Transcriptional regulatory control of mammalian nephron progenitors revealed by multi-factor cistromic analysis and genetic studies

Nephron progenitor number determines nephron endowment; a reduced nephron count is linked to the onset of kidney disease. Several transcriptional regulators including Six2, Wt1, Osr1, Sall1, Eya1, Pax2, and Hox11 paralogues are required for specification and/or maintenance of nephron progenitors. However, little is known about the regulatory intersection of these players. Here, we have mapped nephron progenitor-specific transcriptional networks of Six2, Hoxd11, Osr1, and Wt1. We identified 373 multi-factor associated ‘regulatory hotspots’ around genes closely associated with progenitor programs. To examine their functional significance, we deleted ‘hotspot’ enhancer elements for Six2 and Wnt4. Removal of the distal enhancer for Six2 leads to a ~40% reduction in Six2 expression. When combined with a Six2 null allele, progeny display a premature depletion of nephron progenitors. Loss of the Wnt4 enhancer led to a significant reduction of Wnt4 expression in renal vesicles and a mildly hypoplastic kidney, a phenotype also enhanced in combination with a Wnt4 null mutation. To explore the regulatory landscape that supports proper target gene expression, we performed CTCF ChIP-seq to identify insulator-boundary regions. One such putative boundary lies between the Six2 and Six3 loci. Evidence for the functional significance of this boundary was obtained by deep sequencing of the radiation-induced Brachyrrhine (Br) mutant allele. We identified an inversion of the Six2/Six3 locus around the CTCF-bound boundary, removing Six2 from its distal enhancer regulation, but placed next to Six3 enhancer elements which support ectopic Six2 expression in the lens where Six3 is normally expressed. Six3 is now predicted to fall under control of the Six2 distal enhancer. Consistent with this view, we observed ectopic Six3 in nephron progenitors. 4C-seq supports the model for Six2 distal enhancer interactions in wild-type and Br/+ mouse kidneys. Together, these data expand our view of the regulatory genome and regulatory landscape underpinning mammalian nephrogenesis.
Author summary Nephrons, the filtering units of the kidney, derive from nephron progenitors. Deficiencies in nephron number increases the risk of kidney disease. An understanding of the regulatory programs governing progenitor actions has important translational potential. Several transcription factors regulate the nephron progenitor population. However, their target interactions are largely unknown. Here, we mapped and intersected the genome-wide binding sites for four such factors in mouse nephron progenitor cells in the developing kidney: Six2, Hoxd11, Osr1, and Wt1. The intersectional data highlight a high-value set of putative enhancer elements linked to genes regulating nephron progenitor properties. We validate the function of two such enhancer elements regulating the levels of Six2, a key transcriptional regulatory factor in nephron progenitor maintenance, and Wnt4, a critical signaling factor controlling the mesenchyme to epithelial transition of induced nephron progenitors. Further characterization of the Six2 regulatory landscape identified higher order regulatory interactions that ensure appropriate enhancer-promoter specificity. CTCF-bound sites between Six2 and the adjacent Six3 locus likely act as boundary elements to define topological interactions domains separating enhancer elements thereby providing distinct tissue specificity to each gene’s expression. An inversion of this region in the Brachyrrhine (Br) mutant mouse reverses Six2 and Six3 expression domains, placing Six3 under control of the Six2 enhancer element above resulting in kidney-specific expression, while Six2 expression shifts to the lens, a normal expression domain for Six3. Together, these data expand our view of the regulatory genome and regulatory landscape underpinning mammalian nephrogenesis.