Han Sun

All Publications

• Proteomic predictors of individualized nutrient-specific insulin secretion in health and disease. Cell metabolism Kolic, J., Sun, W. G., Cen, H. H., Ewald, J. D., Rogalski, J. C., Sasaki, S., Sun, H., Rajesh, V., Xia, Y. H., Moravcova, R., Skovsø, S., Spigelman, A. F., Manning Fox, J. E., Lyon, J., Beet, L., Xia, J., Lynn, F. C., Gloyn, A. L., Foster, L. J., MacDonald, P. E., Johnson, J. D. 2024; 36 (7): 1619-1633.e5

  Abstract

  Population-level variation and mechanisms behind insulin secretion in response to carbohydrate, protein, and fat remain uncharacterized. We defined prototypical insulin secretion responses to three macronutrients in islets from 140 cadaveric donors, including those with type 2 diabetes. The majority of donors' islets exhibited the highest insulin response to glucose, moderate response to amino acid, and minimal response to fatty acid. However, 9% of donors' islets had amino acid responses, and 8% had fatty acid responses that were larger than their glucose-stimulated insulin responses. We leveraged this heterogeneity and used multi-omics to identify molecular correlates of nutrient responsiveness, as well as proteins and mRNAs altered in type 2 diabetes. We also examined nutrient-stimulated insulin release from stem cell-derived islets and observed responsiveness to fat but not carbohydrate or protein-potentially a hallmark of immaturity. Understanding the diversity of insulin responses to carbohydrate, protein, and fat lays the groundwork for personalized nutrition.

  View details for DOI 10.1016/j.cmet.2024.06.001

  View details for PubMedID 38959864

• Heterogeneity of increased biological age in type 2 diabetes correlates with differential tissue DNA methylation, biological variables, and pharmacological treatments. GeroScience Cortez, B. N., Pan, H., Hinthorn, S., Sun, H., Neretti, N., Gloyn, A. L., Aguayo-Mazzucato, C. 2023

  Abstract

  Biological age (BA) closely depicts age-related changes at a cellular level. Type 2 diabetes mellitus (T2D) accelerates BA when calculated using clinical biomarkers, but there is a large spread in the magnitude of individuals' age acceleration in T2D suggesting additional factors contributing to BA. Additionally, it is unknown whether BA can be changed with treatment. We hypothesized that potential determinants of the heterogeneous BA distribution in T2D could be due to differential tissue aging as reflected at the DNA methylation (DNAm) level, or biological variables and their respective therapeutic treatments. Publicly available DNAm samples were obtained to calculate BA using the DNAm phenotypic age (DNAmPhenoAge) algorithm. DNAmPhenoAge showed age acceleration in T2D samples of whole blood, pancreatic islets, and liver, but not in adipose tissue or skeletal muscle. Analysis of genes associated with differentially methylated CpG sites found a significant correlation between eight individual CpG methylation sites and gene expression. Clinical biomarkers from participants in the NHANES 2017-2018 and ACCORD cohorts were used to calculate BA using the Klemera and Doubal (KDM) method. Cardiovascular and glycemic biomarkers associated with increased BA while intensive blood pressure and glycemic management reduced BA to CA levels, demonstrating that accelerated BA can be restored in the setting of T2D.

  View details for DOI 10.1007/s11357-023-01009-8

  View details for PubMedID 37987887

• PAX4 loss of function increases diabetes risk by altering human pancreatic endocrine cell development. Nature communications Lau, H. H., Krentz, N. A., Abaitua, F., Perez-Alcantara, M., Chan, J. W., Ajeian, J., Ghosh, S., Lee, Y., Yang, J., Thaman, S., Champon, B., Sun, H., Jha, A., Hoon, S., Tan, N. S., Gardner, D. S., Kao, S. L., Tai, E. S., Gloyn, A. L., Teo, A. K. 2023; 14 (1): 6119

