Age-dependent genes in adipose stem and precursor cells affect regulation of fat cell differentiation and link aging to obesity via cellular and genetic interactions.

BACKGROUND: Age and obesity are dominant risk factors for several common cardiometabolic disorders, and both are known to impair adipose tissue function. However, the underlying cellular and genetic factors linking aging and obesity on adipose tissue function have remained elusive. Adipose stem and precursor cells (ASPCs) are an understudied, yet crucial adipose cell type due to their deterministic adipocyte differentiation potential, which impacts the capacity to store fat in a metabolically healthy manner. METHODS: We integrated subcutaneous adipose tissue (SAT) bulk (n=435) and large single-nucleus RNA sequencing (n=105) data with the UK Biobank (UKB) (n=391,701) data to study age-obesity interactions originating from ASPCs by performing cell-type decomposition, differential expression testing, cell-cell communication analyses, and construction of polygenic risk scores for body mass index (BMI). RESULTS: We found that the SAT ASPC proportions significantly decrease with age in an obesity-dependent way consistently in two independent cohorts, both showing that the age dependency of ASPC proportions is abolished by obesity. We further identified 76 genes (72 SAT ASPC marker genes and 4 transcription factors regulating ASPC marker genes) that are differentially expressed by age in SAT and functionally enriched for developmental processes and adipocyte differentiation (i.e., adipogenesis). The 76 age-perturbed ASPC genes include multiple negative regulators of adipogenesis, such as RORA, SMAD3, TWIST2, and ZNF521, form tight clusters of longitudinally co-expressed genes during human adipogenesis, and show age-based differences in cellular interactions between ASPCs and adipose cell types. Finally, our genetic data demonstrate that cis-regional variants of these genes interact with age as predictors of BMI in an obesity-dependent way in the large UKB, while no such gene-age interaction on BMI is observed with non-age-dependent ASPC marker genes, thus independently confirming our cellular ASPC results at the biobank level. CONCLUSIONS: Overall, we discover that obesity prematurely induces a decrease in ASPC proportions and identify 76 developmentally important ASPC genes that implicate altered negative regulation of fat cell differentiation as a mechanism for aging and directly link aging to obesity via significant cellular and genetic interactions.

Characterizing the landscape of gene expression variance in humans.

Gene expression variance has been linked to organismal function and fitness but remains a commonly neglected aspect of molecular research. As a result, we lack a comprehensive understanding of the patterns of transcriptional variance across genes, and how this variance is linked to context-specific gene regulation and gene function. Here, we use 57 large publicly available RNA-seq data sets to investigate the landscape of gene expression variance. These studies cover a wide range of tissues and allowed us to assess if there are consistently more or less variable genes across tissues and data sets and what mechanisms drive these patterns. We show that gene expression variance is broadly similar across tissues and studies, indicating that the pattern of transcriptional variance is consistent. We use this similarity to create both global and within-tissue rankings of variation, which we use to show that function, sequence variation, and gene regulatory signatures contribute to gene expression variance. Low-variance genes are associated with fundamental cell processes and have lower levels of genetic polymorphisms, have higher gene-gene connectivity, and tend to be associated with chromatin states associated with transcription. In contrast, high-variance genes are enriched for genes involved in immune response, environmentally responsive genes, immediate early genes, and are associated with higher levels of polymorphisms. These results show that the pattern of transcriptional variance is not noise. Instead, it is a consistent gene trait that seems to be functionally constrained in human populations. Furthermore, this commonly neglected aspect of molecular phenotypic variation harbors important information to understand complex traits and disease.

Increased body mass index is linked to systemic inflammation through altered chromatin co-accessibility in human preadipocytes.

Obesity-induced adipose tissue dysfunction can cause low-grade inflammation and downstream obesity comorbidities. Although preadipocytes may contribute to this pro-inflammatory environment, the underlying mechanisms are unclear. We used human primary preadipocytes from body mass index (BMI) -discordant monozygotic (MZ) twin pairs to generate epigenetic (ATAC-sequence) and transcriptomic (RNA-sequence) data for testing whether increased BMI alters the subnuclear compartmentalization of open chromatin in the twins' preadipocytes, causing downstream inflammation. Here we show that the co-accessibility of open chromatin, i.e. compartmentalization of chromatin activity, is altered in the higher vs lower BMI MZ siblings for a large subset ( ~ 88.5 Mb) of the active subnuclear compartments. Using the UK Biobank we show that variants within these regions contribute to systemic inflammation through interactions with BMI on C-reactive protein. In summary, open chromatin co-accessibility in human preadipocytes is disrupted among the higher BMI siblings, suggesting a mechanism how obesity may lead to inflammation via gene-environment interactions.

