Biology subjects

Frazer, K. A.

Publications and source records attributed to Frazer, K. A..

At least 19 recordsLinked to original sources

bioRxiv2025

Genetics of growth rate in induced pluripotent stem cells

Human induced pluripotent stem cells (iPSCs) have transformed biomedical research by enabling the generation of diverse cell types from accessible somatic tissues. However, certain fundamental biological properties, such as the genetic and epigenetic determinants of iPSC proliferation, remain poorly characterized. We measured the growth of iPSC lines derived from 602 unique donors using high-throughput time-lapse imaging, quantified proliferation through a growth Area-Under-the-Curve (gAUC) phenotype, and correlated gAUC with the gene expression and genotype of the cell lines. We identified 3,091 genes associated with gAUC, many of which are well established regulators of cell proliferation. We also found that rare deleterious variants in WDR54 were associated with reduced iPSC growth and that WDR54 was differentially expressed with respect to gAUC. Although no common variants showed a genome-wide association with gAUC, iPSC lines from monozygotic twins were highly correlated, and common genetic variation explained approximately 71-75% of the variance in iPSC growth rates. These results indicate a complex genetic architecture of iPSC growth rates, where rare, large-effect variants in important growth regulators, including WDR54, are layered onto a highly polygenic background. These findings have important implications for the design of pooled iPSC-based studies and disease models, which may be confounded by intrinsic growth differences.

developmental biology↗

bioRxiv2024

IFN-γ activates an immune-like regulatory network in the cardiac vascular endothelium

The regulatory mechanisms underlying the response to pro-inflammatory cytokines in cardiac diseases are poorly understood. Here, we use iPSC-derived cardiovascular progenitor cells (CVPCs) to model the response to interferon gamma (IFN{gamma}) in human cardiac tissue. We generate RNA-seq and ATAC-seq for four CVPCs that were treated with IFN{gamma} and compare them with paired untreated controls. Transcriptional differences after treatment show that IFN{gamma} initiates an innate immune cell-like response, shifts the CVPC transcriptome towards coronary artery and aorta profiles, and stimulates expression of endothelial cell-specific genes. Analysis of the accessible chromatin shows that IFN{gamma} is a potent chromatin remodeler and establishes an IRF-STAT immune-cell like regulatory network. Finally, we show that 11 GWAS risk variants for 8 common cardiac diseases overlap IFN{gamma}-upregulated ATAC-seq peaks. Our findings reveal insights into IFN{gamma}-induced activation of an immune-like regulatory network in the cardiac vascular endothelium and the potential role that regulatory elements in this pathway play in common cardiac diseases. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/592380v2_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@e66966org.highwire.dtl.DTLVardef@111da44org.highwire.dtl.DTLVardef@110a8f5org.highwire.dtl.DTLVardef@c4997b_HPS_FORMAT_FIGEXP M_FIG C_FIG Paired RNA-seq and ATAC-seq was generated for induced pluripotent stem cell derived cardiovascular progenitor cells (CVPCs) treated with interferon-gamma (IFN{gamma}) and matched controls to model the effect of the pro-inflammatory cytokine on human cardiac tissue. Using the RNA-seq, transcriptomic changes were characterized by performing differential gene expression analysis, integrating gene expression data from hundreds of samples of adult cardiac tissues, and using single cell RNA-seq to evaluate cell type-specificity. Using the ATAC-seq, epigenomic changes were characterized by performing differential chromatin accessibility and transcription factor binding analyses, and annotating ATAC-seq peaks with chromatin states from over 800 tissues. Genetic variants in risk loci associated with cardiac diseases were intersected with ATAC-seq peaks to evaluate whether they were in chromatin that is only accessible after IFN{gamma} treatment. The findings demonstrate the utility of using CVPCs to model the effects of cytokines on cardiac tissues and provide the framework for conducting large scale studies to further evaluate GWAS loci that are explained by context-specific regulatory variation.

