A wt1-controlled chromatin switching mechanism underpins tissue-specific wnt4 activation and repression - PubMed (original) (raw)

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. 2011 Sep 13;21(3):559-74.

doi: 10.1016/j.devcel.2011.07.014. Epub 2011 Aug 25.

Anna Webb, Rachel L Berry, Joan Slight, Sally F Burn, Lee Spraggon, Victor Velecela, Ofelia M Martinez-Estrada, John H Wiltshire, Stefan G E Roberts, David Brownstein, Jamie A Davies, Nicholas D Hastie, Peter Hohenstein

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A wt1-controlled chromatin switching mechanism underpins tissue-specific wnt4 activation and repression

Abdelkader Essafi et al. Dev Cell. 2011.

Abstract

Wt1 regulates the epithelial-mesenchymal transition (EMT) in the epicardium and the reverse process (MET) in kidney mesenchyme. The mechanisms underlying these reciprocal functions are unknown. Here, we show in both embryos and cultured cells that Wt1 regulates Wnt4 expression dichotomously. In kidney cells, Wt1 recruits Cbp and p300 as coactivators; in epicardial cells it enlists Basp1 as a corepressor. Surprisingly, in both tissues, Wt1 loss reciprocally switches the chromatin architecture of the entire Ctcf-bounded Wnt4 locus, but not the flanking regions; we term this mode of action "chromatin flip-flop." Ctcf and cohesin are dispensable for Wt1-mediated chromatin flip-flop but essential for maintaining the insulating boundaries. This work demonstrates that a developmental regulator coordinates chromatin boundaries with the transcriptional competence of the flanked region. These findings also have implications for hierarchical transcriptional regulation in development and disease.

Copyright © 2011 Elsevier Inc. All rights reserved.

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Figures

Figure 1

Figure 1

Wt1 is essential for nephron formation at the MET stage. A-F. H&E stained section of E18.5 control (A-C) and mutant (D-F) kidneys. Each section is taken from a different kidney. G. Wt1 immunohistochemistry of E18.5 control (left panel) and mutant (right panel) kidneys. Abbreviations are: UB: ureteric bud; CM: condensed mesenchyme; EM expanded mesenchyme; CS: comma-shaped body; SS: S-shaped body; PT: proximal tubule; DT: distal tubule;G: glomerulus; IPT: immature proximal tubule; IDT: immature distal tubule.*: denotes epithelialised but abnormal structures that have partially lost Wt1.

Figure 2

Figure 2

Wnt4 is directly activated by Wt1 in kidney mesenchyme in vivo and in vitro. A. Wt1 and Wnt4 mRNA in control and Wt1 knockdown M15 cells. B. Wnt4 mRNA expression in FACS sorted control (_Wt1_co/GFP) and Wt1-deficient (_Nes_-Cre _Wt1_co/GFP) e12.5 kidney mesenchyme cells. C. ChIP of putative WREs using Wt1 antibodies in control and Wt1 knockdown M15 cells. D. ChIP of putative WREs using Wt1 antibodies in FACS sorted control (_Wt1_co/GFP) and Wt1-deficient (_Nes_-Cre _Wt1_co/GFP) e12.5 kidney mesenchyme cells. E. Luciferase reporter assays of wild type and mutant putative WREs in M15 cells.

Figure 3

Figure 3

Wnt4 is directly repressed by Wt1 in epicardium in vivo and in vitro. A. mRNA of Wt1 and Wnt4 expression in control and Wt1 knockdown MEEC (epicardium) cells. B. mRNA of Wt1 and Wnt4 expression in FACS sorted control (Wt1co/GFP) and _Wt1_-deficient (_Gata5_-Cre Wt1co/GFP) epicardium. C. ChIP with Wt1 antibodies of putative WREs in control and Wt1 knockdown MEEC cells. D. Luciferase reporter assays of wild type and mutant putative WREs in MEEC cells.

Figure 4

Figure 4

In vitro and in vivo co-factors regulating Wnt4 expression in a tissue-specific manner downstream of Wt1. A. ChIP using p300 antibodies in FACS sorted control (_Wt1_co/GFP) and Wt1-deficient (_Nes_-Cre _Wt1_co/GFP) e12.5 kidney mesenchyme cells. B. ChIP using Cbp antibodies in FACS sorted control (_Wt1_co/GFP) and Wt1-deficient (_Nes_-Cre _Wt1_co/GFP) e12.5 kidney mesenchyme cells. C-E. ChIP with p300 (C), Cbp (D) and Wt1 (E) antibodies in control (lacZ), Wt1 and a combined Cbp-p300 knockdown M15 cells. F. mRNA of indicated genes in control, Cbp, p300 and Cbp/p300 knockdown M15 cells. G. ChIP with Wt1 or Basp1 antibodies of WREs in control, Wt1 and Basp1 knockdown MEEC cells. H. mRNA of indicated genes in control and Basp1 knockdown cells. I. mRNA of indicated genes in control, Basp1 and Cbp/p300 knockdown M15 (kidney) and MEEC (epicardial) cells.

