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Cdx2 Regulates Gene Expression through Recruitment of Brg1-associated Switch-Sucrose Non-fermentable (SWI-SNF) Chromatin Remodeling Activity*

Open AccessPublished:January 12, 2017DOI:https://doi.org/10.1074/jbc.M116.752774
      The packaging of genomic DNA into nucleosomes creates a barrier to transcription that can be relieved through ATP-dependent chromatin remodeling via complexes such as the switch-sucrose non-fermentable (SWI-SNF) chromatin remodeling complex. The SWI-SNF complex remodels chromatin via conformational or positional changes of nucleosomes, thereby altering the access of transcriptional machinery to target genes. The SWI-SNF complex has limited ability to bind to sequence-specific elements, and, therefore, its recruitment to target loci is believed to require interaction with DNA-associated transcription factors. The Cdx family of homeodomain transcript ion factors (Cdx1, Cdx2, and Cdx4) are essential for a number of developmental programs in the mouse. Cdx1 and Cdx2 also regulate intestinal homeostasis throughout life. Although a number of Cdx target genes have been identified, the basis by which Cdx members impact their transcription is poorly understood. We have found that Cdx members interact with the SWI-SNF complex and make direct contact with Brg1, a catalytic member of SWI-SNF. Both Cdx2 and Brg1 co-occupy a number of Cdx target genes, and both factors are necessary for transcriptional regulation of such targets. Finally, Cdx2 and Brg1 occupancy occurs coincident with chromatin remodeling at some of these loci. Taken together, our findings suggest that Cdx transcription factors regulate target gene expression, in part, through recruitment of Brg1-associated SWI-SNF chromatin remodeling activity.

