Patterning and folding of intestinal villi by active mesenchymal dewetting.

Tissue folds are structural motifs critical to organ function. In the intestine, bending of a flat epithelium into a periodic pattern of folds gives rise to villi, finger-like protrusions that enable nutrient absorption. However, the molecular and mechanical processes driving villus morphogenesis remain unclear. Here, we identify an active mechanical mechanism that simultaneously patterns and folds the intestinal epithelium to initiate villus formation. At the cellular level, we find that PDGFRA+ subepithelial mesenchymal cells generate myosin II-dependent forces sufficient to produce patterned curvature in neighboring tissue interfaces. This symmetry-breaking process requires altered cell and extracellular matrix interactions that are enabled by matrix metalloproteinase-mediated tissue fluidization. Computational models, together with in vitro and in vivo experiments, revealed that these cellular features manifest at the tissue level as differences in interfacial tensions that promote mesenchymal aggregation and interface bending through a process analogous to the active dewetting of a thin liquid film.

Genome methylation in D. melanogaster is found at specific short motifs and is independent of DNMT2 activity.

Cytosine methylation in the genome of Drosophila melanogaster has been elusive and controversial: Its location and function have not been established. We have used a novel and highly sensitive genomewide cytosine methylation assay to detect and map genome methylation in stage 5 Drosophila embryos. The methylation we observe with this method is highly localized and strand asymmetrical, limited to regions covering ∼1% of the genome, dynamic in early embryogenesis, and concentrated in specific 5-base sequence motifs that are CA- and CT-rich but depleted of guanine. Gene body methylation is associated with lower expression, and many genes containing methylated regions have developmental or transcriptional functions. The only known DNA methyltransferase in Drosophila is the DNMT2 homolog MT2, but lines deficient for MT2 retain genomic methylation, implying the presence of a novel methyltransferase. The association of methylation with a lower expression of specific developmental genes at stage 5 raises the possibility that it participates in controlling gene expression during the maternal-zygotic transition.

Statin-induced changes in gene expression in EBV-transformed and native B-cells.

Human lymphoblastoid cell lines (LCLs), generated through Epstein-Barr Virus (EBV) transformation of B-lymphocytes (B-cells), are a commonly used model system for identifying genetic influences on human diseases and on drug responses. We have previously used LCLs to examine the cellular effects of genetic variants that modulate the efficacy of statins, the most prescribed class of cholesterol-lowering drugs used for the prevention and treatment of cardiovascular disease. However, statin-induced gene expression differences observed in LCLs may be influenced by their transformation, and thus differ from those observed in native B-cells. To assess this possibility, we prepared LCLs and purified B-cells from the same donors, and compared mRNA profiles after 24 h incubation with simvastatin (2 µm) or sham buffer. Genes involved in cholesterol metabolism were similarly regulated between the two cell types under both the statin and sham-treated conditions, and the statin-induced changes were significantly correlated. Genes whose expression differed between the native and transformed cells were primarily implicated in cell cycle, apoptosis and alternative splicing. We found that ChIP-seq signals for MYC and EBNA2 (an EBV transcriptional co-activator) were significantly enriched in the promoters of genes up-regulated in the LCLs compared with the B-cells, and could be involved in the regulation of cell cycle and alternative splicing. Taken together, the results support the use of LCLs for the study of statin effects on cholesterol metabolism, but suggest that drug effects on cell cycle, apoptosis and alternative splicing may be affected by EBV transformation. This dataset is now uploaded to GEO at the accession number GSE51444.

Now you see it: genome methylation makes a comeback in Drosophila.