  Abstract

  The coding variant (p.Arg192His) in the transcription factor PAX4 is associated with an altered risk for type 2 diabetes (T2D) in East Asian populations. In mice, Pax4 is essential for beta cell formation but its role on human beta cell development and/or function is unknown. Participants carrying the PAX4 p.His192 allele exhibited decreased pancreatic beta cell function compared to homozygotes for the p.192Arg allele in a cross-sectional study in which we carried out an intravenous glucose tolerance test and an oral glucose tolerance test. In a pedigree of a patient with young onset diabetes, several members carry a newly identified p.Tyr186X allele. In the human beta cell model, EndoC-βH1, PAX4 knockdown led to impaired insulin secretion, reduced total insulin content, and altered hormone gene expression. Deletion of PAX4 in human induced pluripotent stem cell (hiPSC)-derived islet-like cells resulted in derepression of alpha cell gene expression. In vitro differentiation of hiPSCs carrying PAX4 p.His192 and p.X186 risk alleles exhibited increased polyhormonal endocrine cell formation and reduced insulin content that can be reversed with gene correction. Together, we demonstrate the role of PAX4 in human endocrine cell development, beta cell function, and its contribution to T2D-risk.

  View details for DOI 10.1038/s41467-023-41860-z

  View details for PubMedID 37777536

  View details for PubMedCentralID 5034897

• Loss of RREB1 in pancreatic beta cells reduces cellular insulin content and affects endocrine cell gene expression. Diabetologia Mattis, K. K., Krentz, N. A., Metzendorf, C., Abaitua, F., Spigelman, A. F., Sun, H., Ikle, J. M., Thaman, S., Rottner, A. K., Bautista, A., Mazzaferro, E., Perez-Alcantara, M., Manning Fox, J. E., Torres, J. M., Wesolowska-Andersen, A., Yu, G. Z., Mahajan, A., Larsson, A., MacDonald, P. E., Davies, B., den Hoed, M., Gloyn, A. L. 2023

  Abstract

  Genome-wide studies have uncovered multiple independent signals at the RREB1 locus associated with altered type 2 diabetes risk and related glycaemic traits. However, little is known about the function of the zinc finger transcription factor Ras-responsive element binding protein 1 (RREB1) in glucose homeostasis or how changes in its expression and/or function influence diabetes risk.A zebrafish model lacking rreb1a and rreb1b was used to study the effect of RREB1 loss in vivo. Using transcriptomic and cellular phenotyping of a human beta cell model (EndoC-βH1) and human induced pluripotent stem cell (hiPSC)-derived beta-like cells, we investigated how loss of RREB1 expression and activity affects pancreatic endocrine cell development and function. Ex vivo measurements of human islet function were performed in donor islets from carriers of RREB1 type 2 diabetes risk alleles.CRISPR/Cas9-mediated loss of rreb1a and rreb1b function in zebrafish supports an in vivo role for the transcription factor in beta cell mass, beta cell insulin expression and glucose levels. Loss of RREB1 also reduced insulin gene expression and cellular insulin content in EndoC-βH1 cells and impaired insulin secretion under prolonged stimulation. Transcriptomic analysis of RREB1 knockdown and knockout EndoC-βH1 cells supports RREB1 as a novel regulator of genes involved in insulin secretion. In vitro differentiation of RREB1KO/KO hiPSCs revealed dysregulation of pro-endocrine cell genes, including RFX family members, suggesting that RREB1 also regulates genes involved in endocrine cell development. Human donor islets from carriers of type 2 diabetes risk alleles in RREB1 have altered glucose-stimulated insulin secretion ex vivo, consistent with a role for RREB1 in regulating islet cell function.Together, our results indicate that RREB1 regulates beta cell function by transcriptionally regulating the expression of genes involved in beta cell development and function.