      Introduction

      The Cdx genes are vertebrate orthologs of Drosophila caudal and encode homeodomain transcriptional factors. In the mouse, the three members of this family (Cdx1, Cdx2, and Cdx4) are co-expressed in the caudal embryo in all three germ layers commencing at mid-gastrulation and play overlapping roles in a number of developmental programs, including axial elongation, endoderm specification, and anterior-posterior vertebral patterning (
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      • Shivdasani R.A.
      Differentiation-specific histone modifications reveal dynamic chromatin interactions and partners for the intestinal transcription factor CDX2.
      ,
      • Beck F.
      • Stringer E.J.
      The role of Cdx genes in the gut and in axial development.
      ,
      • Savory J.G.
      • Pilon N.
      • Grainger S.
      • Sylvestre J.R.
      • Béland M.
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      Cdx1 and Cdx2 are functionally equivalent in vertebral patterning.
      ,
      • Young T.
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      • Deschamps J.
      Cdx and Hox genes differentially regulate posterior axial growth in mammalian embryos.
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      • de Graaff W.
      • Beck F.
      • Meyer B.I.
      • Deschamps J.
      Cdx1 and Cdx2 have overlapping functions in anteroposterior patterning and posterior axis elongation.
      ). Both Cdx1 and Cdx2 are also expressed in the intestinal epithelium throughout life, where they play critical roles in intestinal homeostasis (
      • Hryniuk A.
      • Grainger S.
      • Savory J.G.
      • Lohnes D.
      Cdx function is required for maintenance of intestinal identity in the adult.
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      Homeosis and intestinal tumours in Cdx2 mutant mice.
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      Cdx2 regulates patterning of the intestinal epithelium.
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      Establishment of intestinal identity and epithelial-mesenchymal signaling by Cdx2.
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      • Clevers H.
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      Transformation of intestinal stem cells into gastric stem cells on loss of transcription factor Cdx2.
      ) and can function as tumor suppressors (
      • Hryniuk A.
      • Grainger S.
      • Savory J.G.
      • Lohnes D.
      Cdx1 and Cdx2 function as tumor suppressors.
      • Holik A.Z.
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      ,
      • Bonhomme C.
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      • Chenard M.P.
      • Kedinger M.
      • Beck F.
      • Freund J.N.
      • Domon-Dell C.
      The Cdx2 homeobox gene has a tumour suppressor function in the distal colon in addition to a homeotic role during gut development.
      • Subtil C.
      • Guérin E.
      • Schneider A.
      • Chenard M.P.
      • Martin E.
      • Domon-Dell C.
      • Duluc I.
      • Brabletz T.
      • Kedinger M.
      • Duclos B.
      • Gaub M.P.
      • Freund J.N.
      Frequent rearrangements and amplification of the CDX2 homeobox gene in human sporadic colorectal cancers with chromosomal instability.
      ). Although Cdx members play critical roles in governing gene expression during development and in the adult intestine, little is known about the mechanisms by which Cdx members regulate target gene transcription.
      The packaging of DNA into nucleosomes creates a barrier to transcription by obstructing access of the basal transcription machinery as well as modulating access of other transcriptional regulators to their DNA binding motifs (
      • Kouzarides T.
      Chromatin modifications and their function.
      • Trotter K.W.
      • Archer T.K.
      The BRG1 transcriptional coregulator.
      ,
      • Felsenfeld G.
      • Groudine M.
      Controlling the double helix.
      • Kadoch C.
      • Crabtree G.R.
      Mammalian SWI-SNF chromatin remodeling complexes and cancer: mechanistic insights gained from human genomics.
      ). Alteration of the chromatin structure by ATP-dependent remodeling complexes can alleviate these constraints, and such complexes play important roles in the transcriptional regulation of many eukaryotic genes. Such complexes include the switch-sucrose non-fermentable (SWI-SNF)
      The abbreviations used are: SWI-SNF
      switch-sucrose non-fermentable
      SILAC
      stable isotope labeling by amino acids in culture
      IP
      immunoprecipitation
      CDRE
      Cdx response element
      RIPA
      radioimmune precipitation assay
      qPCR
      quantitative PCR
      CRISPR-Cas9
      clustered regularly interspaced short palindromic repeats-CRISPR-associated system.
      chromatin-remodeling complex (
      • Kouzarides T.
      Chromatin modifications and their function.
      ,
      • Aoyagi S.
      • Trotter K.W.
      • Archer T.K.
      ATP-dependent chromatin remodeling complexes and their role in nuclear receptor-dependent transcription in vivo.
      ,
      • Sif S.
      ATP-dependent nucleosome remodeling complexes: enzymes tailored to deal with chromatin.
      • Vignali M.
      • Hassan A.H.
      • Neely K.E.
      • Workman J.L.
      ATP-dependent chromatin-remodeling complexes.
      ), which remodels the chromatin structure via conformational or positional changes of nucleosomes (
      • Narlikar G.J.
      • Fan H.Y.
      • Kingston R.E.
      Cooperation between complexes that regulate chromatin structure and transcription.
      ,
      • Peterson C.L.
      • Workman J.L.
      Promoter targeting and chromatin remodeling by the SWI-SNF complex.
      ). Because the SWI-SNF complex possesses limited sequence-specific DNA binding activity, its recruitment to relevant target loci is accomplished via interaction with DNA-bound transcription factors (
      • Trotter K.W.
      • Archer T.K.
      The BRG1 transcriptional coregulator.
      ,
      • Narlikar G.J.
      • Fan H.Y.
      • Kingston R.E.
      Cooperation between complexes that regulate chromatin structure and transcription.
      ), including Myc, Tbx5, β-catenin, and p53 (
      • Romero O.A.
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      • John S.
      • Gimenez-Xavier P.
      • Gómez-López G.
      • Pisano D.
      • Condom E.
      • Villanueva A.
      • Hager G.L.
      • Sanchez-Cespedes M.
      The tumour suppressor and chromatin-remodelling factor BRG1 antagonizes Myc activity and promotes cell differentiation in human cancer.
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      • Alexander J.M.
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      • et al.
      Chromatin remodelling complex dosage modulates transcription factor function in heart development.
      ,
      • Barker N.
      • Hurlstone A.
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      • Miles A.
      • Bienz M.
      • Clevers H.
      The chromatin remodelling factor Brg-1 interacts with β-catenin to promote target gene activation.
      • Lee D.
      • Kim J.W.
      • Seo T.
      • Hwang S.G.
      • Choi E.J.
      • Choe J.
      SWI-SNF complex interacts with tumor suppressor p53 and is necessary for the activation of p53-mediated transcription.
      ).
      Although little is known regarding the means by which Cdx members regulate transcription, it is believed that they serve to recruit co-regulators to target genes, and it is the biochemical activity of such co-regulators that impacts the transcription of such targets (
      • Hryniuk A.
      • Grainger S.
      • Savory J.G.
      • Lohnes D.
      Cdx1 and Cdx2 function as tumor suppressors.
      ,
      • Béland M.
      • Pilon N.
      • Houle M.
      • Oh K.
      • Sylvestre J.R.
      • Prinos P.
      • Lohnes D.
      Cdx1 autoregulation is governed by a novel Cdx1-LEF1 transcription complex.
      ,
      • Wang K.
      • Sengupta S.
      • Magnani L.
      • Wilson C.A.
      • Henry R.W.
      • Knott J.G.
      Brg1 is required for Cdx2-mediated repression of Oct4 expression in mouse blastocysts.
      ). Using a quantitative affinity purification/MS approach based on stable isotope labeling by amino acids in culture (SILAC) (
      • Trinkle-Mulcahy L.
      Resolving protein interactions and complexes by affinity purification followed by label-based quantitative mass spectrometry.
      ), we found that Cdx2 associates with multiple members of the Brg1-containing SWI-SNF complex in HEK293 cells. We further found that Cdx2 and Brg1 associated in vitro, suggesting direct interaction, and that both factors co-occupied a number of Cdx target genes. Recruitment of Brg1 to such targets required Cdx2 and occurred coincident with Brg1- or Cdx2-dependent local chromatin remodeling. Taken together, these findings suggest that Cdx members modulate target gene transcription, at least in part, through recruitment of SWI-SNF-mediated chromatin remodeling.

