A randomized phase 2 study of the 5-HT4 receptor agonist felcisetrag for postoperative gastrointestinal dysfunction after bowel surgery.

BACKGROUND: Felcisetrag (5-hydroxytryptamine-4 receptor [5-HT4] agonist) is under investigation as prophylaxis or active treatment for accelerating resolution of gastrointestinal function post-surgery. METHODS: Phase 2, randomized, placebo-controlled, parallel five-arm, double-blind, multicenter study (NCT03827655) in 209 adults undergoing open or laparoscopic-assisted bowel surgery. Patients received intravenous placebo, felcisetrag 0.1 mg/100 â€‹mL or 0.5 mg/100 â€‹mL pre-surgery only, or pre-surgery and daily post-surgery until return of gastrointestinal function or for up to 10 days. PRIMARY ENDPOINT: time to recovery of gastrointestinal function. RESULTS: Median time to recovery of gastrointestinal function was 2.6 days for both felcisetrag 0.5 â€‹mg daily and 0.5 â€‹mg pre-surgery versus 1.9 days for placebo (p â€‹> â€‹0.05). There were no notable differences in adverse events between treatment arms. CONCLUSIONS: Felcisetrag was well tolerated with no new safety concerns. However, no clinically meaningful difference in time to recovery of gastrointestinal function versus placebo was observed. Further investigation of the utility of 5-HT4 agonists in complicated, open abdominal surgeries may be warranted.

NF-κB Inducing Kinase Attenuates Colorectal Cancer by Regulating Noncanonical NF-κB Mediated Colonic Epithelial Cell Regeneration.

BACKGROUND & AIMS: Dysregulated colonic epithelial cell (CEC) proliferation is a critical feature in the development of colorectal cancer. We show that NF-κB-inducing kinase (NIK) attenuates colorectal cancer through coordinating CEC regeneration/differentiation via noncanonical NF-κB signaling that is unique from canonical NF-κB signaling. METHODS: Initial studies evaluated crypt morphology/functionality, organoid generation, transcriptome profiles, and the microbiome. Inflammation and inflammation-induced tumorigenesis was initiated in whole-body NIK knockout mice (Nik-/-)and conditional-knockout mice following administration of azoxymethane and dextran sulfate sodium (AOM/DSS). RESULTS: Human transcriptomic data revealed dysregulated noncanonical NF-κB signaling. In vitro studies evaluating Nik-/- crypts and organoids derived from mature, nondividing CECs and colonic stem cells (CSCs) exhibited increased accumulation and stunted growth, respectively. Transcriptomic analysis of Nik-/- cells revealed gene expression signatures associated with altered differentiation-regeneration. When assessed in vivo, Nik-/- mice exhibited more severe colitis with DSS administration and an altered microbiome characterized by increased colitogenic microbiota. In the inflammation-induced tumorigenesis model, we observed both increased tumor burdens and inflammation in mice where NIK is knocked out in CECs (NikΔCEC). Interestingly, this was not recapitulated when NIK was conditionally knocked out in myeloid cells (NikΔMYE). Surprisingly, conditional knockout of the canonical pathway in myeloid cells (RelAΔMYE) revealed decreased tumor burden and inflammation and no significant changes when conditionally knocked out in CECs (RelAΔCEC). CONCLUSIONS: Dysregulated noncanonical NF-κB signaling is associated with the development of colorectal cancer in a tissue dependent manner and defines a critical role for NIK in regulating gastrointestinal inflammation and regeneration associated with colorectal cancer.

Out with the old, in with the new: Pioneer transcription factors as activators and repressors of lineage specification genes.

In this issue of Molecular Cell, Matsui et al.1 examine lineage determination by pioneer transcription factors, finding that they control cell fate in cooperation with PRDM family members by repressing alternative-lineage and precocious gene expression through establishment of bivalent enhancers.

