A β-catenin-driven switch in TCF/LEF transcription factor binding to DNA target sites promotes commitment of mammalian nephron progenitor cells
- Department of Stem Cell Biology and Regenerative Medicine, Eli and Edythe Broad-CIRM Center for Regenerative Medicine and Stem Cell Research, Keck School of Medicine of the University of Southern California, United States
- The Rogosin Institute, United States
- Center for Molecular Medicine, Maine Medical Center Research Institute, United States
- Ludwig Institute for Cancer Research, Department of Cellular and Molecular Medicine, Institute of Genomic Medicine, Moores Cancer Center, University of California San Diego, United States
Figures
Figure 1 with 1 supplement
Nephron progenitor expansion medium (NPEM) supplemented with differential levels of CHIR99021 (CHIR) models nephron progenitor cell maintenance or differentiation in a plate.
(A) Immunofluorescence (IF) staining showing expression level of Six2, non-phospho (NP) β-catenin and Jag1 in nephron progenitor cell (NPC) cultured in NPEM supplemented with various CHIR dosages. (B…
Figure 1—figure supplement 1
Supplementary RNA-Seq data analysis.
(A) Overview of experiment design and data available (gray). (B) Bar plots show RT-qPCR measurement of relative expression of the indicated genes, as verification of results in Figure 1C. (C) …
Figure 2 with 1 supplement
High dosage of CHIR99021 (CHIR) triggered change of nephron progenitor cell (NPC) epigenome.
(A, B) Genome browser view of RNA-Seq, ATAC-Seq, as well as Six2, H3K27ac, H3K27me3, and Ser5P ChIP-Seq data near Six2 (A) and Wnt4 (B) in low CHIR (left) and high CHIR (right) conditions. Black …
Figure 2—figure supplement 1
Supplementary ATAC-Seq data analysis.
(A) Hierarchical cluster of R-square values between normalized ATAC-Seq reads within merged peaks from all samples. (B) Histograms of distances from differentially accessible (DA) chromatin regions …
Figure 3 with 2 supplements
Differential expression of TCF/LEF family transcription factors in nephron progenitor cell (NPC) in response to distinct level of CHIR99021 (CHIR).
(A) Bar plots showing RNA-Seq measured expression levels of TCF/LEF family factors in NPC cultured in nephron progenitor expansion medium (NPEM) culture supplemented with various concentrations of …
Figure 3—figure supplement 1
Expression of TCF/LEF factors measured by immunoblots.
(A) Immunoblots of TCF/LEF family factors in nephron progenitor cell cultured in nephron progenitor expansion medium with the indicated treatment; red arrows indicate target band. (B) Quantification …
Figure 3—figure supplement 2
Supplementary evidence for differential expression of TCF/LEF factors.
(A) Bar plots show RT-qPCR measurement of relative expression of TCF/LEF family factors, as verification for results in Figure 3A. (B) Bar plots show expression of individual transcripts of TCF/LEF …
Figure 4 with 3 supplements
Increased CHIR99021 (CHIR) dosage induces a switch of TCF/LEF factors binding to the genome.
(A, B) Genome browser view of Wnt4 enhancer locus showing ChIP-Seq signal of TCF/LEF factors in low and high CHIR conditions. (C) Histograms showing binding intensity of TCF/LEF factors and …
Figure 4—figure supplement 1
Supplementary ChIP-Seq data analysis.
(A) Numbers of and overlap between binding sites of the same factors between different conditions. (B) Numbers of and overlap between binding sites of different factors in the same conditions. (C) …
Figure 4—figure supplement 2
Analysis of Tcf7l1 binding in low CHIR99021 (CHIR).
(A) Overlap of direct and indirect Tcf7l1 binding in low CHIR with Lef1 binding in high CHIR (left) and enrichment of ChIP-Seq reads on the two types of binding events. (B) Distribution of ATAC-Seq, …
Figure 4—figure supplement 3
β-Catenin binding sites near (A) Pla2g7 and (B) Tafa5, two β-catenin target genes reported in Karner et al., 2011.
The ChIP-qPCR target sites were marked as red bins on the top track. The highlighted region is where our data is consistent with Karner’s.
Figure 5 with 1 supplement
β-Catenin activates gene expression through both stable and de novo enhancer–promoter loops.
(A) β-Catenin binding sites that overlap with an anchor of chromatin loop in high CHIR99021 (CHIR), the proportion that connects to a transcriptional start site (TSS) (gray in the pie chart) and …
Figure 5—figure supplement 1
Supplementary Hi-C data analysis.