bioinformatics↗

bioRxiv2024

Multi-omic QTL mapping in early developmental tissues reveals phenotypic and temporal complexity of regulatory variants underlying GWAS loci

Most GWAS loci are presumed to affect gene regulation, however, only [~]43% colocalize with expression quantitative trait loci (eQTLs). To address this colocalization gap, we identify eQTLs, chromatin accessibility QTLs (caQTLs), and histone acetylation QTLs (haQTLs) using molecular samples from three early developmental (EDev) tissues. Through colocalization, we annotate 586 GWAS loci for 17 traits by QTL complexity, QTL phenotype, and QTL temporal specificity. We show that GWAS loci are highly enriched for colocalization with complex QTL modules that affect multiple elements (genes and/or peaks). We also demonstrate that caQTLs and haQTLs capture regulatory variations not associated with eQTLs and explain [~]49% of the functionally annotated GWAS loci. Additionally, we show that EDev-unique QTLs are strongly depleted for colocalizing with GWAS loci. By conducting one of the largest multi-omic QTL studies to date, we demonstrate that many GWAS loci exhibit phenotypic complexity and therefore, are missed by traditional eQTL analyses.

genomics↗

bioRxiv2023

High resolution long-read telomere sequencing reveals dynamic mechanisms in aging and cancer

Telomeres are the protective nucleoprotein structures at the end of linear eukaryotic chromosomes. Telomeres repetitive nature and length have traditionally challenged the precise assessment of the composition and length of individual human telomeres. Here, we present Telo-seq to resolve bulk, chromosome arm-specific and allele-specific human telomere lengths using Oxford Nanopore Technologies native long-read sequencing. Telo-seq resolves telomere shortening in five population doubling increments and reveals intrasample, chromosome arm-specific, allele-specific telomere length heterogeneity. Telo-seq can reliably discriminate between telomerase- and ALT-positive cancer cell lines. Thus, Telo-seq is a novel tool to study telomere biology during development, aging, and cancer at unprecedented resolution.

cell biology↗

bioRxiv2023

Unsupervised Machine Learning Identifies Chromatin Accessibility Regulatory Networks that Define Cell State Transitions in Pluripotency

Stem cells exist in vitro in a spectrum of interconvertible pluripotent states. Analyzing hundreds of hiPSCs derived from different individuals, we show the proportions of these pluripotent states vary considerably across lines. We discovered 13 gene network modules (GNMs) and 13 regulatory network modules (RNMs), which were highly correlated with each other suggesting that the coordinated co-accessibility of regulatory elements in the RNMs likely underlied the coordinated expression of genes in the GNMs. Epigenetic analyses revealed that regulatory networks underlying self-renewal and pluripotency have a surprising level of complexity. Genetic analyses identified thousands of regulatory variants that overlapped predicted transcription factor binding sites and were associated with chromatin accessibility in the hiPSCs. We show that the master regulator of pluripotency, the NANOG-OCT4 Complex, and its associated network were significantly enriched for regulatory variants with large effects, suggesting that they may play a role in the varying cellular proportions of pluripotency states between hiPSCs. Our work captures the coordinated activity of tens of thousands of regulatory elements in hiPSCs and bins these elements into discrete functionally characterized regulatory networks, shows that regulatory elements in pluripotency networks harbor variants with large effects, and provides a rich resource for future pluripotent stem cell research.

genomics↗

bioRxiv2022

Affinity-optimizing variants within cardiac enhancers disrupt heart development and contribute to cardiac traits

