Differential regulation of mouse and human nephron progenitors by the Six family of transcriptional regulators

Abstract

Nephron endowment is determined by the self-renewal and induction of a nephron progenitor pool established at the onset of kidney development. In the mouse, the related transcriptional regulators Six1 and Six2 play non-overlapping roles in nephron progenitors. Transient Six1 activity prefigures, and is essential for, active nephrogenesis. By contrast, Six2 maintains later progenitor self-renewal from the onset of nephrogenesis. We compared the regulatory actions of Six2 in mouse and human nephron progenitors by chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq). Surprisingly, SIX1 was identified as a SIX2 target unique to the human nephron progenitors. Furthermore, RNA-seq and immunostaining revealed overlapping SIX1 and SIX2 activity in 16 week human fetal nephron progenitors. Comparative bioinformatic analysis of human SIX1 and SIX2 ChIP-seq showed each factor targeted a similar set of cis-regulatory modules binding an identical target recognition motif. In contrast to the mouse where Six2 binds its own enhancers but does not interact with DNA around Six1, both human SIX1 and SIX2 bind homologous SIX2 enhancers and putative enhancers positioned around SIX1. Transgenic analysis of a putative human SIX1 enhancer in the mouse revealed a transient, mouse-like, pre-nephrogenic, Six1 regulatory pattern. Together, these data demonstrate a divergence in SIX-factor regulation between mouse and human nephron progenitors. In the human, an auto/cross-regulatory loop drives continued SIX1 and SIX2 expression during active nephrogenesis. By contrast, the mouse establishes only an auto-regulatory Six2 loop. These data suggest differential SIX-factor regulation might have contributed to species differences in nephron progenitor programs such as the duration of nephrogenesis and the final nephron count.

Keywords: Nephrogenesis; Nephron; Regulatory network; Six1/2; Transcription.

Fig. 1.

Human SIX2 ChIP-seq reveals a kidney-specific regulatory network. (A) SIX2 and cytokeratin (top), and SIX2, JAG1 and ECAD (bottom) immunostaining of human and mouse fetal kidneys. (B) Genomic view of MEOX1 and WT1 loci showing SIX2 peaks. ‘cons', Phastcon vertebrates conservation score. (C) Distribution of SIX2 peaks relative to TSSs. *P-value represents the significance of peaks falling 50-500 kb in either direction from the TSS. (D) Genomic annotation of SIX2 peaks. (E) Weblogo of the most enriched motif (top) and its conservation (bottom). (F) Distribution of SIX2 motif-peak distances. (G) Functional annotation of SIX2 peaks. Obs., observed; Exp., expected.

Fig. 2.

Mouse and human SIX2 share many common targets but SIX1 represents a unique human target. (A) Comparison of the most enriched motif for Six2/SIX2 peaks. (B) Distribution of Six2 motif-peak distances. (C) Overlap between human SIX2 binding sites and converted mouse Six2 binding sites with mammalian Phylop conservation (left) and peak percentages with motifs (right). (D) Overlap of human and mouse SIX2 target genes. (E) (Left) Human/mouse target genes plotted by their SIX2/Six2 regulatory scores. (Right) Nephron progenitor-specific expression of all conserved genes from human and mouse. Genes identified as species-specific targets with species-specific expression are marked.

Fig. 3.

SIX1 is active and regulated by SIX2 in human but not mouse nephron progenitors. Genomic view of (A) human SIX1 (top) and mouse Six1 (bottom) loci and (B) human SIX2 (top) and mouse Six2 (bottom) loci. Cons, Phastcon vertebrate conservation score. Asterisks indicate samples used for transgenic assays.

Fig. 4.

Six1 expression is transient and independent of Six2 in the mouse whereas it persists in human nephron progenitors. (A) SIX1, SIX2 and cytokeratin immunostaining of sectioned human fetal kidney (HFK) (B) Six1, Six2 and cytokeratin immunostaining of adjacent sections from E10.5, E11.5 and E12.5 mouse kidneys. (C) GFP, Six1, and cytokeration immunostaining of E10.5 Six2^GCE/+ and Six2^GCE/GCE kidneys. Images on far right show zoomed 2views.

Fig. 5.

Transgenic mouse analysis of human SIX1 enhancers shows similar regulation to mouse Six1 in the developing mouse kidney. (A) β-galactosidase (β-gal) activity of the two SIX1 enhancers at E10.5. Number of lacZ⁺ transgenics showing MM expression at E10.5 is indicated. cg, cranial ganglia; ov, otic vesicle; ol, olfactory placode; AER, apical ectodermal ridge; MM, metanephric mesenchyme; ND, nephric duct. (B) β-gal activity of the two Six1 enhancers at E15.5. Number of transgenics showing nephron progenitor expression is indicated. (C) Six2 and cytokeratin staining of kidney sections from B.

Fig. 6.

SIX1 and SIX2 share common targets and show evidence of auto- and cross-regulatory activity. (A) Comparison of the most enriched motif for SIX1 and SIX2 peaks. (B) Distribution of SIX1 motif-peak distances. (C) Overlap of SIX1 and SIX2 binding sites. (D) Overlap of SIX1 and SIX2 target genes. (E) Comparison of raw signals from SIX2 and SIX1 ChIP-Seq data sets. Each point represents a single binding peak. (F) Gene ontology analysis of shared SIX1/SIX2 peaks. Obs., observed; Exp., expected. (G) Genomic view of the human SIX1 (left) and SIX2 (right) gene loci. (H) Western blot of SIX2-3×FLAG co-immunoprecipitations from HEK293 cells. (I) Western blot of SIX1 and SIX2 co-immunoprecipitations from human fetal kidneys.

Fig. 7.

Model of differential regulation of Six1/2 and SIX1/2 in the developing mouse and human kidney. In the metanephric mesenchyme of the mouse (E10.5), Six1 expression is driven by factor(s) ‘X’ and is actively transcribed (Pol II). Six1 can then activate Six2 expression (1), and subsequently Six2 can drive its own expression (2) via an autoregulatory loop. Because both loci are active, they are marked by H3K27ac (ac). However, in the mature nephron progenitors, Six1 is no longer expressed and displays a repressive histone signature of H3K27me3 (me3). Six2 cannot access the Six1 enhancers and continues to drive its own expression. In the human metanephric mesenchyme (∼5 weeks of gestation), the expression and regulation of SIX1 and SIX2 are unknown. In mature nephron progenitors and in contrast to the mouse, SIX1 is active and expression is driven by SIX2 and itself. Similarly, SIX2 expression is driven by SIX1 and itself. SIX1 and SIX2 are likely to regulate expression through discrete complexes.