Genomic transcription factor binding site selection is edited by the chromatin remodeling factor CHD4.

Biologically precise enhancer licensing by lineage-determining transcription factors enables activation of transcripts appropriate to biological demand and prevents deleterious gene activation. This essential process is challenged by the millions of matches to most transcription factor binding motifs present in many eukaryotic genomes, leading to questions about how transcription factors achieve the exquisite specificity required. The importance of chromatin remodeling factors to enhancer activation is highlighted by their frequent mutation in developmental disorders and in cancer. Here, we determine the roles of CHD4 in enhancer licensing and maintenance in breast cancer cells and during cellular reprogramming. In unchallenged basal breast cancer cells, CHD4 modulates chromatin accessibility. Its depletion leads to redistribution of transcription factors to previously unoccupied sites. During cellular reprogramming induced by the pioneer factor GATA3, CHD4 activity is necessary to prevent inappropriate chromatin opening. Mechanistically, CHD4 promotes nucleosome positioning over GATA3 binding motifs to compete with transcription factor-DNA interaction. We propose that CHD4 acts as a chromatin proof-reading enzyme that prevents unnecessary gene expression by editing chromatin binding activities of transcription factors.

Structural and Dynamic Changes of Nucleosome upon GATA3 Binding.

Pioneer factors, which can directly bind to nucleosomes, have been considered to change chromatin conformations. However, the binding impact on the nucleosome is little known. Here, we show how the pioneer factor GATA3 binds to nucleosomal DNA and affects the conformation and dynamics of nucleosomes by using a combination of SAXS, molecular modeling, and molecular dynamics simulations. Our structural models, consistent with the SAXS data, indicate that only one of the two DNA binding domains, N- and C-fingers, of GATA3 binds to an end of the DNA in solution. Our MD simulations further showed that the other unbound end of the DNA increases the fluctuation and enhances the DNA dissociation from the histone core when the N-finger binds to a DNA end, a site near the entry or exit of the nucleosome. However, this was not true for the binding of the C-finger that binds to a location about 15 base pairs distant from the DNA end. In this case, DNA dissociation occurred on the bound end. Taken together, we suggest that the N-finger and C-finger bindings of GATA3 commonly enhance DNA dissociation at one of the two DNA ends (the bound end for the C-finger binding and the unbound end for the N-finger binding), leading to triggering a conformational change in the chromatin.

Beadchip technology to detect DNA methylation in mouse faithfully recapitulates whole-genome bisulfite sequencing.

Aim: To facilitate wide-scale implementation of Illumina Mouse Methylation BeadChip (MMB) technology, array-based measurement of cytosine methylation was compared with the gold-standard assessment of DNA methylation by whole-genome bisulfite sequencing (WGBS). Methods: DNA methylation across two mouse strains (C57B6 and C3H) and both sexes was assessed using the MMB and compared with previously existing deep-coverage WGBS of mice of the same strain and sex. Results & conclusion: The findings demonstrated that 93.3-99.2% of sites had similar measurements of methylation across technologies and that differentially methylated cytosines and regions identified by each technology overlap and enrich for similar biological functions, suggesting that the MMB faithfully recapitulates the findings of WGBS.

Caloric restriction remodels the hepatic chromatin landscape and bile acid metabolism by modulating the gut microbiota.

BACKGROUND: Caloric restriction (CR) has been known to promote health by reprogramming metabolism, yet little is known about how the epigenome and microbiome respond during metabolic adaptation to CR. RESULTS: We investigate chromatin modifications, gene expression, as well as alterations in microbiota in a CR mouse model. Collectively, short-term CR leads to altered gut microbial diversity and bile acid metabolism, improving energy expenditure. CR remodels the hepatic enhancer landscape at genomic loci that are enriched for binding sites for signal-responsive transcription factors, including HNF4α. These alterations reflect a dramatic reprogramming of the liver transcriptional network, including genes involved in bile acid metabolism. Transferring CR gut microbiota into mice fed with an obesogenic diet recapitulates the features of CR-related bile acid metabolism along with attenuated fatty liver. CONCLUSIONS: These findings suggest that CR-induced microbiota shapes the hepatic epigenome followed by altered expression of genes responsible for bile acid metabolism.

