Circadian control of tumor immunosuppression affects efficacy of immune checkpoint blockade.

The circadian clock is a critical regulator of immunity, and this circadian control of immune modulation has an essential function in host defense and tumor immunosurveillance. Here we use a single-cell RNA sequencing approach and a genetic model of colorectal cancer to identify clock-dependent changes to the immune landscape that control the abundance of immunosuppressive cells and consequent suppression of cytotoxic CD8+ T cells. Of these immunosuppressive cell types, PD-L1-expressing myeloid-derived suppressor cells (MDSCs) peak in abundance in a rhythmic manner. Disruption of the epithelial cell clock regulates the secretion of cytokines that promote heightened inflammation, recruitment of neutrophils and the subsequent development of MDSCs. We also show that time-of-day anti-PD-L1 delivery is most effective when synchronized with the abundance of immunosuppressive MDSCs. Collectively, these data indicate that circadian gating of tumor immunosuppression informs the timing and efficacy of immune checkpoint inhibitors.

Colorectal Cancer Stem Cell Subtypes Orchestrate Distinct Tumor Microenvironments.

Several classification systems have been developed to define tumor subtypes in colorectal cancer (CRC). One system proposes that tumor heterogeneity derives in part from distinct cancer stem cell populations that co-exist as admixtures of varying proportions. However, the lack of single cell resolution has prohibited a definitive identification of these types of stem cells and therefore any understanding of how each influence tumor phenotypes. Here were report the isolation and characterization of two cancer stem cell subtypes from the SW480 CRC cell line. We find these cancer stem cells are oncogenic versions of the normal Crypt Base Columnar (CBC) and Regenerative Stem Cell (RSC) populations from intestinal crypts and that their gene signatures are consistent with the "Admixture" and other CRC classification systems. Using publicly available single cell RNA sequencing (scRNAseq) data from CRC patients, we determine that RSC and CBC cancer stem cells are commonly co-present in human CRC. To characterize influences on the tumor microenvironment, we develop subtype-specific xenograft models and we define their tumor microenvironments at high resolution via scRNAseq. RSCs create differentiated, inflammatory, slow growing tumors. CBCs create proliferative, undifferentiated, invasive tumors. With this enhanced resolution, we unify current CRC patient classification schema with TME phenotypes and organization.

Leveraging inter-individual transcriptional correlation structure to infer discrete signaling mechanisms across metabolic tissues.

Inter-organ communication is a vital process to maintain physiologic homeostasis, and its dysregulation contributes to many human diseases. Given that circulating bioactive factors are stable in serum, occur naturally, and are easily assayed from blood, they present obvious focal molecules for therapeutic intervention and biomarker development. Recently, studies have shown that secreted proteins mediating inter-tissue signaling could be identified by 'brute force' surveys of all genes within RNA-sequencing measures across tissues within a population. Expanding on this intuition, we reasoned that parallel strategies could be used to understand how individual genes mediate signaling across metabolic tissues through correlative analyses of gene variation between individuals. Thus, comparison of quantitative levels of gene expression relationships between organs in a population could aid in understanding cross-organ signaling. Here, we surveyed gene-gene correlation structure across 18 metabolic tissues in 310 human individuals and 7 tissues in 103 diverse strains of mice fed a normal chow or high-fat/high-sucrose (HFHS) diet. Variation of genes such as FGF21, ADIPOQ, GCG, and IL6 showed enrichments which recapitulate experimental observations. Further, similar analyses were applied to explore both within-tissue signaling mechanisms (liver PCSK9) and genes encoding enzymes producing metabolites (adipose PNPLA2), where inter-individual correlation structure aligned with known roles for these critical metabolic pathways. Examination of sex hormone receptor correlations in mice highlighted the difference of tissue-specific variation in relationships with metabolic traits. We refer to this resource as gene-derived correlations across tissues (GD-CAT) where all tools and data are built into a web portal enabling users to perform these analyses without a single line of code (gdcat.org). This resource enables querying of any gene in any tissue to find correlated patterns of genes, cell types, pathways, and network architectures across metabolic organs.