Figure 5

Figure 5

Wt1 actively upregulates Wnt4 expression in kidney mesenchyme and actively downregulates its expression in epicardium via the local chromatin, RNAPII elongation and stalling. A. A schematic representation of the 5′end of the Wnt4 gene. B. ChIP with H3K27Me3 (top), H3K4Me3 (middle) and H3Ac (bottom) antibodies in control as well as Wt1 or Cbp/p300 knockdown M15 cells. C. ChIP with H3K27Me3 (top), H3K4Me3 (middle) and H3Ac (bottom) antibodies in control as well as Wt1 or Cbp/p300 knockdown MEEC cells. D. ChIP with RNApII CTDS5, RNAPII CTDS2 and CTDnon phos antibodies of control (top), Wt1 (middle) and Cbp/p300 (bottom) knockdown M15 cells. E. ChIP with RNApII CTDS5, RNAPII CTDS2 and CTDnon phos antibodies of control (top), Wt1 (middle) and Basp1 (bottom) knockdown MEEC cells. F. ChIP with H3K36Me3 antibodies in control, Wt1, Basp1 and Cbp/p300 knockdown M15 cells. G. ChIP with H3K36Me3 antibodies in control, Wt1, Basp1 and Cbp/p300 knockdown MEEC cells.

Figure 6

Figure 6

Wt1-mediated chromatin flip-flop. A. A graphic illustration of the complete Wnt4 locus and neighbouring genes as demarcated by CTCF binding upstream (U1) and downstream (D1). Distances in kb from Wnt4 TSS are indicated. B. ChIP with CTCF, Sa2 and Rad21 antibodies in control, Wt1 and Cbp/p300 knockdown M15 cells at U1 (left panel) and D1 (right panel) sites. C. ChIP with CTCF, Sa2 and Rad21 antibodies in control, Wt1 and Cbp/p300 knockdown MEEC cells at U1 (left panel) and D1 (right panel) sites. D. Occupancy of CTCF and cohesin subunits Rad21 and Sa2 at U1 and D1 in the primary GFP-sorted kidney mesenchymal cells. E. ChIP with H3K27Me3 (top), H3K4Me3 (middle) and H3Ac (bottom) antibodies in control, Wt1 and Cbp/p300 knockdown M15 cells. F. ChIP with H3K27Me3 (top), H3K4Me3 (middle) and H3Ac (bottom) antibodies in control, Wt1 and Basp1 knockdown MEEC cells. G. ChIP with H3K27Me3 (top), H3K4Me3 (middle) and H3Ac (bottom) antibodies in FACS sorted control (_Wt1_co/GFP) and Wt1-deficient (_Nes_-Cre _Wt1_co/GFP) e12.5 kidney mesenchyme cells.

Figure 7

Figure 7

CTCF and cohesin are essential for insulation but are dispensable for Wnt4 expression and Wt1-mediated chromatin flip-flop. A. A graphic illustration of the complete Wnt4 locus and neighbouring genes as demarcated by U1 and D1. B. ChIP with CTCF, Sa2 and Rad21 antibodies of control, Ctcf, Sa2 and Rad21 M15 cells at U1 (left panel) and D1 (right panel) sites. C. ChIP with CTCF, Sa2 and Rad21 antibodies of control, Ctcf, Sa2 and Rad21 MEEC cells at U1 (left panel) and D1 (right panel) sites. D. ChIP using H3K27Me3 (top), H3K4Me3 (middle) and H3Ac (bottom) antibodies in control, Ctcf, Sa2 and Rad21 knockdown M15 cells. E. ChIP using H3K27Me3 (top), H3K4Me3 (middle) and H3Ac (bottom) antibodies in control, Ctcf, Sa2 and Rad21 knockdown MEEC cells. F. mRNA of indicated genes in control, Wt1, Ctcf, Sa2 and Rad21 knockdown M15 cells. G. mRNA of indicated genes in control, Wt1, Ctcf, Sa2 and Rad21 knockdown MEEC cells. H. mRNA of indicated genes in Wt1-deficient kidney mesenchyme and epicardial cells in vivo normalized to control cells.

Figure 8

Figure 8

Wt1-controled chromatin flip-flop in kidney mesenchyme and epicardium. Upper panel: in kidney mesenchyme the Wnt4 locus extending to the CTCF sites U1 and D1 is active (green chromatin). Upon Wt1 loss, but not Cbp-p300 loss, the CTCF-delimited Wnt4 locus is switched to repressive chromatin (red). The flanking regions are unaffected. Upon Ctcf loss the active chromatin signature spreads to neighbouring loci from the Wnt4 locus. Lower panel: in epicardium the Wnt4 locus extending to the CTCF sites U1 and D1 is repressed (red chromatin). Upon Wt1 loss, but not Basp1 loss, the CTCF-delimited Wnt4 locus is switched to active chromatin (green). The flanking regions are unaffected. Upon Ctcf loss the repressive chromatin signature spreads to neighbouring loci from the Wnt4 locus.

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