  View details for DOI 10.1007/s00125-022-05856-6

  View details for PubMedID 36633628

• Inferring causal genes at type 2 diabetes GWAS loci through chromosome interactions in islet cells. Wellcome open research Torres, J. M., Sun, H., Nylander, V., Downes, D. J., van de Bunt, M., McCarthy, M. I., Hughes, J. R., Gloyn, A. L. 2023; 8: 165

  Abstract

  Background: Resolving causal genes for type 2 diabetes at loci implicated by genome-wide association studies (GWAS) requires integrating functional genomic data from relevant cell types. Chromatin features in endocrine cells of the pancreatic islet are particularly informative and recent studies leveraging chromosome conformation capture (3C) with Hi-C based methods have elucidated regulatory mechanisms in human islets. However, these genome-wide approaches are less sensitive and afford lower resolution than methods that target specific loci. Methods: To gauge the extent to which targeted 3C further resolves chromatin-mediated regulatory mechanisms at GWAS loci, we generated interaction profiles at 23 loci using next-generation (NG) capture-C in a human beta cell model (EndoC-betaH1) and contrasted these maps with Hi-C maps in EndoC-betaH1 cells and human islets and a promoter capture Hi-C map in human islets. Results: We found improvements in assay sensitivity of up to 33-fold and resolved ~3.6X more chromatin interactions. At a subset of 18 loci with 25 co-localised GWAS and eQTL signals, NG Capture-C interactions implicated effector transcripts at five additional genetic signals relative to promoter capture Hi-C through physical contact with gene promoters. Conclusions: High resolution chromatin interaction profiles at selectively targeted loci can complement genome- and promoter-wide maps.

  View details for DOI 10.12688/wellcomeopenres.18653.2

  View details for PubMedID 37736013

• A genome-wide CRISPR screen identifies CALCOCO2 as a regulator of beta cell function influencing type 2 diabetes risk. Nature genetics Rottner, A. K., Ye, Y., Navarro-Guerrero, E., Rajesh, V., Pollner, A., Bevacqua, R. J., Yang, J., Spigelman, A. F., Baronio, R., Bautista, A., Thomsen, S. K., Lyon, J., Nawaz, S., Smith, N., Wesolowska-Andersen, A., Fox, J. E., Sun, H., Kim, S. K., Ebner, D., MacDonald, P. E., Gloyn, A. L. 2022

  Abstract

  Identification of the genes and processes mediating genetic association signals for complex diseases represents a major challenge. As many of the genetic signals for type 2 diabetes (T2D) exert their effects through pancreatic islet-cell dysfunction, we performed a genome-wide pooled CRISPR loss-of-function screen in a human pancreatic beta cell line. We assessed the regulation of insulin content as a disease-relevant readout of beta cell function and identified 580 genes influencing this phenotype. Integration with genetic and genomic data provided experimental support for 20 candidate T2D effector transcripts including the autophagy receptor CALCOCO2. Loss of CALCOCO2 was associated with distorted mitochondria, less proinsulin-containing immature granules and accumulation of autophagosomes upon inhibition of late-stage autophagy. Carriers of T2D-associated variants at the CALCOCO2 locus further displayed altered insulin secretion. Our study highlights how cellular screens can augment existing multi-omic efforts to support mechanistic understanding and provide evidence for causal effects at genome-wide association studies loci.

  View details for DOI 10.1038/s41588-022-01261-2

  View details for PubMedID 36543916

• Zmiz1 is required for mature β-cell function and mass expansion upon high fat feeding. Molecular metabolism Alghamdi, T. A., Krentz, N. A., Smith, N., Spigelman, A. F., Rajesh, V., Jha, A., Ferdaoussi, M., Suzuki, K., Yang, J., Manning Fox, J. E., Sun, H., Sun, Z., Gloyn, A. L., MacDonald, P. E. 2022: 101621

  Abstract

  Identifying the transcripts which mediate genetic association signals for type 2 diabetes (T2D) is critical to understand disease mechanisms. Studies in pancreatic islets support the transcription factor ZMIZ1 as a transcript underlying a T2D GWAS signal, but how it influences T2D risk is unknown.β-cell-specific Zmiz1 knockout (Zmiz1βKO) mice were generated and phenotypically characterised. Glucose homeostasis was assessed in Zmiz1βKO mice and their control littermates on chow diet (CD) and high fat diet (HFD). Islet morphology and function were examined by immunocytochemistry and in vitro islet function was assessed by dynamic insulin secretion assay. Transcript and protein expression were assessed by RNA sequencing and Western blotting. In islets isolated from genotyped human donors, we assessed glucose-dependent insulin secretion and islet insulin content by static incubation assay.Male and female Zmiz1βKO mice were glucose intolerant with impaired insulin secretion, compared with control littermates. Transcriptomic profiling of Zmiz1βKO islets identified over 500 differentially expressed genes including those involved in β-cell function and maturity, which we confirmed at the protein level. Upon HFD, Zmiz1βKO mice fail to expand β-cell mass and become severely diabetic. Human islets from carriers of the ZMIZ1-linked T2D-risk alleles have reduced islet insulin content and glucose-stimulated insulin secretion.β-cell Zmiz1 is required for normal glucose homeostasis. Genetic variation at the ZMIZ1 locus may influence T2D-risk by reducing islet mass expansion upon metabolic stress and the ability to maintain a mature β-cell state.

