Dataset Information

Transcriptional Regulation by ATOH1 and its Target SPDEF in the Intestine.

ABSTRACT: BACKGROUND & AIMS:The transcription factor atonal homolog 1 (ATOH1) controls the fate of intestinal progenitors downstream of the Notch signaling pathway. Intestinal progenitors that escape Notch activation express high levels of ATOH1 and commit to a secretory lineage fate, implicating ATOH1 as a gatekeeper for differentiation of intestinal epithelial cells. Although some transcription factors downstream of ATOH1, such as SPDEF, have been identified to specify differentiation and maturation of specific cell types, the bona fide transcriptional targets of ATOH1 still largely are unknown. Here, we aimed to identify ATOH1 targets and to identify transcription factors that are likely to co-regulate gene expression with ATOH1. METHODS:We used a combination of chromatin immunoprecipitation and messenger RNA-based high-throughput sequencing (ChIP-seq and RNA-seq), together with cell sorting and transgenic mice, to identify direct targets of ATOH1, and establish the epistatic relationship between ATOH1 and SPDEF. RESULTS:By using unbiased genome-wide approaches, we identified more than 700 genes as ATOH1 transcriptional targets in adult small intestine and colon. Ontology analysis indicated that ATOH1 directly regulates genes involved in specification and function of secretory cells. De novo motif analysis of ATOH1 targets identified SPDEF as a putative transcriptional co-regulator of ATOH1. Functional epistasis experiments in transgenic mice show that SPDEF amplifies ATOH1-dependent transcription but cannot independently initiate transcription of ATOH1 target genes. CONCLUSIONS:This study unveils the direct targets of ATOH1 in the adult intestines and illuminates the transcriptional events that initiate the specification and function of intestinal secretory lineages.

SUBMITTER: Lo YH

PROVIDER: S-EPMC5247424 | biostudies-literature | 2017 Jan

REPOSITORIES: biostudies-literature

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Transcriptional Regulation by ATOH1 and its Target SPDEF in the Intestine.

Lo Yuan-Hung YH Chung Eunah E Li Zhaohui Z Wan Ying-Wooi YW Mahe Maxime M MM Chen Min-Shan MS Noah Taeko K TK Bell Kristin N KN Yalamanchili Hari Krishna HK Klisch Tiemo J TJ Liu Zhandong Z Park Joo-Seop JS Shroyer Noah F NF

Cellular and molecular gastroenterology and hepatology 20161021 1

<h4>Background & aims</h4>The transcription factor atonal homolog 1 (ATOH1) controls the fate of intestinal progenitors downstream of the Notch signaling pathway. Intestinal progenitors that escape Notch activation express high levels of ATOH1 and commit to a secretory lineage fate, implicating ATOH1 as a gatekeeper for differentiation of intestinal epithelial cells. Although some transcription factors downstream of ATOH1, such as SPDEF, have been identified to specify differentiation and matura ...[more]

PMID: 28174757

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Project description:BackgroundGATA transcription factors are essential for self-renewal of the small intestinal epithelium. Gata4 is expressed in the proximal 85% of small intestine while Gata6 is expressed throughout the length of small intestine. Deletion of intestinal Gata4 and Gata6 results in an altered proliferation/differentiation phenotype, and an up-regulation of SAM pointed domain containing ETS transcription factor (Spdef), a transcription factor recently shown to act as a tumor suppressor. The goal of this study is to determine to what extent SPDEF mediates the downstream functions of GATA4/GATA6 in the small intestine. The hypothesis to be tested is that intestinal GATA4/GATA6 functions through SPDEF by repressing Spdef gene expression. To test this hypothesis, we defined the functions most likely regulated by the overlapping GATA6/SPDEF target gene set in mouse intestine, delineated the relationship between GATA6 chromatin occupancy and Spdef gene regulation in Caco-2 cells, and determined the extent to which prevention of Spdef up-regulation by Spdef knockout rescues the GATA6 phenotype in conditional Gata6 knockout mouse ileum.ResultsUsing publicly available profiling data, we found that 83% of GATA6-regulated genes are also regulated by SPDEF, and that proliferation/cancer is the function most likely to be modulated by this overlapping gene set. In human Caco-2 cells, GATA6 knockdown results in an up-regulation of Spdef gene expression, modeling our mouse Gata6 knockout data. GATA6 occupies a genetic locus located 40 kb upstream of the Spdef transcription start site, consistent with direct regulation of Spdef gene expression by GATA6. Prevention of Spdef up-regulation in conditional Gata6 knockout mouse ileum by the additional deletion of Spdef rescued the crypt cell proliferation defect, but had little effect on altered lineage differentiation or absorptive enterocytes gene expression.ConclusionSPDEF is a key, immediate downstream effecter of the crypt cell proliferation function of GATA4/GATA6 in the small intestine.