Drosophila melanogaster is often considered to lack genomic 5-methylcytosine (m(5) C), an opinion reinforced by two whole genome bisulfite-sequencing studies that failed to find m(5) C. New evidence, however, indicates that genomic methylation is indeed present in the fly, albeit in small quantities and in unusual patterns. At embryonic stage 5, m(5) C occurs in short strand-specific regions that cover ∼1% of the genome, at tissue levels suggesting a distribution restricted to a subset of nuclei. Its function is not obvious, but methylation in subsets of nuclei would obscure functional associations since transcript levels and epigenetic modifications are assayed in whole embryos. Surprisingly, Mt2, the fly's only candidate DNA methyltransferase, is not necessary for the observed methylation. Full evaluation of the functions of genome methylation in Drosophila must await discovery and experimental inactivation of the DNA methyltransferase, as well as a better understanding of the pattern and developmental regulation of genomic m(5) C.

Activation-induced cytidine deaminase (AID) is necessary for the epithelial-mesenchymal transition in mammary epithelial cells.

Activation-induced cytidine deaminase (AID), which functions in antibody diversification, is also expressed in a variety of germ and somatic cells. Evidence that AID promotes DNA demethylation in epigenetic reprogramming phenomena, and that it is induced by inflammatory signals, led us to investigate its role in the epithelial-mesenchymal transition (EMT), a critical process in normal morphogenesis and tumor metastasis. We find that expression of AID is induced by inflammatory signals that induce the EMT in nontransformed mammary epithelial cells and in ZR75.1 breast cancer cells. shRNA-mediated knockdown of AID blocks induction of the EMT and prevents cells from acquiring invasive properties. Knockdown of AID suppresses expression of several key EMT transcriptional regulators and is associated with increased methylation of CpG islands proximal to the promoters of these genes; furthermore, the DNA demethylating agent 5 aza-2'deoxycytidine (5-Aza-dC) antagonizes the effects of AID knockdown on the expression of EMT factors. We conclude that AID is necessary for the EMT in this breast cancer cell model and in nontransformed mammary epithelial cells. Our results suggest that AID may act near the apex of a hierarchy of regulatory steps that drive the EMT, and are consistent with this effect being mediated by cytosine demethylation. This evidence links our findings to other reports of a role for AID in epigenetic reprogramming and control of gene expression.

piRNA-like small RNAs mark extended 3'UTRs present in germ and somatic cells.

BACKGROUND: Piwi-interacting RNAs (piRNAs) are a class of small RNAs; distinct types of piRNAs are expressed in the mammalian testis at different stages of development. The function of piRNAs expressed in the adult testis is not well established. We conducted a detailed characterization of piRNAs aligning at or near the 3' UTRs of protein-coding genes in a deep dataset of small RNAs from adult mouse testis. RESULTS: We identified 2710 piRNA clusters associated with 3' UTRs, including 1600 that overlapped genes not previously associated with piRNAs. 35% of the clusters extend beyond the annotated transcript; we find that these clusters correspond to, and are likely derived from, novel polyadenylated mRNA isoforms that contain previously unannotated extended 3'UTRs. Extended 3' UTRs, and small RNAs derived from them, are also present in somatic tissues; a subset of these somatic 3'UTR small RNA clusters are absent in mice lacking MIWI2, indicating a role for MIWI2 in the metabolism of somatic small RNAs. CONCLUSIONS: The finding that piRNAs are processed from extended 3' UTRs suggests a role for piRNAs in the remodeling of 3' UTRs. The presence of both clusters and extended 3'UTRs in somatic cells, with evidence for involvement of MIWI2, indicates that this pathway is more broadly distributed than currently appreciated.

Functionally conserved enhancers with divergent sequences in distant vertebrates.

