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Developmental gene expression is often controlled by distal regulatory DNA elements called enhancers. Distant enhancer action is restricted to structural chromosomal domains that are flanked by CTCF-associated boundaries and formed through cohesin chromatin loop extrusion. To better understand how enhancers, genes and CTCF boundaries together form structural domains and control expression, we used a bottom-up approach, building series of active regulatory landscapes in inactive chromatin. We demonstrate here that gene transcription levels and activity over time reduce with increased enhancer distance. The enhancer recruits cohesin to stimulate domain formation and engage flanking CTCF sites in loop formation. It requires cohesin exclusively for the activation of distant genes, not of proximal genes, with nearby CTCF boundaries supporting efficient long-range enhancer action. Our work supports a dual activity model for enhancers: its classic role of stimulating transcription initiation and elongation from target gene promoters and a role of recruiting cohesin for the creation of chromosomal domains, the engagement of CTCF sites in chromatin looping and the activation of distal target genes., (© 2022. The Author(s).)

Colorectal cancer stem cells (CSCs) express Lgr5 and display extensive stem cell-like multipotency and self-renewal and are thought to seed metastatic disease. Here, we used a mouse model of colorectal cancer (CRC) and human tumor xenografts to investigate the cell of origin of metastases. We found that most disseminated CRC cells in circulation were Lgr5 - and formed distant metastases in which Lgr5 + CSCs appeared. This plasticity occurred independently of stemness-inducing microenvironmental factors and was indispensable for outgrowth, but not establishment, of metastases. Together, these findings show that most colorectal cancer metastases are seeded by Lgr5 - cells, which display intrinsic capacity to become CSCs in a niche-independent manner and can restore epithelial hierarchies in metastatic tumors., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Various species of the intestinal microbiota have been associated with the development of colorectal cancer 1,2 , but it has not been demonstrated that bacteria have a direct role in the occurrence of oncogenic mutations. Escherichia coli can carry the pathogenicity island pks, which encodes a set of enzymes that synthesize colibactin 3 . This compound is believed to alkylate DNA on adenine residues 4,5 and induces double-strand breaks in cultured cells 3 . Here we expose human intestinal organoids to genotoxic pks + E. coli by repeated luminal injection over five months. Whole-genome sequencing of clonal organoids before and after this exposure revealed a distinct mutational signature that was absent from organoids injected with isogenic pks-mutant bacteria. The same mutational signature was detected in a subset of 5,876 human cancer genomes from two independent cohorts, predominantly in colorectal cancer. Our study describes a distinct mutational signature in colorectal cancer and implies that the underlying mutational process results directly from past exposure to bacteria carrying the colibactin-producing pks pathogenicity island.

The significance of cardiac stem cell (CSC) populations for cardiac regeneration remains disputed. Here, we apply the most direct definition of stem cell function (the ability to replace lost tissue through cell division) to interrogate the existence of CSCs. By single-cell mRNA sequencing and genetic lineage tracing using two Ki67 knockin mouse models, we map all proliferating cells and their progeny in homoeostatic and regenerating murine hearts. Cycling cardiomyocytes were only robustly observed in the early postnatal growth phase, while cycling cells in homoeostatic and damaged adult myocardium represented various noncardiomyocyte cell types. Proliferative postdamage fibroblasts expressing follistatin-like protein 1 (FSTL1) closely resemble neonatal cardiac fibroblasts and form the fibrotic scar. Genetic deletion of Fstl1 in cardiac fibroblasts results in postdamage cardiac rupture. We find no evidence for the existence of a quiescent CSC population, for transdifferentiation of other cell types toward cardiomyocytes, or for proliferation of significant numbers of cardiomyocytes in response to cardiac injury., Competing Interests: Conflict of interest statement: H.C. is co-principal investigator (co-PI) on a Dutch grant on organs-on-a-chip with C.L.M. as co-PI, however, on an unrelated topic., (Copyright © 2018 the Author(s). Published by PNAS.)

