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Broadly permissive intestinal chromatin underlies lateral inhibition and cell plasticity

Cells differentiate when transcription factors (TFs) bind accessible cis-regulatory elements to establish specific gene expression programs. In differentiating embryonic stem (ES) cells, chromatin at lineage-restricted genes becomes sequentially accessible1-4, probably by virtue of “pioneer” TF activity5, but tissues may utilize other strategies in vivo. Lateral inhibition is a pervasive process in which one cell forces a different identity on its neighbors6, 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 signaling mediates lateral inhibition to assign progenitor cells into absorptive or secretory lineages7-9. Transcript profiles in isolated LGR5+ intestinal stem cells (ISC)10 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 DNaseI hypersensitivity - signifying accessible, permissive chromatin - at most of the same cis-elements. Enhancers acting uniquely in progenitors were well-demarcated in LGR5+ ISC, 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 TF, controls lateral inhibition through Delta-like Notch ligand genes and also drives numerous secretory lineage genes. Depletion of ATOH1 from specified secretory cells converted them into functional enterocytes, indicating prolonged responsiveness of marked enhancers to presence or absence of a key TF. Thus, lateral inhibition and intestinal crypt lineage plasticity involve interaction of a lineage-restricted TF with broadly permissive chromatin established in multipotent stem cells.

