Low nephron endowment causes hypertension, chronic kidney disease, and end-stage renal disease requiring renal replacement therapy in children and adults. The premise of multi-potent progenitor cell- based replacement therapy for individuals with low nephron endowment critically depends on scientific and technical advances that foster efficient propagation of native or pluripotent cell-derived nephron progenitor cells (NPC). Genetic and functional analyses in mice indicate that NPC residing in the cap mesenchyme ?age? during maturation, i.e., while Young E13 NPC stay in the niche and engage in self-renewal, Old P0- P2 NPC have a shorter life span because they exit the niche at a higher rate and differentiate into nascent nephrons. The biological basis of NPC aging is not well understood. Changes in the niche microenvironment are not sufficient to explain Old NPC's greater propensity to differentiate since P2-NPC cannot sustain their progenitor state even in optimal growth factor media. Our preliminary studies using genome-wide mapping of open (accessible) chromatin identified intrinsic age-associated chromatin state transitions in Young and Old NPC. This exciting finding prompted us to hypothesize that Old NPC are epigenetically ?primed? for differentiation, which contributes to their limited life span.
Specific Aim 1 will utilize an integrative system biology approach to delineate the active enhancer landscape and transcriptional regulatory network of the Young and Old NPC. We will construct a comprehensive map of the age-dependent chromatin state transitions in freshly isolated Young (E13) and Old (P0-P2) NPC, and integrate this knowledge with enhancer histone signatures, transcriptional profiles, and transcription factor occupancy. We will also determine whether ex vivo expansion triggers remodeling of the chromatin landscape by comparing the epigenomic profiles of native and expanded Young and Old NPC.
In Specific Aim 2, we will test the hypothesis that the Polycomb Repressive Complex 2 (PRC2) restrains remodeling of differentiation gene enhancers in the Young NPC. Using gene targeting and epigenome profiling, we will demonstrate the critical role of PRC2 in controlling access to enhancers of Cdkn2a/p16 and Wnt4. We will utilize CRISPR dCas9-targeted epigenome editing to re-write the histone signature of developmental enhancers and rejuvenate the PRC2-mutant NPC. Successful completion of these aims has the potential to advance the field of kidney development in general, but it will especially benefit the efforts of nephron regeneration. Knowledge of the enhancer landscape of nephron progenitors during maturation, which does not currently exist, can potentially open the way to development of targeted epigenetic therapy to maintain the stemness or rejuvenate the aging NPC, and to refine existing NPC propagation protocols.
Re-building a kidney from synthetic progenitors requires a prior knowledge of the global chromatin landscape and the gene regulatory regions that control expression of tissue-specific developmental regulators. It also requires an understanding of the intrinsic factors that govern the decisions for the nephron progenitor cell to divide and self-renew or differentiate into a functional nephron.