Chromatin Remodelers Interact with Eya1 and Six2 to Target Enhancers to Control Nephron Progenitor Cell Maintenance

Abstract

Background: Eya1 is a critical regulator of nephron progenitor cell specification and interacts with Six2 to promote NPC self-renewal. Haploinsufficiency of these genes causes kidney hypoplasia. However, how the Eya1-centered network operates remains unknown.

Methods: We engineered a 2×HA-3×Flag-Eya1 knock-in mouse line and performed coimmunoprecipitation with anti-HA or -Flag to precipitate the multitagged-Eya1 and its associated proteins. Loss-of-function, transcriptome profiling, and genome-wide binding analyses for Eya1's interacting chromatin-remodeling ATPase Brg1 were carried out. We assayed the activity of the cis-regulatory elements co-occupied by Brg1/Six2 in vivo.

Results: Eya1 and Six2 interact with the Brg1-based SWI/SNF complex during kidney development. Knockout of Brg1 results in failure of metanephric mesenchyme formation and depletion of nephron progenitors, which has been linked to loss of Eya1 expression. Transcriptional profiling shows conspicuous downregulation of important regulators for nephrogenesis in Brg1-deficient cells, including Lin28, Pbx1, and Dchs1-Fat4 signaling, but upregulation of podocyte lineage, oncogenic, and cell death-inducing genes, many of which Brg1 targets. Genome-wide binding analysis identifies Brg1 occupancy to a distal enhancer of Eya1 that drives nephron progenitor-specific expression. We demonstrate that Brg1 enrichment to two distal intronic enhancers of Pbx1 and a proximal promoter region of Mycn requires Six2 activity and that these Brg1/Six2-bound enhancers govern nephron progenitor-specific expression in response to Six2 activity.

Conclusions: Our results reveal an essential role for Brg1, its downstream pathways, and its interaction with Eya1-Six2 in mediating the fine balance among the self-renewal, differentiation, and survival of nephron progenitors.

Keywords: Eya1 enhancers; Eya1/Six2-regulatory network; Mycn proximal enhancer; Pbx1 distal enhancers; interaction of chromatin remodeling SWI/SNF proteins with Eya1/Six2; nephron progenitor cell maintenance.

Figure 1.

Eya1 physically interacts with Brg1-BAFs in developing kidneys. (A) Generation of Eya1^HA-Flag knock-in mice. Southern blot using a 5′-flanking probe detected the WT allele of 20.2 kb and the targeted knock-in allele of 13.7 kb. (B) Coimmunostaining of E13.5 kidney from Eya1^HA-Flag with anti-Flag/Six2 or -HA/Six2. Note that Eya1 expression in RVs (arrows) is higher than Six2, consistent with our previous observation by X-gal staining of Eya1^LacZ reporter. (C) CoIP analysis using nuclear extracts from E13.5 Eya1^HA-Flag kidneys. The input was 5% of the amount used for IP. Anti-Flag was used for IP and anti-HA was used for western blot detection of the multitagged Eya1. Other antibodies used for IP or western blot are indicated. (D) Immunostaining with anti-Brg1 or anti-Brg1/HA on E14.5 WT or Eya1^HA-Flag kidney sections. ES, embryonic stem cells; CB, comma-shaped body; SB, S-shaped body. Scale bars: 30 μm.

Figure 2.

Ablation of Brg1 using Eya1^CreER/+ from E9.0 to E9.5 disrupts MM formation and results in kidney agenesis. (A) Immunostaining showing Brg1 expression in the MM, UB, and nephric duct (ND) in E10.5 Eya1^CreER/+ (n=6) and Eya1^cKO/cKO (Eya1^CreER/+;Eya1^fl/fl; n=6) littermates (tamoxifen administration from approximately E9.0–E9.5). (B) Kidney agenesis in Brg1^cKO/cKO embryo at E16.5 (n=8 for each genotype). a, adrenal gland; k, kidney; o, ovary. (C) Hematoxylin&Eosin-stained (upper panels) and Wt1-immunostained (lower panels) sections showing condensed MM and outgrowth of UB in Eya1^CreER/+ but only residual MM cells (arrows), some of which are positive for Wt1, in Brg1^cKO/cKO (n=6 for each experiment). (D) Medial view of whole-mount embryos hybridized for Eya1 riboprobe. Dorsal is up, and the arrow points to the residual signal of Eya1 in the presumptive MM region (n=6). (E) Medial view of whole-mount embryos hybridized with c-Ret riboprobe (n=6). Dorsal is down, and the arrow points to no UB outgrowth in Brg1^cKO/cKO. Scale bars: 30 μm.

