Abstract P153: Protein Kinase A Inhibition Epigenetically Silences Ren1

Renin-expressing cells are myoendocrine cells crucial for survival which detect changes in blood pressure and release renin to maintain homeostasis. One of the pathways responsible for renin expression includes cAMP as a central factor. cAMP binds to subunits of protein kinase A (PKA), ultimately recruiting both CBP and p300. Binding to the cAMP-responsive element in the renin enhancer region thus amplifies renin transcription. To evaluate transcriptomic and epigenomic changes occurring at the renin locus via the cAMP pathway, we treated As4.1 cells (a tumoral cell line that constitutively expresses renin) with the PKA inhibitor H89 (treated) or DMSO (control). Using scRNA-seq, we confirm that PKA inhibition leads to significant decreases in Ren1 abundance (p=3.18e-25), and Ccl8 , Cilp , Cdkn1a , Lox , Ly6c1 , Ccl7 , Cryab are all significantly increased. Analysis of bulk ATAC-seq of treated and control As4.1 cells revealed 527 significantly increased regions of accessible chromatin and 1523 significantly decreased. Within the increased accessibility regions, motifs for the transcription factors Bach1 , Fos , Fosb , Trp73 , and Gli1 are overrepresented. Within the regions with decreased accessibility are enriched motifs for Zfp161 , Esr2 , Zfa , Maz , Phf21a , Hlf , and Tcf7 . We further identified a cluster of differentially accessible regions composed of 172 chromatin regions that shift concurrently in accessibility following PKA inhibition. Within these regions, the KLF (p= 7.5e-11) and AP-1 (p= 1.4e-5) families of transcription factors are overrepresented. Interestingly, open chromatin around the Ren1 locus is not differentially accessible following H89 treatment. To better understand the effect of PKA inhibition, we evaluated H3K27Ac and P300 binding via ChIP-seq. H3K27Ac is significantly reduced at the renin locus (p=1.99e-08), and P300 binding is also significantly reduced (p=1.79e-13). Taken together, the data suggests that cAMP pathway inhibition controls renin expression through a reduction not in accessibility, but via a switch from an active to poised state of epigenetic control and the rise in immediate early genes regulating cellular gene expression.