Longitudinal Multi-omics Reveals Subset-Specific Mechanisms Underlying Irritable Bowel Syndrome.

The gut microbiome has been implicated in multiple human chronic gastrointestinal (GI) disorders. Determining its mechanistic role in disease has been difficult due to apparent disconnects between animal and human studies and lack of an integrated multi-omics view of disease-specific physiological changes. We integrated longitudinal multi-omics data from the gut microbiome, metabolome, host epigenome, and transcriptome in the context of irritable bowel syndrome (IBS) host physiology. We identified IBS subtype-specific and symptom-related variation in microbial composition and function. A subset of identified changes in microbial metabolites correspond to host physiological mechanisms that are relevant to IBS. By integrating multiple data layers, we identified purine metabolism as a novel host-microbial metabolic pathway in IBS with translational potential. Our study highlights the importance of longitudinal sampling and integrating complementary multi-omics data to identify functional mechanisms that can serve as therapeutic targets in a comprehensive treatment strategy for chronic GI diseases. VIDEO ABSTRACT.

RPA Interacts with HIRA and Regulates H3.3 Deposition at Gene Regulatory Elements in Mammalian Cells.

The histone chaperone HIRA is involved in depositing histone variant H3.3 into distinct genic regions, including promoters, enhancers, and gene bodies. However, how HIRA deposits H3.3 to these regions remains elusive. Through a short hairpin RNA (shRNA) screening, we identified single-stranded DNA binding protein replication protein A (RPA) as a regulator of the deposition of newly synthesized H3.3 into chromatin. We show that RPA physically interacts with HIRA to form RPA-HIRA-H3.3 complexes, and it co-localizes with HIRA and H3.3 at gene promoters and enhancers. Depletion of RPA1, the largest subunit of the RPA complex, dramatically reduces both HIRA association with chromatin and the deposition of newly synthesized H3.3 at promoters and enhancers and leads to altered transcription at gene promoters. These results support a model whereby RPA, best known for its role in DNA replication and repair, recruits HIRA to promoters and enhancers and regulates deposition of newly synthesized H3.3 to these regulatory elements for gene regulation.

Hypoxia-Inducible Factor 1α Stabilization Restores Epigenetic Control of Nitric Oxide Synthase 1 Expression and Reverses Gastroparesis in Female Diabetic Mice.

BACKGROUND & AIMS: Although depletion of neuronal nitric oxide synthase (NOS1)-expressing neurons contributes to gastroparesis, stimulating nitrergic signaling is not an effective therapy. We investigated whether hypoxia-inducible factor 1α (HIF1A), which is activated by high O2 consumption in central neurons, is a Nos1 transcription factor in enteric neurons and whether stabilizing HIF1A reverses gastroparesis. METHODS: Mice with streptozotocin-induced diabetes, human and mouse tissues, NOS1+ mouse neuroblastoma cells, and isolated nitrergic neurons were studied. Gastric emptying of solids and volumes were determined by breath test and single-photon emission computed tomography, respectively. Gene expression was analyzed by RNA-sequencing, microarrays, immunoblotting, and immunofluorescence. Epigenetic assays included chromatin immunoprecipitation sequencing (13 targets), chromosome conformation capture sequencing, and reporter assays. Mechanistic studies used Cre-mediated recombination, RNA interference, and clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-mediated epigenome editing. RESULTS: HIF1A signaling from physiological intracellular hypoxia was active in mouse and human NOS1+ myenteric neurons but reduced in diabetes. Deleting Hif1a in Nos1-expressing neurons reduced NOS1 protein by 50% to 92% and delayed gastric emptying of solids in female but not male mice. Stabilizing HIF1A with roxadustat (FG-4592), which is approved for human use, restored NOS1 and reversed gastroparesis in female diabetic mice. In nitrergic neurons, HIF1A up-regulated Nos1 transcription by binding and activating proximal and distal cis-regulatory elements, including newly discovered super-enhancers, facilitating RNA polymerase loading and pause-release, and by recruiting cohesin to loop anchors to alter chromosome topology. CONCLUSIONS: Pharmacologic HIF1A stabilization is a novel, translatable approach to restoring nitrergic signaling and treating diabetic gastroparesis. The newly recognized effects of HIF1A on chromosome topology may provide insights into physioxia- and ischemia-related organ function.