Figure 3.

Temporal deletion of Brg1 in the induced MM leads to premature epithelialization and depletion of the nephron progenitors. The schedule for tamoxifen treatment is outlined. (A) Kidneys (K) at E14.5 and E17.5 of Eya1^CreER/+ and Brg1^cKO/cKO littermates (n=8). The arrow points to residual kidney tissue. a, adrenal gland. (B) Immunostaining for Wt1 on E14.5 kidney sections (n=8). (C–E) Hematoxylin&Eosin-stained kidney sections of Eya1^CreER/+ and Brg1^cKO/cKO kidneys at (C) E13.5, (D) E12.5, and (E) E11.5 (n=8). The arrow in (C) indicates depletion of the condensed CM, and arrows in (D) and (E) indicate ectopic vesicle–like structures on the outmost region of the kidney. UR, ureter. (F–I) ISH showing (F and G) Wnt4 and (H and I) Pax8 expression in (F and H) PTAs at E11.5 and (G and I) RVs at E12.5 in Eya1^CreER/+ and Brg1^cKO/cKO kidneys. (J and K) ISH showing expression of (J) Eya1 and (K) Six2 (n=6 for each stage). Scale bars: 60 μm.

Figure 4.

Transcriptome profiling analyses reveal the requirement of Brg1 for NPC maintenance. Biologic replicates (samples “1” and “2”) with each RNA-seq library prepared from different 5000-cell populations FACS sorted from control or Brg1^cKO/cKO (cKO) at the same time were sequenced. (A) Volcano plot showing transcripts differentially responding to depletion of Brg1. Genes with P adjusted >0.05 and log₂ fold change <0.58 are displayed in gray. (B) Heat map showing 33 selected DEGs in each sample. Genes in black were found to be statistically insignificant (P adjusted >0.05, in gray in [A]) due to variation between replicates and are excluded from the 3383 DEG list (Supplemental File 1). Blue and red indicate down- and upregulated genes, respectively, in Brg1^cKO/cKO. Genes underlined represent oncogenic and apoptotic factors. (C) Quantitative RT-PCR of Eya1^CreER/+ and Brg1^cKO/cKO kidneys. qPCR was performed in triplicate and repeated three times. *P<0.05; **P<0.01; ***P<0.001.

Figure 5.

ChIP-seq reveals Brg1 and Six2 genome-wide occupancy in the E13.5 kidney. (A) Venn diagram indicating overlap of Brg1 peaks and H3K27ac deposition. (B) Genomic distribution of Brg1 peaks and its overlapping peaks with H3K27ac. UTR, untranslated region. (C) Venn diagram indicating the overlap of Brg1- and Six2-binding sites or targeted genes. (D) Genomic browser visualization of Brg1 peaks at the loci of Fgfr1 and Tcf21 and distal regions co-occupied by Brg1/Six2 (boxed areas). The direction of transcription is shown by the arrow beginning at the transcription start site. Six2E16.5 is from the public database (GSE39837) for comparison. (E) Sequence logos of the significantly enriched top motifs from Homer known motif analysis; letter size indicates nucleotide frequency. Numbers of target sites in Brg1 peaks with the significance of motif occurrence (P value) are indicated. (F) Venn diagrams indicate overlap of Brg1 sites in WT and Six2^−/− kidneys and 596 of 919 sites cobound by Brg1/Six2 absent in Six2^−/− kidney. (G) Heat maps showing Brg1 peaks within a −3-/+3-kb window centered on all Brg1 peaks in WT or Six2^−/− kidneys.

Figure 6.

