The early growth response (Egr) family of transcription factors comprises five members, Egr1‐4 and Wilms Tumor 1 (WT1), which play roles in various cell behaviors including proliferation, apoptosis, and differentiation in a cell type‐ and stimulus‐specific manner. Roles for Egr1‐3 are particularly well‐documented in the nervous system. Egr1 and 3 contribute to learning and memory through regulation of genes that mediate synaptic plasticity and long‐term potentiation. Egr2 controls hindbrain development through regulation of Hox gene expression and drives peripheral nerve myelination through, at least in part, activation of the gene encoding myelin protein zero in Schwann cells. Egr1 and 2 also contribute to transcriptional network that drives neuronal differentiation of PC12 cells. However, the mechanisms that regulate Egr1‐3 activities in the nervous system are not fully understood. One mechanism of regulation involves protein‐protein interactions with co‐regulators NAB1 and 2 through a conserved R1 domain in Egr1‐3. Most reports demonstrate that NABs repress Egr1‐3 transactivation activity when bound, however, NABs can also enhance transactivation in a target gene‐specific manner. The mechanisms controlling the effect of NABs on Egr activities are not clear. Here, we provide evidence that NAB2 regulation may involve its phosphorylation at several sites by ERK. Using PC12 cells as a model system, our data demonstrate that nerve growth factor (NGF) induces a NAB2 mobility shift in SDS‐PAGE within 10 minutes of treatment. The mobility shift was reversed when lysates from NGF‐treated cells were treated with lambda phosphatase, suggesting the shift is due to phosphorylation. Inhibition of ERK signaling during NGF treatment blocked the mobility shift, while recombinant ERK phosphorylated recombinant NAB2 in an in vitro kinase assay, together indicating that direct phosphorylation of NAB2 by ERK may be responsible for the NAB2 mobility shift in response to NGF. Nine potential ERK phosphorylation sites were identified in NAB2, which were mutated individually to alanine within a NAB2 expression construct to evaluate phosphorylation at those sites based on their mobility in SDS‐PAGE. Of the nine mutants, three exhibited clear changes in NAB2 mobility, which were additive in double and triple mutants for those sites. Ongoing experiments are evaluating the effect of NAB2 phosphorylation on its localization and regulation of Egr1‐3. Support or Funding Information Bridgewater State University Undergraduate Research Program

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NGFI‐A‐binding proteins 1 and 2 (NAB1 and 2) are transcriptional coregulators with roles in a variety of cell types and behaviors. Their role in the peripheral nervous system is best established, where they are expressed in Schwann cells and essential for development and maintenance of peripheral nerve myelination. NAB1 and 2 have also been implicated in the regulation of hindbrain development, fibroblast activation in response to TGF‐β, neuronal and hematopoietic differentiation, and thymus cellularity. In addition, misregulation of NAB1 and 2 has been implicated in disease, including the peripheral neuropathy Charcot‐Marie‐Tooth disease and cancers that develop in the form of solitary fibrous tumors. NAB1/2 mediate their effects through interactions with the Egr family of transcription factors, which they bind via their amino terminal NCD1 (NAB‐conserved domain 1). Upon binding Egr, they either repress or potentiate gene expression is a gene‐specific manner. Two NAB domains have been shown to possess transcriptional repression activity, an internal NCD2 domain and a carboxy terminal CID (CHD4‐interacting domain). However, a NAB domain responsible for transcriptional potentiation has not been determined, nor have molecular mechanisms responsible for regulating NAB1/2 activity. Here we provide evidence that NAB2 possesses the ability to undergo liquid‐liquid phase separation (LLPS) to form liquid droplets in cells, which has recently been characterized as critical event through which some transcriptional activators mediate their effects on gene expression. Transfection of multiple cell lines with a NAB2‐GFP fusion resulted in the formation of several puncta within the nucleus that exhibit fluorescence recovery after photo‐bleaching (FRAP). Assembly into puncta is prevented by either truncation of the NAB2 NCD1 domain or by alanine substitution of two arginine residues within that region (Arg‐97 and Arg‐98), indicating that NAB2 droplet formation is dependent on the amino acid composition within NCD1. Examination of the NAB2 amino acid sequence using PONDR predicted 62.5% disordered sequence and 37.5% ordered sequence. Ongoing experiments are evaluating the necessity of additional regions within NAB2 for LLPS as well as whether NAB2 LLPS is affected by phosphorylation as several sites. These data may provide insight into the mechanisms through which NAB2 is regulated and/or mediates its effects on gene expression. Support or Funding Information Bridgewater State University Undergraduate Research Program

PC12 cells are a common model system used to study the molecular events underlying neuronal differentiation due to their ability to differentiate into neuronal cells following treatment with nerve growth factor (NGF). NGF induces neuronal differentiation through activation of the Ras/Raf/MEK/ERK signaling pathway, which in turn activates a transcriptional program leading to up‐regulation of neuronal genes, neurite outgrowth, and establish electrical excitability. Interestingly, treatment with epidermal growth factor (EGF) also activates Ras/Raf/MEK/ERK signaling, but induces proliferation rather than differentiation. The differential effects of NGF versus EGF are due to differences in ERK signal duration; EGF induces more transient ERK signaling that lasts 30–60 min, whereas NGF induces more sustained ERK signaling that lasts 4–6 h. Work in our lab and others indicate that sustained ERK signaling in response to NGF drives differentiation in part through sustained activity of the transcription factor Egr1 and, in turn, sustained expression of Egr1 target genes. Egr1 activity in response to EGF and NGF is regulated at two levels. First, Egr1 levels rapidly rise to peak levels by 1 h after treatment and then decline to baseline levels; similar to ERK signaling, Egr1 levels are sustained longer in response to NGF versus EGF. Second, one Egr1 target gene is its corepressor NAB2, which binds and represses Egr1 transactivation once expressed. This project was designed to further test our hypothesis that sustained Egr1 activity is a major driver of PC12 neuronal differentiation by evaluating whether knockout of NAB2 will lead to sustained Egr1 activity in response to EGF and, in turn, neuronal differentiation. To acquire PC12 cells that lack NAB2 protein, we used CRISPR/Cas9 technology to knockout the NAB2 gene. More specifically, a CRISPR NAB2‐KO plasmid was purchased from Santa Cruz Biotechnology, which encodes a green fluorescent protein (GFP) selectable marker, the Cas9 protein, and the guide RNA (gRNA). The plasmid had three variations for the gRNA which target different locations in the NAB2 gene. One day after transfection, GFP‐positive cells were sorted into 96‐well plates to isolate clones. Following clonal expansion, clones were first screened for NAB2 expression by Western blot, which identified eight putative knockout clones. Genomic DNA was then extracted from the eight clones, the NAB2 gene amplified by PCR, and the PCR project then subjected to Sanger sequencing to identify the INDELs responsible for NAB2 knockout. Ongoing work is examining the effects of NAB2 knockout on Egr1 levels and activity in response to EGF and NGF, as well as neuronal differentiation based on neurite outgrowth with the prediction that NAB2 knockout will result in sustained Egr1 activity and neuronal differentiation in response to both growth factors. Support or Funding Information Bridgewater State University Undergraduate Research Program