Long-range chromosomal interactions increase and mark repressed gene expression during adipogenesis.

Obesity perturbs central functions of human adipose tissue, centred on differentiation of preadipocytes to adipocytes, i.e., adipogenesis. The large environmental component of obesity makes it important to elucidate epigenetic regulatory factors impacting adipogenesis. Promoter Capture Hi-C (pCHi-C) has been used to identify chromosomal interactions between promoters and associated regulatory elements. However, long range interactions (LRIs) greater than 1 Mb are often filtered out of pCHi-C datasets, due to technical challenges and their low prevalence. To elucidate the unknown role of LRIs in adipogenesis, we investigated preadipocyte differentiation to adipocytes using pCHi-C and bulk and single nucleus RNA-seq data. We first show that LRIs are reproducible between biological replicates, and they increase >2-fold in frequency across adipogenesis. We further demonstrate that genomic loci containing LRIs are more epigenetically repressed than regions without LRIs, corresponding to lower gene expression in the LRI regions. Accordingly, as preadipocytes differentiate into adipocytes, LRI regions are more likely to contain repressed preadipocyte marker genes; whereas these same LRI regions are depleted of actively expressed adipocyte marker genes. Finally, we show that LRIs can be used to restrict multiple testing of the long-range cis-eQTL analysis to identify variants that regulate genes via LRIs. We exemplify this by identifying a putative long range cis regulatory mechanism at the LYPLAL1/TGFB2 obesity locus. In summary, we identify LRIs that mark repressed regions of the genome, and these interactions increase across adipogenesis, pinpointing developmental regions that need to be repressed in a cell-type specific way for adipogenesis to proceed.

Identification of 90 NAFLD GWAS loci and establishment of NAFLD PRS and causal role of NAFLD in coronary artery disease.

The prevalence of non-alcoholic fatty liver disease (NAFLD), now also known as metabolic dysfunction-associated fatty liver disease (MAFLD), is rapidly increasing worldwide due to the ongoing obesity epidemic. However, currently the NALFD diagnosis requires non-readily available imaging technologies or liver biopsy, which has drastically limited the sample sizes of NAFLD studies and hampered the discovery of its genetic component. Here we utilized the large UK Biobank (UKB) to accurately estimate the NAFLD status in UKB based on common serum traits and anthropometric measures. Scoring all individuals in UKB for NAFLD risk resulted in 28,396 NAFLD cases and 108,652 healthy individuals at a >90% confidence level. Using this imputed NAFLD status to perform the largest NAFLD genome-wide association study (GWAS) to date, we identified 94 independent (R2 < 0.2) NAFLD GWAS loci, of which 90 have not been identified before; built a polygenic risk score (PRS) model to predict the genetic risk of NAFLD; and used the GWAS variants of imputed NAFLD for a tissue-aware Mendelian randomization analysis that discovered a significant causal effect of NAFLD on coronary artery disease (CAD). In summary, we accurately estimated the NAFLD status in UKB using common serum traits and anthropometric measures, which empowered us to identify 90 GWAS NAFLD loci, build NAFLD PRS, and discover a significant causal effect of NAFLD on CAD.

Identification of TBX15 as an adipose master trans regulator of abdominal obesity genes.

BACKGROUND: Obesity predisposes individuals to multiple cardiometabolic disorders, including type 2 diabetes (T2D). As body mass index (BMI) cannot reliably differentiate fat from lean mass, the metabolically detrimental abdominal obesity has been estimated using waist-hip ratio (WHR). Waist-hip ratio adjusted for body mass index (WHRadjBMI) in turn is a well-established sex-specific marker for abdominal fat and adiposity, and a predictor of adverse metabolic outcomes, such as T2D. However, the underlying genes and regulatory mechanisms orchestrating the sex differences in obesity and body fat distribution in humans are not well understood. METHODS: We searched for genetic master regulators of WHRadjBMI by employing integrative genomics approaches on human subcutaneous adipose RNA sequencing (RNA-seq) data (n ~ 1400) and WHRadjBMI GWAS data (n ~ 700,000) from the WHRadjBMI GWAS cohorts and the UK Biobank (UKB), using co-expression network, transcriptome-wide association study (TWAS), and polygenic risk score (PRS) approaches. Finally, we functionally verified our genomic results using gene knockdown experiments in a human primary cell type that is critical for adipose tissue function. RESULTS: Here, we identified an adipose gene co-expression network that contains 35 obesity GWAS genes and explains a significant amount of polygenic risk for abdominal obesity and T2D in the UKB (n = 392,551) in a sex-dependent way. We showed that this network is preserved in the adipose tissue data from the Finnish Kuopio Obesity Study and Mexican Obesity Study. The network is controlled by a novel adipose master transcription factor (TF), TBX15, a WHRadjBMI GWAS gene that regulates the network in trans. Knockdown of TBX15 in human primary preadipocytes resulted in changes in expression of 130 network genes, including the key adipose TFs, PPARG and KLF15, which were significantly impacted (FDR < 0.05), thus functionally verifying the trans regulatory effect of TBX15 on the WHRadjBMI co-expression network. CONCLUSIONS: Our study discovers a novel key function for the TBX15 TF in trans regulating an adipose co-expression network of 347 adipose, mitochondrial, and metabolically important genes, including PPARG, KLF15, PPARA, ADIPOQ, and 35 obesity GWAS genes. Thus, based on our converging genomic, transcriptional, and functional evidence, we interpret the role of TBX15 to be a main transcriptional regulator in the adipose tissue and discover its importance in human abdominal obesity.