Nuclear RNA catabolism controls endogenous retroviruses, gene expression asymmetry, and dedifferentiation.

Endogenous retroviruses (ERVs) are remnants of ancient parasitic infections and comprise sizable portions of most genomes. Although epigenetic mechanisms silence most ERVs by generating a repressive environment that prevents their expression (heterochromatin), little is known about mechanisms silencing ERVs residing in open regions of the genome (euchromatin). This is particularly important during embryonic development, where induction and repression of distinct classes of ERVs occur in short temporal windows. Here, we demonstrate that transcription-associated RNA degradation by the nuclear RNA exosome and Integrator is a regulatory mechanism that controls the productive transcription of most genes and many ERVs involved in preimplantation development. Disrupting nuclear RNA catabolism promotes dedifferentiation to a totipotent-like state characterized by defects in RNAPII elongation and decreased expression of long genes (gene-length asymmetry). Our results indicate that RNA catabolism is a core regulatory module of gene networks that safeguards RNAPII activity, ERV expression, cell identity, and developmental potency.

Loss of CTRP10 results in female obesity with preserved metabolic health.

Obesity is a major risk factor for type 2 diabetes, dyslipidemia, cardiovascular disease, and hypertension. Intriguingly, there is a subset of metabolically healthy obese (MHO) individuals who are seemingly able to maintain a healthy metabolic profile free of metabolic syndrome. The molecular underpinnings of MHO, however, are not well understood. Here, we report that CTRP10/C1QL2-deficient mice represent a unique female model of MHO. CTRP10 modulates weight gain in a striking and sexually dimorphic manner. Female, but not male, mice lacking CTRP10 develop obesity with age on a low-fat diet while maintaining an otherwise healthy metabolic profile. When fed an obesogenic diet, female Ctrp10 knockout (KO) mice show rapid weight gain. Despite pronounced obesity, Ctrp10 KO female mice do not develop steatosis, dyslipidemia, glucose intolerance, insulin resistance, oxidative stress, or low-grade inflammation. Obesity is largely uncoupled from metabolic dysregulation in female KO mice. Multi-tissue transcriptomic analyses highlighted gene expression changes and pathways associated with insulin-sensitive obesity. Transcriptional correlation of the differentially expressed gene (DEG) orthologous in humans also show sex differences in gene connectivity within and across metabolic tissues, underscoring the conserved sex-dependent function of CTRP10. Collectively, our findings suggest that CTRP10 negatively regulates body weight in females, and that loss of CTRP10 results in benign obesity with largely preserved insulin sensitivity and metabolic health. This female MHO mouse model is valuable for understanding sex-biased mechanisms that uncouple obesity from metabolic dysfunction.

Systems genetics approaches for understanding complex traits with relevance for human disease.

Quantitative traits are often complex because of the contribution of many loci, with further complexity added by environmental factors. In medical research, systems genetics is a powerful approach for the study of complex traits, as it integrates intermediate phenotypes, such as RNA, protein, and metabolite levels, to understand molecular and physiological phenotypes linking discrete DNA sequence variation to complex clinical and physiological traits. The primary purpose of this review is to describe some of the resources and tools of systems genetics in humans and rodent models, so that researchers in many areas of biology and medicine can make use of the data.

Increased AID Results in Mutations at the CRLF2 Locus Implicated in Latin American ALL Health Disparities.

