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Supplementary Figure Legends.

Supplementary Figure 1. Experimental Schema: Conditional ablation of Dnmt1 in the ApcMin/+ intestinal epithelium ApcMin/+;Dnmt1loxP/loxP mice (controls) and ApcMin/+;Dnmt1loxP/loxP;Villin-Cre-ERT2 mice (mutants) were injected with tamoxifen at 4 weeks of age. Intestines were harvested one week or two months following tamoxifen treatment.

Sequences of primers used for the study.

Supplementary Figure 3. Neoplastic lesions found in Dnmt1 mutant mice are predominantly adenomas. (A,B) Histopathological scoring methods were used to count total number of lesions displaying gastrointestinal intraepithelial neoplasia (GIN) and adenoma, in mutant and control mice one-month (A) and two months (B) following tamoxifen injection (n=3-8 per group). Intestines were isolated, fixed and embedded in paraffin. One representative section was stained with H&E for each animal. Histopathological scoring was performed using the guidelines published by Boivin et al. (25) by the Comparative Pathology Core at the University of Pennsylvania School of Veterinary Medicine. Data are presented as average{plus minus}SEM; *P

Supplementary Figure 2. Dnmt1 deletion causes increased incidence of microadenomas in the colon at three months. (A,B) Dnmt1 deletion was maintained two months after tamoxifen treatment in the adult mouse colonic epithelium. Immunohistochemical staining of Dnmt1 protein is evident in the crypts of control colonic epithelia (A), but is completely absent in mutant colon (B). (C,D) Increased incidence of neoplastic crypts observed in the Dnmt1-deficient colon, two months after tamoxifen injection. β-catenin staining shows an example of a microadenoma with nuclear accumulation of β-catenin in mutant colon (D) compared to localization on the cell membrane in the nontransformed colonic epithelia of control mice (C). Scale bars are 50μm. (E) Mutant animals have increased incidence of neoplastic transformation in the colon at 2 months post-tamoxifen. Table listing I.D. of individual mice and total number of lesions observed in the colon. Control and mutant colons were isolated, fixed and embedded in paraffin. A representative section from the middle of the specimen was stained with β-catenin. Lesions were defined by the nuclear accumulation of β-catenin. (F) Mutant colons have increased number of neoplastic lesions at 2 months post-tamoxifen. Total lesions observed using data in (E) are presented as average{plus minus}SEM; *P

Methylated cytosines are associated with gene silencing. The ten-eleven translocation (TET) hydroxylases, which oxidize methylated cytosines to 5-hydroxymethylcytosine (5hmC), are essential for cytosine demethylation. Gene silencing and activation are critical for intestinal stem cell (ISC) maintenance and differentiation, but the potential role of TET hydroxylases in these processes has not yet been examined. Here, we generated genome-wide maps of the 5hmC mark in ISCs and their differentiated progeny. Genes with high levels of hydroxymethylation in ISCs are strongly associated with Wnt signaling and developmental processes. We found Tet1 to be the most abundantly expressed Tet gene in ISCs; therefore, we analyzed intestinal development in Tet1-deficient mice and determined that these mice are growth-retarded, exhibit partial postnatal lethality, and have significantly reduced numbers of proliferative cells in the intestinal epithelium. In addition, the Tet1-deficient intestine displays reduced organoid-forming capacity. In the Tet1-deficient crypt, decreased expression of Wnt target genes such as Axin2 and Lgr5 correlates with lower 5hmC levels at their promoters. These data demonstrate that Tet1-mediated DNA hydroxymethylation plays a critical role in the epigenetic regulation of the Wnt pathway in intestinal stem and progenitor cells and consequently in the self-renewal of the intestinal epithelium.