Integrative analysis of liver-specific non-coding regulatory SNPs associated with the risk of coronary artery disease.

Genetic factors underlying coronary artery disease (CAD) have been widely studied using genome-wide association studies (GWASs). However, the functional understanding of the CAD loci has been limited by the fact that a majority of GWAS variants are located within non-coding regions with no functional role. High cholesterol and dysregulation of the liver metabolism such as non-alcoholic fatty liver disease confer an increased risk of CAD. Here, we studied the function of non-coding single-nucleotide polymorphisms in CAD GWAS loci located within liver-specific enhancer elements by identifying their potential target genes using liver cis-eQTL analysis and promoter Capture Hi-C in HepG2 cells. Altogether, 734 target genes were identified of which 121 exhibited correlations to liver-related traits. To identify potentially causal regulatory SNPs, the allele-specific enhancer activity was analyzed by (1) sequence-based computational predictions, (2) quantification of allele-specific transcription factor binding, and (3) STARR-seq massively parallel reporter assay. Altogether, our analysis identified 1,277 unique SNPs that display allele-specific regulatory activity. Among these, susceptibility enhancers near important cholesterol homeostasis genes (APOB, APOC1, APOE, and LIPA) were identified, suggesting that altered gene regulatory activity could represent another way by which genetic variation regulates serum lipoprotein levels. Using CRISPR-based perturbation, we demonstrate how the deletion/activation of a single enhancer leads to changes in the expression of many target genes located in a shared chromatin interaction domain. Our integrative genomics approach represents a comprehensive effort in identifying putative causal regulatory regions and target genes that could predispose to clinical manifestation of CAD by affecting liver function.

Further evidence supporting a potential role for ADH1B in obesity.

Insulin is an essential hormone that regulates glucose homeostasis and metabolism. Insulin resistance (IR) arises when tissues fail to respond to insulin, and it leads to serious health problems including Type 2 Diabetes (T2D). Obesity is a major contributor to the development of IR and T2D. We previously showed that gene expression of alcohol dehydrogenase 1B (ADH1B) was inversely correlated with obesity and IR in subcutaneous adipose tissue of Mexican Americans. In the current study, a meta-analysis of the relationship between ADH1B expression and BMI in Mexican Americans, African Americans, Europeans, and Pima Indians verified that BMI was increased with decreased ADH1B expression. Using established human subcutaneous pre-adipocyte cell lines derived from lean (BMI < 30 kg m-2) or obese (BMI ≥ 30 kg m-2) donors, we found that ADH1B protein expression increased substantially during differentiation, and overexpression of ADH1B inhibited fatty acid binding protein expression. Mature adipocytes from lean donors expressed ADH1B at higher levels than obese donors. Insulin further induced ADH1B protein expression as well as enzyme activity. Knockdown of ADH1B expression decreased insulin-stimulated glucose uptake. Our findings suggest that ADH1B is involved in the proper development and metabolic activity of adipose tissues and this function is suppressed by obesity.

Enhancing droplet-based single-nucleus RNA-seq resolution using the semi-supervised machine learning classifier DIEM.

