To characterize somatic alterations in colorectal carcinoma, we conducted a genome-scale analysis of 276 samples, analysing exome sequence, DNA copy number, promoter methylation and messenger RNA and microRNA expression. A subset of these samples (97) underwent low-depth-of-coverage whole-genome sequencing. In total, 16% of colorectal carcinomas were found to be hypermutated: three-quarters of these had the expected high microsatellite instability, usually with hypermethylation and MLH1 silencing, and one-quarter had somatic mismatch-repair gene and polymerase e (POLE) mutations. Excluding the hypermutated cancers, colon and rectum cancers were found to have considerably similar patterns of genomic alteration. Twenty-four genes were significantly mutated, and in addition to the expected APC, TP53, SMAD4, PIK3CA and KRAS mutations, we found frequent mutations in ARID1A, SOX9 and FAM123B. Recurrent copy-number alterations include potentially drug-targetable amplifications of ERBB2 and newly discovered amplification of IGF2. Recurrent chromosomal translocations include the fusion of NAV2 and WNT pathway member TCF7L1. Integrative analyses suggest new markers for aggressive colorectal carcinoma and an important role for MYC-directed transcriptional activation and repression.

https://cdr.lib.unc.edu/downloads/m900nw503

Comprehensive molecular characterization of human colon and rectal cancer

Wnt/β-catenin signaling: components, mechanisms, and diseases

https://web.stanford.edu/group/nusselab/cgi-bin/wnt/sites/default/files/reviews/Cell%202012%20Clevers.pdf

Wnt/β-catenin signaling and disease.

MicroRNAs (miRNAs) are 21–23 nucleotide RNA molecules that regulate the stability or translational efficiency of target messenger RNAs. miRNAs have diverse functions, including the regulation of cellular differentiation, proliferation and apoptosis. Although strict tissue- and developmental-stage-specific expression is critical for appropriate miRNA function, mammalian transcription factors that regulate miRNAs have not yet been identified. The proto-oncogene c-MYC encodes a transcription factor that regulates cell proliferation, growth and apoptosis. Dysregulated expression or function of c-Myc is one of the most common abnormalities in human malignancy. Here we show that c-Myc activates expression of a cluster of six miRNAs on human chromosome 13. Chromatin immunoprecipation experiments show that c-Myc binds directly to this locus. The transcription factor E2F1 is an additional target of c-Myc that promotes cell cycle progression. We find that expression of E2F1 is negatively regulated by two miRNAs in this cluster, miR-17-5p and miR-20a. These findings expand the known classes of transcripts within the c-Myc target gene network, and reveal a mechanism through which c-Myc simultaneously activates E2F1 transcription and limits its translation, allowing a tightly controlled proliferative signal.

c-MYC-regulated micro RNAs modulate E2F1 expression

MicroRNAs (miRNAs) are 21-23 nucleotide RNA molecules that regulate the stability or translational efficiency of target messenger RNAs. miRNAs have diverse functions, including the regulation of cellular differentiation, proliferation and apoptosis. Although strict tissue- and developmental-stage-specific expression is critical for appropriate miRNA function, mammalian transcription factors that regulate miRNAs have not yet been identified. The proto-oncogene c-MYC encodes a transcription factor that regulates cell proliferation, growth and apoptosis. Dysregulated expression or function of c-Myc is one of the most common abnormalities in human malignancy. Here we show that c-Myc activates expression of a cluster of six miRNAs on human chromosome 13. Chromatin immunoprecipation experiments show that c-Myc binds directly to this locus. The transcription factor E2F1 is an additional target of c-Myc that promotes cell cycle progression. We find that expression of E2F1 is negatively regulated by two miRNAs in this cluster, miR-17-5p and miR-20a. These findings expand the known classes of transcripts within the c-Myc target gene network, and reveal a mechanism through which c-Myc simultaneously activates E2F1 transcription and limits its translation, allowing a tightly controlled proliferative signal.

c-Myc-regulated microRNAs modulate E2F1 expression.

