Fig 1: Conceptual approach and gene expression dynamics following TCF7L2 silencing. (A) Schematic summary of the experimental approach. We sought to explore how silencing TCF7L2 impacted the colon cancer gene network in terms of chromatin structure and gene expression. (B) qPCR demonstrated progressive silencing of TCF7L2 over time with a 75% reduction in TCF7L2 transcript levels after 72 hours. Each dot represents a biological replicate, with 3 replicates plotted per time point (n = 3). The green line represents the mean, while the green-shaded ribbon represents the standard deviation. (C) Western blot analysis demonstrated a 70% decrease in TCF4 protein abundance by the last time point with ß-actin as loading control. (D) PCA of gene expression (TPM) over the time course, with time points differentiated by color (n = 2). (E) Volcano plots show differential gene expression genome-wide with thresholds set at log2FC = 2, and P = 0.05 (equivalent to 1.3 on the –log10P scale). Genes in red are significantly down-regulated and undergo a >4-fold decrease in expression, while those in green are significantly up-regulated and experience a >4-fold increase in expression. The number of genes which fall within these regions are shown. (F) Expression profile of SOX2, which increases dramatically across the time series. The dots correspond to each biological replicate (n = 2), the shaded ribbon represents the standard deviation. (G) Western blot for active ß-catenin over time with ß-actin as loading control. No noticeable change in active ß-catenin protein levels occurs during the time series. (Color version of figure is available online.)
Fig 2: Changes in global and local Hi-C partitioning over time. (A) Algebraic difference between genome-wide Hi-C contact maps at 0 and 72 hours demonstrates an overall increase in contact frequency over time. (B) Global partitioning (A/B compartmentalization) dynamics revealed that 1091 100-kb genomic bins switched compartments at one or more time points genome-wide. The Fiedler number (see Methods) of each loci are k-means clustered into 8 groups (denoted to the left) exhibiting different switching dynamics. The number of genomic bins in each cluster is specified on the right. A 95% change in Fiedler number from one time point to another was set as a threshold to remove inherent noise. (C) Local partitioning (TAD organization) of the region on Chromosome 19 (34.6 - 45.2 Mb) containing two CEACAM gene groups. Hi-C contact maps are shown at 100kb resolution with TAD domains at 0h denoted by solid, black lines and at 72h denoted by dashed, black lines. The CEACAM gene groups are denoted by blue lines in the expression array. ChIPseq data for TCF4 and SP1 binding demonstrated that TCF4 and SP1 may bind within the TAD boundary region. (D) qPCR was performed on TCF7L2 silenced cells at the 72-hour time point to determine the expression of CEACAM1 in various colon cancer cell lines. Expression of TCF7L2 and CEACAM1 was normalized to the negative control (Time 0 - not plotted), which was uniformly set to 1 for each cell line. All cell lines tested demonstrated an up-regulation of CEACAM1, however the weakest response was observed in COLO201. (E) Diagram illustrating coupled chromosome structure and gene expression between two conditions as well as a possible explanation for this coupling, i.e., condition specific transcription factor activity. (F) Transcription factor enrichment analysis for the 64 genes found in the differentially conformed region of chromosome 19 (see 2C) showed enrichment for SP1, KLF4, ZFX, and MZF1 when compared against a background of 24,752 genes.
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