Activation-induced cytidine deaminase (AID) is a B cell-specific base editor required during class switch recombination and somatic hypermutation for B cell maturation and antibody diversification. However, it has also been implicated as a factor in the etiology of several B cell malignancies. Evaluating the AID-induced mutation load in patients at-risk for certain types of blood cancers is critical in assessing disease severity and treatment options. Here, we have developed a digital PCR (dPCR) assay that allows us to track the mutational landscape resulting from AID modification or DNA double-strand break (DSB) formation and repair at sites known to be prone to DSBs. Implementation of this new assay showed that increased AID levels in immature B cells increases genome instability at loci linked to translocation formation. This included the CRLF2 locus that is often involved in chromosomal translocations associated with a subtype of acute lymphoblastic leukemia (ALL) that disproportionately affects Latin Americans (LAs). To support this LA-specific identification of AID mutation signatures, we characterized DNA from immature B cells isolated from the bone marrow of ALL patients. Our ability to detect and quantify these mutation signatures will potentiate future risk identification, early detection of cancers, and reduction of associated cancer health disparities.

Feeding neurons integrate metabolic and reproductive states in mice.

Balance between metabolic and reproductive processes is important for survival, particularly in mammals that gestate their young. How the nervous system coordinates this balance is an active area of study. Herein, we demonstrate that somatostatin (SST) neurons of the tuberal hypothalamus alter feeding in a manner sensitive to metabolic and reproductive states in mice. Whereas chemogenetic activation of SST neurons increased food intake across sexes, ablation decreased food intake only in female mice during proestrus. This ablation effect was only apparent in animals with low body mass. Fat transplantation and bioinformatics analysis of SST neuronal transcriptomes revealed white adipose as a key modulator of these effects. These studies indicate that SST hypothalamic neurons integrate metabolic and reproductive cues by responding to varying levels of circulating estrogens to modulate feeding differentially based on energy stores. Thus, gonadal steroid modulation of neuronal circuits can be context dependent and gated by metabolic status.

CTRP13 ablation improves systemic glucose and lipid metabolism.

OBJECTIVE: Tissue crosstalk mediated by secreted hormones underlies the integrative control of metabolism. We previously showed that CTRP13/C1QL3, a secreted protein of the C1q family, can improve glucose metabolism and insulin action in vitro and reduce food intake and body weight in mice when centrally delivered. A role for CTRP13 in regulating insulin secretion in isolated islets has also been demonstrated. It remains unclear, however, whether the effects of CTRP13 on cultured cells and in mice reflect the physiological function of the protein. Here, we use a loss-of-function mouse model to address whether CTRP13 is required for metabolic homeostasis. METHODS: WT and Ctrp13 knockout (KO) mice fed a standard chow or a high-fat diet were subjected to comprehensive metabolic phenotyping. Transcriptomic analyses were carried out on visceral and subcutaneous fat, liver, and skeletal muscle to identify pathways altered by CTRP13 deficiency. RNA-seq data was further integrated with the Metabolic Syndrome in Man (METSIM) cohort data. Adjusted regression analysis was used to demonstrate that genetic variation of CTRP13 expression accounts for a significant proportion of variance between differentially expressed genes (DEGs) in adipose tissue and metabolic traits in humans. RESULTS: Contrary to expectation, chow-fed Ctrp13-KO male mice had elevated physical activity, lower body weight, and improved lipid handling. On a high-fat diet (HFD), Ctrp13-KO mice of either sex were consistently more active and leaner. Loss of CTRP13 reduced hepatic glucose output and improved glucose tolerance, insulin sensitivity, and triglyceride clearance, though with notable sex differences. Consistent with the lean phenotype, transcriptomic analyses revealed a lower inflammatory profile in visceral fat and liver. Reduced hepatic steatosis was correlated with the suppression of lipid synthesis and enhanced lipid catabolism gene expression. Visceral fat had the largest number of DEGs and mediation analyses on the human orthologs of the DEGs suggested the potential causal contribution of CTRP13 to human metabolic syndrome. CONCLUSIONS: Our results suggest that CTRP13 is a negative metabolic regulator, and its deficiency improves systemic metabolic profiles. Our data also suggest the reduction in circulating human CTRP13 levels seen in obesity and diabetes may reflect a compensatory physiologic response to counteract insulin resistance.