Enhancers direct precise gene expression patterns during development and harbor the majority of variants associated with disease. We find that suboptimal affinity ETS transcription factor binding sites are prevalent within Ciona and human developmental heart enhancers. Here we demonstrate in two diverse systems, Ciona intestinalis and human iPSC-derived cardiomyocytes (iPSC-CMs), that single nucleotide changes can optimize the affinity of ETS binding sites, leading to gain-of-function gene expression associated with heart phenotypes. In Ciona, ETS affinity-optimizing SNVs lead to ectopic expression and phenotypic changes including two beating hearts. In human iPSC-CMs, an affinity-optimizing SNV associated with QRS duration occurs within an SCN5A enhancer and leads to increased enhancer activity. Our mechanistic approach provides a much-needed systematic framework that works across different enhancers, cell types and species to pinpoint causal enhancer variants contributing to enhanceropathies, phenotypic diversity and evolutionary changes. In BriefThe prevalent use of low-affinity ETS sites within developmental heart enhancers creates vulnerability within genomes whereby single nucleotide changes can dramatically increase binding affinity, causing gain-of-function enhancer activity that impacts heart development. HighlightsETS affinity-optimizing SNVs can lead to migration defects and a multi-chambered heart. An ETS affinity-optimizing human SNV within an SCN5A enhancer increases expression and is associated with QRS duration. Searching for ETS affinity-optimizing variants is a systematic and generalizable approach to pinpoint causal enhancer variants.

genomics↗

bioRxiv2021

Type 1 diabetes risk genes mediate pancreatic beta cell survival in response to proinflammatory cytokines

Beta cells intrinsically contribute to the pathogenesis of type 1 diabetes (T1D), but the genes and molecular processes that mediate beta cell survival in T1D remain largely unknown. We combined high throughput functional genomics and human genetics to identify T1D risk loci regulating genes affecting beta cell survival in response to the proinflammatory cytokines IL-1{beta}, IFN{gamma}, and TNF. We mapped 38,931 cytokine-responsive candidate cis-regulatory elements (cCREs) active in beta cells using ATAC-seq and single nuclear ATAC-seq (snATAC-seq), and linked cytokine-responsive beta cell cCREs to putative target genes using single cell co-accessibility and HiChIP. We performed a genome-wide pooled CRISPR loss-of-function screen in EndoC-{beta}H1 cells, which identified 867 genes affecting cytokine-induced beta cell loss. Genes that promoted beta cell survival and had up-regulated expression in cytokine exposure were specifically enriched at T1D loci, and these genes were preferentially involved in inhibiting inflammatory response, ubiquitin-mediated proteolysis, mitophagy and autophagy. We identified 2,229 variants in cytokine-responsive beta cell cCREs altering transcription factor (TF) binding using high-throughput SNP-SELEX, and variants altering binding of TF families regulating stress, inflammation and apoptosis were broadly enriched for T1D association. Finally, through integration with genetic fine mapping, we annotated T1D loci regulating beta cell survival in cytokine exposure. At the 16p13 locus, a T1D variant affected TF binding in a cytokine-induced beta cell cCRE that physically interacted with the SOCS1 promoter, and increased SOCS1 activity promoted beta cell survival in cytokine exposure. Together our findings reveal processes and genes acting in beta cells during cytokine exposure that intrinsically modulate risk of T1D.

genomics↗

bioRxiv2021

Regulatory variants active in iPSC-derived pancreatic progenitor cells are associated with Type 2 Diabetes in adults

Pancreatic progenitor cells (PPC) are an early developmental multipotent cell type that give rise to mature endocrine, exocrine, and ductal cells. To investigate the extent to which regulatory variants active in PPC contribute to pancreatic complex traits and disease in the adult, we derived PPC from induced pluripotent stem cells (iPSCs) of nine unrelated individuals and generated single cell profiles of chromatin accessibility (snATAC- seq) and transcriptome (scRNA-seq). While iPSC-PPC differentiation was asynchronous and included cell types from early to late developmental stages, we found that the predominant cell type consisted of NKX6-1+ progenitors. Genetic characterization using snATAC-seq identified 86,261 regulatory variants that either displayed chromatin allelic bias and/or were predicted to affect active transcription factor (TF) binding sites. Integration of these regulatory variants with 380 fine-mapped type 2 diabetes (T2D) risk loci identified regulatory variants in 209 of these loci that are functional in iPSC-PPC, either by affecting transcription factor binding or through association with allelic effects on chromatin accessibility. The PPC active regulatory variants in 65 of these loci showed strong evidence of causally underlying the association with T2D. Our study shows that studying the functional associations of regulatory variation in iPSC-PPC enables the identification and characterization of causal SNPs for adult Type 2 Diabetes.