BACKGROUND: To examine the contributions of sequence and function conservation in the evolution of enhancers, we systematically identified enhancers whose sequences are not conserved among distant groups of vertebrate species, but have homologous function and are likely to be derived from a common ancestral sequence. Our approach combined comparative genomics and epigenomics to identify potential enhancer sequences in the genomes of three groups of distantly related vertebrate species. RESULTS: We searched for sequences that were conserved within groups of closely related species but not between groups of more distant species, and were associated with an epigenetic mark of enhancer activity. To facilitate inferring orthology between non-conserved sequences, we limited our search to introns whose orthology could be unambiguously established by mapping the bracketing exons. We show that a subset of these non-conserved but syntenic sequences from the mouse and zebrafish genomes have homologous functions in a zebrafish transgenic enhancer assay. The conserved expression patterns driven by these enhancers are probably associated with short transcription factor-binding motifs present in the divergent sequences. CONCLUSIONS: We have identified numerous potential enhancers with divergent sequences but a conserved function. These results indicate that selection on function, rather than sequence, may be a common mode of enhancer evolution; evidence for selection at the sequence level is not a necessary criterion to define a gene regulatory element.

Phyloepigenomic comparison of great apes reveals a correlation between somatic and germline methylation states.

We have determined methylation state differences in the epigenomes of uncultured cells purified from human, chimpanzee, and orangutan, using digestion with a methylation-sensitive enzyme, deep sequencing, and computational analysis of the sequence data. The methylomes show a high degree of conservation, but the methylation states of ~10% of CpG island-like regions differ significantly between human and chimp. The differences are not associated with changes in CG content and recapitulate the known phylogenetic relationship of the three species, indicating that they are stably maintained within each species. Inferences about the relationship between somatic and germline methylation states can be made by an analysis of CG decay, derived from methylation and sequence data. This indicates that somatic methylation states are highly related to germline states and that the methylation differences between human and chimp have occurred in the germline. These results provide evidence for epigenetic changes that occur in the germline and distinguish closely related species and suggest that germline epigenetic states might constrain somatic states.

Epithelial zonation along the mouse and human small intestine defines five discrete metabolic domains.

A key aspect of nutrient absorption is the exquisite division of labour across the length of the small intestine, with individual nutrients taken up at different proximal:distal positions. For millennia, the small intestine was thought to comprise three segments with indefinite borders: the duodenum, jejunum and ileum. By examining the fine-scale longitudinal transcriptional patterns that span the mouse and human small intestine, we instead identified five domains of nutrient absorption that mount distinct responses to dietary changes, and three regional stem cell populations. Molecular domain identity can be detected with machine learning, which provides a systematic method to computationally identify intestinal domains in mice. We generated a predictive model of transcriptional control of domain identity and validated the roles of Ppar-δ and Cdx1 in patterning lipid metabolism-associated genes. These findings represent a foundational framework for the zonation of absorption across the mammalian small intestine.

5'-YRNA fragments derived by processing of transcripts from specific YRNA genes and pseudogenes are abundant in human serum and plasma.

Small noncoding RNAs carry out a variety of functions in eukaryotic cells, and in multiple species they can travel between cells, thus serving as signaling molecules. In mammals multiple small RNAs have been found to circulate in the blood, although in most cases the targets of these RNAs, and even their functions, are not well understood. YRNAs are small (84-112 nt) RNAs with poorly characterized functions, best known because they make up part of the Ro ribonucleoprotein autoantigens in connective tissue diseases. In surveying small RNAs present in the serum of healthy adult humans, we have found YRNA fragments of lengths 27 nt and 30-33 nt, derived from the 5'-ends of specific YRNAs and generated by cleavage within a predicted internal loop. Many of the YRNAs from which these fragments are derived were previously annotated only as pseudogenes, or predicted informatically. These 5'-YRNA fragments make up a large proportion of all small RNAs (including miRNAs) present in human serum. They are also present in plasma, are not present in exosomes or microvesicles, and circulate as part of a complex with a mass between 100 and 300 kDa. Mouse serum contains far fewer 5'-YRNA fragments, possibly reflecting the much greater copy number of YRNA genes and pseudogenes in humans. The function of the 5'-YRNA fragments is at present unknown, but the processing and secretion of specific YRNAs to produce 5'-end fragments that circulate in stable complexes are consistent with a signaling function.