prominently marked in progenitor cells, but H3K4me2 showed strikingly similar distributions in Sec-Pro and Ent-Pro (Group 1 in Fig. 2a; Extended Data Fig. 5b). Marks were evident at these sites even in LGR5 + ISC, distinct from the Group 2 profile, and aggregate analysis verified significant overlap of H3K4me2 enhancers among crypt populations (Fig. 2b). Sites that seemed superficially better marked in DBZ-derived Sec-Pro (Group 1b) lacked TF motif enrichment or other distinguishing features and Sec-Prorestricted genes were not enriched near these sites but distributed among enhancers marked in all 3 crypt populations ( Fig. 2a and Supplementary Information Tables 1 and 2).
As H3K4 is methylated at both poised and active cis-elements in ES cells 1 , the striking overlap in progenitor cell marks might reflect enhancer potential, distinct from enhancer activity; H3K27ac is thought to mark active enhancers specifically 22,23 . Though many H3K4me2-marked enhancers lacked H3K27ac, global and local distributions of the latter were remarkably similar in Sec-Pro and Ent-Pro ( Fig. 2c-d). DNaseI-hypersensitivity (DHS -an independent, direct measure of chromatin access) 24 was evident at most H3K4me2 sites and its distribution also highly similar in Sec-Pro and Ent-Pro ( Fig. 2c-d), distinct from DHS in other mouse tissues (Extended Data Fig. 5c). Thus, thousands of enhancers for lineagerestricted genes show comparable histone activation and tissue-specific chromatin access in distinct, specified crypt progenitors and most such sites carry H3K4me2 in ISC. This early, indiscriminate delineation of enhancers offers a compelling explanation for crypt cell equipotency, a key feature of lateral inhibition, and reveals that intestinal lineage separation does not require differential chromatin priming in daughter cells and may instead rely principally on TF activity. ATOH1, a Sec-Pro-specific TF, is an ideal candidate to confer selectivity (Extended data Fig. 6a). FLAG ChIP-seq in Sec-Pro isolated from Atoh1 Flag mice 25 identified thousands of binding sites, most of them in highly conserved, distant enhancers carrying ATOH1 consensus motifs (Extended Data Figs. 4b, 6b) and different from ATOH1 binding sites in cerebellar neurons 25 (Fig. 3a). Intestinal and neuronal binding sites coincided with tissuespecific H3K4 methylation (Extended Data Fig. 6c), attesting to the regulatory significance of this histone modification. Lateral inhibition requires Atoh1 repression in Notch-recipient cells and ATOH1 activity in Sec-Pro 12,14 , and the rapidity of ATOH1-induced lineage divergence 9 hints that it may directly regulate genes driving lateral inhibition. Indeed, ATOH1 bound strongly to its own locus and those for Notch ligands Dll1 and Dll4 (Fig. 3b), but not Ent-Pro-specific loci such as Bcl215 (Extended Data Fig. 6d). These findings support a cell-intrinsic, ATOH1-dependent mechanism for lateral inhibition and a simple, direct means for feed-forward maintenance of this bistable system 26 . ATOH1 also bound numerous loci implicated in secretory differentiation, including determinants of each sublineage, and the corresponding transcripts are low or absent in Atoh1 −/− crypts ( Fig. 3b; Extended Data Fig. 6e-f). Strong, specific association of ATOH1 binding with Sec-Prorestricted transcripts further implies that it activates secretory genes without binding or directly repressing ENT genes (Extended Data Fig. 6g).
H3K4me2, H3K27ac, and DHS signals at ATOH1-bound enhancers were, on average, equally strong in ATOH1 + Sec-Pro and Ent-Pro, which categorically lack ATOH1 (Fig. 3c). Few secretory loci showed modestly stronger features in Sec-Pro or GOB (Extended Data Fig. 5d), but most profiles were similar in LGR5 + ISC and both progenitors (Fig. 3d). Although in villus cells ATOH1 preferred sites marked only in GOB over those marked only in ENT, in crypts it overwhelmingly favored enhancers bearing H3K4me2 in all populations (Extended Data Fig. 7). The pervasive demarcation of ATOH1-bound enhancers in Atoh1 −/− Ent-Pro reveals that this TF does not initiate or maintain chromatin access in crypt cells.
The extreme overlap of Sec-Pro and Ent-Pro enhancer profiles indicates persistence of ciselement marks after lineage specification. Intestinal lineages might therefore remain plastic beyond the period of lateral inhibition and sustained chromatin access might explain why some differentiated crypt cells readily assume stem-cell properties 17,27,28 . Our findings particularly suggest that chromatin in specified cells may stay responsive to presence or absence of ATOH1. In Atoh1-null fetal intestines, an Atoh1 LacZ allele is expressed in ENT 14 , but this finding could represent a non-cell-autonomous effect of abortive lateral inhibition. We postulated prolonged, cell-autonomous ATOH1 control of lineage identity in specified cells.
To test this hypothesis, we suppressed Notch in Atoh1 Fl/Fl ;Villin-Cre ER mice and waited past the period of widespread Sec-Pro differentiation before administering tamoxifen to induce Atoh1 deletion; we also injected bromodeoxyuridine (BrdU) to monitor cell replication and turnover (Fig. 4a). Crypts and villus bases in control Atoh1 Fl/Fl ;Cre − mice became filled with non-replicating GOB 3 days after Notch inhibition, as expected ( Fig. 4bd), whereas ATOH1-depleted crypts showed substantial proliferation and many fewer Alcian blue-avid cells ( Fig. 4e-g). To demonstrate that this marked difference from control mice did not represent rescue by a few ISC that might have eluded Notch inhibition but rather secretory cell conversion to ENT, we followed cells for 3 additional days (Fig. 4a). By this time Notch-inhibited control Atoh1 Fl/Fl ;Cre − mice became moribund and had short villi containing only GOB that showed no BrdU labeling ( Fig. 4h-k). Meanwhile, in the absence of Notch suppression, BrdU tracer in wild-type or Atoh1 −/− intestines had moved from crypts into villus tips (Extended Data Fig. 8a-d), as expected. Atoh1 Fl/Fl ;Villin Cre-ER mice remained fit and carried only alkaline phosphatase (ALPI)-expressing ENT on tall villi with abundant BrdU signal at the tips ( Fig. 4l-o). Thus, ATOH1 loss had overcome secretory fate and replication arrest more than 2 days after Notch inhibition to become Ent-Pro that proliferated and matured into functional ENT.
To verify cell-autonomous fate conversion, we used progesterone/RU486-responsive Atoh1 Cre-PR mice 29 . Following RU486 injection, YFP signals in control Atoh1 Cre-PR/+ ;Rosa26 YFP mice were confined to few GOB and enteroendocrine cells, indicating weak but restricted Cre activity; 4 independent mice never labeled ENT or cell stripes, crucially confirming absence of leaky Cre activity in ISC or multipotent progenitors (Extended Data Fig. 8e-f). We suppressed Notch in Atoh1 Cre-PR/Fl; Rosa26 YFP mice, then activated Cre selectively in ATOH1 + secretory cells, thus deleting the floxed Atoh1 allele and simultaneously activating a fluorescent lineage tracer (Fig. 4p). Four days later, stripes of YFP + cells emanated from crypts and 95% ± s.d Mathematical modeling suggests that bifurcating valleys in Waddington's famous epigenetic landscape best represent reversible nodes, a hallmark of lateral inhibition 26 . The broad priming of ISC and progenitor cell enhancers offers a satisfying explanation for this property in intestinal crypts and accounts for ATOH1 activity in both lateral inhibition and secretory differentiation. ATOH1 reinforces its own expression and directly regulates Dll1 and Dll4, thus enabling Notch-dependent Atoh1 repression in neighboring cells; its subsequent activity at primed cis-elements triggers numerous secretory genes. Sustained enhancer priming allows ATOH1 activity or its absence, which respectively promotes or represses secretory differentiation 13 , to affect the fate of specified cells, and DNA methylation also barely changes as ISC differentiate 30 . True lineage commitment thus occurs late, if ever, in the intestinal epithelium. Indeed, various stimuli revert differentiated cells into ISC with surprising ease 17,27,28 , implying that the environment, and not some cell-intrinsic feature, determines crypt cell potential. Broadly delineated enhancers likely underlie this remarkable plasticity.