Genomic transcription factor binding site selection is edited by the chromatin remodeling factor CHD4.

Biologically precise enhancer licensing by lineage-determining transcription factors enables activation of transcripts appropriate to biological demand and prevents deleterious gene activation. This essential process is challenged by the millions of matches to most transcription factor binding motifs present in many eukaryotic genomes, leading to questions about how transcription factors achieve the exquisite specificity required. The importance of chromatin remodeling factors to enhancer activation is highlighted by their frequent mutation in developmental disorders and in cancer. Here we determine the roles of CHD4 to enhancer licensing and maintenance in breast cancer cells and during cellular reprogramming. In unchallenged basal breast cancer cells, CHD4 modulates chromatin accessibility at transcription factor binding sites; its depletion leads to altered motif scanning and redistribution of transcription factors to sites not previously occupied. During GATA3-mediated cellular reprogramming, CHD4 activity is necessary to prevent inappropriate chromatin opening and enhancer licensing. Mechanistically, CHD4 competes with transcription factor-DNA interaction by promoting nucleosome positioning over binding motifs. We propose that CHD4 acts as a chromatin proof-reading enzyme that prevents inappropriate gene expression by editing binding site selection by transcription factors.

Multiple tissue-specific epigenetic alterations regulate persistent gene expression changes following developmental DES exposure in mouse reproductive tissues.

Clinically, developmental exposure to the endocrine disrupting chemical, diethylstilboestrol (DES), results in long-term male and female infertility. Experimentally, developmental exposure to DES results in abnormal reproductive tract phenotypes in male and female mice. Previously, we reported that neonatal DES exposure causes ERα-mediated aberrations in the transcriptome and in DNA methylation in seminal vesicles (SVs) of adult mice. However, only a subset of DES-altered genes could be explained by changes in DNA methylation. We hypothesized that alterations in histone modification may also contribute to the altered transcriptome during SV development. To test this idea, we performed a series of genome-wide analyses of mouse SVs at pubertal and adult developmental stages in control and DES-exposed wild-type and ERα knockout mice. Neonatal DES exposure altered ERα-mediated mRNA and lncRNA expression in adult SV, including genes encoding chromatin-modifying proteins that can impact histone H3K27ac modification. H3K27ac patterns, particularly at enhancers, and DNA methylation were reprogrammed over time during normal SV development and after DES exposure. Some of these reprogramming changes were ERα-dependent, but others were ERα-independent. A substantial number of DES-altered genes had differential H3K27ac peaks at nearby enhancers. Comparison of gene expression changes, H3K27ac marks and DNA methylation marks between adult SV and adult uterine tissue from ovariectomized mice neonatally exposed to DES revealed that most of the epigenetic changes and altered genes were distinct in the two tissues. These findings indicate that the effects of developmental DES exposure cause reprogramming of reproductive tract tissue differentiation through multiple epigenetic mechanisms.

Proteins That Read DNA Methylation.

Covalent modification of DNA via deposition of a methyl group at the 5' position on cytosine residues alters the chemical groups available for interaction in the major groove of DNA. This modification, thereby, alters the affinity and specificity of DNA-binding proteins; some of them favor interaction with methylated DNA, and others disfavor it. Molecular recognition of cytosine methylation by proteins often initiates sequential regulatory events that impact gene expression and chromatin structure. The known methyl-DNA-binding proteins have unique domains responsible for DNA methylation recognition: (1) the methyl-CpG-binding domain (MBD), (2) the SET- and RING finger-associated domain (SRA), and (3) some of TF families, such as the C2H2 zinc finger domain, basic helix-loop-helix (bHLH), basic leucine-zipper (bZIP), and homeodomain proteins. Structural analyses have revealed that each domain has a characteristic methylated DNA-binding pattern, and the difference in the recognition mechanisms renders the DNA methylation mark able to transmit complicated biological information. Recent genetic and genomic studies have revealed novel functions of methyl-DNA-binding proteins. These emerging data have also provided glimpses into how methyl-DNA-binding proteins possess unique features and, presumably, functions. In this chapter, we summarize structural and biochemical analyses elucidating the mechanisms for recognition of DNA methylation and correlate this information with emerging genomic and functional data.