      Discussion

      Cdx members play critical roles in many developmental processes and impact intestinal homeostasis and tumorigenesis in the adult (
      • Beck F.
      • Stringer E.J.
      The role of Cdx genes in the gut and in axial development.
      ,
      • Young T.
      • Rowland J.E.
      • van de Ven C.
      • Bialecka M.
      • Novoa A.
      • Carapuco M.
      • van Nes J.
      • de Graaff W.
      • Duluc I.
      • Freund J.N.
      • Beck F.
      • Mallo M.
      • Deschamps J.
      Cdx and Hox genes differentially regulate posterior axial growth in mammalian embryos.
      ,
      • Hryniuk A.
      • Grainger S.
      • Savory J.G.
      • Lohnes D.
      Cdx function is required for maintenance of intestinal identity in the adult.
      • Chawengsaksophak K.
      • James R.
      • Hammond V.E.
      • Köntgen F.
      • Beck F.
      Homeosis and intestinal tumours in Cdx2 mutant mice.
      ,
      • Grainger S.
      • Savory J.G.
      • Lohnes D.
      Cdx2 regulates patterning of the intestinal epithelium.
      • Gao N.
      • White P.
      • Kaestner K.H.
      Establishment of intestinal identity and epithelial-mesenchymal signaling by Cdx2.
      ,
      • Savory J.G.
      • Mansfield M.
      • Rijli F.M.
      • Lohnes D.
      Cdx mediates neural tube closure through transcriptional regulation of the planar cell polarity gene Ptk7.
      ,
      • Strumpf D.
      • Mao C.A.
      • Yamanaka Y.
      • Ralston A.
      • Chawengsaksophak K.
      • Beck F.
      • Rossant J.
      Cdx2 is required for correct cell fate specification and differentiation of trophectoderm in the mouse blastocyst.
      ). Little is known, however, regarding the mechanisms by which Cdx members regulate target gene expression. In an effort to better understand this, we utilized a quantitative mass spectrometry approach to identify proteins associated with Cdx2 from HEK293 cells. This cell line expresses abundant levels of Cdx2 and is capable of supporting Cdx-dependent transcription. This exercise recovered multiple members of the SWI-SNF chromatin remodeling complex, including the catalytic subunit Brg1. Additional protein-protein association assays, co-localization analysis, and chromatin occupancy and DNA accessibility assays suggest that Cdx2 (and likely other Cdx members) regulate target gene expression, at least in part, through recruitment of SWI-SNF-mediated chromatin remodeling. These findings extend prior work indicative of Brg1-Cdx interactions (
      • Wang K.
      • Sengupta S.
      • Magnani L.
      • Wilson C.A.
      • Henry R.W.
      • Knott J.G.
      Brg1 is required for Cdx2-mediated repression of Oct4 expression in mouse blastocysts.
      ) and suggest that this is a more general mechanism of transcriptional regulation by Cdx family members.