Wilms tumor is a pediatric kidney cancer associated with inactivation of the WT1 tumor-suppressor gene in 5 to 10% of cases. Using a high-resolution screen for DNA copy-number alterations in Wilms tumor, we identified somatic deletions targeting a previously uncharacterized gene on the X chromosome. This gene, which we call WTX, is inactivated in approximately one-third of Wilms tumors (15 of 51 tumors). Tumors with mutations in WTX lack WT1 mutations, and both genes share a restricted temporal and spatial expression pattern in normal renal precursors. In contrast to biallelic inactivation of autosomal tumor-suppressor genes, WTX is inactivated by a monoallelic "single-hit" event targeting the single X chromosome in tumors from males and the active X chromosome in tumors from females.

https://science.sciencemag.org/content/sci/315/5812/642.full.pdf

An X chromosome gene, WTX, is commonly inactivated in Wilms tumor.

Uncertainty as to which member of a family of DNA-binding transcription factors regulates a specific promoter in intact cells is a problem common to many investigators. Determining target gene specificity requires both an analysis of protein binding to the endogenous promoter as well as a characterization of the functional consequences of transcription factor binding. By using a formaldehyde crosslinking procedure and Gal4 fusion proteins, we have analyzed the timing and functional consequences of binding of Myc and upstream stimulatory factor (USF)1 to endogenous cellular genes. We demonstrate that the endogenous cad promoter can be immunoprecipitated with antibodies against Myc and USF1. We further demonstrate that although both Myc and USF1 can bind to cad, the cad promoter can respond only to the Myc transactivation domain. We also show that the amount of Myc bound to the cad promoter fluctuates in a growth-dependent manner. Thus, our data analyzing both DNA binding and promoter activity in intact cells suggest that cad is a Myc target gene. In addition, we show that Myc binding can occur at many sites in vivo but that the position of the binding site determines the functional consequences of this binding. Our data indicate that a post-DNA-binding mechanism determines Myc target gene specificity. Importantly, we have demonstrated the feasibility of analyzing the binding of site-specific transcription factors in vivo to single copy mammalian genes.

https://www.pnas.org/content/pnas/95/23/13887.full.pdf

c-Myc target gene specificity is determined by a post-DNAbinding mechanism

Characterizing transcription factor binding sites using formaldehyde crosslinking and immunoprecipitation.

The E2F family of transcription factors plays an important role in the regulation of gene expression at the G1/S-phase transition of the mammalian cell cycle (see reference 16 for an extensive review of the E2F regulatory pathway). E2F binding sites are found in the promoters of genes whose products are required for nucleotide synthesis (e.g., dihydrofolate reductase [DHFR] and thymidine kinase [TK]), for DNA replication (e.g., DNA polymerase α and cdc6), and for cell cycle progression (e.g., cyclin E, cyclin D1, c-myc, b-myb, and cdc2). Transcription from each of these promoters increases during late G1 or early S phase, and this regulation is mediated by protein binding to one or more E2F binding sites (3, 11, 22, 30, 34, 43, 44). To date, eight members of the E2F family have been identified: six E2F proteins (E2F1 to -6) and two DP proteins (DP1 and DP2). The E2F and DP proteins bind to DNA as a heterodimer and can function as activators of transcription. Alternatively, E2F-DP heterodimers can also repress transcription when complexed with members of the Rb family of pocket proteins (pRb, p107, and p130) due to the ability of the pocket proteins to bind to and mask the E2F transactivation domain and to recruit histone deacetylases (6, 17, 25). Individual E2Fs preferentially bind to different pocket proteins. E2F1, E2F2, and E2F3, for example, bind to pRb, while E2F4 predominantly binds to p130 and p107 and E2F5 binds to p130.

