Biology subjects

Kursawe, R.

Publications and source records attributed to Kursawe, R..

9 recordsLinked to original sources

bioRxiv2025

Single-cell decoding of human islet cell type-specific alterations in type 2 diabetes reveals converging genetic- and state-driven β-cell gene expression defects

Pancreatic islets maintain glucose homeostasis through coordinated action of their constituent endocrine and affiliate cell types and are central to type 2 diabetes (T2D) genetics and pathophysiology. Our understanding of robust human islet cell type-specific alterations in T2D remains limited. Here, we report comprehensive single cell transcriptome profiling of 245,878 human islet cells from a 48-donor cohort spanning non-diabetic (ND), pre-diabetic (PD), and T2D states, identifying 14 distinct cell types detected in every donor from each glycemic state. Cohort analysis reveals [~]25-30% loss of functional beta cell mass in T2D vs. ND or PD donors resulting from (1) reduced total beta cell numbers/proportions and (2) reciprocal loss of high function and gain of senescent {beta}-cell subpopulations. We identify in T2D {beta}-cells 511 differentially expressed genes (DEGs), including new (66.5%) and validated genes (e.g., FXYD2, SLC2A2, SYT1), and significant neuronal transmission and vitamin A metabolism pathway alterations. Importantly, we demonstrate newly identified DEG roles in human {beta}-cell viability and/or insulin secretion and link 47 DEGs to diabetes-relevant phenotypes in knockout mice, implicating them as potential causal islet dysfunction genes. Additionally, we nominate as candidate T2D causal genes and therapeutic targets 27 DEGs for which T2D genetic risk variants (GWAS SNPs) and pathophysiology (T2D vs. ND) exert concordant expression effects. We provide this freely accessible atlas for data exploration, analysis, and hypothesis testing. Together, this study provides new genomic resources for and insights into T2D pathophysiology and human islet dysfunction.

genomics↗

bioRxiv2024

LONP1 regulation of mitochondrial protein folding provides insight into beta cell failure in type 2 diabetes

Proteotoxicity is a contributor to the development of type 2 diabetes (T2D), but it is unknown whether protein misfolding in T2D is generalized or has special features. Here, we report a robust accumulation of misfolded proteins within the mitochondria of human pancreatic islets in T2D and elucidate its impact on {beta} cell viability. Surprisingly, quantitative proteomics studies of protein aggregates reveal that human islets from donors with T2D have a signature more closely resembling mitochondrial rather than ER protein misfolding. The matrix protease LonP1 and its chaperone partner mtHSP70 were among the proteins enriched in protein aggregates. Deletion of LONP1 in mice yields mitochondrial protein misfolding and reduced respiratory function, ultimately leading to {beta} cell apoptosis and hyperglycemia. Intriguingly, LONP1 gain of function ameliorates mitochondrial protein misfolding and restores human {beta} cell survival following glucolipotoxicity via a protease-independent effect requiring LONP1-mtHSP70 chaperone activity. Thus, LONP1 promotes {beta} cell survival and prevents hyperglycemia by facilitating mitochondrial protein folding. These observations may open novel insights into the nature of impaired proteostasis on {beta} cell loss in the pathogenesis of T2D that could be considered as future therapeutic targets.

cell biology↗

bioRxiv2024

Comparative Multi-omic Mapping of Human Pancreatic Islet Endoplasmic Reticulum and Cytokine Stress Responses Provides Insights into Type 2 Diabetes Genetics

Endoplasmic reticulum (ER) and inflammatory stress responses are two pathophysiologic factors contributing to islet dysfunction and failure in Type 2 Diabetes (T2D). However, how human islet cells respond to these stressors and whether T2D-associated genetic variants modulate these responses is unknown. To fill this knowledge gap, we profiled transcriptional (RNA-seq) and epigenetic (ATAC-seq) remodeling in human islets exposed to ex vivo ER (thapsigargin) or inflammatory (IL-1{beta}+IFN-{gamma}) stress. 5,427 genes ([~]32%) were associated with stress responses; most were stressor-specific, including upregulation of genes mediating unfolded protein response (e.g. DDIT3, ATF4) and NFKB signaling (e.g. NFKB1, NFKBIA) in ER stress and cytokine-induced inflammation respectively. Islet single-cell RNA-seq profiling revealed strong but heterogeneous beta cell ER stress responses, including a distinct beta cell subset that highly expressed apoptotic genes. Epigenetic profiling uncovered 14,968 stress-responsive cis-regulatory elements (CREs; [~]14%), the majority of which were stressor-specific, and revealed increased accessibility at binding sites of transcription factors that were induced upon stress (e.g. ATF4 for ER stress, IRF8 for cytokine-induced inflammation). Eighty-six stress-responsive CREs overlapped known T2D-associated variants, including 20 residing within CREs that were more accessible upon ER stress. Among these, we linked the rs6917676 T2D risk allele (T) to increased in vivo accessibility of an islet ER stress-responsive CRE and allele-specific beta cell nuclear factor binding in vitro. We showed that MAP3K5, the only ER stress-responsive gene in this locus, promotes beta cell apoptosis. Consistent with its pro-apoptotic and putative diabetogenic roles, MAP3K5 expression inversely correlated with beta cell abundance in human islets and was induced in beta cells from T2D donors. Together, this study provides new genome-wide insights into human islet stress responses and putative mechanisms of T2D genetic variants.