      Cdx Members Interact with Brg1

      The SWI-SNF complexes are large multimeric protein complexes comprised of over a dozen subunits, the composition of which can vary according to developmental stage and/or cell type (
      • Ho L.
      • Crabtree G.R.
      Chromatin remodelling during development.
      ). Although the Brg1 SWI-SNF subunit contains DNA binding motifs (
      • Trotter K.W.
      • Archer T.K.
      The BRG1 transcriptional coregulator.
      ,
      • Kadam S.
      • Emerson B.M.
      Transcriptional specificity of human SWI-SNF BRG1 and BRM chromatin remodeling complexes.
      ), these do not appear to be capable of directing the complex to specific genomic targets. Rather, recruitment of SWI-SNF to specific loci is believed to rely on association with DNA-bound transcription factors (
      • Felsenfeld G.
      • Groudine M.
      Controlling the double helix.
      ,
      • Peterson C.L.
      • Workman J.L.
      Promoter targeting and chromatin remodeling by the SWI-SNF complex.
      ). Our finding that a number of SWI-SNF members, including Brg1, associated with Cdx2 in HEK293 cells and embryos suggests that Cdx2 may serve to recruit SWI-SNF to specific target genes.
      Of particular note was the finding that Brg1 was significantly enriched and relatively abundant in CDX2 immunoprecipitates from cultured cells. Furthermore, in vitro GST pulldown assays revealed that Brg1 interacted with all three Cdx members. Taken together, these findings suggest that Cdx transcription factors interface with the SWI-SNF complex through direct association with Brg1, likely through the Brg1-B2 domain, although interaction with other SWI-SNF components or regions of Brg1 or additional scaffold proteins cannot be ruled out at present. The promiscuous interaction between Cdx members and Brg1 is also consistent with the functional overlap between family members, as evidenced by the interaction between mutant Cdx alleles (
      • Young T.
      • Rowland J.E.
      • van de Ven C.
      • Bialecka M.
      • Novoa A.
      • Carapuco M.
      • van Nes J.
      • de Graaff W.
      • Duluc I.
      • Freund J.N.
      • Beck F.
      • Mallo M.
      • Deschamps J.
      Cdx and Hox genes differentially regulate posterior axial growth in mammalian embryos.
      ,
      • van den Akker E.
      • Forlani S.
      • Chawengsaksophak K.
      • de Graaff W.
      • Beck F.
      • Meyer B.I.
      • Deschamps J.
      Cdx1 and Cdx2 have overlapping functions in anteroposterior patterning and posterior axis elongation.
      ,
      • Houle M.
      • Allan D.
      • Lohnes D.
      ,
      • Davidson A.J.
      • Zon L.I.
      The caudal-related homeobox genes cdx1a and cdx4 act redundantly to regulate hox gene expression and the formation of putative hematopoietic stem cells during zebrafish embryogenesis.
      ) and the ability of Cdx2 to completely complement Cdx1 function in vertebral patterning (
      • Savory J.G.
      • Pilon N.
      • Grainger S.
      • Sylvestre J.R.
      • Béland M.
      • Houle M.
      • Oh K.
      • Lohnes D.
      Cdx1 and Cdx2 are functionally equivalent in vertebral patterning.
      ). Moreover, all three Cdx members appear equivalent in their ability to occupy target genes (
      • Savory J.G.
      • Pilon N.
      • Grainger S.
      • Sylvestre J.R.
      • Béland M.
      • Houle M.
      • Oh K.
      • Lohnes D.
      Cdx1 and Cdx2 are functionally equivalent in vertebral patterning.
      ,
      • Grainger S.
      • Lam J.
      • Savory J.G.
      • Mears A.J.
      • Rijli F.M.
      • Lohnes D.
      Cdx regulates Dll1 in multiple lineages.
      ,
      • Savory J.G.
      • Mansfield M.
      • Rijli F.M.
      • Lohnes D.
      Cdx mediates neural tube closure through transcriptional regulation of the planar cell polarity gene Ptk7.
      ), further supporting a common mechanistic basis for their impact on transcription.