Rising Obesity-Associated Mortality in Men: Exploration of Gender Disparity from the Global Burden of Disease Study, 1990-2019.

OBJECTIVES: The global rise in overweight, obesity, and related diseases is undeniable; however, the pathogenesis of obesity and obesity-associated diseases is heterogeneous, with varied complications and a discordant response to treatment. Intriguingly, men have a shorter lifespan than women, despite being half as likely to be obese. This paradox suggests a potential gender disparity in the impact of obesity on mortality, with men potentially being more vulnerable to obesity-associated health risks. METHODS: This retrospective study utilized Global Burden of Diseases data from 204 countries/territories to bridge the knowledge gap in understanding gender disparities in obesity-related mortality. Outcomes were obesity-associated mortality, years of life lost, years lived with disability, and disability-adjusted life years (DALYs). RESULTS: In 2019, the global overweight/obesity-related disease burden reached 160.2 million DALYs, with 5.02 million associated deaths. From 1990 to 2019, the age-standardized death rates increased in males (from 58.19 to 66.55 per 100,000 person-years, APC = 0.36%, 95% CI: 0.30 to 0.42%, P < 0.001), while females experienced a decrease in age-standardized death rates (from 59.31 to 58.14 per 100,000 person-years, APC = -0.22%, 95% CI: -0.29% to -0.14%, P < 0.001). Age-standardized DALYs increased more in males (1632.5 to 2070.34 per 100,000 years, APC = 0.74%, 95% CI: 0.70% to 0.78%, P < .001) compared to females (1618.26 to 1789.67 per 100,000 years, APC = 0.24%, 95% CI: 0.19% to 0.29%, P < 0.001). Disparities were more pronounced in countries with a higher socioeconomic status and predominantly affected younger populations. CONCLUSIONS: Overweight/obesity-related morbidity and mortality are higher among male sex. Identifying differences in pathogenesis, complications and treatment response is crucial to develop targeted interventions and equitable public health policies to combat this global burden.

Glucocorticoids unmask silent non-coding genetic risk variants for common diseases.

Understanding the function of non-coding genomic sequence variants represents a challenge for biomedicine. Many diseases are products of gene-by-environment interactions with complex mechanisms. This study addresses these themes by mechanistic characterization of non-coding variants that influence gene expression only after drug or hormone exposure. Using glucocorticoid signaling as a model system, we integrated genomic, transcriptomic, and epigenomic approaches to unravel mechanisms by which variant function could be revealed by hormones or drugs. Specifically, we identified cis-regulatory elements and 3D interactions underlying ligand-dependent associations between variants and gene expression. One-quarter of the glucocorticoid-modulated variants that we identified had already been associated with clinical phenotypes. However, their affected genes were 'unmasked' only after glucocorticoid exposure and often with function relevant to the disease phenotypes. These diseases involved glucocorticoids as risk factors or therapeutic agents and included autoimmunity, metabolic and mood disorders, osteoporosis and cancer. For example, we identified a novel breast cancer risk gene, MAST4, with expression that was repressed by glucocorticoids in cells carrying the risk genotype, repression that correlated with MAST4 expression in breast cancer and treatment outcomes. These observations provide a mechanistic framework for understanding non-coding genetic variant-chemical environment interactions and their role in disease risk and drug response.

USP51 deubiquitylates H2AK13,15ub and regulates DNA damage response.

Dynamic regulation of RNF168-mediated ubiquitylation of histone H2A Lys13,15 (H2AK13,15ub) at DNA double-strand breaks (DSBs) is crucial for preventing aberrant DNA repair and maintaining genome stability. However, it remains unclear which deubiquitylating enzyme (DUB) removes H2AK13,15ub. Here we show that USP51, a previously uncharacterized DUB, deubiquitylates H2AK13,15ub and regulates DNA damage response. USP51 depletion results in increased spontaneous DNA damage foci and elevated levels of H2AK15ub and impairs DNA damage response. USP51 overexpression suppresses the formation of ionizing radiation-induced 53BP1 and BRCA1 but not RNF168 foci, suggesting that USP51 functions downstream from RNF168 in DNA damage response. In vitro, USP51 binds to H2A-H2B directly and deubiquitylates H2AK13,15ub. In cells, USP51 is recruited to chromatin after DNA damage and regulates the dynamic assembly/disassembly of 53BP1 and BRCA1 foci. These results show that USP51 is the DUB for H2AK13,15ub and regulates DNA damage response.