Single-nucleus RNA sequencing (snRNA-seq) measures gene expression in individual nuclei instead of cells, allowing for unbiased cell type characterization in solid tissues. We observe that snRNA-seq is commonly subject to contamination by high amounts of ambient RNA, which can lead to biased downstream analyses, such as identification of spurious cell types if overlooked. We present a novel approach to quantify contamination and filter droplets in snRNA-seq experiments, called Debris Identification using Expectation Maximization (DIEM). Our likelihood-based approach models the gene expression distribution of debris and cell types, which are estimated using EM. We evaluated DIEM using three snRNA-seq data sets: (1) human differentiating preadipocytes in vitro, (2) fresh mouse brain tissue, and (3) human frozen adipose tissue (AT) from six individuals. All three data sets showed evidence of extranuclear RNA contamination, and we observed that existing methods fail to account for contaminated droplets and led to spurious cell types. When compared to filtering using these state of the art methods, DIEM better removed droplets containing high levels of extranuclear RNA and led to higher quality clusters. Although DIEM was designed for snRNA-seq, our clustering strategy also successfully filtered single-cell RNA-seq data. To conclude, our novel method DIEM removes debris-contaminated droplets from single-cell-based data fast and effectively, leading to cleaner downstream analysis. Our code is freely available for use at https://github.com/marcalva/diem.

Publisher Correction: Accurate estimation of cell composition in bulk expression through robust integration of single-cell information.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Accurate estimation of cell composition in bulk expression through robust integration of single-cell information.

We present Bisque, a tool for estimating cell type proportions in bulk expression. Bisque implements a regression-based approach that utilizes single-cell RNA-seq (scRNA-seq) or single-nucleus RNA-seq (snRNA-seq) data to generate a reference expression profile and learn gene-specific bulk expression transformations to robustly decompose RNA-seq data. These transformations significantly improve decomposition performance compared to existing methods when there is significant technical variation in the generation of the reference profile and observed bulk expression. Importantly, compared to existing methods, our approach is extremely efficient, making it suitable for the analysis of large genomic datasets that are becoming ubiquitous. When applied to subcutaneous adipose and dorsolateral prefrontal cortex expression datasets with both bulk RNA-seq and snRNA-seq data, Bisque replicates previously reported associations between cell type proportions and measured phenotypes across abundant and rare cell types. We further propose an additional mode of operation that merely requires a set of known marker genes.

Novel Lipid Long Intervening Noncoding RNA, Oligodendrocyte Maturation-Associated Long Intergenic Noncoding RNA, Regulates the Liver Steatosis Gene Stearoyl-Coenzyme A Desaturase As an Enhancer RNA.

The global obesity epidemic is driving the concomitant rise in nonalcoholic fatty liver disease (NAFLD). To identify new genes involved in central liver functions, we examined liver RNA-sequence data from 259 patients who underwent morbidly obese bariatric surgery. Of these patients, 84 had normal liver histology, 40 simple steatosis, 43 nonalcoholic steatohepatitis, and the remaining 92 patients had varying degrees of NAFLD based on liver histology. We discovered oligodendrocyte maturation-associated long intergenic noncoding RNA (OLMALINC), a long intervening noncoding RNA (lincRNA) in a human liver co-expression network (n = 75 genes) that was strongly associated with statin use and serum triglycerides (TGs). OLMALINC liver expression was highly correlated with the expression of known cholesterol biosynthesis genes and stearoyl-coenzyme A desaturase (SCD). SCD is the rate-limiting enzyme in monounsaturated fatty acids and a key TG gene that is known to be up-regulated in liver steatosis and NAFLD and resides adjacent to OLMALINC on the human chromosome 10q24.31. Next, we functionally demonstrated that OLMALINC regulates SCD as an enhancer-RNA (eRNA), thus describing the first lincRNA that functions as an eRNA to regulate lipid metabolism. Specifically, we show that OLMALINC promotes liver expression of SCD in cis through regional chromosomal DNA-DNA looping interactions. Conclusion: The primate-specific lincRNA OLMALINC is a novel epigenetic regulator of the key TG and NAFLD gene SCD.

Reverse gene-environment interaction approach to identify variants influencing body-mass index in humans.

Identifying gene-environment interactions (GxEs) contributing to human cardiometabolic disorders is challenging. Here we apply a reverse GxE candidate search by deriving candidate variants from promoter-enhancer interactions that respond to dietary fatty acid challenge through altered chromatin accessibility in human primary adipocytes. We then test all variants residing in the lipid-responsive open chromatin sites within adipocyte promoter-enhancer contacts for interaction effects between the genotype and dietary saturated fat intake on body mass index (BMI) in the UK Biobank. We discover 14 novel GxE variants in 12 lipid-responsive promoters, including well-known lipid genes (LIPE, CARM1, and PLIN2) and novel genes, such as LDB3, for which we provide further functional and integrative genomics evidence. We further identify 24 GxE variants in enhancers, totaling 38 new GxE variants for BMI in the UK Biobank, demonstrating that molecular genomics data produced in physiologically relevant contexts can discover new functional GxE mechanisms in humans.

