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. 2008 Apr;28(8):2732-44.
doi: 10.1128/MCB.02175-07. Epub 2008 Feb 11.

Genome-wide pattern of TCF7L2/TCF4 chromatin occupancy in colorectal cancer cells

Pantelis Hatzis  1 , Laurens G van der Flier, Marc A van Driel, Victor Guryev, Fiona Nielsen, Sergei Denissov, Isaäc J Nijman, Jan Koster, Evan E Santo, Willem Welboren, Rogier Versteeg, Edwin Cuppen, Marc van de Wetering, Hans Clevers, Hendrik G Stunnenberg
Affiliations

Genome-wide pattern of TCF7L2/TCF4 chromatin occupancy in colorectal cancer cells

Pantelis Hatzis et al. Mol Cell Biol. 2008 Apr.

Abstract

Wnt signaling activates gene expression through the induced formation of complexes between DNA-binding T-cell factors (TCFs) and the transcriptional coactivator beta-catenin. In colorectal cancer, activating Wnt pathway mutations transform epithelial cells through the inappropriate activation of a TCF7L2/TCF4 target gene program. Through a DNA array-based genome-wide analysis of TCF4 chromatin occupancy, we have identified 6,868 high-confidence TCF4-binding sites in the LS174T colorectal cancer cell line. Most TCF4-binding sites are located at large distances from transcription start sites, while target genes are frequently "decorated" by multiple binding sites. Motif discovery algorithms define the in vivo-occupied TCF4-binding site as evolutionarily conserved A-C/G-A/T-T-C-A-A-A-G motifs. The TCF4-binding regions significantly correlate with Wnt-responsive gene expression profiles derived from primary human adenomas and often behave as beta-catenin/TCF4-dependent enhancers in transient reporter assays.

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Figures

FIG. 1.
FIG. 1.
ChIPs over regions bound by TCF4 and genomic distribution of TCF4-bound regions. (a) Association of TCF4 with the proximal promoters of SP5 and c-Myc was determined by single (light blue) or sequential (dark blue) ChIP followed by qPCR and expressed as relative enrichment over the nonbound exon 2 of the myoglobin gene. Error bars represent standard deviations for three independent experiments. (b) Schematic illustration delineating the criteria for binding-site classification with respect to a gene locus. (c) Localization of TCF4-binding sites in relation to annotation to nearest transcription units. Shown are percentages of binding sites in the different location categories as defined in panel b. (d) Distribution in categories, defined as in panel b, of TCF4-bound regions (light blue) or random genomic regions (dark blue). Error bars represent standard deviations of 100 random groups. (e) Distribution in 100-bp intervals of TCF4-bound regions located within 10 kb of annotated TSSs. (f) Venn diagrams depicting the number of TCF4-bound regions within 1 kb of CpG islands, annotated transcription start sites of protein-coding genes, or both (top) and the number of TCF4-bound regions within 1 kb of CAGE tags, annotated transcription start sites of protein-coding genes, or both (bottom). (g) Distribution, in categories defined as in panel f, of TCF4-bound regions (light blue) or random genomic regions (dark blue). Error bars represent standard deviations of 250 random groups.
FIG. 2.
FIG. 2.
TCF4-binding-site clustering around target genes. (a) TCF4-binding-site distribution around the AXIN2 gene. Depicted is the binding pattern of TCF4 around AXIN2 as revealed by the genome-wide experiment and the three independent biological replicates on the dedicated array, including the mean and variance tracks from the three replicates. High-confidence peaks are highlighted in magenta and numbered 1 to 11, low-confidence peaks in light blue. (b) Numbers of genes bound within 100 kb of their TSSs by three, four, or five or more TCF4-binding sites (light blue) or random genomic regions (dark blue). Error bars represent standard deviations of 100 random groups.
FIG. 3.
FIG. 3.
(a) Sequence logos illustrating the nucleotide distribution for the in vivo TCF4 consensus sites of 7, 11, and 15 bp, as defined by MDscan. (b) Number of TCF4-bound (light blue) or random genomic (dark blue) regions containing the indicated TCF4-binding motif, as depicted in panel a. Error bars represent standard deviations of 100 random groups. (c) Percent identities of TCF4-bound regions (light blue), random genomic regions (dark blue), and the 7-mer TCF4-binding motif (red, as depicted in panel a) for mouse-human and rat-human pairwise genomic comparisons. Error bars represent standard deviations of 100 random groups.
FIG. 4.
FIG. 4.
Correlation of TCF4 binding and TCF4/β-catenin-controlled gene expression. Differential expression rank analysis for genes bound within 100 kb of TSS by TCF4 or random groups from genes upregulated in human primary adenomas, using a step size of 100. Error bars represent standard deviations of 100 random groups.
FIG. 5.
FIG. 5.
Transcriptional activity of TCF4-bound regions in CRC cells. (a) TCF4-binding regions were cloned into the pGL3b or pGL3/AdMLTATA vector, in the case of TSS-proximal or non-TSS-proximal regions, respectively, and transfected into Ls174T with cotransfection of the CMV-Renilla vector as the normalizing control and with or without cotransfection of ΔNTCF4. Values are expressed as activity relative to that of the respective empty pGL3 vectors. Error bars represent standard deviations for three independent experiments. (b) Eleven TCF4-binding regions surrounding the AXIN2 gene within 100 kb of the TSS were cloned into the pGL4.10 or pGL4.10/TATA vector, in the case of TSS-proximal or non-TSS-proximal regions, respectively, and transfected into LS174T/ΔNTCF4 cells with cotransfection of the CMV-Renilla vector as the normalizing control and with or without doxycycline treatment to induce ΔNTCF4 expression. Values are expressed as activity relative to that of the respective empty pGL4.10 vectors. Error bars represent standard deviations for three independent experiments.
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