genetics↗

bioRxiv2021

Fine mapping spatiotemporal mechanisms of genetic variants underlying cardiac traits and disease

The causal variants and genes underlying thousands of cardiac GWAS signals have yet to be identified. Here, we leveraged spatiotemporal information on 966 RNA-seq cardiac samples and performed an expression quantitative trait locus (eQTL) analysis detecting eQTLs considering both eGenes and eIsoforms. We identified 2,578 eQTLs associated with a specific developmental stage-, tissue- and/or cell type. Colocalization between eQTL and GWAS signals of five cardiac traits identified variants with high posterior probabilities for being causal in 210 GWAS loci. Pulse pressure GWAS loci were enriched for colocalization with fetal- and smooth muscle- eQTLs; pulse rate with adult- and cardiac muscle- eQTLs; and atrial fibrillation with cardiac muscle- eQTLs. Fine mapping identified 79 credible sets with five or fewer SNPs, of which 15 were associated with spatiotemporal eQTLs. Our study shows that many cardiac GWAS variants impact traits and disease in a developmental stage-, tissue- and/or cell type-specific fashion.

genetics↗

bioRxiv2021

In heart failure reactivation of RNA-binding proteins drives the transcriptome into a fetal state

Transcriptome-wide expression changes occur during heart failure, including reactivation of fetal-specific isoforms. However, the underlying molecular mechanisms and the extent to which a fetal gene program switch occurs remains unclear. Limitations hindering transcriptome-wide analyses of alternative splicing differences (i.e. isoform switching) in cardiovascular system (CVS) tissues between fetal and adult (healthy and diseased) stages have included both cellular heterogeneity across bulk RNA-seq samples and limited availability of fetal tissue for research. To overcome these limitations, we have deconvoluted the cellular compositions of 996 RNA-seq samples representing heart failure, healthy adult (heart and arteria), and fetal-like (iPSC-derived cardiovascular progenitor cells) CVS tissues. Comparison of the expression profiles revealed that RNA-binding proteins (RBPs) are highly overexpressed in fetal-like compared with healthy adult and are reactivated in heart failure, which results in expression of thousands fetal-specific isoforms. Of note, isoforms for 20 different RBPs were among those that reverted in heart failure to the fetal-like expression pattern. We determined that, compared with adult-specific isoforms, fetal-specific isoforms are more likely to bind RBPs, have canonical sequences at their splice sites and encode proteins with more functions. Our findings suggest targeting RBP fetal-specific isoforms could result in novel therapeutics for heart failure.

genomics↗

bioRxiv2021

iPSC-derived pancreatic progenitors are an optimal model system to study T2D regulatory variants active during fetal development of the pancreas

The impact of genetic regulatory variation active in early pancreatic development on adult pancreatic disease and traits is not well understood. Here, we generate a panel of 107 fetal-like iPSC-derived pancreatic progenitor cells (iPSC-PPCs) from whole genome-sequenced individuals and identify 4,065 genes and 4,016 isoforms whose expression and/or alternative splicing are affected by regulatory variation. We integrate eQTLs identified in adult islets and whole pancreas samples, which reveal 1,805 eQTL associations that are unique to the fetal-like iPSC-PPCs and 1,043 eQTLs that exhibit regulatory plasticity across the fetal-like and adult pancreas tissues. Colocalization with GWAS risk loci for pancreatic diseases and traits show that some putative causal regulatory variants are active only in the fetal-like iPSC-PPCs and likely influence disease by modulating expression of disease-associated genes in early development, while others with regulatory plasticity likely exert their effects in both the fetal and adult pancreas by modulating expression of different disease genes in the two developmental stages.

genomics↗

bioRxiv2019

Systematic assessment of regulatory effects of human disease variants in pluripotent cells

