Abstract 15512: Disrupting the NelfA Dependent RNA pol II Paused Complex in Heart Precipitates Dilated Cardiomyopathy

Abstract

RNA polymerase II (pol II) pausing, a wide-spread genomic phenomenon regulates pol II dependent transcription and plays a critical role in gene expression during development and disease. Negative elongation factor (Nelf) complex consisting of five subunits, namely NelfA, NelfB, NelfC/D and NelfE, in coordination with Drb sensitive inducing factor (DISF) complex has been implicated in mediating pol II pausing. Structural studies show NelfA is indispensable for nucleation of paused complex. We have reported widespread promoter NelfA occupancy on cardiac genome, and showed that it is required for compensatory gene expression in hearts during cardiac hypertrophy. Here, we examine paused complex, with respect to its interactome, influence on gene expression and cardiac function. Immunoprecipitation of chromatin bound NelfA, followed by mass spectrometry in sham and TAC induced hypertrophied hearts identified Nelf proteins, pol II and Supt5, along with novel partners like Trim28 (chromatin remodeler), Adprhl1 (cardiac restricted, nuclear enriched, involved in DNA repair) and Numa1 (nuclear structural protein). Conditional NelfA knockout mice developed progressive dilated cardiomyopathy (LVID’d: 4.72 ±0.1, LVID;s: 3.96±0.13, %EF: 33.7±2.12, %FS: 16.15±1.12, LV vol;d: 104.5±21.9, LV vol;s: 70.26±5.5) compared to Wt-Cre (LVID’d: 4.16 ±0.09, LVID;s: 3.14±0.12, %EF: 49.1±2.3, %FS: 24.6±1.3, LV vol;d: 77.5±13.9, LV vol;s: 40.9±3.9), with sudden death by 3mths of age, in males and females KO mice compared to Wt-Cre. Interestingly, absence of NelfA resulted in decrease in cardiomyocyte size and increase in apoptosis. Single nuclei RNAseq on these hearts show decrease in cardiac-enriched sarcomeric genes, along with increase in genes involved in fibrosis and endothelial dysfunction. Further, ChIP-seq showed reduced Trim28 and NelfE promoter occupancy on essential gene, while below threshold or absent on cardiac-enriched and selected inducible promoters suggesting disruption of paused complex. Thus, we conclude that NelfA induced paused complex plays an essential role in efficient pol II dependent gene transcription and expression in heart, and deconstruction of the paused complex precipitates cardiac dysfunction and dilated cardiomyopathy