Liquid‐Liquid Phase Separation by the Egr Transcriptional Coregulator NAB2

Abstract

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 InformationBridgewater State University Undergraduate Research Program