Phenotype-Specific Enrichment of Mendelian Disorder Genes near GWAS Regions across 62 Complex Traits.

Although recent studies provide evidence for a common genetic basis between complex traits and Mendelian disorders, a thorough quantification of their overlap in a phenotype-specific manner remains elusive. Here, we have quantified the overlap of genes identified through large-scale genome-wide association studies (GWASs) for 62 complex traits and diseases with genes containing mutations known to cause 20 broad categories of Mendelian disorders. We identified a significant enrichment of genes linked to phenotypically matched Mendelian disorders in GWAS gene sets; of the total 1,240 comparisons, a higher proportion of phenotypically matched or related pairs (n = 50 of 92 [54%]) than phenotypically unmatched pairs (n = 27 of 1,148 [2%]) demonstrated significant overlap, confirming a phenotype-specific enrichment pattern. Further, we observed elevated GWAS effect sizes near genes linked to phenotypically matched Mendelian disorders. Finally, we report examples of GWAS variants localized at the transcription start site or physically interacting with the promoters of genes linked to phenotypically matched Mendelian disorders. Our results are consistent with the hypothesis that genes that are disrupted in Mendelian disorders are dysregulated by non-coding variants in complex traits and demonstrate how leveraging findings from related Mendelian disorders and functional genomic datasets can prioritize genes that are putatively dysregulated by local and distal non-coding GWAS variants.

Author Correction: Integration of human adipocyte chromosomal interactions with adipose gene expression prioritizes obesity-related genes from GWAS.

In the original version of this Article, Supplementary Table 10 contained incorrect primer sequences for the mobility shift assay for SNP rs4776984. These errors have now been fixed and the corrected version of the Supplementary Information PDF is available to download from the HTML version of the Article.

Integration of human adipocyte chromosomal interactions with adipose gene expression prioritizes obesity-related genes from GWAS.

Increased adiposity is a hallmark of obesity and overweight, which affect 2.2 billion people world-wide. Understanding the genetic and molecular mechanisms that underlie obesity-related phenotypes can help to improve treatment options and drug development. Here we perform promoter Capture Hi-C in human adipocytes to investigate interactions between gene promoters and distal elements as a transcription-regulating mechanism contributing to these phenotypes. We find that promoter-interacting elements in human adipocytes are enriched for adipose-related transcription factor motifs, such as PPARG and CEBPB, and contribute to heritability of cis-regulated gene expression. We further intersect these data with published genome-wide association studies for BMI and BMI-related metabolic traits to identify the genes that are under genetic cis regulation in human adipocytes via chromosomal interactions. This integrative genomics approach identifies four cis-eQTL-eGene relationships associated with BMI or obesity-related traits, including rs4776984 and MAP2K5, which we further confirm by EMSA, and highlights 38 additional candidate genes.

Hand2 elevates cardiomyocyte production during zebrafish heart development and regeneration.

Embryonic heart formation requires the production of an appropriate number of cardiomyocytes; likewise, cardiac regeneration following injury relies upon the recovery of lost cardiomyocytes. The basic helix-loop-helix (bHLH) transcription factor Hand2 has been implicated in promoting cardiomyocyte formation. It is unclear, however, whether Hand2 plays an instructive or permissive role during this process. Here, we find that overexpression of hand2 in the early zebrafish embryo is able to enhance cardiomyocyte production, resulting in an enlarged heart with a striking increase in the size of the outflow tract. Our evidence indicates that these increases are dependent on the interactions of Hand2 in multimeric complexes and are independent of direct DNA binding by Hand2. Proliferation assays reveal that hand2 can impact cardiomyocyte production by promoting division of late-differentiating cardiac progenitors within the second heart field. Additionally, our data suggest that hand2 can influence cardiomyocyte production by altering the patterning of the anterior lateral plate mesoderm, potentially favoring formation of the first heart field at the expense of hematopoietic and vascular lineages. The potency of hand2 during embryonic cardiogenesis suggested that hand2 could also impact cardiac regeneration in adult zebrafish; indeed, we find that overexpression of hand2 can augment the regenerative proliferation of cardiomyocytes in response to injury. Together, our studies demonstrate that hand2 can drive cardiomyocyte production in multiple contexts and through multiple mechanisms. These results contribute to our understanding of the potential origins of congenital heart disease and inform future strategies in regenerative medicine.