Genetic architecture of heart mitochondrial proteome influencing cardiac hypertrophy.

Mitochondria play an important role in both normal heart function and disease etiology. We report analysis of common genetic variations contributing to mitochondrial and heart functions using an integrative proteomics approach in a panel of inbred mouse strains called the Hybrid Mouse Diversity Panel (HMDP). We performed a whole heart proteome study in the HMDP (72 strains, n=2-3 mice) and retrieved 848 mitochondrial proteins (quantified in ≥50 strains). High-resolution association mapping on their relative abundance levels revealed three trans-acting genetic loci on chromosomes (chr) 7, 13 and 17 that regulate distinct classes of mitochondrial proteins as well as cardiac hypertrophy. DAVID enrichment analyses of genes regulated by each of the loci revealed that the chr13 locus was highly enriched for complex-I proteins (24 proteins, P=2.2E-61), the chr17 locus for mitochondrial ribonucleoprotein complex (17 proteins, P=3.1E-25) and the chr7 locus for ubiquinone biosynthesis (3 proteins, P=6.9E-05). Follow-up high resolution regional mapping identified NDUFS4, LRPPRC and COQ7 as the candidate genes for chr13, chr17 and chr7 loci, respectively, and both experimental and statistical analyses supported their causal roles. Furthermore, a large cohort of Diversity Outbred mice was used to corroborate Lrpprc gene as a driver of mitochondrial DNA (mtDNA)-encoded gene regulation, and to show that the chr17 locus is specific to heart. Variations in all three loci were associated with heart mass in at least one of two independent heart stress models, namely, isoproterenol-induced heart failure and diet-induced obesity. These findings suggest that common variations in certain mitochondrial proteins can act in trans to influence tissue-specific mitochondrial functions and contribute to heart hypertrophy, elucidating mechanisms that may underlie genetic susceptibility to heart failure in human populations.

Conserved multi-tissue transcriptomic adaptations to exercise training in humans and mice.

Physical activity is associated with beneficial adaptations in human and rodent metabolism. We studied over 50 complex traits before and after exercise intervention in middle-aged men and a panel of 100 diverse strains of female mice. Candidate gene analyses in three brain regions, muscle, liver, heart, and adipose tissue of mice indicate genetic drivers of clinically relevant traits, including volitional exercise volume, muscle metabolism, adiposity, and hepatic lipids. Although ∼33% of genes differentially expressed in skeletal muscle following the exercise intervention are similar in mice and humans independent of BMI, responsiveness of adipose tissue to exercise-stimulated weight loss appears controlled by species and underlying genotype. We leveraged genetic diversity to generate prediction models of metabolic trait responsiveness to volitional activity offering a framework for advancing personalized exercise prescription. The human and mouse data are publicly available via a user-friendly Web-based application to enhance data mining and hypothesis development.

RNF130 Regulates LDLR Availability and Plasma LDL Cholesterol Levels.

BACKGROUND: Removal of circulating plasma low-density lipoprotein cholesterol (LDL-C) by the liver relies on efficient endocytosis and intracellular vesicle trafficking. Increasing the availability of hepatic LDL receptors (LDLRs) remains a major clinical target for reducing LDL-C levels. Here, we describe a novel role for RNF130 (ring finger containing protein 130) in regulating plasma membrane availability of LDLR. METHODS: We performed a combination of gain-of-function and loss-of-function experiments to determine the effect of RNF130 on LDL-C and LDLR recycling. We overexpressed RNF130 and a nonfunctional mutant RNF130 in vivo and measured plasma LDL-C and hepatic LDLR protein levels. We performed in vitro ubiquitination assays and immunohistochemical staining to measure levels and cellular distribution of LDLR. We supplement these experiments with 3 separate in vivo models of RNF130 loss-of-function where we disrupted Rnf130 using either ASO (antisense oligonucleotides), germline deletion, or AAV CRISPR (adeno-associated virus clustered regularly interspaced short palindromic repeats) and measured hepatic LDLR and plasma LDL-C. RESULTS: We demonstrate that RNF130 is an E3 ubiquitin ligase that ubiquitinates LDLR resulting in redistribution of the receptor away from the plasma membrane. Overexpression of RNF130 decreases hepatic LDLR and increases plasma LDL-C levels. Further, in vitro ubiquitination assays demonstrate RNF130-dependent regulation of LDLR abundance at the plasma membrane. Finally, in vivo disruption of Rnf130 using ASO, germline deletion, or AAV CRISPR results in increased hepatic LDLR abundance and availability and decreased plasma LDL-C levels. CONCLUSIONS: Our studies identify RNF130 as a novel posttranslational regulator of LDL-C levels via modulation of LDLR availability, thus providing important insight into the complex regulation of hepatic LDLR protein levels.

