Transcription Factor Affinity at Dorsal Root Ganglia Enhancers after Nerve Injury

Abstract

Misregulation of gene expression in persistent pain supports a role for context-dependent regulation of gene expression in primary sensory neurons through coordinated transcription factor binding to cis-regulatory elements. In this study, we used a multiomic approach to identify regulatory regions and transcription factors (TFs) that may be involved in the development of nerve injury-induced persistent pain. Adult Sprague Dawley rats were randomized to Chronic Constriction Injury (CCI) to the sciatic nerve or no treatment (naïve). The ipsilateral L4-L6 dorsal root ganglia (DRG)s were removed on Day 14 and used for ChIP-seq for H3K4me1, ATAC-seq, and RNA-seq. Computational integration of this sequencing data generated a comprehensive map of chromatin accessibility changes in the DRG after CCI at cis-regulatory elements. We then used luciferase assays to determine the functional significance of these sequences and determined their ability to bind to TFs using a custom human TF array and EMSA. In vitro validation of binding was confirmed by ChIP-PCR and physiological relevance informed by shRNA knockdown in the 50B11 (nociceptor) cell line. Of 58,446 putative regulatory regions identified genome-wide, 2145 (3.67%) were differentially accessible after CCI. Gene ontology analysis identified molecular functions and biological processes associated with neuronal activation and synaptic signaling. Luciferase assays confirmed their regulatory potential in 50B11 cells which suggests that altered chromatin structure at these regions contributes to the development of mechanical hypersensitivity after nerve injury. Several TFs showed significant binding to one of these regions. CEBPG binding was then confirmed by EMSA and ChIP-qPCR with a fold enrichment of 2.72 ±0.47 over IgG. RNA-seq then identified pathways altered by CEBPG shRNA knockdown. Our data provides a comprehensive map of chromatin accessibility changes in the DRG after CCI and emphasizes the importance of chromatin structure at cis-regulatory elements in persistent pain. This study was supported by grants from National Institutes of Health (Bethesda, Maryland, USA) F32NR015728 (KES), KL2TR003108 (KES), NS110598 (YG), NS117761 (YG), R01GM118760 (SDT), the Arkansas Children's Research Institute (KES), the Arkansas Breast Cancer Research Program (KES) as well as a seed grant from the Johns Hopkins Blaustein Pain Research Fund (SDT).