14-3-3 binding motif phosphorylation disrupts Hdac4 organized condensates to stimulate cardiac reprogramming

Cell fate conversion is associated with extensive post translational modifications (PTMs) and architectural changes of sub-organelles, yet how these events are interconnected remains unknown. We report here the identification of a phosphorylation code in 14-3-3 binding motifs (PC14-3-3) that greatly stimulates induced cardiomyocyte (iCM) formation from fibroblasts. PC14-3-3 is identified in pivotal functional proteins for iCM reprogramming, including transcription factors and epigenetic factors. Akt1 kinase and PP2A phosphatase are key writer and eraser of the PC14-3-3 code, respectively. PC14-3-3 activation induces iCM formation with the presence of only Tbx5. In contrast, PC14-3-3 inhibition by mutagenesis or inhibitor-mediated code removal abolishes reprogramming. We discover that key PC14-3-3 embedded factors, such as Hdac4, Mef2c and Foxo1, form Hdac4 organized inhibitory nuclear condensates. PC14-3-3 activation disrupts Hdac4 condensates to promote cardiac gene expression. Our study suggests that sub-organelle dynamics regulated by a PTM code could be a general mechanism for stimulating cell reprogramming. To investigate the effect of the HDAC4 inhibitor TMP269 on cardiac reprogramming, we utilized TMP269 to treat neonatal cardiac fibroblasts transfected with MGT. Additionally, cells infected with the MGT virus or DsRed were employed as positive and negative controls, respectively. We subsequently conducted gene expression profiling analysis using data obtained from RNA sequencing of three different cell types 14 days after virus infection.