genomics↗

bioRxiv2022

Variation in histone configurations correlates with gene expression across nine inbred strains of mice

It is well established that epigenetic features, such as histone modifications and DNA methylation, are associated with variation in gene expression across cell types. Less well known is the extent to which epigenetic states vary across genetically diverse individuals, and whether such variation corresponds to inter-individual variation in gene expression. To investigate genetically driven variation in epigenetics, we conducted a survey of epigenetic modifications and gene expression in hepatocytes of nine inbred mouse strains. We surveyed four histone modifications (H3K4me1, H3K4me3, H3K27me3, and H3K27ac), and DNA methylation. We used ChromHMM to identify 14 chromatin states, each of which represented a distinct combination of the four histone modifications. We found that chromatin states varied widely across the nine strains and that epigenetic state was strongly correlated with local gene expression. We replicated this correspondence between chromatin state and gene expression in an independent population of Diversity Outbred mice in which we imputed local chromatin state. In contrast, we found that DNA methylation did not vary across the inbred strains and was not correlated with variation in gene expression in DO mice. This work suggests that chromatin state is highly influenced by local genotype and may be a primary mode through which expression quantitative trait loci (eQTLs) are mediated. Through examples, we demonstrate that naturally occurring chromatin state variation, in conjunction with gene expression, can aid in functional annotation of the mouse genome. Finally, we provide a data resource that documents variation in chromatin state in hepatocytes across genetically distinct mice.

genomics↗

bioRxiv2021

A read count-based method to detect multiplets and their cellular origins from snATAC-seq data

Similar to other droplet-based single cell assays, single nucleus ATAC-seq (snATAC-seq) data harbor multiplets that confound downstream analyses. Detecting multiplets in snATAC-seq data is particularly challenging due to its sparsity and trinary nature (0 reads: closed chromatin, 1: open in one allele, 2: open in both alleles), yet offers a unique opportunity to infer multiplets when >2 uniquely aligned reads are observed at multiple loci. Here, we implemented the first read count-based multiplet detection method, ATAC-DoubletDetector, that detects multiplets independently of cell-type. Using PBMC and pancreatic islet datasets, ATAC-DoubletDetector captured simulated heterotypic multiplets (different cell-types) with [~]0.60 recall, showing [~]24% improvement over state of the art. ATAC-DoubletDetector detected homotypic multiplets with [~]0.61 recall, representing the first method to detect multiplets originating from the same cell type. Using our novel clustering-based algorithm, multiplets were annotated to their cellular origins with [~]85% accuracy. Application of ATAC-DoubletDetector will improve downstream analysis of snATAC-seq.

bioinformatics↗

bioRxiv2020

Tet2 Controls Beta cells Responses to Inflammation in Type 1 Diabetes

{beta} cells may participate and contribute to their own demise during Type 1 diabetes (T1D). We identified a novel role of Tet2 in regulating immune killing of {beta} cells. Tet2 is induced in murine and human {beta} cells with inflammation but its expression is reduced in surviving {beta} cells. Tet2-KO mice that receive WT bone marrow transplants develop insulitis but not diabetes and islet infiltrates do not eliminate {beta} cells even though immune cells from the mice can transfer diabetes to NOD/scid recipients. Tet2-KO {beta} cells show reduced expression of inflammatory genes, associated with closed transcription factor binding sites. Tet2-KO recipients are protected from transfer of disease by diabetogenic immune cells. We conclude that Tet2 regulates pathologic interactions between {beta} cells and immune cells and controls intrinsic protective pathways. Modulating TET2 may enable survival of {beta} cells or their replacements in the setting of pathologic immune cells.

immunology↗

bioRxiv2020

Functional characterization of thousands of type 2 diabetes-associated and chromatin-modulating variants under steady state and endoplasmic reticulum stress

