFIX may have a dual role in adult neurogenesis: preserving stem cell quiescence and cell fate determination to neurons and astrocytes. Previously, we found that NFIX expression is downregulated in differentiating neural stem cells in vitro. In order to identify novel upstream regulators of NFIX, we first analyzed miRNA binding sites on the NFIX 3'UTR. We selected miRNAs which are highly expressed in neural progenitor cells for further analysis: miR25, miR92b, miR124 and miR363. Additionally, miR1915 which was reported to supress NFIX is selected as positive control. NFIX 3'UTR fragments which contain binding sites of these miRNAs were isolated, fragment I (nts ~1300-2600) was inserted in the downstream of the luciferase reporter gene. Then, supression of reporter gene translation by miRNA mimics was assayed. miR25 and miR363 repress luciferase activity from the NFIX 3'UTR I, while preliminary data indicate that miR92b and miR124 did not have such an effect. Interestingly, miR1915 could not repress translation of reporter genes fused to NFIX 3'UTR I. However, the activity of this mimic has not been validated yet. We are in the process of mutating the miR25 and miR363 binding sites on the NFIX 3'UTR to ascertain the specificity of these interactions. Future studies will explore the physiological significance of potential supression of NFIX by these miRNAs.