Background: Genome-wide transcriptome analysis has greatly advanced our understanding of the regulatory networks underlying basic cardiac biology and mechanisms driving disease. However, so far, the resolution of studying gene expression patterns in the adult heart has been limited to the level of extracts from whole tissues. The use of tissue homogenates inherently causes the loss of any information on cellular origin or cell type-specific changes in gene expression. Recent developments in RNA amplification strategies provide a unique opportunity to use small amounts of input RNA for genome-wide sequencing of single cells., Methods: Here, we present a method to obtain high-quality RNA from digested cardiac tissue from adult mice for automated single-cell sequencing of both the healthy and diseased heart., Results: After optimization, we were able to perform single-cell sequencing on adult cardiac tissue under both homeostatic conditions and after ischemic injury. Clustering analysis based on differential gene expression unveiled known and novel markers of all main cardiac cell types. Based on differential gene expression, we could identify multiple subpopulations within a certain cell type. Furthermore, applying single-cell sequencing on both the healthy and injured heart indicated the presence of disease-specific cell subpopulations. As such, we identified cytoskeleton-associated protein 4 as a novel marker for activated fibroblasts that positively correlates with known myofibroblast markers in both mouse and human cardiac tissue. Cytoskeleton-associated protein 4 inhibition in activated fibroblasts treated with transforming growth factor β triggered a greater increase in the expression of genes related to activated fibroblasts compared with control, suggesting a role of cytoskeleton-associated protein 4 in modulating fibroblast activation in the injured heart., Conclusions: Single-cell sequencing on both the healthy and diseased adult heart allows us to study transcriptomic differences between cardiac cells, as well as cell type-specific changes in gene expression during cardiac disease. This new approach provides a wealth of novel insights into molecular changes that underlie the cellular processes relevant for cardiac biology and pathophysiology. Applying this technology could lead to the discovery of new therapeutic targets relevant for heart disease., (© 2018 American Heart Association, Inc.)

The regeneration-capable flatworm Macrostomum lignano is a powerful model organism to study the biology of stem cells in vivo. As a flatworm amenable to transgenesis, it complements the historically used planarian flatworm models, such as Schmidtea mediterranea . However, information on the transcriptome and markers of stem cells in M. lignano is limited. We generated a de novo transcriptome assembly and performed the first comprehensive characterization of gene expression in the proliferating cells of M. lignano , represented by somatic stem cells, called neoblasts, and germline cells. Knockdown of a selected set of neoblast genes, including Mlig-ddx39 , Mlig-rrm1 , Mlig-rpa3 , Mlig-cdk1 , and Mlig-h2a , confirmed their crucial role for the functionality of somatic neoblasts during homeostasis and regeneration. The generated M. lignano transcriptome assembly and gene expression signatures of somatic neoblasts and germline cells will be a valuable resource for future molecular studies in M. lignano ., Competing Interests: The authors declare that no competing interests exist.

Cycling Lgr5+ stem cells fuel the rapid turnover of the adult intestinal epithelium. The existence of quiescent Lgr5+ cells has been reported, while an alternative quiescent stem cell population is believed to reside at crypt position +4. Here, we generated a novel Ki67RFP knock-in allele that identifies dividing cells. Using Lgr5-GFP;Ki67RFP mice, we isolated crypt stem and progenitor cells with distinct Wnt signaling levels and cell cycle features and generated their molecular signature using microarrays. Stem cell potential of these populations was further characterized using the intestinal organoid culture. We found that Lgr5high stem cells are continuously in cell cycle, while a fraction of Lgr5low progenitors that reside predominantly at +4 position exit the cell cycle. Unlike fast dividing CBCs, Lgr5low Ki67- cells have lost their ability to initiate organoid cultures, are enriched in secretory differentiation factors, and resemble the Dll1 secretory precursors and the label-retaining cells of Winton and colleagues. Our findings support the cycling stem cell hypothesis and highlight the cell cycle heterogeneity of early progenitors during lineage commitment., (© 2014 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

Objectives: With the aging of HIV populations, vascular contributions to neuropathogenesis are increasingly important. Indirect analyses of cerebral small vessel disease have been performed, but there have been no direct studies of human brain to elucidate risk factors for arteriolar sclerosis., Methods: Mean arteriolar wall thickness (sclerotic index, SI) was measured in the deep cerebral white matter of 126 brains (96 HIV+, 30 HIV-). Correlations with SI were performed for age, sex, race, hypertension, hyperlipidemia, diabetes, obesity, cirrhosis, hepatitis C virus (HCV) infection, herpes infection, HIV infection, HIV risk, cocaine use, CD4 count, plasma HIV load, and combination antiretroviral therapy (cART) at the time of death., Results: Age, hypertension, race, HCV, and cirrhosis were associated with SI; of the HIV variables, only cART at death was associated with SI. To address colinearity, partial correlations were run with HCV and cirrhosis, hypertension and race, and hypertension and age. With HCV controlled, cirrhosis lost significance; with hypertension controlled, age lost significance. For the entire sample, HCV, African American race, and hypertension accounted for 15% of SI variance in multivariate analysis. Each was independently associated with SI, and HCV had the largest effect. For the HIV sample, inclusion of cART in the model increased R (2) to 0.205, with only HCV, hypertension, and cART remaining significant or trend level., Conclusions: This tissue-based analysis of cerebral arteriolar disease demonstrates that HCV constitutes an independent risk, in addition to African American race, hypertension, and cART. Further study is needed to understand what aspects of HCV and cART contribute to cerebrovascular neuropathogenesis.