Identifying regulatory genetic effects in pluripotent cells provides important insights into disease variants with potentially transient or developmental origins. Combining existing and newly-generated data, we characterized 1,367 iPSC lines from 948 unique donors, collectively analyzed within the \"Integrated iPSC QTL\" (i2QTL) Consortium. The sample size of our study allowed us to derive the most comprehensive map of quantitative trait loci (QTL) in pluripotent human cells to date. We mapped the effects of nearby common genetic variants on five expression phenotypes, identifying cis-QTL at gene-, exon-level and transcript-, splicing-, alternative polyadenylation-ratio (APA) for a total of 18,556 genes. For gene-level, we further quantified the effects of rare and singleton variants, and the effect of distal variants that act in trans (trans-eQTL), which we replicated in independent samples. Our data are a valuable community resource, uncovering novel regulatory effects that have not previously been described in differentiated cells and tissues. Building on this regulatory map, we functionally explore GWAS signals for over 4,336 trait loci, finding evidence for colocalization with common and rare iPSC QTL for traits such as height and BMI, and diseases, such as cancer and coronary artery disease.

genomics↗

bioRxiv2019

Structural variant classes and short tandem repeats differentially impact gene expression and complex traits

Structural variants (SVs) and short tandem repeats (STRs) comprise a broad group of diverse DNA variants which vastly differ in their sizes and distributions across the genome. Here, we show that different SV classes and STRs differentially impact gene expression and complex traits. Functional differences between SV classes and STRs include their genomic locations relative to eGenes, likelihood of being associated with multiple eGenes, associated eGene types (e.g., coding, noncoding, level of evolutionary constraint), effect sizes, linkage disequilibrium with tagging single nucleotide variants used in GWAS, and likelihood of being associated with GWAS traits. We also identified a set of high-impact SVs/STRs associated with the expression of three or more eGenes via chromatin loops and showed they are highly enriched for being associated with GWAS traits. Our study provides insights into the genomic properties of structural variant classes and short tandem repeats that impact gene expression and human traits.

genomics↗

bioRxiv2019

Discovery and Quality Analysis of a Comprehensive Set of Structural Variants and Short Tandem Repeats using Deep Whole Genome Sequencing Data

Structural variants (SVs) and short tandem repeats (STRs) are important sources of genetic diversity but are not routinely analyzed in genetic studies because they are difficult to accurately identify and genotype. Because SVs and STRs range in size and type, it is necessary to apply multiple algorithms that incorporate different types of evidence from sequencing data and employ complex filtering strategies to discover a comprehensive set of high-quality and reproducible variants. Here we assembled a set of 719 deep whole genome sequencing (WGS) samples (mean 42x) from 477 distinct individuals which we used to discover and genotype a wide spectrum of SV and STR variants using five algorithms. We used 177 unique pairs of genetic replicates to identify factors that affect variant call reproducibility and developed a systematic filtering strategy to create of one of the most complete and well characterized maps of SVs and STRs to date.

genomics↗

bioRxiv2019

Cellular deconvolution of GTEx tissues powers eQTL studies to discover thousands of novel disease and cell-type associated regulatory variants

The Genotype-Tissue Expression (GTEx) resource has contributed a wealth of novel insights into the regulatory impact of genetic variation on gene expression across human tissues, however thus far has not been utilized to study how variation acts at the resolution of the different cell types composing the tissues. To address this gap, using liver and skin as a proof-of-concept tissues, we show that readily available signature genes based on expression profiles of mouse cell types can be used to deconvolute the cellular composition of human GTEx tissues. We then deconvoluted 6,829 bulk RNA-seq samples corresponding to 28 GTEx tissues and show that we are able to quantify cellular heterogeneity, determining both the different cell types present in each of the tissues and how their proportions vary between samples of the same tissue type. Conducting eQTL analyses for GTEx liver and skin samples using cell type composition estimates as interaction terms, we identified thousands of novel genetic associations that had lower effect sizes and were cell-type-associated. We further show that cell-type-associated eQTLs in skin colocalize with melanoma, malignant neoplasm, and infection signatures, indicating variants that influence gene expression in distinct skin cell types play important roles in skin traits and disease. Overall, our study provides a framework to estimate the relative fractions of different cell types in GTEx tissues using signature genes from mouse cell types and functionally characterize human genetic variation that impacts gene expression in a cell-type-specific manner.