The potential of integrating human and mouse discovery platforms to advance our understanding of cardiometabolic diseases.

Cardiometabolic diseases encompass a range of interrelated conditions that arise from underlying metabolic perturbations precipitated by genetic, environmental, and lifestyle factors. While obesity, dyslipidaemia, smoking, and insulin resistance are major risk factors for cardiometabolic diseases, individuals still present in the absence of such traditional risk factors, making it difficult to determine those at greatest risk of disease. Thus, it is crucial to elucidate the genetic, environmental, and molecular underpinnings to better understand, diagnose, and treat cardiometabolic diseases. Much of this information can be garnered using systems genetics, which takes population-based approaches to investigate how genetic variance contributes to complex traits. Despite the important advances made by human genome-wide association studies (GWAS) in this space, corroboration of these findings has been hampered by limitations including the inability to control environmental influence, limited access to pertinent metabolic tissues, and often, poor classification of diseases or phenotypes. A complementary approach to human GWAS is the utilisation of model systems such as genetically diverse mouse panels to study natural genetic and phenotypic variation in a controlled environment. Here, we review mouse genetic reference panels and the opportunities they provide for the study of cardiometabolic diseases and related traits. We discuss how the post-GWAS era has prompted a shift in focus from discovery of novel genetic variants to understanding gene function. Finally, we highlight key advantages and challenges of integrating complementary genetic and multi-omics data from human and mouse populations to advance biological discovery.

Feeding Neurons Integrate Metabolic and Reproductive States in Mice.

Trade-offs between metabolic and reproductive processes are important for survival, particularly in mammals that gestate their young. Puberty and reproduction, as energetically taxing life stages, are often gated by metabolic availability in animals with ovaries. How the nervous system coordinates these trade-offs is an active area of study. We identify somatostatin neurons of the tuberal nucleus (TNSST) as a node of the feeding circuit that alters feeding in a manner sensitive to metabolic and reproductive states in mice. Whereas chemogenetic activation of TNSST neurons increased food intake across sexes, selective ablation decreased food intake only in female mice during proestrus. Interestingly, this ablation effect was only apparent in animals with a low body mass. Fat transplantation and bioinformatics analysis of TNSST neuronal transcriptomes revealed white adipose as a key modulator of the effects of TNSST neurons on food intake. Together, these studies point to a mechanism whereby TNSST hypothalamic neurons modulate feeding by responding to varying levels of circulating estrogens differentially based on energy stores. This research provides insight into how neural circuits integrate reproductive and metabolic signals, and illustrates how gonadal steroid modulation of neuronal circuits can be context-dependent and gated by metabolic status.

APOBEC3B drives PKR-mediated translation shutdown and protects stress granules in response to viral infection.

