High-Resolution mRNA and Secretome Atlas of Human Enteroendocrine Cells.

Enteroendocrine cells (EECs) sense intestinal content and release hormones to regulate gastrointestinal activity, systemic metabolism, and food intake. Little is known about the molecular make-up of human EEC subtypes and the regulated secretion of individual hormones. Here, we describe an organoid-based platform for functional studies of human EECs. EEC formation is induced in vitro by transient expression of NEUROG3. A set of gut organoids was engineered in which the major hormones are fluorescently tagged. A single-cell mRNA atlas was generated for the different EEC subtypes, and their secreted products were recorded by mass-spectrometry. We note key differences to murine EECs, including hormones, sensory receptors, and transcription factors. Notably, several hormone-like molecules were identified. Inter-EEC communication is exemplified by secretin-induced GLP-1 secretion. Indeed, individual EEC subtypes carry receptors for various EEC hormones. This study provides a rich resource to study human EEC development and function.

Transcription shapes 3D chromatin organization by interacting with loop extrusion.

Cohesin folds mammalian interphase chromosomes by extruding the chromatin fiber into numerous loops. "Loop extrusion" can be impeded by chromatin-bound factors, such as CTCF, which generates characteristic and functional chromatin organization patterns. It has been proposed that transcription relocalizes or interferes with cohesin and that active promoters are cohesin loading sites. However, the effects of transcription on cohesin have not been reconciled with observations of active extrusion by cohesin. To determine how transcription modulates extrusion, we studied mouse cells in which we could alter cohesin abundance, dynamics, and localization by genetic "knockouts" of the cohesin regulators CTCF and Wapl. Through Hi-C experiments, we discovered intricate, cohesin-dependent contact patterns near active genes. Chromatin organization around active genes exhibited hallmarks of interactions between transcribing RNA polymerases (RNAPs) and extruding cohesins. These observations could be reproduced by polymer simulations in which RNAPs were moving barriers to extrusion that obstructed, slowed, and pushed cohesins. The simulations predicted that preferential loading of cohesin at promoters is inconsistent with our experimental data. Additional ChIP-seq experiments showed that the putative cohesin loader Nipbl is not predominantly enriched at promoters. Therefore, we propose that cohesin is not preferentially loaded at promoters and that the barrier function of RNAP accounts for cohesin accumulation at active promoters. Altogether, we find that RNAP is an extrusion barrier that is not stationary, but rather, translocates and relocalizes cohesin. Loop extrusion and transcription might interact to dynamically generate and maintain gene interactions with regulatory elements and shape functional genomic organization.

Cell division-dependent dissemination following E-cadherin loss underlies initiation of diffuse-type gastric cancer.

Loss of the cell-cell adhesion protein E-cadherin underlies the development of diffuse-type gastric cancer (DGC), which is characterized by the gradual accumulation of tumor cells originating from the gastric epithelium in the surrounding stroma. How E-cadherin deficiency drives DGC formation remains elusive. Therefore, we investigated the consequences of E-cadherin loss on gastric epithelial organization utilizing a human gastric organoid model and histological analyses of early-stage DGC lesions. E-cadherin depletion from gastric organoids recapitulates DGC initiation, with progressive loss of a single-layered architecture and detachment of individual cells. We found that E-cadherin deficiency in gastric epithelia does not lead to a general loss of epithelial cohesion but disrupts the spindle orientation machinery. This leads to a loss of planar cell division orientation and, consequently, daughter cells are positioned outside of the gastric epithelial layer. Although basally delaminated cells fail to detach and instead reintegrate into the epithelium, apically mispositioned daughter cells can trigger the gradual loss of the single-layered epithelial architecture. This impaired architecture hampers reintegration of mispositioned daughter cells and enables basally delaminated cells to disseminate into the surrounding matrix. Taken together, our findings describe how E-cadherin deficiency disrupts gastric epithelial architecture through displacement of dividing cells and provide new insights in the onset of DGC. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Molecular characterization of Barrett's esophagus at single-cell resolution.

Barrett's esophagus (BE) is categorized, based on morphological appearance, into different stages, which correlate with the risk of developing esophageal adenocarcinoma. More advanced stages are more likely to acquire chromosomal instabilities, but stage-specific markers remain elusive. Here, we performed single-cell DNA-sequencing experiments (scDNAseq) with fresh BE biopsies. Dysplastic BE cells frequently contained chromosomal instability (CIN) regions, and these CIN cells carried mutations corresponding to the COSMIC mutational signature SBS17, which were not present in biopsy-matched chromosomally stable (CS) cells or patient-matched nondiseased control cells. CS cells were predominantly found in nondysplastic BE biopsies. The single-base substitution (SBS) signatures of all CS BE cells analyzed were indistinguishable from those of nondiseased esophageal or gastric cells. Single-cell RNA-sequencing (scRNAseq) experiments with BE biopsies identified two sets of marker genes which facilitate the distinction between columnar BE epithelium and nondysplastic/dysplastic stages. Moreover, histological validation confirmed a correlation between increased CLDN2 expression and the presence of dysplastic BE stages. Our scDNAseq and scRNAseq datasets, which are a useful resource for the community, provide insight into the mutational landscape and gene expression pattern at different stages of BE development.

