Investigation of NFIB binding to CDO and FGF19 gene regulatory regions by chromatin immunoprecipitation

Nuclear factor one (NFI) family of transcription factors play important roles in regulation of gene expression during nervous system development. NFI family has four members: NFIA, NFIB, NFIC and NFIX. NFIs bind to the consensus sequence, TTGGC(N5)GCCAA on double-stranded DNA as homodimers or heterodimers. NFI family members contain an N-terminal DNA binding and dimerization domain and a C-terminal transcriptional activation and repression domain. The N-terminal domain is highly conserved among members and includes an MH1 motif, also found in SMAD proteins of the TGF-β signalling pathway. The proline rich C-terminal domain is less conserved compared to the N-terminal domain. Each NFI member has multiple alternative splicing isoforms; however, functions of these isoforms of NFIs have not been well described. NFIA, NFIB and NFIX are all expressed in the central nervous system and are required for neural development. NFIA, NFIB and NFIX knock-out mice exhibit distinct phenotypes, where different developmental processes such as differentiation, neurogenesis or gliogenesis, are affected. Previously, we found that among the three neural NFI family members, NFIB is specifically required for development of the precerebellar neurons in the mouse hindbrain. As the defect in precerebellar development appeared to be due to a delay in neurogenesis, we looked for changes in gene expression of precerebellar neuroprogenitors by mRNA profiling. Of the 33 genes that were misregulated in NFIB-/- progenitors, CDO and FGF19 were initially selected for further study. Both genes are also implicated in regulation of neurogenesis. CDO is a cell surface glycoprotein of the Ig superfamily, is found in a signalling complex with BOC and N-cadherin and can trigger gene expression changes required for myogenesis and neurogenesis by enhancing heterodimerization of basic helix loop helix transcription factors with E proteins. FGF19 belongs to the fibroblast growth factor (FGF) family; it promotes neurogenesis by controlling the expression of neurogenic and proneural genes in the cerebral cortex and midbrain. In order find out whether these genes are direct targets of NFIB, we performed Chromatin Immunoprecipitation (ChIP) experiments that test whether NFIB indeed binds to regulatory regions of these genes in vitro. First, we identified potential NFI binding sites in the 5 kb promoter region using the previously reported NFI specific position weight matrix. Scanning this region with the Find Individual Motif Analysis (FIMO) algorithm discerned seven binding sites for CDO and eight sites for FGF19. We selected the highest scoring five putative NFI binding sites for further study. To optimize the ChIP protocol, experiments were initially performed on HEK293T cells that overexpress epitope tagged NFIB. Binding of exogenous protein to the NFI sites were determined using ChIP followed by PCR. We found that exogenous NFIB reproducibly bound to site 2 (at about 6-fold enrichment) and site3, which are located within the 1kb region proximal to the transcription start site (TSS). Analysis of NFI occupancy in human neural stem cells (hNSCs, Gibco) found that endogenous NFI binds to the same two sequences, with 2.5-4 fold enrichment in ChIP. We also started to examine NFI occupancy of the FGF19 promoter in hNSCs. Here, endogenous NFI was found to bind to site 5, the NFI motif most proximal to the TSS. Next, we overexpressed epitope tagged NFIB in hNSCs and analysed binding to the same promoter sequences. Interestingly, preliminary results indicate that exogenous NFIB can bind to one additional site on each promoter. Namely, overexpressed NFIB binds to sites 2, 3 and 5, found in the 1 kb promoter region of CDO. On the FGF19 promoter, site 1 in addition to site 5, was found to be occupied by exogenous NFIB. Both of these NFI sites are also located in the 1 kb upstream region of FGF19. We are in the process of verifying the preliminary data and exploring the functionality of NFI occupancy of these sites by loss and gain of function experiments in hNSCs as well as luciferase reporter assays that measure NFI's effects on promoter activity.