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Broadly permissive intestinal chromatin underlies lateral inhibition and cell plasticity

Nature volume 506, pages 511–515 (2014)Cite this article

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Abstract

Cells differentiate when transcription factors bind accessible cis-regulatory elements to establish specific gene expression programs. In differentiating embryonic stem cells, chromatin at lineage-restricted genes becomes sequentially accessible1,2,3,4, probably by means of ‘pioneer’ transcription factor activity5, but tissues may use other strategies in vivo. Lateral inhibition is a pervasive process in which one cell forces a different identity on its neighbours6, and it is unclear how chromatin in equipotent progenitors undergoing lateral inhibition quickly enables distinct, transiently reversible cell fates. Here we report the chromatin and transcriptional underpinnings of differentiation in mouse small intestine crypts, where notch signalling mediates lateral inhibition to assign progenitor cells into absorptive or secretory lineages7,8,9. Transcript profiles in isolated LGR5+ intestinal stem cells10 and secretory and absorptive progenitors indicated that each cell population was distinct and the progenitors specified. Nevertheless, secretory and absorptive progenitors showed comparable levels of H3K4me2 and H3K27ac histone marks and DNase I hypersensitivity—signifying accessible, permissive chromatin—at most of the same cis-elements. Enhancers acting uniquely in progenitors were well demarcated in LGR5+ intestinal stem cells, revealing early priming of chromatin for divergent transcriptional programs, and retained active marks well after lineages were specified. On this chromatin background, ATOH1, a secretory-specific transcription factor, controls lateral inhibition through delta-like notch ligand genes and also drives the expression of numerous secretory lineage genes. Depletion of ATOH1 from specified secretory cells converted them into functional enterocytes, indicating prolonged responsiveness of marked enhancers to the presence or absence of a key transcription factor. Thus, lateral inhibition and intestinal crypt lineage plasticity involve interaction of a lineage-restricted transcription factor with broadly permissive chromatin established in multipotent stem cells.

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Figure 1: Enrichment of Sec-Pro and Ent-Pro cells.
Figure 2: Broadly permissive chromatin in intestinal crypt progenitors.
Figure 3: ATOH1 binding and chromatin interactions.
Figure 4: Conversion of specified secretory cells to enterocytes.

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Gene Expression Omnibus

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Data are deposited in the Gene Expression Omnibus under accession number GSE51464.

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Acknowledgements

This work was supported by NIH grants R01DK082889 and R01DK081113 (R.A.S.), K99DK095983 (T.-H.K.), K01DK088868 (M.V.), and P50CA127003; a North American Neuroendocrine Tumor Society fellowship (T.-H.K.); and the Caring For Carcinoid Foundation (R.A.S.). We thank S. Robine for villin-CreER-T2 mice; T. Honjo and S. Artavanis-Tsakonas for Rbpjfl mice; J. Johnson for ATOH1 antibody; D. Podolsky for TFF3 antibody; and M. Brown and P. Cejas for discussions.

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Authors and Affiliations

  1. Department of Medical Oncology and Center for Functional Cancer Epigenetics, Dana-Farber Cancer Institute, Boston, 02215, Massachusetts, USA

    Tae-Hee Kim, Fugen Li, Isabel Ferreiro-Neira, Li-Lun Ho, Annouck Luyten, Kodandaramireddy Nalapareddy, Henry Long, Michael Verzi & Ramesh A. Shivdasani

  2. Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, 02215, Massachusetts, USA

    Tae-Hee Kim, Isabel Ferreiro-Neira, Li-Lun Ho, Annouck Luyten, Kodandaramireddy Nalapareddy, Michael Verzi & Ramesh A. Shivdasani

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Contributions

T.-H.K. conceived and designed experiments, collected and analysed data, and drafted the manuscript; F.L. performed computational analyses; I.F.-N. performed some mouse experiments; L.-L.H, A.L., K.N. and M.V. prepared some ChIP-seq libraries; H.L. supervised computational analyses; R.A.S. conceived and supervised the study, analysed data and wrote the manuscript.

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Correspondence to Ramesh A. Shivdasani.