  View details for DOI 10.1016/j.molmet.2022.101621

  View details for PubMedID 36307047

• Patient-derived gene and protein expression signatures of NGLY1 deficiency. Journal of biochemistry Rauscher, B., Mueller, W. F., Clauder-Munster, S., Jakob, P., Islam, M. S., Sun, H., Ghidelli-Disse, S., Boesche, M., Bantscheff, M., Pflaumer, H., Collier, P., Haase, B., Chen, S., Hoffman, R., Wang, G., Benes, V., Drewes, G., Snyder, M., Steinmetz, L. M. 2021

  Abstract

  N-Glycanase 1 (NGLY1) deficiency is a rare and complex genetic disorder. Although recent studies have shed light on the molecular underpinnings of NGLY1 deficiency, a systematic characterization of gene and protein expression changes in patient-derived cells has been lacking. Here, we performed RNA-sequencing and mass spectrometry to determine the transcriptomes and proteomes of 66 cell lines representing 4 different cell types derived from 14 NGLY1 deficient patients and 17 controls. Although NGLY1 protein levels were up to 9.5-fold downregulated in patients compared to parents, residual and likely non-functional NGLY1 protein was detectable in all patient-derived lymphoblastoid cell lines. Consistent with the role of NGLY1 as a regulator of the transcription factor Nrf1, we observed a cell type-independent downregulation of proteasomal genes in NGLY1 deficient cells. In contrast, genes involved in ribosome biogenesis and mRNA processing were upregulated in multiple cell types. In addition, we observed cell type-specific effects. For example, genes and proteins involved in glutathione synthesis, such as the glutamate-cysteine ligase subunits GCLC and GCLM, were downregulated specifically in lymphoblastoid cells. We provide a web application that enables access to all results generated in this study at https://apps.embl.de/ngly1browser. This resource will guide future studies of NGLY1 deficiency in directions that are most relevant to patients.

  View details for DOI 10.1093/jb/mvab131

  View details for PubMedID 34878535

• Single-molecule, full-length transcript isoform sequencing reveals disease-associated RNA isoforms in cardiomyocytes. Nature communications Zhu, C., Wu, J., Sun, H., Briganti, F., Meder, B., Wei, W., Steinmetz, L. M. 2021; 12 (1): 4203

  Abstract

  Alternative splicing generates differing RNA isoforms that govern phenotypic complexity of eukaryotes. Its malfunction underlies many diseases, including cancer and cardiovascular diseases. Comparative analysis of RNA isoforms at the genome-wide scale has been difficult. Here, we establish an experimental and computational pipeline that performs de novo transcript annotation and accurately quantifies transcript isoforms from cDNA sequences with a full-length isoform detection accuracy of 97.6%. We generate a searchable, quantitative human transcriptome annotation with 31,025 known and 5,740 novel transcript isoforms ( http://steinmetzlab.embl.de/iBrowser/ ). By analyzing the isoforms in the presence of RNA Binding Motif Protein 20 (RBM20) mutations associated with aggressive dilated cardiomyopathy (DCM), we identify 121 differentially expressed transcript isoforms in 107 cardiac genes. Our approach enables quantitative dissection of complex transcript architecture instead of mere identification of inclusion or exclusion of individual exons, as exemplified by the discovery of IMMT isoforms mis-spliced by RBM20 mutations. Thereby we achieve a path to direct differential expression testing independent of an existing annotation of transcript isoforms, providing more immediate biological interpretation and higher resolution transcriptome comparisons.