      Brg1-dependent Regulation of Cdx Target Genes

      Cdx members occupy a number of target genes in the developing embryo (
      • Grainger S.
      • Lam J.
      • Savory J.G.
      • Mears A.J.
      • Rijli F.M.
      • Lohnes D.
      Cdx regulates Dll1 in multiple lineages.
      ,
      • Savory J.G.
      • Bouchard N.
      • Pierre V.
      • Rijli F.M.
      • De Repentigny Y.
      • Kothary R.
      • Lohnes D.
      Cdx2 regulation of posterior development through non-Hox targets.
      ) and the intestine (
      • Grainger S.
      • Savory J.G.
      • Lohnes D.
      Cdx2 regulates patterning of the intestinal epithelium.
      ,
      • Gao N.
      • White P.
      • Kaestner K.H.
      Establishment of intestinal identity and epithelial-mesenchymal signaling by Cdx2.
      ,
      • San Roman A.K.
      • Tovaglieri A.
      • Breault D.T.
      • Shivdasani R.A.
      Distinct processes and transcriptional targets underlie CDX2 requirements in intestinal stem cells and differentiated villus cells.
      ,
      • Verzi M.P.
      • Shin H.
      • Ho L.L.
      • Liu X.S.
      • Shivdasani R.A.
      Essential and redundant functions of caudal family proteins in activating adult intestinal genes.
      ), and this occupation is often predictive of Cdx-dependent expression. ChIP sequencing analyses from embryonic stem cells undergoing mesodermal differentiation (
      • Alexander J.M.
      • Hota S.K.
      • He D.
      • Thomas S.
      • Ho L.
      • Pennacchio L.A.
      • Bruneau B.G.
      Brg1 modulates enhancer activation in mesoderm lineage commitment.
      ) suggests that Brg1 is also resident on a number of these genes in a manner that overlaps known (or potential) CDREs. Co-occupation of a number of these loci by Brg1 and Cdx2 was confirmed by ChIP analysis in HEK293 cells. Moreover, and consistent with our model, occupancy of Brg1 at these loci was lost in Cdx2-null cells. The specificity of this relationship was further supported by recovery of Brg1 chromatin occupancy upon re-expression of wild-type Cdx2 into the knockout cell lines.
      A comparison of Cdx target gene mRNA levels between wild-type and Cdx2- or Brg1-null cell lines suggests that Brg1 is essential for the normal expression of a number of such targets. The finding that loss of Cdx2 resulted in a comparable reduction in the expression of many of these target genes, together with Cdx-dependent recruitment of Brg1 to these loci, is consistent with a functional requirement for Brg1 in Cdx-dependent gene expression. Finally, a limited number of target genes, although exhibiting co-occupation by Cdx2 and Brg1, were not impacted by loss of one or the other factor. This may be indicative of fortuitous, but non-functional, binding or context-dependent regulation by Cdx that is not faithfully recapitulated in HEK293 cells.