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Extended data figures and tables

Extended Data Figure 1 Isolation of intestinal stem cells and other intestinal cells.

a, Crypt-villus unit structure and components of crypt lateral inhibition. Terminally differentiated enterocytes and secretory cells line intestinal villi and depend on self-renewing crypt populations of LGR5+ intestinal stem cells, bipotential (Bi-Pro), and specified secretory (Sec-Pro) and enterocyte (Ent-Pro) progenitors. Stochastic ATOH1 expression in one Bi-Pro induces secretory differentiation and delta ligand expression, allowing notch signal delivery to neighbouring cells, which use HES1 to repress Atoh1 and become enterocytes. b, Intestinal epithelial cell isolation strategies. LGR5+ intestinal stem cells were isolated from Lgr5GFP-creER mice10; Sec-Pro and goblet cells were isolated from Rbpj−/− intestines7; additional Sec-Pro cells were collected from DBZ-treated wild-type mouse crypts; Ent-Pro and enterocytes were collected from Atoh1−/− intestines14. c, Crypt cells expressing high LGR5/GFP were isolated by flow cytometry and pooled from 12 mice. Gates to distinguish GFPhi intestinal stem cells from their GFPlo and GFP− progeny are shown in the representative scatter plot. d, e, Alcian blue staining of Atoh1−/− intestines at low (d) and high (e) magnification, demonstrating global absence of goblet cells. Inset in e shows the boxed region at higher magnification. f, Rbpjfl/fl;villin-creER intestines rendered null for notch activity 6 days after tamoxifen injection, showing severe goblet cell metaplasia, comparable to DBZ-treated wild-type intestines (See Extended Data Fig. 2d). Scale bars, 50 µm. Duplicate samples were examined from three (Rbpj−/−) or seven (Atoh1−/−) mice.

Extended Data Figure 2 Enrichment of Sec-Pro by chemical inhibition of notch signalling.

a, Quantitative RT–PCR data showing peak Atoh1 expression ∼38 h after treatment of wild-type mice with dibenzazepine (DBZ). b–d, Low magnification views show no increase in mature, Alcian-blue-avid goblet cells 38 h after DBZ treatment (b), appearance of goblet cells in villus bases and crypts 72 h after DBZ treatment (c), and migration of these cells to occupy villi almost fully within 6 days (d). Inset in b shows a magnified view of the boxed area. e, Within 72 h of DBZ treatment, crypt cells virtually cease to replicate (E), retain ATOH1 (C), and stain with Alcian blue (A), a sign of goblet cell maturation. By 120 h after DBZ exposure, these ATOH1+ post-mitotic goblet cells occupy short villi (B, D, F). Scale bars, 50 μm. Tissue analyses were done in duplicate on seven (DBZ+38 h) or three (DBZ+72 h, DBZ+144 h) mice.

Extended Data Figure 3 Distinct expression profiles in LGR5+ intestinal stem cells, Sec-Pro, Ent-Pro, and villus enterocytes.

Differential transcripts in purified intestinal cell types, arranged by k-means clustering of normalized expression values from Affymetrix microarrays. Representative known genes in each cell type are listed alongside the Gene Ontology functions most enriched in each group (false discovery rates in parentheses), determined using Gene Set Enrichment Analysis tools (http://www.broadinstitute.org/gsea/msigdb/annotate.jsp).

Extended Data Figure 4 Summary of ChIP-seq and DHS-seq experiments.

a, Summary of sites identified by MACS II in ChIP-seq and DHS-seq data from intestinal cell populations. b, Distribution of sites marked in promoters (≤2 kb upstream or ≤1 kb downstream of any TSS), coding regions, introns and intergenic regions. c, Correlation analysis with Spearman’s rank coefficient (left) and sample traces (right) of H3K4me2-marked enhancers in independent ChIP-seq replicates of Ent-Pro, showing very high concordance. d, H3K4me2 ChIP-seq profiles at arbitrarily selected regions of mouse chromosome 17 illustrate the highly concordant signals in Sec-Pro produced by Rbpj loss (top traces) or DBZ treatment (bottom traces), as heat maps and clustering analysis (Fig. 2a, b) demonstrate throughout the genome.

Extended Data Figure 5 Accessible, marked enhancers in intestinal crypt progenitors.

a, b, ChIP-seq and DHS-seq traces at sample secretory loci illustrate selective intron marking in goblet cells at Tpsg1 (a), representing the profiles in group 2 enhancers (Fig. 2a), and highly similar distribution of H3K4me2, H3K27ac and DHS signals (within dotted, asterisked lines) across cell populations at Foxa1 (b), representing the prevalent group 1 pattern. c, Heat maps of DHS at 116,324 to 139,312 enhancers showing accessible chromatin in Sec-Pro and Ent-Pro cells, compared to DNase I insensitivity at the same sites in adipocytes, cardiac and skeletal muscle (data from the ENCODE Project Consortium41), and lack of chromatin access in crypt progenitors at enhancers that are active in these heterologous cells. d, At a few secretory loci, such as Spdef, signs of enhancer activity and access are higher in Rbpj−/− Sec-Pro or goblet cells than in other intestinal cells. Asterisked intervals mark ATOH1 binding regions.

