In vitro and in silico investigation of NFIB-SUMO interactions

Abstract

Nuclear Factor I family of transcription factors are involved in regulation in diverse physiological processes, including neuronal terminal differentiation, gliogenesis, stem cell quiescence as well as in pathologies such as tumorigenesis and cancer progression. NFI family is encoded by four genes: NFIA, NFIB, NFIC and NFIX. NFI proteins contain a highly conserved N-terminal DNA binding and dimerization domain, while their C-terminal domains diverge. Because of their highly similar DNA binding and dimerization domain, NFIs bind to a palindromic consensus site with similar affinity in vitro, potentially regulating the same set of target genes in vivo. Any functional differences between the family members may arise from the more diverse C-terminal domain provides which can promote either transcriptional activation or repression. NFIs may regulate target gene expression via different mechanisms of action. NFI can bind directly to DNA and regulate the expression of the target gene or interact with another protein to affect gene expression indirectly. Moreover, NFIs can interact with histone proteins and cause alterations in the nucleosome structure, thereby being involved in the formation of the transcription complex. NFIs can directly interact with and facilitate recruitment of basal transcription factors. NFIs can also bind co-activator or co-repressors to control transcriptional activation. In addition, NFIs can, along with other transcription factors, co-regulate target gene expression. Finally, NFIs can promote dissociation of DNA methyltransferase from target gene promoters and activate transcription. Any alterations in the production or action mechanisms of NFI proteins lead to important developmental defects as well as cancer. One member of the NFI family, NFIB, is an essential gene as demonstrated by studies on knockout on mice: silencing of NFIB leads to perinatal death due to lung defects. NFIB controls stem cell differentiation in different cell types such as, adipocytes, megakaryocytes, melanocytes and hippocampal neural progenitors. Interestingly, in humans, mutation of one copy of the NFIB gene can result in intellectual disability and brain malformations. These findings underscore the importance of NFIB as a transcriptional regulator, however, the mechanism by which NFIB acts or regulatory events upstream of NFIB have not been fully elucidated. Indeed, scarce data exists regarding NFI post-translational modifications. Phosphorylation, glycosylation, acetylation, sumoylation may regulate the activity of NFI. Among these modifications, sumoylation is conserved by eukaryotic organisms. Sumoylation regulates many cellular mechanisms such as nuclear transport, chromosome segregation, and transcription activation/repression. SUMO (small ubiquitin like modifiers), which is generally observed as a suppressor in transcriptional regulation, can be conjugated to many transcription factors and affects the activity of these factors. The SUMO gene family has five mammalian isoforms: SUMO1, SUMO2, SUMO3, SUMO4 and SUMO5. SUMO peptides are activated by a series of enzymatic processes. These processes are required to form mature SUMO, which is active and may able to conjugate specifically to the target protein. The sumoylation consensus sites and SIM (SUMO interacting motif) on target proteins enable SUMO to specifically recognize and bind to these proteins and regulate their activities. Sumoylation can affect transcription factors in several ways. SUMO can compete with other modifications, may interact with co-activators, and can control the binding of the transcription factor to its target site on chromatin. In addition, sumoylation can control intracellular localization of transcription factors. Sumoylation of NFI has been shown in vitro. Interestingly, a study on neuroblastoma cells exposed to oxidative stress, identified NFIB among sumoylated proteins modified on sumoylation consensus sites. However, sumoylation of NFIs have not been further explored, in silico and in cell culture. In this study, we set out to investigate the functionality of sumoylation consensus site and SUMO interacting motifs of NFIB, using in silico methods and forced expression in cell culture. Currently, there is no experimental or modeled 3D structure of NFI proteins. Information about NFI protein structure is quite limited. As mentioned above, NFI proteins contain an N-terminal DNA binding and dimerization domain and C-terminal transactivation domain. NFI proteins carry four conserved cysteine residues in their DNA binding and dimerization domains, three of which are required for DNA binding activity. Another piece of evidence regarding NFI structure comes from the homology with the MH1 domain of SMAD3. Both proteins have highly similar Cys-His box motifs consisting of three cysteine residues and one histidine residue. Nevertheless, this homology is below 30%. Here, due to the lack of high homology and also fold similarity, we used ab initio modeling method to predict structure of NFIB DNA binding and dimerization domain. Subsequently, these predictions were compared to each other. For this comparison, we focused on the cysteine residues which are required for DNA binding activity as well as the conserved MH1 Cys-His box motif. Then, selected structure prediction models were assessed by molecular dynamic simulations. Finally, with REMD (Replica Exchange Molecular Dynamics) the model that showed higher stability and quality was verified. We performed molecular docking simulations to investigate NFIB SIM-SUMO1 interactions. We found that SUMO1 would preferentially bind to a specific NFIB SIM. Meanwhile, to investigate NFIB-SUMO1 conjugation in vitro; site-directed mutagenesis was performed for generation of a sumoylation consensus site mutant and a SIM mutant. HEK293T cells were co-transfected with SUMO1 and wild-type NFIB or NFIB mutants and NFIB was immunoprecipitated for analysis of NFIB-SUMO1 conjugation. Future experiments are required to validate the putative NFIB sumoylation consensus site and SIMs in cell culture.