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Single-cell chromatin and gene-regulatory dynamics of mouse nephron progenitors

Background Cis-regulatory elements (CREs), such as enhancers and promoters, and their cognate transcription factors play a central role in cell fate specification. Bulk analysis of CREs has provided insights into gene regulation in nephron progenitor cells (NPCs). However, the cellular resolution required to unravel the dynamic changes in regulatory elements associated with cell fate choices remains to be defined. Methods We integrated single-cell chromatin accessibility (scATAC-seq) and gene expression (scRNA-seq) in embryonic E16.5 (self-renewing) and postnatal P2 (primed) mouse Six2GFP NPCs. This analysis revealed NPC diversity and identified candidate CREs. To validate these findings and gain additional insights into more differentiated cell types, we performed a multiome analysis of E16.5 and P2 kidneys. Results CRE accessibility recovered the diverse states of NPCs and precursors of differentiated cells. Single-cell types such as podocytes, proximal and distal precursors are marked by differentially accessible CREs. Domains of regulatory chromatin as defined by rich CRE-gene associations identified NPC fate-determining transcription factors (TF). Likewise, key TF expression correlates well with its regulon activity. Young NPCs exhibited enrichment in accessible motifs for bHLH, homeobox, and Forkhead TFs, while older NPCs were enriched in AP-1, HNF1, and HNF4 motif activity. A subset of Forkhead factors exhibiting high chromatin activity in podocyte precursors. Conclusion Defining the regulatory landscape of nephrogenesis at single-cell resolution informs the basic mechanisms of nephrogenesis and provides a foundation for future studies in disease states characterized by abnormal nephrogenesis. Significance Statement Nephron progenitor cells (NPCs) are a multipotent population giving rise to all cell types of the nephron. At any given time, the NPC’s choice to self-renew or differentiate is determined not only by its transcription factor (TF) repertoire but also by the genome accessibility of the cognate cis-regulatory elements. Using single-cell analysis, we demonstrate the heterogeneity of NPCs at the epigenetic level and observe dynamic and cell type-specific changes in chromatin accessibility. Fate-determining TFs harbor domains rich in interactive chromatin that are established prior to gene activation. These findings illustrate the importance of chromatin-based mechanisms in the regulation of nephrogenesis and may have implications for nephron regeneration and repair.