genetics↗

bioRxiv2019

Human iPSC gene signatures and X chromosome dosage impact response to WNT inhibition and cardiac differentiation fate

Non-genetic variability in human induced pluripotent stem cell (iPSC) lines impacts their differentiation outcome, limiting their utility for genetic studies and clinical applications. Despite the importance of understanding how non-genetic molecular variability influences iPSC differentiation outcome, large-scale studies capable of addressing this question have not yet been conducted. Here, we performed 258 directed differentiations of 191 iPSC lines using established protocols to generate iPSC-derived cardiovascular progenitor cells (iPSC-CVPCs). We observed cellular heterogeneity across the iPSC-CVPC samples due to varying fractions of two cell types: cardiomyocytes (CMs) and epicardium-derived cells (EPDCs). Analyzing the transcriptomes of CM-fated and EPDC-fated iPSCs discovered that 91 signature genes and X chromosome dosage differences influence WNT inhibition response during differentiation and are associated with cardiac fate. Analysis of an independent set of 39 iPSCs differentiated to the cardiac lineage confirmed shared sex and transcriptional differences that impact cardiac fate outcome. The scale and systematic approach of our study enabled novel insights into how iPSC transcriptional and X chromosome gene dosage differences influence WNT signaling during differentiation and hence cardiac cell fate.

genomics↗

bioRxiv2019

Chromatin co-accessibility is highly structured, spans entire chromosomes, and mediates long range regulatory genetic effects

Chromatin accessibility identifies active regions of the genome, often at transcription factor (TF) binding sites, enhancers, and promoters, and contains regulatory genetic variation. Functionally related accessible sites have been reported to be co-accessible; however, the prevalence and range of co-accessibility is unknown. We perform ATAC-seq in induced pluripotent stem cells from 134 individuals and integrate it with RNA-seq, WGS, and ChIP-seq, providing the first long-range chromosome-length analysis of co-accessibility. We show that co-accessibility is highly connected, with sites having a median of 24 co-accessible partners up to 250Mb away. We also show that co-accessibility can de novo identify known and novel co-expressed genes, and co-regulatory TFs and chromatin states. We perform a cis and trans-caQTL, a trans-eQTL, and examine allelic effects of co-accessibility, identifying tens of thousands of trans-caQTLs, and showing that trans genetic effects can be propagated through co-accessibility to gene expression for cell-type and disease relevant genes.

genomics↗

bioRxiv2019

Glioblastomas derived from genetically modified pluripotent stem cells recapitulate pathobiology

Many current cellular models aimed at elucidating cancer biology do not recapitulate pathobiology including tumor heterogeneity, an inherent feature of cancer that underlies treatment resistance. Here we introduce a new cancer modeling paradigm using genetically engineered human pluripotent stem cells (hiPSCs) that capture authentic cancer pathobiology. Orthotopic engraftment of neural progenitor cells derived from hiPSCs that have been genome-edited to contain tumor-associated genetic driver mutations revealed by The Cancer Genome Atlas project for glioblastoma (GBM) result in formation of high-grade gliomas. As observed in GBM patient samples, these models harbor inter-tumor heterogeneity resembling different GBM molecular subtypes, and intra-tumor heterogeneity. Further, re-engraftment of primary tumor neurospheres generates secondary tumors with features characteristic of patient samples and present mutation-dependent patterns of tumor evolution. Thus, these cancer avatar models provide a platform for a comprehensive longitudinal assessment of human tumor development as governed by molecular subtype mutations and lineage-restricted differentiation.

cancer biology↗