Brg1 occupancy to two distal-intronic enhancer elements of Pbx1 depends on Six2 activity, and both enhancers govern NPC-specific expression in response to binding to Six2. (A) Genome browser visualization of overlapping occupancy of Brg1and Six2 to the Pbx1 gene. Two conserved regions approximately +49 and +39 kb with strong H3K27ac deposition are co-occupied by Brg1/Six2 in the kidney and by Six1 in the E10.5 embryo, and Brg1 enrichment at these regions was reduced in Six2^−/− kidney. (B) ChIP-qPCR in the E13.5 kidney confirming disruption of Brg1 enrichment at both enhancer regions in the Six2^−/− kidney. The enrichment of mock IP was considered one-fold. ***P<0.001. (C) Both enhancer regions contain two SIX motifs, and each mutant reporter transgene was generated by introducing mutations into both predicted SIX motifs. (D) ChIP-qPCR confirming disruption of these SIXmt mutations to Six2 binding assessed by using chromatin prepared from HEK293 cells cotransfected with His-Six2 expression plasmid and each WT or mutant reporter. The enrichment of mock IP was considered one-fold. **P<0.001. (E) Coimmunostaining for Pbx1 (red)/Six2 (green) in the E16.5 kidney showing Pbx1 expression in stromal cells (SCs), CM, and RV. (F–I) G0 Tg analysis of the LacZ transgene driven by 51 -bp of Pbx1+49000, 907 bp of Pbx1+39000, or each SIXmt reporter in the E16.5 kidney. (F) β-Gal⁺ cells were detected in CM and RVs of transgenic kidneys driven by the enhancer region at +49 kb (n=7/7 Tg lines), whereas (G) the enhancer at +39 kb drove transgene expression with more β-Gal⁺ cells around the UB tip and migrating toward ventral side to the UB branching and fewer in the CM peripheral to the UB branching (n=2/2 Tg lines). (H and I) X-gal–stained kidney sections of each transgene driven by each SIXmt enhancer (n=8/8 in [H] or n=10/10 in [I]). Scale bars: 30 μm.

Figure 7.

Enhancers/CREs of Mycn and Eya1 loci occupied by Brg1 and/or Six2 drive expression in MM lineage. (A) Genome browser visualization of overlapping occupancy of Brg1 and Six2/Six1 to the Mycn promoter (red blocks; −333 to −962 bp). Peak calling did not detect Brg1 binding to this region in the Six2^−/− kidney. The arrow indicates the direction of transcription beginning at the transcription start site. Black asterisks indicate nonspecific peaks as seen in the IgG control. (B) ChIP-qPCR confirmed disruption of Brg1 binding to the Mycn promoter region in the Six2^−/− kidney. (C) The Mycn proximal-promoter fragment (631 bp; −333 to −962 bp) contains three SIX motifs, and a mutant reporter transgene was generated by introducing mutations into these predicted SIX motifs. (D) ChIP-qPCR confirming disruption of Six2 binding to the mutant reporter assessed using chromatin prepared from HEK293 cells cotransfected with His-Six2 expression plasmid and reporter Mycn -631-EGFP and Mycn-631SIXmt-EGFP. The enrichment of mock IP was considered one-fold. ***P<0.001. (E) Anti-Mycn immunostaining and G0 transgenic analysis of EGFP transgene driven by Mycn-631 and SIXmt in the E16.5 kidney showing EGFP activity in the NPCs and RVs (n=2/2 transgenic lines), but SIXmt abolished EGFP expression (n=12/12 Tg lines). (F) Genomic browser visualization showing comparison of Brg1, Six2 (2012, GSE39837 and 2016, GSE73867¹⁵), and Six1 in E10.5 embryo (GSE108130) and H3K27ac and H3K27me3 enrichment (GSE166588) at three distal regions of the Eya1 locus. The left panel shows the Six2-bound peak approximately +520 kb (red asterisks), which is also bound by Six1 in E10.5 embryo. The center panel shows the Brg1-bound −195-kb region with H3K27me3 at E10.5 but with H3K27ac at E13.5. The red asterisks indicate weak Six2 enrichment and reduced Brg1 binding in Six2^−/−. The right panel shows two Six2-bound regions without Brg1 peak or H3K27ac deposition. The one approximately −316 kb (double gray asterisks) is untested, whereas the other approximately −325 kb (double black asterisks) was previously shown to drive transgene expression in the NPCs. G0 transgenic analysis of transgenes driven by (G) a 1043-bp fragment approximately +520 kb bound by Six2 and Six1 (Eya1 + 520kb-GFP) and (H) a 2263-bp fragment approximately −195 kb occupied by Brg1 (Eya1–195kb-LacZ) in mouse embryos. For Eya1 + 520kb-GFP, all Tg lines showed consistent expression in uninduced MM progenitors at E10.5 (n=3) and in CM, PTA, and RV at E14.5 (n=3). For Eya1–195kb-LacZ, five Tg embryos at E14.5 were analyzed, and all showed consistent expression restricting to the NPCs. ND, nephric duct. Scale bars: 30 μm.