Intestinal cancer is a heterogeneous disease driven by genetic mutations and epigenetic changes. Approximately 80% of sporadic colorectal cancers are initiated by mutation and inactivation of the adenomatous polyposis coli (APC) gene, which results in unrestrained intestinal epithelial growth and formation of adenomas. Aberrant DNA methylation promotes cancer progression by the inactivation of tumor suppressor genes via promoter methylation. In addition, global DNA hypomethylation is often seen before the formation of adenomas, suggesting that it contributes to neoplastic transformation. Previous studies employed mice with a hypomorphic mutation in DNA methyltransferase 1 (Dnmt1), which exhibited constitutive global DNA hypomethylation and decreased tumorigenesis in the ApcMin/+ mouse model of intestinal cancer. However, the consequences of intestinal epithelial-specific acute hypomethylation during ApcMin/+ tumor initiation have not been reported. Using temporally controlled intestinal epithelial-specific gene ablation, we show that total loss of Dnmt1 in the ApcMin/+ mouse model of intestinal cancer causes accelerated adenoma initiation. Deletion of Dnmt1 precipitates an acute response characterized by hypomethylation of repetitive elements and genomic instability, which surprisingly is followed by remethylation with time. Two months post-Dnmt1 ablation, mice display increased macroadenoma load, consistent with a role for Dnmt1 and DNA methylation in maintaining genomic stability. These data suggest that DNA hypomethylation plays a previously unappreciated role in intestinal adenoma initiation. Cancer Prev Res; 9(7); 534–46. ©2016 AACR. See related article by Lee and Laird, p. 509

The DNA methyltransferase Dnmt1 maintains DNA methylation patterns and genomic stability in several in vitro cell systems. Ablation of Dnmt1 in mouse embryos causes death at the post-gastrulation stage; however, the functions of Dnmt1 and DNA methylation in organogenesis remain unclear. Here, we report that Dnmt1 is crucial during perinatal intestinal development. Loss of Dnmt1 in intervillus progenitor cells causes global hypomethylation, DNA damage, premature differentiation, apoptosis and, consequently, loss of nascent villi. We further confirm the crucial role of Dnmt1 during crypt development using the in vitro organoid culture system, and illustrate a clear differential requirement for Dnmt1 in immature versus mature organoids. These results demonstrate an essential role for Dnmt1 in maintaining genomic stability during intestinal development and the establishment of intestinal crypts.

The mammalian intestinal epithelium has a unique organization in which crypts harboring stem cells produce progenitors and finally clonal populations of differentiated cells. Remarkably, the epithelium is replaced every 3–5 d throughout adult life. Disrupted maintenance of the intricate balance of proliferation and differentiation leads to loss of epithelial integrity or barrier function or to cancer. There is a tight correlation between the epigenetic status of genes and expression changes during differentiation; however, the mechanism of how changes in DNA methylation direct gene expression and the progression from stem cells to their differentiated descendants is unclear. Using conditional gene ablation of the maintenance methyltransferase Dnmt1, we demonstrate that reducing DNA methylation causes intestinal crypt expansion in vivo. Determination of the base-resolution DNA methylome in intestinal stem cells and their differentiated descendants shows that DNA methylation is dynamic at enhancers, which are often associated with genes important for both stem cell maintenance and differentiation. We establish that the loss of DNA methylation at intestinal stem cell gene enhancers causes inappropriate gene expression and delayed differentiation.

Chromatin immunoprecipitation with massively parallel DNA sequencing (ChIP-Seq) has been used extensively to determine the genome-wide location of DNA-binding factors, such as transcription factors, posttranscriptionally modified histones, and members of the transcription complex, to assess regulatory input, epigenetic modifications, and transcriptional activity, respectively. Here we describe methods to isolate chromatin from tissues, immunoprecipitate DNA bound to a protein of interest, and perform next-generation sequencing to identify a genome-wide DNA-binding pattern.

Chromatin immunoprecipitation with massively parallel DNA sequencing (ChIP-Seq) has been used extensively to determine the genome-wide location of DNA-binding factors, such as transcription factors, posttranscriptionally modified histones, and members of the transcription complex, to assess regulatory input, epigenetic modifications, and transcriptional activity, respectively. Here we describe methods to isolate chromatin from tissues, immunoprecipitate DNA bound to a protein of interest, and perform next-generation sequencing to identify a genome-wide DNA-binding pattern.

