Genome-Wide Pattern of TCF7L2/TCF4 Chromatin Occupancy in Colorectal Cancer Cells

Abstract

Physiological Wnt signaling is required for the maintenance of the crypt progenitor phenotype and controls the proliferation/differentiation switch in the adult, self-renewing intestinal epithelium (33). A constitutively active Tcf/␤-catenin transcription complex, resulting from mutations in adenomatous polyposis coli (APC), Axin, or ␤-catenin, is the primary transforming factor in colorectal cancer (CRC) (25,26,32); aberrant Tcf/␤-catenin activity results in a transcriptional profile in CRC cells similar to that which is physiologically driven by Tcf/␤-catenin in the crypt stem/progenitor cells of the intestine (49). Through candidate gene approaches and microarray technology, a large number of genes have been uncovered whose expression levels are altered upon abrogation or activation of the Wnt pathway (for references, see http://www .stanford.edu/ϳrnusse/pathways/targets.html). It remains unclear whether the affected genes are direct or indirect targets of the Tcf/␤-catenin transcription factor complex. cis-regulatory elements directly bound by Tcf have been identified for only a few candidate genes. Such studies have been mostly limited to regulatory regions close to the transcription start site (TSS) of candidate genes (e.g., see reference 17). A comprehensive identification of regulatory elements is essential for a more complete understanding of the transcriptional repertoire driven by the Wnt pathway and the elucidation of the molecular mechanisms by which Tcf and ␤-catenin control the transcription of their target genes.A recent approach taken to achieve such goals is chromatin immunoprecipitation (ChIP)-coupled DNA microarray analysis (ChIP-on-chip), which couples the immunoprecipitation of chromatin-bound transcription factors with the identification of the bound DNA sequences through hybridization on DNA microarrays (35). This approach has been used to generate, among others, a comprehensive map of active, preinitiation complex-bound promoters in human fibroblast cells (24). Microarrays covering the nonrepetitive sequence of chromosomes 21 and 22 have allowed the study of histone H3 methylation and acetylation patterns in human hepatoma cells (5) and estrogen receptor binding sites in breast cancer cells (8). The latter study revealed selective binding of estrogen receptor (ER) to a limited number of sites, most of which were distant from the TSSs of ER-regulated genes (8). Similar conclusions were put forth by work examining the in vivo binding of transcription factors Sp1, c-Myc, and p53 along chromosomes 21 and 22: most binding sites identified do not correspond to the proximal promoters of protein-coding genes but rather lie within or immediately 3Ј to well-characterized genes or are significantly correlated with noncoding RNAs (10). Collectively these studies point to the necessity of interrogating entire genomes for the comprehensive determination of in vivo-occupied binding sites (9,23,52,54).In the present work, we used a combination of ChIP and location analysis with genome-wide tiling arrays...

Attenuation of the beta-catenin/TCF4 complex in colorectal cancer cells induces several growth-suppressive microRNAs that target cancer promoting genes

“…Correlation between TCF4 chromatin occupancy and dnTCF4-responsive miRNAs We next asked if there was a correlation between dnTCF4-induced miRNA changes and chromatin occupancy of beta-catenin/TCF4 complexes as inferred from published ChIP-chip data obtained in LS174T CRC cells and estimated to share 96% overlap in DLD1 cells (Hatzis et al, 2008). Because the architecture of most miRNA transcriptional units is still unknown and TCF4s often seem to work by symmetrically located distal ( ± 10 to 100 kb) enhancer elements (Hatzis et al, 2008), we searched for experimentally validated TCF4 binding sites (n ¼ 6868) within a 200 kb window centered on the miRNA hairpin ( Figure 1c; Supplementary Table S3).…”

“…Because the architecture of most miRNA transcriptional units is still unknown and TCF4s often seem to work by symmetrically located distal ( ± 10 to 100 kb) enhancer elements (Hatzis et al, 2008), we searched for experimentally validated TCF4 binding sites (n ¼ 6868) within a 200 kb window centered on the miRNA hairpin ( Figure 1c; Supplementary Table S3). In all, 35% (242 out of 701) of all hairpins was located within 100 kb of at least one TCF4 site, and 5% (38 out of 701) near multiple (at least three) TCF4 sites (Figure 1d), which is a hallmark of many protein-coding genes directly regulated by beta-catenin/TCF4 (Hatzis et al, 2008). Of the 60 dnTCF4-responsive miRNAs, 29 miRNAs (encoded by 28 hairpins) were in proximity of at least one TCF4 site, including miR-30e-3p (also known as miR-30e*), the promoter of which is regulated by beta-catenin/TCF4 complexes (Liao and Lonnerdal, 2010).…”

“…(b) Expression fold changes of dnTCF4-responsive miRNAs and overlapping host genes exhibiting at least twofold change (log2 scale). (c, d) Schematic representation and results of search strategy for miRNA hairpins within 100 kb of TCF4-bound chromatin regions originally identified using ChIP-chip by Hatzis (Hatzis et al, 2008). (e) Schematic representation of TCF4-bound chromatin regions near selected miRNA hairpins.…”

“…¼ positive control region; enhancer region located at 3 0 of the MYC gene and previously reported to be bound by TCF4 (Hatzis et al, 2008;Yochum et al, 2008). Myo ex2 ¼ negative control region; exon 2 of the myoglobin gene not bound by TCF4 (Hatzis et al, 2008). The CD58 antibody was used as a negative control antibody.…”

The ubiquitin-like modifier FAT10 in cancer development

“…4). As a result, the expression of Homeobox B9 (HOXB9), a member of the class I homeobox (HOX) genes and a target of ␤-catenin/TCF4 (Hatzis et al, 2008), was found to be highly upregulated in HCC and the HOXB9 mRNA and protein expression levels correlated with the mRNA and protein expression levels of FAT10. HOX genes are normally involved in embryogenesis but a role in cancer development has been proposed (Abate-Shen, 2002).…”