Extended Data Figure 6 ATOH1 transcriptional control of secretory differentiation.

a, Model for cell-specific ATOH1 binding to permissive chromatin in crypt progenitors. b, Phastcons plot showing high evolutionary conservation of ATOH1-occupied sites in Sec-Pro cells and maximal enrichment of the consensus ATOH1 recognition motif near their peaks. c, Composite profiles of average H3K4me density near ATOH1 binding sites in Sec-Pro cells (left) and mouse brain (right). H3K4me marks are highly correlated with ATOH1 occupancy in the same, but not in the heterologous, tissue, as representative ChIP-seq data illustrate below. d, e, Examples of ATOH1 occupancy at loci for key secretory lineage transcription factors and secretory-specific genes: Nkx2-2 and Foxa2 (enteroendocrine cells), Xbp1 (Paneth cells), Tff3 (goblet cells), Spink4 and Hgfac (goblet and Paneth cells) and Sstr1 (endocrine cells). ATOH1 does not bind near Ent-Pro-specific genes, as shown at a sample locus, Bcl2l15. f, Schematic representation of ATOH1’s dual function in direct control of two gene groups: delta ligands for lateral inhibition and key secretory-specific transcription factors to promote secretory lineage differentiation. g, Relation of ATOH1 occupancy at enhancers with Sec-Pro-specific transcription. Bins of 200 genes, ranked by differential expression in Sec-Pro (left) or Ent-Pro (right), are represented along the x axis, and normalized ATOH1 binding in Sec-Pro (red) or brain (blue) at enhancers within 20 kb of the genes in each bin is represented on the y axis. The graphs show the first (Sec-Pro-specific), middle (no differential expression), and last (Ent-Pro-specific) three bins of 200 genes each. Error bars indicate standard errors of the mean and pair-wise t-tests support a role for ATOH1 in activating Sec-Pro genes, without an overt role in silencing Ent-Pro genes.

Extended Data Figure 7 ATOH1 binding at H3K4me2-marked enhancers.

Representation of ATOH1 binding sites in relation to the heat map of H3K4me2 marking at 57,481 non-promoter sites (from Fig. 2a), showing that ATOH1 occupies an abundance of sites marked in all crypt populations. Only in mature villus cells does ATOH1 preferentially bind enhancers that are selectively marked in goblet cells.

Extended Data Figure 8 ATOH1-dependent lineage conversion.

a–d, BrdU tracing 1 and 3 days after label injection, showing similar turnover of intestinal epithelial cells in wild-type and Atoh1-null intestines in the absence of Notch inhibition. Nearly all the label is in the villus base at 1 day and in the top halves of villi at 3 days. e, Lineage tracing 3 days after induction of Cre activity by tamoxifen in Lgr5cre-ER;Rosa26YFP mice (left) or by RU486 in Atoh1cre-PR;Rosa26YFP mice (right). YFP is detected in numerous whole crypts (except Paneth cells) in the former, and never in crypts, ribbons or cell pairs in the latter, confirming absence of leaky CrePR expression in multipotent progenitors. f, YFP signal in Atoh1cre-PR;Rosa26YFP mice 3 days after RU486-induced Cre activation is confined to scattered ChgA+ enteroendocrine and Muc2+ goblet cells. g, Two additional representative examples of YFP-traced cell groups that had converted into TFF3-negative enterocytes after Atoh1 deletion in secretory cells. Arrows indicate rare (∼5%, see text) YFP+ cells that still express TFF3. Scale bars, 50 μm. Tissues were analysed in duplicate from four (WT, Atoh1−/−, Atohcre-PR/+;Rosa26YFP) or six (Lgr5cre-ER;Rosa26YFP) mice.

Extended Data Table 1 Mouse strains used in this study
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Supplementary information

Supplementary Table 1

This file shows transcripts enriched in Sec-Pro and Ent-Pro. (XLSX 58 kb)

Supplementary Table 2

This file shows genes and TF motifs associated with cell type-specific enhancers. (XLSX 567 kb)

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Kim, TH., Li, F., Ferreiro-Neira, I. et al. Broadly permissive intestinal chromatin underlies lateral inhibition and cell plasticity. Nature 506, 511–515 (2014). https://doi.org/10.1038/nature12903

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