Summary Background FoxA factors are critical regulators of embryonic development and postembryonic life, but little is know about the upstream pathways that modulate their activity [1]. C. elegans pha-4 encodes a FoxA transcription factor that is required to establish the foregut in embryos and to control growth and longevity after birth [2–5]. We previously identified the AAA+ ATPase homolog ruvb-1 as a potent suppressor of pha-4 mutations [6]. Results Here we show that ruvb-1 is a component of the Target of Rapamycin (TOR) pathway in C. elegans (CeTOR). Both ruvb-1 and let-363/TOR control nucleolar size and promote localization of box C/D snoRNPs to nucleoli, suggesting a role in rRNA maturation. Inactivation of let-363/TOR or ruvb-1 suppresses the lethality associated with reduced pha-4 activity. The CeTOR pathway controls protein homeostasis and also contributes to adult longevity [7, 8]. We find that pha-4 is required to extend adult lifespan in response to reduced CeTOR signaling. Mutations in the predicted CeTOR target rsks-1/S6 kinase or in ife-2/eIF4E also reduce protein biosynthesis and extend lifespan [9–11], but only rsks-1 mutations require pha-4 for adult longevity. In addition, rsks-1 , but not ife-2 , can suppress the larval lethality associated with pha-4 loss-of-function mutations. Conclusions The data suggest that pha-4 and the CeTOR pathway antagonize one another to regulate postembryonic development and adult longevity. We suggest a model in which nutrients promote TOR and S6 kinase signaling, which represses pha-4/FoxA , leading to a shorter lifespan. A similar regulatory hierarchy may function in other animals to modulate metabolism, longevity, or disease.

The development of the gastrointestinal tract is a complex process that integrates signaling processes with downstream transcriptional responses. Here, we discuss the regionalization of the primitive gut and formation of the intestine and liver. Anterior-posterior position in the primitive gut is important for establishing regions that will become functional organs. Coordination of signaling between the epithelium and mesenchyme and downstream transcriptional responses is required for intestinal development and homeostasis. Liver development uses a complex transcriptional network that controls the establishment of organ domains, cell differentiation, and adult function. Discussion of these transcriptional mechanisms gives us insight into how the primitive gut, composed of simple endodermal cells, develops into multiple diverse cell types that are organized into complex mature organs.

Transcription factors are key components of regulatory networks that control development, as well as the response to environmental stimuli. We have established an experimental pipeline in Caenorhabditis elegans that permits global identification of the binding sites for transcription factors using chromatin immunoprecipitation and deep sequencing. We describe and validate this strategy, and apply it to the transcription factor PHA-4, which plays critical roles in organ development and other cellular processes. We identified thousands of binding sites for PHA-4 during formation of the embryonic pharynx, and also found a role for this factor during the starvation response. Many binding sites were found to shift dramatically between embryos and starved larvae, from developmentally regulated genes to genes involved in metabolism. These results indicate distinct roles for this regulator in two different biological processes and demonstrate the versatility of transcription factors in mediating diverse biological roles., Author Summary The C. elegans transcription factor PHA-4 is a member of the highly conserved FOXA family of transcription factors. These factors act as master regulators of organ development by controlling how genes are turned off and on as tissues are formed. Additionally they regulate genes in response to nutrient levels and control both longevity and survival of the organism. However, the extent to which these factors control similar or distinct gene targets for each of these functions is unknown. For this reason, we have used the technique of chromatin immunoprecipitation followed by deep sequencing (ChIP–Seq), to define the target binding sites of PHA-4 on a genome-wide scale, when it is either functioning as an organ identity regulator or in response to environmental stress. Our data clearly demonstrate distinct sets of biologically relevant target genes for the transcription factor PHA-4 under these two different conditions. Not only have we defined PHA-4 targets, but we established an experimental ChIP–Seq pipeline to facilitate the identification of binding sites for many transcription factors in the future.

Background DNA methylation has emerged as an important regulator of development and disease, necessitating the design of more efficient and cost-effective methods for detecting and quantifying this epigenetic modification. Next-generation sequencing (NGS) techniques offer single base resolution of CpG methylation levels with high statistical significance, but are also high cost if performed genome-wide. Here, we describe a simplified targeted bisulfite sequencing approach in which DNA sequencing libraries are prepared following sodium bisulfite conversion and two rounds of PCR for target enrichment and sample barcoding, termed BisPCR2. Results We have applied the BisPCR2 technique to validate differential methylation at several type 2 diabetes risk loci identified in genome-wide studies of human islets. We confirmed some previous findings while not others, in addition to identifying novel differentially methylated CpGs at these genes of interest, due to the much higher depth of sequencing coverage in BisPCR2 compared to prior array-based approaches. Conclusion This study presents a robust, efficient, and cost-effective technique for targeted bisulfite NGS, and illustrates its utility by reanalysis of prior findings from genome-wide studies. Electronic supplementary material The online version of this article (doi:10.1186/s13072-015-0020-x) contains supplementary material, which is available to authorized users.