Abstract 9746: Rig-1 Agonists Regulate Cardiac Reprogramming via Yy1

Abstract

We have discovered that innate immunity plays an important role in the reprogramming of fibroblasts into cardiomyocytes. Previously, we have focused on the role of the TLR3-NFkB pathway. We have now found evidence for an alternative Rig-1:YY1 pathway. Specific Rig-1 agonists such as 3p-hpRNA were found to enhance the efficacy of cardiac reprogramming with cardiomyocyte-specific gene expression increasing by 10 to 20 fold (Actn2, Myh6, Tnni3; N=6; P<0.01). Similarly, formation of new cardiomyocytes was increased 5 fold (N=3, P<0.01). To determine the mechanism of action, MNase-seq was performed. MNase-seq indicated that Rig-1 activation induced transcription factor binding to genomic regions immediately downstream of the transcription start site of cardiomyocyte-specific genes (N=3, P<0.01). Further analysis of the binding regions revealed an overabundance of YY1 binding sequences (N=3, P<0.001). To understand the role of YY1, knockdown experiments were performed. YY1 knockdown completely ablated the effects of Rig-1 agonists on cardiac reprogramming (N=5, P<0.01). To understand how YY1 was activating cardiomyocyte-specific genes, ChIP-seq and ChIP-qPCR was carried out. ChIP-seq and ChIP-qPCR indicated that following Rig-1 activation, YY1 associated with Kdm6 and P300. Kdm6 and P300 were found to work in concert; activating cardiomyocyte-specific gene expression via the removal of the inhibitory epigenetic mark tri-methylated lysine-27 of Histone-H3 and deposition of the stimulatory epigenetic mark acetylated lysine-4 of Histone-H3 (N=3, P<0.01). To conclude, the Rig-1:YY1 pathway plays a critical role in cardiac reprogramming. Manipulation of the Rig-1:YY1 pathway may enhance the effectiveness of cardiac reprogramming in vivo.