NLRX1 Deficiency Alters the Gut Microbiome and Is Further Exacerbated by Adherence to a Gluten-Free Diet.

Patients with gluten sensitivities present with dysbiosis of the gut microbiome that is further exacerbated by a strict adherence to a gluten-free diet (GFD). A subtype of patients genetically susceptible to gluten sensitivities are Celiac Disease (CeD) patients, who are carriers of the HLA DR3/DQ2 or HLA DR4/DQ8 haplotypes. Although 85-95% of all CeD patients carry HLA DQ2, up to 25-50% of the world population carry this haplotype with only a minority developing CeD. This suggests that CeD and other gluten sensitivities are mediated by factors beyond genetics. The contribution of innate immune system signaling has been generally understudied in the context of gluten sensitivities. Thus, here we examined the role of NOD-like receptors (NLRs), a subtype of pattern recognition receptors, in maintaining the composition of the gut microbiome in animals maintained on a GFD. Human transcriptomics data revealed significant increases in the gene expression of multiple NLR family members, across functional groups, in patients with active CeD compared to control specimens. However, NLRX1 was uniquely down-regulated during active disease. NLRX1 is a negative regulatory NLR that functions to suppress inflammatory signaling and has been postulate to prevent inflammation-induced dysbiosis. Using Nlrx1-/- mice maintained on either a normal or gluten-free diet, we show that loss of NLRX1 alters the microbiome composition, and a distinctive shift further ensues following adherence to a GFD, including a reciprocal loss of beneficial microbes and increase in opportunistic bacterial populations. Finally, we evaluated the functional impact of an altered gut microbiome by assessing short- and medium-chain fatty acid production. These studies revealed significant differences in a selection of metabolic markers that when paired with 16S rRNA sequencing data could reflect an overall imbalance and loss of immune system homeostasis in the gastrointestinal system.

Beluga whale (Delphinapterus leucas) acoustic foraging behavior and applications for long term monitoring.

Cook Inlet, Alaska, is home to an endangered and declining population of 279 belugas (Delphinapterus leucas). Recovery efforts highlight a paucity of basic ecological knowledge, impeding the correct assessment of threats and the development of recovery actions. In particular, information on diet and foraging habitat is very limited for this population. Passive acoustic monitoring has proven to be an efficient approach to monitor beluga distribution and seasonal occurrence. Identifying acoustic foraging behavior could help address the current gap in information on diet and foraging habitat. To address this conservation challenge, eight belugas from a comparative, healthy population in Bristol Bay, Alaska, were instrumented with a multi-sensor tag (DTAG), a satellite tag, and a stomach temperature transmitter in August 2014 and May 2016. DTAG deployments provided 129.6 hours of data including foraging and social behavioral states. A total of 68 echolocation click trains ending in terminal buzzes were identified during successful prey chasing and capture, as well as during social interactions. Of these, 37 click trains were successfully processed to measure inter-click intervals (ICI) and ICI trend in their buzzing section. Terminal buzzes with short ICI (minimum ICI <8.98 ms) and consistently decreasing ICI trend (ICI increment range <1.49 ms) were exclusively associated with feeding behavior. This dual metric was applied to acoustic data from one acoustic mooring within the Cook Inlet beluga critical habitat as an example of the application of detecting feeding in long-term passive acoustic monitoring data. This approach allowed description of the relationship between beluga presence, feeding occurrence, and the timing of spawning runs by different species of anadromous fish. Results reflected a clear preference for the Susitna River delta during eulachon (Thaleichthys pacificus), Chinook (Oncorhynchus tshawytscha), pink (Oncorhynchus gorbuscha), and coho (Oncorhynchus kisutch) salmon spawning run periods, with increased feeding occurrence at the peak of the Chinook and pink salmon runs.