Cohesin is positioned in mammalian genomes by transcription, CTCF and Wapl.

Mammalian genomes are spatially organized by CCCTC-binding factor (CTCF) and cohesin into chromatin loops and topologically associated domains, which have important roles in gene regulation and recombination. By binding to specific sequences, CTCF defines contact points for cohesin-mediated long-range chromosomal cis-interactions. Cohesin is also present at these sites, but has been proposed to be loaded onto DNA elsewhere and to extrude chromatin loops until it encounters CTCF bound to DNA. How cohesin is recruited to CTCF sites, according to this or other models, is unknown. Here we show that the distribution of cohesin in the mouse genome depends on transcription, CTCF and the cohesin release factor Wings apart-like (Wapl). In CTCF-depleted fibroblasts, cohesin cannot be properly recruited to CTCF sites but instead accumulates at transcription start sites of active genes, where the cohesin-loading complex is located. In the absence of both CTCF and Wapl, cohesin accumulates in up to 70 kilobase-long regions at 3'-ends of active genes, in particular if these converge on each other. Changing gene expression modulates the position of these 'cohesin islands'. These findings indicate that transcription can relocate mammalian cohesin over long distances on DNA, as previously reported for yeast cohesin, that this translocation contributes to positioning cohesin at CTCF sites, and that active genes can be freed from cohesin either by transcription-mediated translocation or by Wapl-mediated release.

Sororin actively maintains sister chromatid cohesion.

Cohesion between sister chromatids is established during DNA replication but needs to be maintained to enable proper chromosome-spindle attachments in mitosis or meiosis. Cohesion is mediated by cohesin, but also depends on cohesin acetylation and sororin. Sororin contributes to cohesion by stabilizing cohesin on DNA. Sororin achieves this by inhibiting WAPL, which otherwise releases cohesin from DNA and destroys cohesion. Here we describe mouse models which enable the controlled depletion of sororin by gene deletion or auxin-induced degradation. We show that sororin is essential for embryonic development, cohesion maintenance, and proper chromosome segregation. We further show that the acetyltransferases ESCO1 and ESCO2 are essential for stabilizing cohesin on chromatin, that their only function in this process is to acetylate cohesin's SMC3 subunit, and that DNA replication is also required for stable cohesin-chromatin interactions. Unexpectedly, we find that sororin interacts dynamically with the cohesin complexes it stabilizes. This implies that sororin recruitment to cohesin does not depend on the DNA replication machinery or process itself, but on a property that cohesin acquires during cohesion establishment.

Mutational landscape of intestinal crypt cells after long-term in vivo exposure to high fat diet.

Obesity is a modifiable risk factor in cancer development, especially for gastrointestinal cancer. While the etiology of colorectal cancer is well characterized by the adenoma-carcinoma sequence, it remains unclear how obesity influences colorectal cancer development. Dietary components of a high fat diet along with obesity have been shown to modulate the cancer risk by perturbing the homeostasis of intestinal stem cells, yet how adiposity impacts the development of genomic instability has not been studied. Mutational signatures are a powerful way to understand how a complex biological response impacts genomic stability. We utilized a mouse model of diet-induced obesity to study the mutational landscape of intestinal crypt cells after a 48-week exposure to an experimental high fat diet in vivo. By clonally enriching single crypt derived cells in organoid culture and obtaining whole genome sequences, we analyzed and compared the mutational landscape of intestinal epithelial cells from normal diet and high fat diet mice. Single nucleotide substitution signatures and indel signatures present in our cohort are found equally active in both diet groups and reflect biological processes of normal aging, cellular replication, and oxidative stress induced during organoid culturing. Thus, we demonstrate that in the absence of activating mutations or chemical exposure, high fat diet alone is not sufficient to increase genomic instability.

Barrett's esophagus stages: their correlation with SBS17-associated DNA mutations and the identification of histological marker genes.

We have recently reported a correlation between the accumulation of specific T > C and T > G mutations and the chromosomal instability in cells of Barrett's esophagus (BE), which represents a premalignant condition of esophageal adenocarcinoma. Additionally, we identified seven marker genes that facilitate the distinction of individual BE stages by histopathological examination.

