Differentiation-Specific Histone Modifications Reveal Dynamic Chromatin Interactions and Partners for the Intestinal Transcription Factor CDX2

Abstract

SUMMARY Cell differentiation requires remodeling of tissue-specific gene loci and activities of key transcriptional regulators, which are recognized for their dominant control over cellular programs. Using epigenomic methods, we characterized enhancer elements specifically modified in differentiating intestinal epithelial cells and found enrichment of transcription factor-binding motifs corresponding to CDX2, a critical regulator of the intestine. Directed investigation revealed surprising lability in CDX2 occupancy of the genome, with redistribution from hundreds of sites occupied only in proliferating cells to thousands of new sites in differentiated cells. Knockout mice confirmed distinct Cdx2 requirements in dividing and mature adult intestinal cells, including responsibility for the active enhancer configuration associated with maturity. Dynamic CDX2 occupancy corresponds with condition-specific gene expression and, importantly, to differential co-occupancy with other tissue-restricted transcription factors such as GATA6 and HNF4A. These results reveal dynamic, context-specific functions and mechanisms of a prominent transcriptional regulator within a cell lineage.

Chromatin Profiling Reveals Regulatory Network Shifts and a Protective Role for Hepatocyte Nuclear Factor 4α during Colitis

“…The intestinal epithelium is an integral component of inflammatory bowel disease, but the impact of acute inflammation on the transcriptional regulatory networks of the colonic epithelium is uncharacterized. The NSD methodology score was developed and has been applied to detect changes in enhancer chromatin structure and to discover new transcriptional regulatory networks (8,9,14). The NSD scoring algorithm identifies changes in regulatory chromatin by accounting for two properties of transcriptional enhancers: DNA accessibility where transcription factors bind and flanking nucleosomes with elevated levels of posttranslational modifications that are associated with active transcriptional enhancers (H3K27ac, H3K4me2, and H3K4me1) (8,9,17).…”

“…Nucleosomes containing histone 3, lysine 27 acetylation (H3K27ac) can be used to identify regions that have distal regulatory activity (transcriptional enhancers), flank chromatin-accessible transcription factor binding regions, and are predictive of active transcription in a conditionspecific manner (4,6,7). Changes in H3K27ac levels and DNA accessibility predict changes in transcription factor occupancy (8,9); dynamic enhancer chromatin structures can thus serve as a discovery tool to identify shifts in transcription factor regulatory networks induced by disease.…”

“…H3K27ac histone ChIPs were done as described previously (9,14). Isolated colon epithelial cells were first permeabilized with digestion buffer (50 mM Tris-HCl [pH7.6], 1 mM CaCl 2 , 0.2% Triton X-100, 5 mM Na-butyrate, 1ϫ protease inhibitor cocktail, 0.5 mM PMSF) for 5 min on ice and then incubated with 0.2 to 0.4 unit of micrococcal nuclease (MNase; Sigma) until most of the genome was reduced to mononucleosome length (ϳ146 bp).…”

Differential Effects of Hepatocyte Nuclear Factor 4α Isoforms on Tumor Growth and T-Cell Factor 4/AP-1 Interactions in Human Colorectal Cancer Cells

“…In Fig. 7, another custom-designed array was ordered from Agilent (SurePrint G3 Custom GE 1X1M), which contained an ϳ60-base oligonucleotide corresponding to sequences within 100 bp of the center of published HNF4␣ ChIP-seq peaks from HepG2 and CaCo2 cells (52,53). A total of ϳ125,000 loci, including SNP alleles from dbSNP version 132, were spotted in quadruplicate (ϳ125,000 loci ϫ ϳ2 alleles ϫ 4 replicates ϭ 1 million spots of DNA) on the slide as singlestranded DNA.…”

“…To identify additional binding motifs shared by HNF4␣ and TCFs, we designed a second PBM that contained ϳ1 million spots of DNA corresponding to 250,000 sequences (in four replicates) that we mined from published HNF4␣ ChIP-seq data from a human liver cancer cell line, HepG2 (52), which expresses predominantly P1-HNF4␣, and the colon cancer line, CaCO2 (53), which expresses predominantly P2-HNF4␣ (17). We found 741 DNA sequences bound by both TCF4 and HNF4␣8, the majority of which also bound HNF4␣2 (Fig.…”