Here we describe Protein Interaction Quantitation (PIQ), a computational method that models the magnitude and shape of genome-wide DNase profiles to facilitate the identification of transcription factor (TF) binding sites. Through the use of machine learning techniques, PIQ identified binding sites for >700 TFs from one DNase-seq experiment with accuracy comparable to ChIP-seq for motif-associated TFs (median AUC=0.93 across 303 TFs). We applied PIQ to analyze DNase-seq data from mouse embryonic stem cells differentiating into pre-pancreatic and intestinal endoderm. We identified (n=120) and experimentally validated eight ‘pioneer’ TF families that dynamically open chromatin, enabling other TFs to bind to adjacent DNA. Four pioneer TF families only open chromatin in one direction from their motifs. Furthermore, we identified a class of ‘settler’ TFs whose genomic binding is principally governed by proximity to open chromatin. Our results support a model of hierarchical TF binding in which directional and non-directional pioneer activity shapes the chromatin landscape for population by settler TFs.
The full-text may be used and/or reproduced, and given to third parties in any format or medium, without prior permission or charge, for personal research or study, educational, or not-for-pro t purposes provided that:• a full bibliographic reference is made to the original source • a link is made to the metadata record in DRO • the full-text is not changed in any way The full-text must not be sold in any format or medium without the formal permission of the copyright holders.Please consult the full DRO policy for further details. 59apically and are in contact with the intestinal lumen (Fig. 1A, Supplementary Fig. 1A). 60Therefore, we hypothesized that age-related changes in SJ could directly contribute to 74and an enriched analysis of the dataset revealed that the "cell adhesion" gene ontology 75(GO) category was one of the most representative GO categories that change with age 76(Supplementary Table1). The expression level of the majority of these genes (16 of 18) 77was up-regulated in old flies, indicating that decreased transcription is not a primary 78 mechanism contributing to age-related changes in SJs in the midgut. 79In Drosophila, SJ are divided into two classes based on morphological 95One striking and consistent age-related change in SJs was observed at tricellular 96 junctions (TCJ) (arrowheads, Fig. 1D-I, L-M, P-T), the specialized junction at the 101In the adult posterior midgut, Gli co-localized with Dlg ( Fig. 2A-B'; Supplementary 102 Fig. 2A-A''), as described previously in wing discs 16, 17 . Gli protein was clearly detected 103at EC-EC and EC-EE cell TCJ (Fig. 2 A,C,F); however, no Gli protein was detected in 104ISCs/EBs (Fig. 2D). In midguts from aged flies, Gli localization was largely absent from 105 the TCJ, and protein levels were increased in the cytoplasm (Fig. 2F-H). In hindguts, no 106 changes in Gli localization or protein levels were observed, similar to our observation for 107 other SJ proteins ( Supplementary Fig. 1P-Q). Interestingly, Dlg appeared cytoplasmic, 108rather than membrane-localized, in ISC/EB 'nests', suggesting that definitive SJ may be 109 absent between ISC/EBs and that formation of SJ is coordinated with differentiation. 110Consistent with this hypothesis, SJ were not apparent between ISCs and EBs via EM 111( Fig. 2E). 112Given the significant changes in TCJ (Fig. 1 D-M, P-T) and the striking loss of Gli 113from TCJ in older animals ( Fig. 2G-H age was a significant factor contributing to changes in TCJ (Fig. 2I, J) 118To determine whether compromised TCJ function could contribute to age-related 119 changes in the intestine, Gli was depleted from TCJs using a drug-inducible version of 120 the GAL4-UAS system 19, 20 . Targeted gene expression using the 5966 GS GAL4 "driver" 125Depletion of Gli from ECs resulted in an accelerated loss of barrier integrity (Fig. 1263A ; Supplementary Fig. 2E). Integrity of the intestinal barrier can be assayed by feeding 127 flies a non-absorbable blue food dye. When the intestinal barrier is intact, the dye is 128 retaine...
We found that human intestinal organoids maintain the age of the patient from whom they are derived, as measured by the epigenetic clock. Unexpectedly, we found that crypts and spheroids derived from small intestine showed striking epigenetic age reduction, relative to the colon. BACKGROUND & AIMS:The epithelia of the intestine and colon turn over rapidly and are maintained by adult stem cells at the base of crypts. Although the small intestine and colon have distinct, well-characterized physiological functions, it remains unclear if there are fundamental regional differences in stem cell behavior or region-dependent degenerative changes during aging. Mesenchyme-free organoids provide useful tools for investigating intestinal stem cell biology in vitro and have started to be used for investigating agerelated changes in stem cell function. However, it is unknown whether organoids maintain hallmarks of age in the absence of an aging niche. We tested whether stem cell-enriched organoids preserved the DNA methylationbased aging profiles associated with the tissues and crypts from which they were derived. METHODS:To address this, we used standard human methylation arrays and the human epigenetic clock as a biomarker of age to analyze in vitro-derived, 3-dimensional, stem cell-enriched intestinal organoids. RESULTS:We found that human stem cell-enriched organoids maintained segmental differences in methylation patterns and that age, as measured by the epigenetic clock, also was maintained in vitro. Surprisingly, we found that stem cell-enriched organoids derived from the small intestine showed striking epigenetic age reduction relative to organoids derived from colon. CONCLUSIONS:Our data validate the use of organoids as a model for studying human intestinal aging and introduce methods that can be used when modeling aging or age-onset diseases in vitro. (Cell Mol Gastroenterol Hepatol 2020;9:527-541; https://doi.
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