5' tRNA halves are present as abundant complexes in serum, concentrated in blood cells, and modulated by aging and calorie restriction.

BACKGROUND: Small RNAs complex with proteins to mediate a variety of functions in animals and plants. Some small RNAs, particularly miRNAs, circulate in mammalian blood and may carry out a signaling function by entering target cells and modulating gene expression. The subject of this study is a set of circulating 30-33 nt RNAs that are processed derivatives of the 5' ends of a small subset of tRNA genes, and closely resemble cellular tRNA derivatives (tRFs, tiRNAs, half-tRNAs, 5' tRNA halves) previously shown to inhibit translation initiation in response to stress in cultured cells. RESULTS: In sequencing small RNAs extracted from mouse serum, we identified abundant 5' tRNA halves derived from a small subset of tRNAs, implying that they are produced by tRNA type-specific biogenesis and/or release. The 5' tRNA halves are not in exosomes or microvesicles, but circulate as particles of 100-300 kDa. The size of these particles suggest that the 5' tRNA halves are a component of a macromolecular complex; this is supported by the loss of 5' tRNA halves from serum or plasma treated with EDTA, a chelating agent, but their retention in plasma anticoagulated with heparin or citrate. A survey of somatic tissues reveals that 5' tRNA halves are concentrated within blood cells and hematopoietic tissues, but scant in other tissues, suggesting that they may be produced by blood cells. Serum levels of specific subtypes of 5' tRNA halves change markedly with age, either up or down, and these changes can be prevented by calorie restriction. CONCLUSIONS: We demonstrate that 5' tRNA halves circulate in the blood in a stable form, most likely as part of a nucleoprotein complex, and their serum levels are subject to regulation by age and calorie restriction. They may be produced by blood cells, but their cellular targets are not yet known. The characteristics of these circulating molecules, and their known function in suppression of translation initiation, suggest that they are a novel form of signaling molecule.

Graded BMP signaling within intestinal crypt architecture directs self-organization of the Wnt-secreting stem cell niche.

Signals from the surrounding niche drive proliferation and suppress differentiation of intestinal stem cells (ISCs) at the bottom of intestinal crypts. Among sub-epithelial support cells, deep sub-cryptal CD81+ PDGFRAlo trophocytes capably sustain ISC functions ex vivo. Here, we show that mRNA and chromatin profiles of abundant CD81- PDGFRAlo mouse stromal cells resemble those of trophocytes and that both populations provide crucial canonical Wnt ligands. Mesenchymal expression of key ISC-supportive factors extends along a spatial and molecular continuum from trophocytes into peri-cryptal CD81- CD55hi cells, which mimic trophocyte activity in organoid co-cultures. Graded expression of essential niche factors is not cell-autonomous but dictated by the distance from bone morphogenetic protein (BMP)-secreting PDGFRAhi myofibroblast aggregates. BMP signaling inhibits ISC-trophic genes in PDGFRAlo cells near high crypt tiers; that suppression is relieved in stromal cells near and below the crypt base, including trophocytes. Cell distances thus underlie a self-organized and polar ISC niche.

Epithelial TNF controls cell differentiation and CFTR activity to maintain intestinal mucin homeostasis.

The gastrointestinal tract relies on the production, maturation, and transit of mucin to protect against pathogens and to lubricate the epithelial lining. Although the molecular and cellular mechanisms that regulate mucin production and movement are beginning to be understood, the upstream epithelial signals that contribute to mucin regulation remain unclear. Here, we report that the inflammatory cytokine tumor necrosis factor (TNF), generated by the epithelium, contributes to mucin homeostasis by regulating both cell differentiation and cystic fibrosis transmembrane conductance regulator (CFTR) activity. We used genetic mouse models and noninflamed samples from patients with inflammatory bowel disease (IBD) undergoing anti-TNF therapy to assess the effect of in vivo perturbation of TNF. We found that inhibition of epithelial TNF promotes the differentiation of secretory progenitor cells into mucus-producing goblet cells. Furthermore, TNF treatment and CFTR inhibition in intestinal organoids demonstrated that TNF promotes ion transport and luminal flow via CFTR. The absence of TNF led to slower gut transit times, which we propose results from increased mucus accumulation coupled with decreased luminal fluid pumping. These findings point to a TNF/CFTR signaling axis in the adult intestine and identify epithelial cell-derived TNF as an upstream regulator of mucin homeostasis.