Human gastrointestinal epithelia of the esophagus, stomach, and duodenum resolved at single-cell resolution.

The upper gastrointestinal tract, consisting of the esophagus, stomach, and duodenum, controls food transport, digestion, nutrient uptake, and hormone production. By single-cell analysis of healthy epithelia of these human organs, we molecularly define their distinct cell types. We identify a quiescent COL17A1high KRT15high stem/progenitor cell population in the most basal cell layer of the esophagus and detect substantial gene expression differences between identical cell types of the human and mouse stomach. Selective expression of BEST4, CFTR, guanylin, and uroguanylin identifies a rare duodenal cell type, referred to as BCHE cell, which likely mediates high-volume fluid secretion because of continual activation of the CFTR channel by guanylin/uroguanylin-mediated autocrine signaling. Serotonin-producing enterochromaffin cells in the antral stomach significantly differ in gene expression from duodenal enterochromaffin cells. We, furthermore, discover that the histamine-producing enterochromaffin-like cells in the oxyntic stomach express the luteinizing hormone, yet another member of the enteroendocrine hormone family.

The potential and challenges of patient-derived organoids in guiding the multimodality treatment of upper gastrointestinal malignancies.

The incidence of adenocarcinoma at the gastrooesophageal junction increased over the last years. Curative treatment for patients with upper gastrointestinal (UGI) malignancies, such as oesophageal and gastric tumours, is challenging and requires a multidisciplinary approach. Radical surgical resection with complete lymphadenectomy is the cornerstone of UGI cancer treatment. Combined with peri-operative treatment (i.e. by applying CROSS, EOX or FLOT regimen), the survival is even better than with surgery alone. However, peri-operative treatment is not effective in all patients, and the most effective strategy is a topic of active debate, as is reflected by varying treatment guidelines between countries. UGI cancers are (epi)genetically highly heterogeneous. It is thus not likely that a uniform treatment will benefit all patients equally well. Over recent years, patient-derived organoids (PDOs) gained more and more interest as an in vitro prediction model that may assist as a diagnostic tool in the future to select and eventually optimize the best peri-operative treatments for each patient. PDOs can be derived from endoscopic tumour biopsies, which maintain heterogeneity in culture. They can be rapidly established and expanded in a relatively short time for in vitro drug screening experiments. This review summarizes the clinical and molecular aspects of oesophageal and gastric tumours, as well as the current progress and remaining challenges in the use of PDOs for drug and radiation screens.

Absolute quantification of cohesin, CTCF and their regulators in human cells.

The organisation of mammalian genomes into loops and topologically associating domains (TADs) contributes to chromatin structure, gene expression and recombination. TADs and many loops are formed by cohesin and positioned by CTCF. In proliferating cells, cohesin also mediates sister chromatid cohesion, which is essential for chromosome segregation. Current models of chromatin folding and cohesion are based on assumptions of how many cohesin and CTCF molecules organise the genome. Here we have measured absolute copy numbers and dynamics of cohesin, CTCF, NIPBL, WAPL and sororin by mass spectrometry, fluorescence-correlation spectroscopy and fluorescence recovery after photobleaching in HeLa cells. In G1-phase, there are ~250,000 nuclear cohesin complexes, of which ~ 160,000 are chromatin-bound. Comparison with chromatin immunoprecipitation-sequencing data implies that some genomic cohesin and CTCF enrichment sites are unoccupied in single cells at any one time. We discuss the implications of these findings for how cohesin can contribute to genome organisation and cohesion.

Transcription factor Pax5 activates the chromatin of key genes involved in B cell signaling, adhesion, migration, and immune function.

The transcription factor Pax5 represses B lineage-inappropriate genes and activates B cell-specific genes in B lymphocytes. Here we have identified 170 Pax5-activated genes. Conditional mutagenesis demonstrated that the Pax5-regulated genes require continuous Pax5 activity for normal expression in pro-B and mature B cells. Expression of half of the Pax5-activated genes is either absent or substantially reduced upon Pax5 loss in plasma cells. Direct Pax5 target genes were identified based on their protein synthesis-independent activation by a Pax5-estrogen receptor fusion protein. Chromatin immunoprecipitation (ChIP) of Pax5 together with chromatin profiling by ChIP-on-chip analysis demonstrated that Pax5 directly activates the chromatin at promoters or putative enhancers of Pax5 target genes. The Pax5-activated genes code for key regulatory and structural proteins involved in B cell signaling, adhesion, migration, antigen presentation, and germinal-center B cell formation, thus revealing a complex regulatory network that is activated by Pax5 to control B cell development and function.