(A) Reproducible loops between replicates. (B) Bar plots showing the percentage of loops in the corresponding category (high CHIR99021 [CHIR] only, low CHIR only or share) that are linked to …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | Rabbit polyclonal anti-Six2 | ProteinTech | 11562-1-AP; RRID:AB_2189084 | ChIP (1:200) |
| Antibody | Mouse monoclonal IgG1 anti-Six2 | Abnova | H00010736-M01; RRID:AB_436993 | IF (1:1000) |
| Antibody | Rabbit polyclonal anti-β-catenin | ThermoFisher | 71–2700; RRID:AB_2533982 | ChIP (1:200) |
| Antibody | Rabbit monoclonal anti-non-phopho-β-catenin | Cell Signaling Technology | 8814; RRID:AB_11127203 | ChIP (1:200), IF (1:500) and western blot (1:1000) |
| Antibody | Mouse monoclonal anti-Tcf7l1 | Santa Cruz | sc-166411; RRID:AB_2302942 | IF (1:1000), western blot (1:1000) |
| Antibody | Rabbit polyclonal anti-Tcf7l1 | Thermo Scientific | PA5-40327; RRID:AB_2577173 | ChIP (1:200) |
| Antibody | Rabbit monoclonal anti-Tcf7l2 | Cell Signaling Technology | 2569; RRID:AB_2199816 | ChIP (1:200), IF (1:500), western blot (1:1000) |
| Antibody | Rabbit monoclonal anti-Tcf7 | Cell Signaling Technology | 2203; RRID:AB_2199302 | ChIP (1:200), western blot (1:1000) |
| Antibody | Rat monoclonal IgG2b anti-Tcf7 | R and D Systems | MAB8224 | IF (1:100) |
| Antibody | Rabbit monoclonal anti-Lef1 | Cell Signaling Technology | 2230; RRID:AB_823558 | ChIP (1:200), IF (1:500), western blot (1:1000) |
| Antibody | Rabbit monoclonal anti-histone H3 | Abcam | ab1791; RRID:AB_302613 | Western blot (1:1000) |
| Antibody | Rabbit monoclonal anti-histone H3K27ac | Abcam | ab4729; RRID:AB_2118291 | ChIP (1:200) |
| Antibody | Mouse monoclonal anti-histone H3K27me2me3 | Active Motif | 39536; RRID:AB_2793247 | ChIP (1:200) |
| Antibody | Mouse monoclonal anti-Ser5P-RNAPII | Millipore | 05–623; RRID:AB_309852 | ChIP (1:200) |
| Antibody | Goat polyclonal anti-Jag1 | R and D Systems | AF599; RRID:AB_2128257 | IF (1:50) |
| Antibody | HRP-conjugated goat anti-mouse IgG1 | Thermo Scientific | A10551 | Western blot (1:1000) |
| Antibody | HRP-conjugated goat anti-rabbit IgG | Cell Signaling Technology | 7074 | Western blot (1:1000) |
| Antibody | Alexa647 mouse anti-rat IgG2b | Abcam | ab172335 | IF (1:500) |
| Antibody | Alexa555 donkey anti-rabbit IgG | Abcam | ab150074 | IF (1:500) |
| Antibody | Alexa488 goat anti-mouse IgG1 | Thermo Scientific | A10551 | IF (1:500) |
| Commercial assay or kit | SuperScript IV VILO Master Mix with ezDNase Enzyme | Thermo Fisher Scientific | 11766050 | |
| Commercial assay or kit | Luna Universal qPCR Master Mix | New England Biolab | M3003 | |
| Commercial assay or kit | RNeasy micro kit | Qiagen | 74004 | |
| Commercial assay or kit | KAPA Stranded mRNA-Seq Kit | Kapa Biosystems | KK8420 | |
| Commercial assay or kit | SimpleChIP chromatin IP buffers | Cell Signaling Technology | 14231 | |
| Commercial assay or kit | Protein A/G agarose beads | Thermo Fisher Scientific | 20423 | |
| Commercial assay or kit | minElute reaction cleanup kit | Qiagen | 28204 | |
| Commercial assay or kit | SEA block | Thermo Fisher Scientific | 107452659 | |
| Commercial assay or kit | Thruplex DNA library prep kit | Clontech | R400523 | |
| Software, algorithm | Partek Flow platform | Partek (https://www.partek.com/partek-flow/) | ||
| Software, algorithm | DESeq2 | Love et al., 2014 | RRID:SCR_015687 | |
| Software, algorithm | DAVID | Huang et al., 2009 (http://david.abcc.ncifcrf.gov/) | RRID:SCR_001881 | |
| Software, algorithm | Homer | Heinz et al., 2010 (http://homer.ucsd.edu/) | RRID:SCR_010881 | |
| Software, algorithm | QuEST | Valouev et al., 2008 | ||
| Software, algorithm | GREAT | McLean et al., 2010 |
Additional files
-
Supplementary file 1
Differentially expressed genes (DEG) between conditions.
- https://cdn.elifesciences.org/articles/64444/elife-64444-supp1-v2.xlsx
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Supplementary file 2
Differentially accessible regions (DAR) between conditions.
- https://cdn.elifesciences.org/articles/64444/elife-64444-supp2-v2.xlsx
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Supplementary file 3
Tcf7l1 and Lef1 peak coordinates by category as in Figure 4.
- https://cdn.elifesciences.org/articles/64444/elife-64444-supp3-v2.xlsx
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Supplementary file 4
Hi-C loops to transcriptional start site (TSS).
- https://cdn.elifesciences.org/articles/64444/elife-64444-supp4-v2.xlsx
-
Supplementary file 5
Hi-C loops that are anchored on β-catenin binding sites in high CHIR99021 (CHIR) condition.
- https://cdn.elifesciences.org/articles/64444/elife-64444-supp5-v2.xlsx
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Supplementary file 6
Expression of Wnt target genes published in Karner et al., 2011).
- https://cdn.elifesciences.org/articles/64444/elife-64444-supp6-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/64444/elife-64444-transrepform-v2.docx
