Epigenetic Control of Nephron Progenitor Cell Lifespan - SUMMARY In mice and humans, nephrogenesis ceases perinatally and so is the opportunity to generate new nephrons following congenital or acquired loss of renal mass. A significant barrier to progress in the renal regeneration field is insufficient knowledge surrounding the mechanisms driving cessation of nephrogenesis. Recent genetic and epigenetic evidence support the notion that the lifespan of nephron progenitor cells (NPCs) is not fixed. The mechanisms whereby perinatal NPCs cease to self-renew and exhibit distinct metabolic fuel requirement and enhanced responsiveness to differentiation factors are under intense investigations. Initial comparative analysis of the chromatin landscape and gene expression at single-cell resolution has unraveled age-dependent chromatin remodeling affecting the accessibility of candidate cis-regulatory elements (cCREs), thus implicating epigenetic mechanisms in NPC age. The current proposal is designed to test the overall hypothesis that age- related redistribution of transcription factor-binding sites (TFBS) decommissions cell identity/renewal cCREs and engages AP-1-linked chromatin triggering the NPC differentiation program. We further propose that maintenance of the bivalent chromatin state by H3K27 methylation restrains AP-1-linked chromatin activity and can potentially be exploited to maintain NPC stemness. We present a comprehensive experimental design that combines cutting-edge multiomics, epigenomics, and mouse genetics to dissect the complex regulation of NPC lifespan. Aim 1 will test the hypothesis that redistribution of TFBS from cell identity to AP-1-linked cCREs underpins chromatin remodeling in NPC age. Leveraging deep multiome profiling, we will uncover TF/TFBS pairs affected by age-related chromatin remodeling. CUT&RUN will elucidate the AP-1 TF subunit(s) that physically occupy differentially accessible cCREs. Preliminary evidence implicates the transcription factor ATF4. Aim 2 will test the hypothesis that age-related enrichment in ATF4/TFBS drives NPC differentiation. Conditional gain and loss of function approaches will establish the necessity and sufficiency of ATF4 in driving NPC differentiation. This will be followed by mapping the ATF4-dependent regulome and downstream gene-regulatory networks. Aim 3 will test the hypothesis that histone bivalency controls NPC lifespan by restraining AP-1 activity. We will first demonstrate that resolution of bivalent chromatin in perinatal cCREs is mediated by the H3K27 demethylase, Kdm6b. Next, we will demonstrate that maintenance of chromatin bivalency in vivo, via Kdm6b inactivation, closes AP-1-linked cCREs and sustains NPC lifespan. Lastly, we will demonstrate that targeted closure of AP- 1 TFBS in cultured NPCs by epigenome editing maintains NPC stemness. Successful completion of this project will have broad impacts on defining the rules of epigenetic control of nephron progenitor maintenance. This knowledge may be translated to optimizing nephron regeneration and understanding epigenetic programming of renal disease.
