Adrianna K. San Roman

ARID1A loss impairs enhancer-mediated gene regulation and drives colon cancer in mice

Genes encoding subunits of SWI/SNF (BAF) chromatin-remodeling complexes are collectively mutated in ∼20% of all human cancers. Although ARID1A is the most frequent target of mutations, the mechanism by which its inactivation promotes tumorigenesis is unclear. Here we demonstrate that Arid1a functions as a tumor suppressor in the mouse colon, but not the small intestine, and that invasive ARID1A-deficient adenocarcinomas resemble human colorectal cancer (CRC). These tumors lack deregulation of APC/β-catenin signaling components, which are crucial gatekeepers in common forms of intestinal cancer. We find that ARID1A normally targets SWI/SNF complexes to enhancers, where they function in coordination with transcription factors to facilitate gene activation. ARID1B preserves SWI/SNF function in ARID1A-deficient cells, but defects in SWI/SNF targeting and control of enhancer activity cause extensive dysregulation of gene expression. These findings represent an advance in colon cancer modeling and implicate enhancer-mediated gene regulation as a principal tumor-suppressor function of ARID1A.

Intestinal Master Transcription Factor CDX2 Controls Chromatin Access for Partner Transcription Factor Binding

ABSTRACT Tissue-specific gene expression requires modulation of nucleosomes, allowing transcription factors to occupy cis elements that are accessible only in selected tissues. Master transcription factors control cell-specific genes and define cellular identities, but it is unclear if they possess special abilities to regulate cell-specific chromatin and if such abilities might underlie lineage determination and maintenance. One prevailing view is that several transcription factors enable chromatin access in combination. The homeodomain protein CDX2 specifies the embryonic intestinal epithelium, through unknown mechanisms, and partners with transcription factors such as HNF4A in the adult intestine. We examined enhancer chromatin and gene expression following Cdx2 or Hnf4a excision in mouse intestines. HNF4A loss did not affect CDX2 binding or chromatin, whereas CDX2 depletion modified chromatin significantly at CDX2-bound enhancers, disrupted HNF4A occupancy, and abrogated expression of neighboring genes. Thus, CDX2 maintains transcription-permissive chromatin, illustrating a powerful and dominant effect on enhancer configuration in an adult tissue. Similar, hierarchical control of cell-specific chromatin states is probably a general property of master transcription factors.

Boundaries, junctions and transitions in the gastrointestinal tract

Contiguous regions along the mammalian gastrointestinal tract, from the esophagus to the rectum, serve distinct digestive functions. Some organs, such as the esophagus and glandular stomach or the small bowel and colon, are separated by sharp boundaries. The duodenal, jejunal and ileal segments of the small intestine, by contrast, have imprecise borders. Because human esophageal and gastric cancers frequently arise in a background of tissue metaplasia and some intestinal disorders are confined to discrete regions, it is useful to appreciate the molecular and cellular basis of boundary formation and preservation. Here we review the anatomy and determinants of boundaries and transitions in the alimentary canal with respect to tissue morphology, gene expression, and, especially, transcriptional control of epithelial identity. We discuss the evidence for established and candidate molecular mechanisms of boundary formation, including the solitary and combinatorial actions of tissue-restricted transcription factors. Although the understanding remains sparse, genetic studies in mice do provide insights into dominant mechanisms and point the way for future investigation.

Transcription Factors GATA4 and HNF4A Control Distinct Aspects of Intestinal Homeostasis in Conjunction with Transcription Factor CDX2

Background: Different transcription factor combinations may control distinct or overlapping cellular functions. Results: Intestines lacking tissue-restricted CDX2 and broadly expressed GATA4 or HNF4A show unique defects. Conclusion: Combined with CDX2, GATA4 controls crypt cell replication, whereas HNF4A regulates enterocyte maturation and a cohort of functional enterocyte genes. Significance: Combinatorial mechanisms for intestine-specific gene regulation may apply generally to other tissues. Distinct groups of transcription factors (TFs) assemble at tissue-specific cis-regulatory sites, implying that different TF combinations may control different genes and cellular functions. Within such combinations, TFs that specify or maintain a lineage and are therefore considered master regulators may play a key role. Gene enhancers often attract these tissue-restricted TFs, as well as TFs that are expressed more broadly. However, the contributions of the individual TFs to combinatorial regulatory activity have not been examined critically in many cases in vivo. We address this question using a genetic approach in mice to inactivate the intestine-specifying and intestine-restricted factor CDX2 alone or in combination with its more broadly expressed partner factors, GATA4 and HNF4A. Compared with single mutants, each combination produced significantly greater defects and rapid lethality through distinct anomalies. Intestines lacking Gata4 and Cdx2 were deficient in crypt cell replication, whereas combined loss of Hnf4a and Cdx2 specifically impaired viability and maturation of villus enterocytes. Integrated analysis of TF binding and of transcripts affected in Hnf4a;Cdx2 compound-mutant intestines indicated that this TF pair controls genes required to construct the apical brush border and absorb nutrients, including dietary lipids. This study thus defines combinatorial TF activities, their specific requirements during tissue homeostasis, and modules of transcriptional targets in intestinal epithelial cells in vivo.