  View details for DOI 10.1038/s41467-021-24484-z

  View details for PubMedID 34244519

• A Circulating Bioreactor Reprograms Cancer Cells Toward a More Mesenchymal Niche. Advanced biosystems Calamak, S., Ermis, M., Sun, H., Islam, S., Sikora, M., Nguyen, M., Hasirci, V., Steinmetz, L. M., Demirci, U. 2020; 4 (2): e1900139

  Abstract

  Cancer is a complex and heterogeneous disease, and cancer cells dynamically interact with the mechanical microenvironment such as hydrostatic pressure, fluid shear, and interstitial flow. These factors play an essential role in cell fate and circulating tumor cell heterogeneity, and can influence the cellular phenotype. In this study, a peristaltic continuous flow reactor is designed and applied to HCT-116 colorectal carcinoma cells to mimic the fluid dynamics of circulation. With this intervention, a CD44/CD24-cell subpopulation emerges, and 100 genes are significantly regulated. The expression of cells at 4 h in the flow reactor is very similar to TGF-ß treatment, which is an inducer of epithelial-mesenchymal transition. ATF3 and SERPINE1 are significantly upregulated in these groups, suggesting that the mesenchymal transition is induced through this signaling pathway. This flow reactor model is satisfactory on its own to reprogram colorectal cancer cells toward a more mesenchymal niche mimicking circulation of the blood.

  View details for DOI 10.1002/adbi.201900139

  View details for PubMedID 32293132

• iPSC Modeling of RBM20-Deficient DCM Identifies Upregulation of RBM20 as a Therapeutic Strategy. Cell reports Briganti, F. n., Sun, H. n., Wei, W. n., Wu, J. n., Zhu, C. n., Liss, M. n., Karakikes, I. n., Rego, S. n., Cipriano, A. n., Snyder, M. n., Meder, B. n., Xu, Z. n., Millat, G. n., Gotthardt, M. n., Mercola, M. n., Steinmetz, L. M. 2020; 32 (10): 108117

  Abstract

  Recent advances in induced pluripotent stem cell (iPSC) technology and directed differentiation of iPSCs into cardiomyocytes (iPSC-CMs) make it possible to model genetic heart disease in vitro. We apply CRISPR/Cas9 genome editing technology to introduce three RBM20 mutations in iPSCs and differentiate them into iPSC-CMs to establish an in vitro model of RBM20 mutant dilated cardiomyopathy (DCM). In iPSC-CMs harboring a known causal RBM20 variant, the splicing of RBM20 target genes, calcium handling, and contractility are impaired consistent with the disease manifestation in patients. A variant (Pro633Leu) identified by exome sequencing of patient genomes displays the same disease phenotypes, thus establishing this variant as disease causing. We find that all-trans retinoic acid upregulates RBM20 expression and reverts the splicing, calcium handling, and contractility defects in iPSC-CMs with different causal RBM20 mutations. These results suggest that pharmacological upregulation of RBM20 expression is a promising therapeutic strategy for DCM patients with a heterozygous mutation in RBM20.

  View details for DOI 10.1016/j.celrep.2020.108117

  View details for PubMedID 32905764

• Loss of N-glycanase 1 Alters Transcriptional and Translational Regulation in K562 Cell Lines. G3 (Bethesda, Md.) Mueller, W. F., Jakob, P. n., Sun, H. n., Clauder-Münster, S. n., Ghidelli-Disse, S. n., Ordonez, D. n., Boesche, M. n., Bantscheff, M. n., Collier, P. n., Haase, B. n., Benes, V. n., Paulsen, M. n., Sehr, P. n., Lewis, J. n., Drewes, G. n., Steinmetz, L. M. 2020

  Abstract

  N-Glycanase 1 (NGLY1) deficiency is an ultra-rare, complex and devastating neuromuscular disease, with multi-organ symptoms. NGLY1 is a deglycosylating protein involved in the degradation of misfolded proteins retrotranslocated from the endoplasmic reticulum (ER). We show that the loss of NGLY1 causes substantial changes in the RNA and protein landscape of K562 cells. We employed the CMap database to predict compounds that can modulate NGLY1 activity. Utilizing our robust K562 screening system, we demonstrate that the compound NVP-BEZ235 promotes degradation of NGLY1-dependent substrates, concurrent with increased autophagic flux, suggesting that autophagy may assist in clearing aberrant substrates during NGLY1 deficiency.