A popular model for how E2F family members regulate G1/S-phase-specific gene expression invokes a complex pattern of protein-protein and protein-DNA interactions that change as cells progress through the cell cycle (Fig. ​(Fig.1A).1A). As depicted, transcription from E2F site-containing promoters is thought to be repressed in G0 phase due to the binding of a trimolecular E2F-DP-pocket protein complex and recruitment of histone deacetylase activity by the pocket protein component (6, 17, 25). As cells progress through the cell cycle, various cyclin–cyclin-dependent kinase (cdk) complexes phosphorylate the pocket proteins, causing release of the hyperphosphorylated pocket protein and associated proteins from the DNA-bound E2F-DP heterodimer (1, 2, 7). Finally, traversal through S phase is thought to be accompanied by cyclin-cdk-mediated phosphorylation of the DP subunit of E2F1-3–DP complexes, resulting in release of the heterodimers from the promoter DNA (15, 21, 42). In some cells, E2F4-containing complexes are thought to be inactivated by relocation to the cytoplasm (28, 38). 



FIG. 1

E2F-mediated transcriptional regulation. (A) The current model of E2F-mediated transcriptional regulation is thought to involve binding of an E2F-pocket protein complex to promoters in G0 phase of the cell cycle and release of the pocket protein in late ...



Although this model is attractive, it is largely based upon circumstantial data, and several important questions remain unanswered. For example, the transcriptional activity of complexes containing E2F4 and E2F5 may be shut off during mid- to late S phase by association with unphosphorylated p107 or p130, rather than by relocation to the cytoplasm (10). Additionally, it is not known if E2F target gene specificity exists or if all six E2Fs bind to and regulate every target gene or if Rb family members display target gene specificity. Determination of target gene specificity has been difficult due to the fact that most cells studied to date contain all of the E2Fs and pocket proteins. Thus, most analyses of E2F and pocket protein binding specificity have been performed using in vitro systems or by overexpression of an individual E2F or pocket protein in cells. In vitro systems, however, cannot recapitulate the complex environment of living cells, and altering the relative amounts of individual proteins through overexpression may abolish important protein-protein interactions. Therefore, we wished to determine which E2Fs and pocket proteins bind to and regulate expression of specific target genes in intact cells under physiological conditions. We felt that the use of an unperturbed in vivo system was of particular importance in the analysis of E2F target gene specificity since regulation occurs in the context of cell cycle progression which cannot be mimicked in vitro and which is often altered when individual E2F proteins are overexpressed. Toward this goal, we have used a formaldehyde cross-linking and immunoprecipitation system to monitor protein-DNA and protein-protein interactions on E2F target genes in living cells. Importantly, this procedure has allowed us to determine the in vivo patterns of E2F and pocket protein binding to specific E2F target genes as cells progress through a cell cycle. Our results indicate that, while some promoters fit the proposed model for E2F-mediated transcriptional regulation, others do not, necessitating individualization of the current model. Furthermore, our data suggest that cell cycle-regulated activity is a stochastic event, as cells have the ability to form several different E2F-pocket protein complexes on a given promoter at each stage of the cell cycle.

Target gene specificity of E2F and pocket protein family members in living cells.

We show that the Mre11 complex associates with E2F family members via the Nbs1 N terminus This association and Nbs1 phosphorylation are correlated with S-phase checkpoint proficiency, whereas neither is sufficient individually for checkpoint activation The Nbs1 E2F interaction occurred near the Epstein-Barr virus origin of replication as well as near a chromosomal replication origin in the c-myc promoter region and was restricted to S-phase cells The Mre11 complex colocalized with PCNA at replication forks throughout S phase, both prior to and coincident with the appearance of nascent DNA These data suggest that the Mre11 complex suppresses genomic instability through its influence on both the regulation and progression of DNA replication

Julie Wells

Papers

An X chromosome gene, WTX, is commonly inactivated in Wilms tumor.

c-Myc target gene specificity is determined by a post-DNAbinding mechanism

Characterizing transcription factor binding sites using formaldehyde crosslinking and immunoprecipitation.

Target gene specificity of E2F and pocket protein family members in living cells.

Mre11 complex and DNA replication: linkage to E2F and sites of DNA synthesis.