Patterning and folding of intestinal villi by active mesenchymal dewetting.

Tissue folding generates structural motifs critical to organ function. In the intestine, bending of a flat epithelium into a periodic pattern of folds gives rise to villi, the numerous finger-like protrusions that are essential for nutrient absorption. However, the molecular and mechanical mechanisms driving the initiation and morphogenesis of villi remain a matter of debate. Here, we identify an active mechanical mechanism that simultaneously patterns and folds intestinal villi. We find that PDGFRA+ subepithelial mesenchymal cells generate myosin II-dependent forces sufficient to produce patterned curvature in neighboring tissue interfaces. At the cell-level, this occurs through a process dependent upon matrix metalloproteinase-mediated tissue fluidization and altered cell-ECM adhesion. By combining computational models with in vivo experiments, we reveal these cellular features manifest at the tissue-level as differences in interfacial tensions that promote mesenchymal aggregation and interface bending through a process analogous to the active de-wetting of a thin liquid film.

Epithelial zonation along the mouse and human small intestine defines five discrete metabolic domains.

A key aspect of nutrient absorption is the exquisite division of labor across the length of the small intestine, with individual classes of micronutrients taken up at different positions. For millennia, the small intestine was thought to comprise three segments with indefinite borders: the duodenum, jejunum, and ileum. By examining fine-scale longitudinal segmentation of the mouse and human small intestines, we identified transcriptional signatures and upstream regulatory factors that define five domains of nutrient absorption, distinct from the three traditional sections. Spatially restricted expression programs were most prominent in nutrient-absorbing enterocytes but initially arose in intestinal stem cells residing in three regional populations. While a core signature was maintained across mice and humans with different diets and environments, domain properties were influenced by dietary changes. We established the functions of Ppar-ẟ and Cdx1 in patterning lipid metabolism in distal domains and generated a predictive model of additional transcription factors that direct domain identity. Molecular domain identity can be detected with machine learning, representing the first systematic method to computationally identify specific intestinal regions in mice. These findings provide a foundational framework for the identity and control of longitudinal zonation of absorption along the proximal:distal small intestinal axis.

mRNA-Seq reveals complex patterns of gene regulation and expression in the mouse skeletal muscle transcriptome associated with calorie restriction.

Sarcopenia is an age-associated loss of skeletal muscle mass and strength that increases the risk of disability. Calorie restriction (CR), the consumption of fewer calories while maintaining adequate nutrition, mitigates sarcopenia and many other age-related diseases. To identify potential mechanisms by which CR preserves skeletal muscle integrity during aging, we used mRNA-Seq for deep characterization of gene regulation and mRNA abundance in skeletal muscle of old mice compared with old mice subjected to CR. mRNA-Seq revealed complex CR-associated changes in expression of mRNA isoforms, many of which occur without a change in total message abundance and thus would not be detected by methods other than mRNA-Seq. Functional annotation of differentially expressed genes reveals CR-associated upregulation of pathways involved in energy metabolism and lipid biosynthesis, and downregulation of pathways mediating protein breakdown and oxidative stress, consistent with earlier microarray-based studies. CR-associated changes not noted in previous studies involved downregulation of genes controlling actin cytoskeletal structures and muscle development. These CR-associated changes reflect generally healthier muscle, consistent with CR's mitigation of sarcopenia. mRNA-Seq generates a rich picture of the changes in gene expression associated with CR, and may facilitate identification of genes that are primary mediators of CR's effects.