Titration-based normalization of antibody amount improves consistency of ChIP-seq experiments.

Chromatin immunoprecipitation (ChIP) is an antibody-based approach that is frequently utilized in chromatin biology and epigenetics. The challenge in experimental variability by unpredictable nature of usable input amounts from samples and undefined antibody titer in ChIP reaction still remains to be addressed. Here, we introduce a simple and quick method to quantify chromatin inputs and demonstrate its utility for normalizing antibody amounts to the optimal titer in individual ChIP reactions. For a proof of concept, we utilized ChIP-seq validated antibodies against the key enhancer mark, acetylation of histone H3 on lysine 27 (H3K27ac), in the experiments. The results indicate that the titration-based normalization of antibody amounts improves assay outcomes including the consistency among samples both within and across experiments for a broad range of input amounts.

Specialized Mechanosensory Epithelial Cells in Mouse Gut Intrinsic Tactile Sensitivity.

BACKGROUND AND AIMS: The gastrointestinal (GI) tract extracts nutrients from ingested meals while protecting the organism from infectious agents frequently present in meals. Consequently, most animals conduct the entire digestive process within the GI tract while keeping the luminal contents entirely outside the body, separated by the tightly sealed GI epithelium. Therefore, like the skin and oral cavity, the GI tract must sense the chemical and physical properties of the its external interface to optimize its function. Specialized sensory enteroendocrine cells (EECs) in GI epithelium interact intimately with luminal contents. A subpopulation of EECs express the mechanically gated ion channel Piezo2 and are developmentally and functionally like the skin's touch sensor- the Merkel cell. We hypothesized that Piezo2+ EECs endow the gut with intrinsic tactile sensitivity. METHODS: We generated transgenic mouse models with optogenetic activators in EECs and Piezo2 conditional knockouts. We used a range of reference standard and novel techniques from single cells to living animals, including single-cell RNA sequencing and opto-electrophysiology, opto-organ baths with luminal shear forces, and in vivo studies that assayed GI transit while manipulating the physical properties of luminal contents. RESULTS: Piezo2+ EECs have transcriptomic features of synaptically connected, mechanosensory epithelial cells. EEC activation by optogenetics and forces led to Piezo2-dependent alterations in colonic propagating contractions driven by intrinsic circuitry, with Piezo2+ EECs detecting the small luminal forces and physical properties of the luminal contents to regulate transit times in the small and large bowel. CONCLUSIONS: The GI tract has intrinsic tactile sensitivity that depends on Piezo2+ EECs and allows it to detect luminal forces and physical properties of luminal contents to modulate physiology.

TCF7L2 lncRNA: a link between bipolar disorder and body mass index through glucocorticoid signaling.

Bipolar disorder (BD) and obesity are highly comorbid. We previously performed a genome-wide association study (GWAS) for BD risk accounting for the effect of body mass index (BMI), which identified a genome-wide significant single-nucleotide polymorphism (SNP) in the gene encoding the transcription factor 7 like 2 (TCF7L2). However, the molecular function of TCF7L2 in the central nervous system (CNS) and its possible role in the BD and BMI interaction remained unclear. In the present study, we demonstrated by studying human induced pluripotent stem cell (hiPSC)-derived astrocytes, cells that highly express TCF7L2 in the CNS, that the BD-BMI GWAS risk SNP is associated with glucocorticoid-dependent repression of the expression of a previously uncharacterized TCF7L2 transcript variant. That transcript is a long non-coding RNA (lncRNA-TCF7L2) that is highly expressed in the CNS but not in peripheral tissues such as the liver and pancreas that are involved in metabolism. In astrocytes, knockdown of the lncRNA-TCF7L2 resulted in decreased expression of the parent gene, TCF7L2, as well as alterations in the expression of a series of genes involved in insulin signaling and diabetes. We also studied the function of TCF7L2 in hiPSC-derived astrocytes by integrating RNA sequencing data after TCF7L2 knockdown with TCF7L2 chromatin-immunoprecipitation sequencing (ChIP-seq) data. Those studies showed that TCF7L2 directly regulated a series of BD risk genes. In summary, these results support the existence of a CNS-based mechanism underlying BD-BMI genetic risk, a mechanism based on a glucocorticoid-dependent expression quantitative trait locus that regulates the expression of a novel TCF7L2 non-coding transcript.