Distinct Processes and Transcriptional Targets Underlie CDX2 Requirements in Intestinal Stem Cells and Differentiated Villus Cells

Summary Lgr5-expressing intestinal stem cells (ISCs) renew the adult gut epithelium by producing mature villus cells (VCs); the transcriptional basis for ISC functions remains unclear. RNA sequencing analysis identified transcripts modulated during differentiation of Lgr5+ ISCs into VCs, with high expression of the intestine-restricted transcription factor (TF) gene Cdx2 in both populations. Cdx2-deleted mouse ISCs showed impaired proliferation and long-term inability to produce mature lineages, revealing essential ISC functions. Chromatin immunoprecipitation sequencing analysis of CDX2 in Lgr5+ ISCs, coupled with mRNA profiling of control and Cdx2−/− ISCs, identified features of CDX2 regulation distinct from VCs. Most CDX2 binding in ISCs occurs in anticipation of future gene expression, but whereas CDX2 primarily activates VC genes, direct ISC targets are activated and repressed. Diverse CDX2 requirements in stem and differentiated cells may reflect the versatility of TFs that specify a tissue in development and control the same tissue in adults.

Abstract LB-286: ARID1A loss impairs enhancer-mediated gene regulation and drives colon cancer in mice

Genes encoding subunits of SWI/SNF (BAF) chromatin remodeling complexes are collectively mutated in ~20% of all human cancers, but the mechanism by which these mutations promote tumorigenesis is unclear. In this study, we investigate the tumor suppressor role of ARID1A, the subunit of SWI/SNF complexes that is the most frequent target of mutations. We develop an inducible Arid1a-knockout mouse model and find that mice develop invasive ARID1A-deficient colon adenocarcinoma. These cancers show prominent mucinous differentiation and tumor-infiltrating lymphocytes, features associated particularly with microsatellite-instable (MSI) human colorectal cancer - the subtype with the highest frequency of inactivating ARID1A mutations (37-39%). Although deregulation of Wnt signaling components (APC/β-catenin) is commonly utilized to model intestinal tumorigenesis, Arid1a-knockout mice reflect human colorectal cancer with greater accuracy. These mice do not show deregulation of Wnt signaling components, thus demonstrating a novel pathway to colon tumorigenesis that is independent of established models. Chromatin immunoprecipitation sequencing reveals that SWI/SNF complexes are targeted primarily to enhancers, where they function in coordination with transcription factors to activate gene expression. ARID1A loss impairs the targeting of SWI/SNF complexes to thousands of enhancers, which subsequently lose activity - showing reduced levels of H3K27ac and expression of nearest genes. Residual SWI/SNF complexes in ARID1A-deficient cells bind enhancers that remain active; these complexes contain ARID1B, the subunit that is mutually exclusive with ARID1A and has been identified as a therapeutic target in ARID1A-deficient cancers. Enhancers associated with developmental gene expression programs are most affected by ARID1A loss, both in the mouse colonic epithelium and in human colon cancer cells. Collectively, these findings represent an advance in colon cancer modeling and implicate enhancer-mediated gene regulation as the principal tumor suppressor function of ARID1A. Citation Format: Radhika Mathur, Burak H. Alver, Adrianna K. San Roman, Boris G. Wilson, Xiaofeng Wang, Agoston T. Agoston, Peter Park, Ramesh Shivdasani, Charles W. Roberts. ARID1A loss impairs enhancer-mediated gene regulation and drives colon cancer in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-286. doi:10.1158/1538-7445.AM2017-LB-286

The Alimentary Canal

In progressing from a simple tube to mature digestive organs, the alimentary tract is patterned along distinct axes. Transcription factors pattern the gut tube along the rostrocaudal axis, delineating a foregut, a midgut, and a hindgut. Reciprocal signals along the radial axis then enable differentiation of the mesoderm-derived mesenchyme and muscle cells from diverse endoderm-derived epithelia. Inductive interactions further specify discrete organs such as the stomach, liver, and pancreas, which show dorsoventral and left–right asymmetries. Finally, patterning of the radially symmetric small bowel along a luminal–mural axis separates progenitors in submucosal crypts from differentiated cells restricted to the villi. Diverse forces drive morphogenesis: radial cell intercalation, cell shape changes, asymmetric tissue growth, and tension from surrounding structures. Much remains unclear about how positional and inductive cues activate particular genes to implement this developmental program.