VISTA Region Viewer (RViewer)--a computational system for prioritizing genomic intervals for biomedical studies.

SUMMARY: Current genome browsers are designed for linear browsing of individual genomic regions, but the high-throughput nature of experiments aiming to elucidate the genetic component of human disease makes it very important to develop user-friendly tools for comparing several genomic regions in parallel and prioritizing them based on their functional content. We introduce VISTA Region Viewer (RViewer), an interactive online tool that allows for efficient screening and prioritization of regions of the human genome for follow-up studies. The tool takes as input genetic variation data from different biomedical studies, determines a number of various functional parameters for both coding and non-coding sequences in each region and allows for sorting and searching the results of the analysis in multiple ways. AVAILABILITY AND IMPLEMENTATION: The tool is implemented as a web application and is freely accessible on the Web at http://rviewer.lbl.gov CONTACT: rviewer@lbl.gov; ildubchak@lbl.gov.

Symbiosis insights through metagenomic analysis of a microbial consortium.

Symbioses between bacteria and eukaryotes are ubiquitous, yet our understanding of the interactions driving these associations is hampered by our inability to cultivate most host-associated microbes. Here we use a metagenomic approach to describe four co-occurring symbionts from the marine oligochaete Olavius algarvensis, a worm lacking a mouth, gut and nephridia. Shotgun sequencing and metabolic pathway reconstruction revealed that the symbionts are sulphur-oxidizing and sulphate-reducing bacteria, all of which are capable of carbon fixation, thus providing the host with multiple sources of nutrition. Molecular evidence for the uptake and recycling of worm waste products by the symbionts suggests how the worm could eliminate its excretory system, an adaptation unique among annelid worms. We propose a model that describes how the versatile metabolism within this symbiotic consortium provides the host with an optimal energy supply as it shuttles between the upper oxic and lower anoxic coastal sediments that it inhabits.

Lymphangiocrine signals are required for proper intestinal repair after cytotoxic injury.

The intestinal epithelium undergoes continuous renewal and has an exceptional capacity to regenerate after injury. Maintenance and proliferation of intestinal stem cells (ISCs) are regulated by their surrounding niche, largely through Wnt signaling. However, it remains unclear which niche cells produce signals during different injury states, and the role of endothelial cells (ECs) as a component of the ISC niche during homeostasis and after injury has been underappreciated. Here, we show that lymphatic endothelial cells (LECs) reside in proximity to crypt epithelial cells and secrete molecules that support epithelial renewal and repair. LECs are an essential source of Wnt signaling in the small intestine, as loss of LEC-derived Rspo3 leads to a lower number of stem and progenitor cells and hinders recovery after cytotoxic injury. Together, our findings identify LECs as an essential niche component for optimal intestinal recovery after cytotoxic injury.

High-level correction of the sickle mutation is amplified in vivo during erythroid differentiation.

Background: A point mutation in sickle cell disease (SCD) alters one amino acid in the ß-globin subunit of hemoglobin, with resultant anemia and multiorgan damage that typically shortens lifespan by decades. Because SCD is caused by a single mutation, and hematopoietic stem cells (HSCs) can be harvested, manipulated, and returned to an individual, it is an attractive target for gene correction. Results: An optimized Cas9 ribonucleoprotein (RNP) with an ssDNA oligonucleotide donor together generated correction of at least one ß-globin allele in more than 30% of long-term engrafting human HSCs. After adopting a high-fidelity Cas9 variant, efficient correction with minimal off-target events also was observed. In vivo erythroid differentiation markedly enriches for corrected ß-globin alleles, indicating that erythroblasts carrying one or more corrected alleles have a survival advantage. Significance: These findings indicate that the sickle mutation can be corrected in autologous HSCs with an optimized protocol suitable for clinical translation.