Investigation of the interactions between NFIB and potential binding partners MAD2B and ATXN3

Nuclear Factor I (NFI) family of transcription factors are site-specific DNA-binding proteins that play roles in viral DNA replication and regulation of gene expression. NFI proteins bind with high affinity to the dyad symmetric consensus sequence TTGGC(N5)GCCAA on duplex DNA as homo or heterodimers. Four NFI genes (NFIA, NFIB, NFIC and NFIX) have been identified in every studied vertebrate species from Xenopus to human. NFIs contain an N-terminal DNA binding and dimerization domain and a C-terminal transcriptional activation and repression domain. The N-terminal DNA-binding/dimerization domain (DBD) is highly conserved between the four genes and includes an MH1 motif also found in SMAD proteins of the TGF-β signaling pathway. In contrast, the proline rich C-terminal domain is less conserved. Furthermore, C-terminal domain, which encodes the transcription modulation domain, varies among the NFI family members. NFI transcription factors are expressed in a cell-type specific fashion and may regulate tissue-specific gene expression during mammalian development. NFIA, NFIB, and NFIX are expressed in the ventricular zones in the developing nervous system, and in the adult neural stem cell niches. NFIs can both activate and repress transcription. Previously, a yeast two hybrid screen of a fetal human brain cDNA library using NFIB as bait identified ATXN3 and MAD2B as potential NFIB binding partners. ATXN3 belongs to the family of polyglutamine proteins which are implicated in nine different genetic neurodegenerative disorders. ATXN3 is generally a cytoplasmic protein, widely expressed in neural and non-neural human tissues. ATXN3 has been associated with the ubiquitin–proteasome system as well as transcriptional regulation. MAD2B is also known as MAD2L2 and Rev7 and is homologous to the spindle checkpoint gene MAD2. MAD2B is an important intermediary of the spindle assembly checkpoint. MAD2B is also involved in cell cycle regulation where it was shown to inhibit the Anaphase Promoting Complex (APC) by binding to Cdc20/Cdh1. MAD2B also acts in the DNA damage response in mammalian cells. Moreover, this protein carries the HORMA domain, responsible for protein-protein interactions. Both proteins can regulate developmental processes by transcriptional regulation. In mice that are deficient in MAD2B, primordial germ cells (PGCs) die after being specified due to misregulation of epigenetic reprogramming. Deubiquitinating activity of ATXN3 is critical for the nervous system. ATXN3 null mice exhibit neuronal dysfunction and neurodegeneration. In this study, in order to explore interactions between NFIB and ATXN3 or MAD2B, we performed co-immunoprecipitation experiments and GST pull down assays. To this end, epitope tagged MAD2B, ATXN3 and NFIs were overexpressed in HEK293T cells and binding was assessed via co-immunoprecipitation. We found that MAD2B and NFIB could be co-immunoprecipitated with each other. Other NFI family members (NFIA, NFIC, NFIX) did not appear to bind MAD2B. In addition, we also tested for binding of several mutant NFIB constructs that comprise only the NFIB N-terminal DNA binding and dimerization domain (NFIB_N) or C-terminal transactivation-repression domain (NFIB_C) to MAD2B. Both mutant NFIB proteins could not precipitate with MAD2B. On the other hand, ATXN3 did not co-immunoprecipitate with NFIB and NFIB did not co-immunoprecipitate with ATXN3. Furthermore, we analyzed binding of these proteins using an alternative approach, the Glutathione-S transferase (GST) pull down assay. GST pulldown experiments with NFIs and ATXN3 showed that ATXN3 pulled down NFIA and NFIB. To find out which NFIB domains are required for ATXN3 binding, we performed GST pulldown experiments using mutant NFIB proteins. Once again, these mutant constructs were NFIB_C, NFIB_N and finally, NFIB_E which encodes for NFIB DBD-engrailed repressor fusion protein. Preliminary data indicate that GST-ATXN3 precipitated NFI_N and NFIB_E but not NFIB_C. We, therefore, surmised that NFIB may interact with ATXN3 through its N terminal domain in these experiments. Further experiments that examine these interactions in vivo are required to verify these results. NFIB and binding partner domains that are involved in these putative interactions, can then be dissected by site-directed mutagenesis. In conclusion, the results obtained from this study will help elucidate the regulation of NFIB by various interaction partners, which in turn will help uncover the role of NFI family in neural development.