      Cdx2-dependent Chromatin Remodeling

      The SWI-SNF complex remodels the chromatin structure via conformational or positional changes of nucleosomes (
      • Ho L.
      • Crabtree G.R.
      Chromatin remodelling during development.
      ). Enzyme accessibility assays have been used as a surrogate measure of such activity and have revealed, for example, Brg1-dependent changes in chromatin structure (
      • Ohkawa Y.
      • Yoshimura S.
      • Higashi C.
      • Marfella C.G.
      • Dacwag C.S.
      • Tachibana T.
      • Imbalzano A.N.
      Myogenin and the SWI-SNF ATPase Brg1 maintain myogenic gene expression at different stages of skeletal myogenesis.
      ,
      • de la Serna I.L.
      • Ohkawa Y.
      • Berkes C.A.
      • Bergstrom D.A.
      • Dacwag C.S.
      • Tapscott S.J.
      • Imbalzano A.N.
      MyoD targets chromatin remodeling complexes to the myogenin locus prior to forming a stable DNA-bound complex.
      ). Using such an assay, we assessed the consequence of Brg1 or Cdx2 loss of function on the chromatin structure at the Dll1 locus and found that chromatin accessibility was comparably affected by loss of either factor. Similarly altered chromatin accessibility was also seen in Brg1- and Cdx-deficient murine intestinal epithelial cells. Moreover, wild-type Brg1, but not a catalytically inert mutant, restored both target gene expression and chromatin accessibility in Brg1-null cells, consistent with a requirement for Brg1-dependent chromatin remodeling in Cdx-mediated transcription. Finally, the finding of non-complementation of Cdx and Brg1 mutant alleles regarding goblet cell hypertrophy further supports the relevance of this interaction in vivo. In this regard, mice null for Muc2 exhibit changes in goblet cell morphology (
      • Birchenough G.M.
      • Johansson M.E.
      • Gustafsson J.K.
      • Bergström J.H.
      • Hansson G.C.
      New developments in goblet cell mucus secretion and function.
      ), and MUC2 is a CDX2 target (
      • Yamamoto H.
      • Bai Y.Q.
      • Yuasa Y.
      Homeodomain protein CDX2 regulates goblet-specific MUC2 gene expression.
      ). It is therefore tempting to speculate that Muc2 may be co-regulated by Cdx and SWI-SNF.
      Gene expression is tightly regulated at multiple levels to ensure appropriate transcriptomes; the chromatin state is a major determinant of transcription (
      • Meier K.
      • Brehm A.
      Chromatin regulation: how complex does it get?.
      ). Our findings are consistent with a model wherein Cdx members impact the chromatin state and target gene expression through recruitment of Brg1 and associated SWI-SNF-dependent chromatin remodeling (Fig. 8). In this regard, prior work has shown Cdx2 occupancy of a number of genes in intestinal progenitor cells. Many of these targets, however, are not perturbed in this progenitor population by loss of Cdx2 but were impacted in the differentiated progeny thereof (
      • San Roman A.K.
      • Tovaglieri A.
      • Breault D.T.
      • Shivdasani R.A.
      Distinct processes and transcriptional targets underlie CDX2 requirements in intestinal stem cells and differentiated villus cells.
      ). Taken together, these observations suggest that one mechanism of action of Cdx2 may be to recruit SWI-SNF to establish the appropriate chromatin landscape in progenitor cells permissive for subsequent transcription in descendant lineages.
      Figure thumbnail gr8
      FIGURE 8Proposed model. CDX2 interacts with BRG1, the central ATPase subunit of SWI-SNF, recruiting this complex to target loci, altering the local chromatin structure and facilitating transcription of CDX2 target genes via the transcriptional machinery (T.M.).

      Author Contributions

      D. L., T. T. N., L. T. M., and T. B. B. conceived and coordinated the study. T. T. N., T. B. B., R. R., and D. L. wrote the paper. L. T. M., R. R., and T. B. B. designed, performed, and analyzed the experiment shown in Fig. 1. J. G. A. S. designed, performed, and analyzed the experiment shown in Fig. 2. T. B. B. and T. M. designed, performed, and analyzed the experiment shown in Fig. 3. T. M. and T. M. M. designed, performed, and analyzed the experiment shown in Fig. 4. T. T. N. designed, performed, and analyzed the experiments shown in FIGURE 5, FIGURE 6. T. E. F. designed, performed, and analyzed the experiment shown in Fig. 7. B. L. H. and K. J. M. derived performed initial characterization of CRISPR-Cas9 mutant cell lines. All authors reviewed and approved the final version of the manuscript.

      Acknowledgments

      We thank D. Metzger and P. Chambon for the Brg1F/F mouse line, M. Mansfield for mouse husbandry, and the histology core for excellent service.

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