Double-stranded RNA produced during viral replication and transcription activates both protein kinase R (PKR) and ribonuclease L (RNase L), which limits viral gene expression and replication through host shutoff of translation. In this study, we find that APOBEC3B forms a complex with PABPC1 to stimulate PKR and counterbalances the PKR-suppressing activity of ADAR1 in response to infection by many types of viruses. This leads to translational blockage and the formation of stress granules. Furthermore, we show that APOBEC3B localizes to stress granules through the interaction with PABPC1. APOBEC3B facilitates the formation of protein-RNA condensates with stress granule assembly factor (G3BP1) by protecting mRNA associated with stress granules from RNAse L-induced RNA cleavage during viral infection. These results not only reveal that APOBEC3B is a key regulator of different steps of the innate immune response throughout viral infection but also highlight an alternative mechanism by which APOBEC3B can impact virus replication without editing viral genomes.

Genetic mapping of microbial and host traits reveals production of immunomodulatory lipids by Akkermansia muciniphila in the murine gut.

The molecular bases of how host genetic variation impacts the gut microbiome remain largely unknown. Here we used a genetically diverse mouse population and applied systems genetics strategies to identify interactions between host and microbe phenotypes including microbial functions, using faecal metagenomics, small intestinal transcripts and caecal lipids that influence microbe-host dynamics. Quantitative trait locus (QTL) mapping identified murine genomic regions associated with variations in bacterial taxa; bacterial functions including motility, sporulation and lipopolysaccharide production and levels of bacterial- and host-derived lipids. We found overlapping QTL for the abundance of Akkermansia muciniphila and caecal levels of ornithine lipids. Follow-up in vitro and in vivo studies revealed that A. muciniphila is a major source of these lipids in the gut, provided evidence that ornithine lipids have immunomodulatory effects and identified intestinal transcripts co-regulated with these traits including Atf3, which encodes for a transcription factor that plays vital roles in modulating metabolism and immunity. Collectively, these results suggest that ornithine lipids are potentially important for A. muciniphila-host interactions and support the role of host genetics as a determinant of responses to gut microbes.

Dysregulated systemic metabolism in a Down syndrome mouse model.

OBJECTIVE: Trisomy 21 is one of the most complex genetic perturbations compatible with postnatal survival. Dosage imbalance arising from the triplication of genes on human chromosome 21 (Hsa21) affects multiple organ systems. Much of Down syndrome (DS) research, however, has focused on addressing how aneuploidy dysregulates CNS function leading to cognitive deficit. Although obesity, diabetes, and associated sequelae such as fatty liver and dyslipidemia are well documented in the DS population, only limited studies have been conducted to determine how gene dosage imbalance affects whole-body metabolism. Here, we conduct a comprehensive and systematic analysis of key metabolic parameters across different physiological states in the Ts65Dn trisomic mouse model of DS. METHODS: Ts65Dn mice and euploid littermates were subjected to comprehensive metabolic phenotyping under basal (chow-fed) state and the pathophysiological state of obesity induced by a high-fat diet (HFD). RNA sequencing of liver, skeletal muscle, and two major fat depots were conducted to determine the impact of aneuploidy on tissue transcriptome. Pathway enrichments, gene-centrality, and key driver estimates were performed to provide insights into tissue autonomous and non-autonomous mechanisms contributing to the dysregulation of systemic metabolism. RESULTS: Under the basal state, chow-fed Ts65Dn mice of both sexes had elevated locomotor activity and energy expenditure, reduced fasting serum cholesterol levels, and mild glucose intolerance. Sexually dimorphic deterioration in metabolic homeostasis became apparent when mice were challenged with a high-fat diet. While obese Ts65Dn mice of both sexes exhibited dyslipidemia, male mice also showed impaired systemic insulin sensitivity, reduced mitochondrial activity, and elevated fibrotic and inflammatory gene signatures in the liver and adipose tissue. Systems-level analysis highlighted conserved pathways and potential endocrine drivers of adipose-liver crosstalk that contribute to dysregulated glucose and lipid metabolism. CONCLUSIONS: A combined alteration in the expression of trisomic and disomic genes in peripheral tissues contribute to metabolic dysregulations in Ts65Dn mice. These data lay the groundwork for understanding the impact of aneuploidy on in vivo metabolism.