A major goal in functional genomics and complex disease genetics is to identify functional cis-regulatory elements (CREs) and single nucleotide polymorphisms (SNPs) altering CRE activity in disease-relevant cell types and environmental conditions. We tested >13,000 sequences containing each allele of 6,628 SNPs associated with altered in vivo chromatin accessibility in human islets and/or type 2 diabetes risk (T2D GWAS SNPs) for transcriptional activity in {beta} cell under steady state and endoplasmic reticulum (ER) stress conditions using the massively parallel reporter assay (MPRA). Approximately 30% (n=1,983) of putative CREs were active in at least one condition. SNP allelic effects on in vitro MPRA activity strongly correlated with their effects on in vivo islet chromatin accessibility (Pearson r=0.52), i.e., alleles associated with increased chromatin accessibility exhibited higher MPRA activity. Importantly, MPRA identified 220/2500 T2D GWAS SNPs, representing 104 distinct association signals, that significantly altered transcriptional activity in {beta} cells. This study has thus identified functional {beta} cell transcription-activating sequences with in vivo relevance, uncovered regulatory features that modulate transcriptional activity in {beta} cells under steady state and ER stress conditions, and substantially expanded the set of putative functional variants that modulate transcriptional activity in {beta} cells from thousands of genetically-linked T2D GWAS SNPs.

genomics↗

bioRxiv2018

EndoC-βH1 multi-genomic profiling defines gene regulatory programs governing human pancreatic β cell identity and function

EndoC-{beta}H1 is emerging as a critical human beta cell model to study the genetic and environmental etiologies of beta cell function, especially in the context of diabetes. Comprehensive knowledge of its molecular landscape is lacking yet required to fully take advantage of this model. Here, we report extensive chromosomal (spectral karyotyping), genetic (genotyping), epigenetic (ChIP-seq, ATAC-seq), chromatin interaction (Hi-C, Pol2 ChIA-PET), and transcriptomic (RNA-seq, miRNA-seq) maps of this cell model. Integrated analyses of these maps define known (e.g., PDX1, ISL1) and putative (e.g., PCSK1, mir-375) beta cell-specific chromatin interactions and transcriptional cis-regulatory networks, and identify allelic effects on cis-regulatory element use and expression.\n\nImportantly, comparative analyses with maps generated in primary human islets/beta cells indicate substantial preservation of chromatin looping, but also highlight chromosomal heterogeneity and fetal genomic signatures in EndoC-{beta}H1. Together, these maps, and an interactive web application we have created for their exploration, provide important tools for the broad community in the design and success of experiments to probe and manipulate the genetic programs governing beta cell identity and (dys)function in diabetes.

genomics↗

bioRxiv2017

Chromatin accessibility profiling uncovers genetic- and T2D disease state-associated changes in cis-regulatory element use in human islets

Genetic and environmental factors both contribute to islet dysfunction and failure, resulting in type 2 diabetes (T2D). The islet epigenome integrates these cues and can be remodeled by genetic and environmental variation. However, our knowledge of how genetic variants and T2D disease state alter human islet chromatin landscape and cis-regulatory element (RE) use is lacking. To fill this gap, we profiled and analyzed human islet chromatin accessibility maps from 19 genotyped individuals (5 with T2D) using ATAC-seq technology. Chromatin accessibility quantitative trait locus (caQTL) analyses identified 3001 sequence variants (FDR<10%) altering putative cis-RE use/activity. Islet caQTL were significantly and specifically enriched in islet stretch enhancers and islet-specific transcription factor binding motifs, such as FOXA2, NKX6.1, RFX5/6 and PDX1. Importantly, these analyses identified putative functional single nucleotide variants (SNVs) in 13 T2D-associated GWAS loci, including those previously associated with altered ZMIZ1, MTNR1B, RNF6, and ADCY5 islet expression, and linked the risk alleles to increased (n=8) or decreased (n=5) islet chromatin accessibility. Luciferase reporter assays confirmed allelic differences in cis-RE activity for 5/9 caQTL sequences tested, including a T2D-associated SNV in the IL20RA locus. Comparison of T2D and non-diabetic islets revealed 1882 open chromatin sites exhibiting T2D-associated chromatin accessibility changes (FDR<10%). Together, this study provides new insights into genetic variant and T2D disease state effects on islet cis-RE use and serves as an important resource to identify putative functional variants in T2D-and islet dysfunction-associated GWAS loci and link their risk allele to in vivo loss or gain of chromatin accessibility.

genomics↗