Low-input ATAC&mRNA-seq protocol for simultaneous profiling of chromatin accessibility and gene expression.

We present a simple, fast, and robust protocol (low-input ATAC&mRNA-seq) to simultaneously generate ATAC-seq and mRNA-seq libraries from the same cells in limited cell numbers by coupling a simplified ATAC procedure using whole cells with a novel mRNA-seq approach that features a seamless on-bead process including direct mRNA isolation from the cell lysate, solid-phase cDNA synthesis, and direct tagmentation of mRNA/cDNA hybrids for library preparation. It enables dual-omics profiling from limited material when joint epigenome and transcriptome analyses are needed. For complete details on the use and execution of this protocol, please refer to Li et al. (2021).

A simple and robust method for simultaneous dual-omics profiling with limited numbers of cells.

Deciphering epigenetic regulation of gene expression requires measuring the epigenome and transcriptome jointly. Single-cell multi-omics technologies have been developed for concurrent profiling of chromatin accessibility and gene expression. However, multi-omics profiling of low-input bulk samples remains challenging. Therefore, we developed low-input ATAC&mRNA-seq, a simple and robust method for studying the role of chromatin structure in gene regulation in a single experiment with thousands of cells, to maximize insights from limited input material by obtaining ATAC-seq and mRNA-seq data simultaneously from the same cells with data quality comparable to that of conventional mono-omics assays. Integrative data analysis revealed similar strong association between promoter accessibility and gene expression when using the data of low-input ATAC&mRNA-seq as when using single-assay data, underscoring the accuracy and reliability of our dual-omics assay to generate both datum types simultaneously with just thousands of cells. We envision our method to be widely applied in many biological disciplines with limited materials.

Runs of homozygosity in killer whale genomes provide a global record of demographic histories.

Runs of homozygosity (ROH) occur when offspring inherit haplotypes that are identical by descent from each parent. Length distributions of ROH are informative about population history; specifically, the probability of inbreeding mediated by mating system and/or population demography. Here, we investigated whether variation in killer whale (Orcinus orca) demographic history is reflected in genome-wide heterozygosity and ROH length distributions, using a global data set of 26 genomes representative of geographic and ecotypic variation in this species, and two F1 admixed individuals with Pacific-Atlantic parentage. We first reconstructed demographic history for each population as changes in effective population size through time using the pairwise sequential Markovian coalescent (PSMC) method. We found a subset of populations declined in effective population size during the Late Pleistocene, while others had more stable demography. Genomes inferred to have undergone ancestral declines in effective population size, were autozygous at hundreds of short ROH (<1 Mb), reflecting high background relatedness due to coalescence of haplotypes deep within the pedigree. In contrast, longer and therefore younger ROH (>1.5 Mb) were found in low latitude populations, and populations of known conservation concern. These include a Scottish killer whale, for which 37.8% of the autosomes were comprised of ROH >1.5 Mb in length. The fate of this population, in which only two adult males have been sighted in the past five years, and zero fecundity over the last two decades, may be inextricably linked to its demographic history and consequential inbreeding depression.

The estrogen receptor/GATA3/FOXA1 transcriptional network: lessons learned from breast cancer.

