ELK1 binding sites are located slightly upstream of the transcriptional start site of target genes.(56) To determine if the gene expression changes could be attributed to decreased transcriptional activity of ELK1 directly at the target loci, we used ChIP to assess the occupancy of ELK1 at the promoter regions of downstream targets in the NAc of rats that underwent the self-administration paradigm. We examined published ChIP sequencing data obtained using ELK1, CREB, and SRF antibodies to guide our selection of ELK1 target genes.(56, 57) Such studies suggested Bag1 and Use1 are regulated by ELK1 in a CREB- and SRF-independent manner.(56, 57) We observed no enrichment of ELK1 binding over nonspecific IgG controls at any region of Bag1 studied (data not shown). In animals that were sacrificed 1 hour after the last heroin self-administration session, ELK1 occupancy was significantly decreased at the promoter of Use1 versus saline animals (0.034± 0.007% versus 0.058±0.006; p<.05; Fig 6C), indicating a direct functional regulation of ELK1 on downstream gene targets as a consequence of heroin use. At the 24-hour time-point, ELK1 binding at the Use1 promoter was not significantly different from controls, indicating that heroin did not induce a prolonged decrease in ELK1 occupancy.