Project description:IntroductionProstate cancer (PCa) presents a significant health challenge in men, with a substantial number of deaths attributed to metastatic castration resistant PCa (mCRPC). Moreover, African American men experience disproportionately high mortality rates due to PCa. This study delves into the pivotal role of SPDEF, a prostate specific Ets transcription factor, and its regulation by DNA methylation in the context of PCa progression.MethodsWe performed Epigenetic reprogramming using daily treatment with non-toxic dose of 5Aza-2-deoxycytidine (5Aza-dC) for two weeks to assess its impact on PDEF expression in prostate cancer cells. Next, we conducted functional studies on reprogrammed cells, including cell migration (wound-healing assay), invasion (Boyden-Chamber test), and proliferation (MTT assay) to comprehensively evaluate the consequences of altered PDEF expression. We used bisulfite sequencing (BSP) to examine DNA methylation at SPDEF promoter. Simultaneously, we utilized siRNA-mediated targeting of key DNMTs (DNMT1, DNMT3A, and DNMT3B) to elucidate their specific role in regulating PDEF. We measured mRNA and protein expressions using qRT-PCR and immune-blotting techniques, respectively.ResultsIn this report, we observed that: a) there is a gradual decrease in SPDEF expression with a concomitant increase in methylated CpG sites within the SPDEF gene during prostate cancer progression from lower to higher Gleason grade; b) Expression of DNMT's (DNMT1, 3a and 3b) is increased during prostate cancer progression, and there is an inverse correlation between SPDEF and DNMT expression; c) SPDEF levels are decreased in RC77/T, a line of PCa cells from African American origin similar to PC3 and DU145 cells (CRPC cells), as compared to LNCaP cells , a line of androgen dependent cells,; d) the 5' CpG island of SPDEF gene are hypermethylated in SPDEF-negative CRPC ( PC3, DU145 and RC77/T) cell lines but the same regions are hypomethylated in SPDEF-positive castrate sensitive (LNCaP) cell line ; (e) expression of SPDEF in PCa cells lacking SPDEF decreases cell migration and invasion, but has no significant effect on cell proliferation, and; (f) treatment with the demethylating agent, 5-aza-2'-deoxycytidine, or silencing of the DNMT's by siRNA, partially restores SPDEF expression in SPDEF-negative PCa cell lines, and decreases cell migration and invasion.DiscussionThese results indicate hypermethylation is a prevalent mechanism for decreasing SPDEF expression during prostate cancer progression. The data demonstrate that loss of SPDEF expression in prostate cancer cells, a critical step in cellular plasticity, results from a potentially reversible process of aberrant DNA methylation. These studies suggest DMNT activity as a potential therapeutic vulnerability that can be exploited for limiting cellular plasticity, tumor progression, and therapy resistance in prostate cancer.

Project description:Background & aimsThe canonical Wnt signaling pathway activates the transcriptional activity of β-catenin. This pathway is often activated in colorectal cancer cells, but strategies to block it in tumors have not been effective. The SAM pointed domain containing ETS transcription factor (SPDEF) suppresses formation of colon tumors by unclear mechanisms. We investigated these mechanisms and the effects of SPDEF on β-catenin activity in mouse models of colorectal cancer (CRC), CRC cell lines, and mouse and human normal and cancer colonoids.MethodsWe performed studies of Lgr5CreERT2; β-cateninexon3; Rosa26LSL-rtta-ires-EGFP; TRE-Spdef mice, which express an oncogenic form of β-catenin in Lgr5-positive ISCs upon administration of tamoxifen and SPDEF upon administration of tetracycline. CRC lines (HCT116 and SW480) were engineered to express inducible tagged SPDEF or vector (control) and subcutaneously injected into immunodeficient NSG mice. We generated SPDEF-inducible human colonoids, including a line derived from normal rectal mucosa (control) and an adenocarcinoma line derived from a patient with germline MUTYH mutation. Full-length and truncated forms of SPDEF were expressed in CRC cells; cells were assayed for β-catenin activity and studied in immunoprecipitation and chromatin immunoprecipitation assays.ResultsExpression of SPDEF was sufficient to inhibit intestinal tumorigenesis by activated β-catenin, block tumor cell proliferation, and restrict growth of established tumors. In tumor cells with activated β -catenin, expression of SPDEF induced a quiescent state, which was reversed when SPDEF expression was stopped. In mouse and human normal and tumor-derived enteroids/colonoids, those that expressed SPDEF for 3 days were significantly smaller. SPDEF inhibited the transcriptional activity of β-catenin via a protein-protein interaction, independent of SPDEF DNA binding capacity. SPDEF disrupted β-catenin binding to TCF1 and TCF3, displacing β-catenin from enhancer regions of genes that regulate the cell cycle but not genes that regulate stem cell activities.ConclusionsIn studies of mice and human CRC, we found that SPDEF induces a quiescent state in CRC cells by disrupting binding of β-catenin to TCF1 and TCF3 and regulation of genes that control the cell cycle. In this model, β-catenin activity determines the proliferation or quiescence of CRC cells based on the absence or presence of SPDEF.

Dataset Information

Transcriptional Regulation by ATOH1 and its Target SPDEF in the Intestine.

Publications

Transcriptional Regulation by ATOH1 and its Target SPDEF in the Intestine.

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