Transcriptional regulation of human <i>NMNAT2</i> : insights from <scp>3D</scp> genome sequencing and bioinformatics

Nicotinamide/nicotinic acid mononucleotide adenylyltransferase 2 (NMNAT2) is a crucial enzyme for synthesizing nicotinamide adenine dinucleotide (NAD) and plays a vital role in neuronal health. NMNAT2 mRNA levels correlate positively with cognitive function in older adults but decline after injuries or proteinopathies. In this study, we used chromosome conformation capture followed by high-throughput sequencing (4C-seq) to unbiasedly identify NMNAT2 regulatory regions throughout the human genome. Using various bioinformatics analyses with these genomic regions, referred to as interactomes, we identified NMNAT2-associated genes and putative transcription factors (TFs). NMNAT2 transcription increases in SH-SY5Y cells when they differentiate into a neuron-like state. Excitingly, our 4C-seq data revealed distinct sets of interactomes interacting with the NMNAT2 promoter in undifferentiated versus neuron-like SH-SY5Y cells. Using the Religious Orders Study and the Rush Memory and Aging Project (ROSMAP) snRNA-seq data, we showed that the expression levels of many NMNAT2-associated genes are significantly correlated with NMNAT2 transcription in human neurons. Our biological validation studies confirmed the requirement of two specific genomic regions and four TFs, including cyclic AMP-dependent transcription factor ATF4, cyclic AMP-dependent transcription factor ATF-6 alpha (ATF6), transcription factor SOX11, and heat shock factor protein 1 (HSF1), in NMNAT2 transcription. ATF4 has been identified as an injury-responsive TF, whereas HSF1 is modulated by protein stress. Together, our study identifies distinctive genomic loci containing NMNAT2 regulatory elements in undifferentiated versus neuron-like SH-SY5Y cells, NMNAT2-associated genes, and putative NMNAT2-TFs.