Cellular identity and physiologic function in mammary epithelial cells and in many breast cancers flow from the action of a network of master transcriptional regulators including estrogen receptor alpha, GATA3, and FOXA1. The last decade has seen the completion of multiple large sequencing projects focusing on breast cancer. These massive compendia of sequence data have provided a wealth of new information linking mutation in these transcription factors to alterations in tumor biology and transcriptional program. The emerging details on mutation in cancer, and direct experimental exploration of hypotheses based on it, are now providing a wealth of new information on the roles played by estrogen receptor alpha, GATA3, and FOXA1 in regulating gene transcription and how their combined action contributes to a network shaping cell function in both physiologic and disease states.

An epigenetic clock to estimate the age of living beluga whales.

DNA methylation data facilitate the development of accurate molecular estimators of chronological age or "epigenetic clocks." We present a robust epigenetic clock for the beluga whale, Delphinapterus leucas, developed for an endangered population in Cook Inlet, Alaska, USA. We used a custom methylation array to measure methylation levels at 37,491 cytosine-guanine sites (CpGs) from skin samples of dead whales (n = 67) whose chronological ages were estimated based on tooth growth layer groups. Using these calibration data, a penalized regression model selected 23 CpGs, providing an R 2 = 0.92 for the training data; and an R 2 = 0.74 and median absolute age error = 2.9 years for the leave one out cross-validation. We applied the epigenetic clock to an independent dataset of 38 skin samples collected with a biopsy dart from living whales between 2016 and 2018. Age estimates ranged from 11 to 27 years. We also report sex correlations in CpG data and describe an approach of identifying the sex of an animal using DNA methylation. The epigenetic estimators of age and sex presented here have broad applications for conservation and management of Cook Inlet beluga whales and potentially other cetaceans.

DNMT1 reads heterochromatic H4K20me3 to reinforce LINE-1 DNA methylation.

DNA methylation and trimethylated histone H4 Lysine 20 (H4K20me3) constitute two important heterochromatin-enriched marks that frequently cooperate in silencing repetitive elements of the mammalian genome. However, it remains elusive how these two chromatin modifications crosstalk. Here, we report that DNA methyltransferase 1 (DNMT1) specifically 'recognizes' H4K20me3 via its first bromo-adjacent-homology domain (DNMT1BAH1). Engagement of DNMT1BAH1-H4K20me3 ensures heterochromatin targeting of DNMT1 and DNA methylation at LINE-1 retrotransposons, and cooperates with the previously reported readout of histone H3 tail modifications (i.e., H3K9me3 and H3 ubiquitylation) by the RFTS domain to allosterically regulate DNMT1's activity. Interplay between RFTS and BAH1 domains of DNMT1 profoundly impacts DNA methylation at both global and focal levels and genomic resistance to radiation-induced damage. Together, our study establishes a direct link between H4K20me3 and DNA methylation, providing a mechanism in which multivalent recognition of repressive histone modifications by DNMT1 ensures appropriate DNA methylation patterning and genomic stability.

A deepening understanding of animal culture suggests lessons for conservation.

A key goal of conservation is to protect biodiversity by supporting the long-term persistence of viable, natural populations of wild species. Conservation practice has long been guided by genetic, ecological and demographic indicators of risk. Emerging evidence of animal culture across diverse taxa and its role as a driver of evolutionary diversification, population structure and demographic processes may be essential for augmenting these conventional conservation approaches and decision-making. Animal culture was the focus of a ground-breaking resolution under the Convention on the Conservation of Migratory Species of Wild Animals (CMS), an international treaty operating under the UN Environment Programme. Here, we synthesize existing evidence to demonstrate how social learning and animal culture interact with processes important to conservation management. Specifically, we explore how social learning might influence population viability and be an important resource in response to anthropogenic change, and provide examples of how it can result in phenotypically distinct units with different, socially learnt behavioural strategies. While identifying culture and social learning can be challenging, indirect identification and parsimonious inferences may be informative. Finally, we identify relevant methodologies and provide a framework for viewing behavioural data through a cultural lens which might provide new insights for conservation management.