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

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.Peer reviewe

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.Peer reviewe

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.Peer reviewe

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

Family-specific aggregation of lipid GWAS variants confers the susceptibility to familial hypercholesterolemia in a large Austrian family

Background and aims: Hypercholesterolemia confers susceptibility to cardiovascular disease (CVD). Both serum total cholesterol (TC) and LDL-cholesterol (LDL-C) exhibit a strong genetic component (heritability estimates 0.41-0.50). However, a large part of this heritability cannot be explained by the variants identified in recent extensive genome-wide association studies (GWAS) on lipids. Our aim was to find genetic causes leading to high LDL-C levels and ultimately CVD in a large Austrian family presenting with what appears to be autosomal dominant inheritance for familial hypercholesterolemia (FH). Methods: We utilized linkage analysis followed by whole-exome sequencing and genetic risk score analysis using an Austrian multi-generational family with various dyslipidemias, including elevated TC and LDL-C, and one family branch with elevated lipoprotein (a) (Lp(a)). Results: We did not find evidence for genome-wide significant linkage for LDL-C or apparent causative variants in the known FH genes rather, we discovered a particular family-specific combination of nine GWAS LDL-C SNPs (p = 0.02 by permutation), and putative less severe familial hypercholesterolemia mutations in the LDLR and APOB genes in a subset of the affected family members. Separately, high Lp(a) levels observed in one branch of the family were explained primarily by the LPA locus, including short (<23) Kringle IV repeats and rs3798220. Conclusions: Taken together, some forms of FH may be explained by family-specific combinations of LDL-C GWAS SNPs. (c) 2017 Elsevier B.V. All rights reserved.Peer reviewe

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

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 similar to 1400) and WHRadjBMI GWAS data (n similar to 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.Peer reviewe