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Open chromatin and single-cell sequencing analysis reveals the contribution of Wnt4-lineage cells and the importance of chromatin regulators in renal tubular and interstitial differentiation

ABSTRACT: Although Wnt4 is an early marker of nephron differentiation, how Wnt4-lineage cells differentiate into the renal interstitium and other cells types is unclear. Cell fate, proliferation, migration, and differentiation are controlled by precise spatiotemporal gene expression programs that are coordinately regulated by cell-cycle, lineage-specific transcription and chromatin regulatory factors. However, little is known about the roles of epigenetic/chromatin regulators in renal tubular and interstitial differentiation and diseases. We deleted the chromatin regulator Brg1 in Wnt4-expressing cells and FACS-purified Wnt4-lineage cells of wild-type and Brg1-deficient kidneys. We then carried out assay for transposase-accessible chromatin sequencing (ATAC-seq) to determine how Brg1-deficiency alters chromatin accessibility. We further performed single-cell RNA sequencing (scRNA-seq) to define the population and the effects of Brg1-deficiency in the differentiation of Wnt4-lineage cells. Our ATAC-seq analysis shows that Brg1-deficiency alters accessibility at thousands of active enhancers harboring motifs for an array of transcription factors. Unbiased scRNA-seq analyses identify 35 clusters of Wnt4-descendant cells and uncover novel cell types and genes that have so far not been linked to Wnt4-lineage cells. Brg1-deficiency leads to increased endothelial, stromal, and smooth-muscle cells and decreased nephron tubular cells. In Brg1-deficient kidneys, the expression of Pttg1 and proliferation are increased in Wnt4-expressing precursors, which cannot transition to a characteristic tubular state and appear to undergo fibrosis. Our results provide crucial insight into chromatin remodeling events and Wnt4-lineage cell-associated gene expression dynamics, highlighting the importance of chromatin regulators in controlling the differentiation of these cells and the expression of disease-associated genes.

ORGANISM(S): Mus musculus

PROVIDER: GSE199188 | GEO | 2022/04/08

REPOSITORIES: GEO

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Project description:Bulk ATAC_seq on GFP expressing FACS-isolated cells from E16.5 and P0 Six2TGC and compound Ezh1 and Ezh2 mutant kidneys( Six2TGC_Ezh2-/- , Ezh1+/-; Six2TGC_Ezh2-/-; and Ezh1-/- ; scRNA-seq analysis of NPCs (Six2/GFP+ cells) from E16.5 as well as P2 kidneys. Genotypes analyzed: Six2TGC (E16.5&P2), Six2TGCEzh2-/- (E16.5), Ezh1+/-;Six2TGCEzh2-/- (E16.5), and Ezh2-/-;Six2TGCEzh2-/- (E16.5) Six2/GFP+ nephron progenitor cells (NPCs) give rise to all epithelial cell types of the nephron, the filtering unit of the kidney. NPCs have a limited lifespan and are consumed near the time of birth. Pre-term birth or prenatal stress further shorten the lifespan of NPCs and result in nephron deficit and chronic kidney disease. Accordingly, there is a pressing need to better understand the factors that regulate NPC lifespan in order to develop novel regenerative strategies. Epigenetic factors are implicated in maintenance of organ-restricted progenitors such as NPCs, but the chromatin-based mechanisms are not well understood. In this study, we examined the role of the H3K27 methyltransferases, Enhancer of zeste (Ezh1 and Ezh2), in NPC maintenance using a combination of gene targeting, chromatin profiling, and single-cell RNA analysis. We find that Ezh2 expression correlates with NPC growth potential, and that Ezh2 is the dominant H3K27 methyltransferase in NPCs and epithelial descendants. Surprisingly, NPCs lacking H3K27me3 maintain their progenitor state albeit cycle slowly leading to formation of fewer nephrons. Unlike Ezh2 loss-of-function, dual inactivation of Ezh1 and Ezh2 triggers overexpression of the transcriptional repressor Hey1 and downregulation of Six2 and result in unscheduled activation of Wnt4-driven differentiation, early termination of nephrogenesis and severe renal dysgenesis. Double-mutant NPCs also overexpress the Six family member, Six1. However, in this context, Six1 is unable to access the closed Six2 enhancer and fails to maintain NPC stemness. At the chromatin level, Ezh1 and Ezh2 act by restricting accessibility of AP1 factors to their genomic binding motifs and their absence provokes a regulatory landscape akin of differentiated and non-lineage cells. We conclude that Ezh2 is required for NPC renewal potential, while maintenance of NPC lifespan and tempering the differentiation program require cooperation of both Ezh1 and Ezh2.

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Open chromatin and single-cell sequencing analysis reveals the contribution of Wnt4-lineage cells and the importance of chromatin regulators in renal tubular and interstitial differentiation

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