  View details for DOI 10.1534/g3.119.401031

  View details for PubMedID 32265286

• Dysregulated ribonucleoprotein granules promote cardiomyopathy in RBM20 gene-edited pigs. Nature medicine Schneider, J. W., Oommen, S. n., Qureshi, M. Y., Goetsch, S. C., Pease, D. R., Sundsbak, R. S., Guo, W. n., Sun, M. n., Sun, H. n., Kuroyanagi, H. n., Webster, D. A., Coutts, A. W., Holst, K. A., Edwards, B. S., Newville, N. n., Hathcock, M. A., Melkamu, T. n., Briganti, F. n., Wei, W. n., Romanelli, M. G., Fahrenkrug, S. C., Frantz, D. E., Olson, T. M., Steinmetz, L. M., Carlson, D. F., Nelson, T. J. 2020

  Abstract

  Ribonucleoprotein (RNP) granules are biomolecular condensates-liquid-liquid phase-separated droplets that organize and manage messenger RNA metabolism, cell signaling, biopolymer assembly, biochemical reactions and stress granule responses to cellular adversity. Dysregulated RNP granules drive neuromuscular degenerative disease but have not previously been linked to heart failure. By exploring the molecular basis of congenital dilated cardiomyopathy (DCM) in genome-edited pigs homozygous for an RBM20 allele encoding the pathogenic R636S variant of human RNA-binding motif protein-20 (RBM20), we discovered that RNP granules accumulated abnormally in the sarcoplasm, and we confirmed this finding in myocardium and reprogrammed cardiomyocytes from patients with DCM carrying the R636S allele. Dysregulated sarcoplasmic RBM20 RNP granules displayed liquid-like material properties, docked at precisely spaced intervals along cytoskeletal elements, promoted phase partitioning of cardiac biomolecules and fused with stress granules. Our results link dysregulated RNP granules to myocardial cellular pathobiology and heart failure in gene-edited pigs and patients with DCM caused by RBM20 mutation.

  View details for DOI 10.1038/s41591-020-1087-x

  View details for PubMedID 33188278

• Biological plasticity rescues target activity in CRISPR knock outs. Nature methods Smits, A. H., Ziebell, F. n., Joberty, G. n., Zinn, N. n., Mueller, W. F., Clauder-Münster, S. n., Eberhard, D. n., Fälth Savitski, M. n., Grandi, P. n., Jakob, P. n., Michon, A. M., Sun, H. n., Tessmer, K. n., Bürckstümmer, T. n., Bantscheff, M. n., Steinmetz, L. M., Drewes, G. n., Huber, W. n. 2019

  Abstract

  Gene knock outs (KOs) are efficiently engineered through CRISPR-Cas9-induced frameshift mutations. While the efficiency of DNA editing is readily verified by DNA sequencing, a systematic understanding of the efficiency of protein elimination has been lacking. Here we devised an experimental strategy combining RNA sequencing and triple-stage mass spectrometry to characterize 193 genetically verified deletions targeting 136 distinct genes generated by CRISPR-induced frameshifts in HAP1 cells. We observed residual protein expression for about one third of the quantified targets, at variable levels from low to original, and identified two causal mechanisms, translation reinitiation leading to N-terminally truncated target proteins or skipping of the edited exon leading to protein isoforms with internal sequence deletions. Detailed analysis of three truncated targets, BRD4, DNMT1 and NGLY1, revealed partial preservation of protein function. Our results imply that systematic characterization of residual protein expression or function in CRISPR-Cas9-generated KO lines is necessary for phenotype interpretation.

  View details for DOI 10.1038/s41592-019-0614-5

  View details for PubMedID 31659326