Animals and drug administration
Mouse strains were purchased from The Jackson Laboratory or received as gifts and are referenced in Extended Data Table 1. Mice, maintained under specific pathogen-free conditions, were handled in accordance with protocols approved and monitored by the Dana-Farber Cancer Institute Animal Care and Use Committee. Male and female C57BL/6 mice were used between 2 and 6 months of age. Dibenzazepine (DBZ, Syncom, Groningen, The Netherlands) was suspended in 0.5% (w/v) hydroxypropylmethyl-cellulose (Methocel E4M, Dow Chemicals) and 0.1% (w/v) Tween 80 (Bio-Rad) in water and 100 μmol/kg was administered by intraperitoneal (IP) injection. We activated Cre-ER recombinase by IP injection of 1 mg tamoxifen (Invitrogen) for 5 days or one dose of 4 mg tamoxifen. Cre-PR recombinase was induced by 2 IP injections of 2 mg RU486 (Sigma) approximately 8 h apart. BrdU (B-D Biosciences, 50 mg/kg) was injected IP and mice were sacrificed 1 h or 3 days later.

Cell isolation
Epigenomes were characterized in cells isolated from the first half of the small intestine.
LGR5 + ISC: Intestinal villi in Lgr5 GFP-IRES-Cre mice 10 were scraped away using glass slides, crypt epithelium was collected by shaking in 5 mM EDTA for 50 min at 4°C, and single-cell suspensions prepared by digestion in 4X TrypLE (Invitrogen) for 50 min at 37°C. GFP hi (LGR5 + ) cells were sorted using a MoFlo instrument (Beckman Coulter). Sec-Pro and GOB: Villi were scraped from intestines of wild-type mice 38 h after intraperitoneal (IP) injection of DBZ (Syncom, 100 μmol/kg i.p.) and crypt cells isolated by shaking residual, crypt-enriched tissue in 5 mM EDTA for 50 min at 4°C. Rbpj Fl/Fl ;Villin-Cre ER mice were injected with 2 daily IP doses of 2 mg tamoxifen, followed by isolation of crypt Sec-Pro as above on the third day, or with 5 daily doses of 1 mg tamoxifen, followed by isolation of villus GOB on the 10 th day. The latter were collected by shaking intestines gently in 5 mM EDTA for 30 min at 4°C, followed by passage through 70 μm filters to eliminate contaminating crypts. Ent-Pro and ENT: Crypt epithelial cells and mature ENT were collected from Atoh1 Fl/Fl ;Villin-Cre ER intestines 14 days after tamoxifen treatment, as described for other mice.

ChIP-Seq and DHS-Seq analyses
Sequence tags were aligned to the Mus musculus genome, build mm9 and uniquely mapped, non-redundant reads were retained 34,35 . Genomic profiles of DNaseI hypersensitivity (DHS) and histone marks or ATOH1 binding sites were generated using Model-Based Analysis of ChIP-seq 2 (MACS2), with a q-value (false discovery rate, FDR) threshold of 0.01 and default parameters 36,37 . Peaks were refined by requiring >5-fold enrichment in comparisons with input DNA for ChIP-seq and local backgrounds for DHS-seq. For analyses centered on ChIP peaks, non-promoter (>2 kb upstream or <1 kb downstream from any TSS) peaks from all study samples were merged to create a union set. Read densities (in reads per 1M tags within fixed, 200-bp bins, RPM) were calculated by normalizing counts in each peak region in the union set based on sample sequencing depth. Meta-analyses around ATOH1 binding sites were performed in the Cistrome pipeline 38 . Genomic wiggle traces were normalized for sample sequence depth. Similarities among H3K4me2-marked enhancers in different cell types were determined by hierarchal clustering using Spearman's rank correlation.

Expression analyses and correlation with Atoh1 occupancy
RNA was extracted from LGR5 + ISC, Sec-Pro, Ent-Pro, GOB and ENT with Trizol (Invitrogen) and RNeasy Mini Kits (Qiagen), processed, and hybridized to Mouse Genome 430A 2.0 microarrays (Affymetrix). Data quality was verified using Affy QC Report in R bio-conductor. mRNA profiles were analyzed by RMA to normalize expression indices 39 , and differential expression across cell types was identified using LIMMA 40 by F test with p <0.002. Values in the expression heatmap (Extended Data Fig. 3) were normalized by magnitude normalization and cell-type specificity was determined by k-means clustering of differentially expressed genes. ATOH1 binding at enhancers was used as a function to determine if ATOH1 activates Sec-Pro-specific genes. Each ATOH1 enhancer (>2 kb upstream or <1 kb downstream from a TSS) was assigned to a nearby gene (TSS <20 kb from an ATOH1 summit). After normalization of tags for sequence depth and peak width, the sum of tag densities for each gene was used to measure its correlation with differential expression.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.