Role of ATF3 in the transcription of GI-SINEs (growth inducing B2-SINEs) in DRG neurons in response to sciatic nerve crush

Injury of the sciatic nerve upregulates a specific subset of B2-SINE RNAs in Dorsal Root Ganglia (DRG) neurons that enhances axon regeneration, which we termed GI-SINEs (growth inducing SINEs). Based on ATAC-seq and transcription factor binding site analysis and in-culture perturbation of AP-1 TFs, we hypotheiszed that GI-SINEs specifically regulated by injury activated AP-1 TFs. Here we experimentally tested the role of ATF3, a CREB TF that belongs to the AP-1 TF superfamily, in the transcriptional upregulation of GI-SINEs. ATF3 was previously identified and characterized as a key activator of nerve-injury induced regeneration associated genes (RAGs) mRNA. Cre recombinase expressed under the Advillin promoter was used for ATF3-exon3 excision in a broad and specific manner in the DRG. Sciatic nerve injury was carried on on mice with a conditional knockout of ATF3 in sensory neurons and ATF3-wt mice and total RNAseq was carried on RNA extracred from on sciatic innervating DRGs (L4-L6) from the injured side ("Inj") and non-injured side (naive / "Na"). Overall design: Homozygous ATF3(exon3)-floxed (Cre-negative) female mice were bred with Advillin-Cre/ heterozygous ATF3(exon3)-flox mice to yield sensory neuron specific knockout of ATF3 ("ATF3 cKO": Adv-Cre+/ATF3-fl_homo). As control mice, either Cre-negative or ATF3-flox null from the same colony were used ("ATF3 wt"). Sciatic nerve crush injury was carried on the left side of 8-12 weeks old: 8 mice were included in the ATF3 cKO group and 9 mice in the ATF3-wt control group. Mice were euthanized 3 days later and sciatic-innervating DRGs L4-L6 were excised from both injured and non-injured (naive) sides. Dissociated DRG cells were passed on Percoll gradient for neuronal enrichment and RNA was extracted and purified using Qiagen RNeasy micro kit.