scholarly journals Chromatin compartment dynamics in a haploinsufficient model of cardiac laminopathy

2019 ◽  
Vol 218 (9) ◽  
pp. 2919-2944 ◽  
Author(s):  
Alessandro Bertero ◽  
Paul A. Fields ◽  
Alec S.T. Smith ◽  
Andrea Leonard ◽  
Kevin Beussman ◽  
...  
Keyword(s):  
Gene Expression ◽  
Large Scale ◽  
Induced Pluripotent Stem Cell ◽  
Nuclear Lamina ◽  
Lamin A ◽  
Chromosome Conformation ◽  
Nuclear Lamins ◽  
Genome Wide ◽  
Induced Pluripotent ◽  
Gene Expression Alterations

Mutations in A-type nuclear lamins cause dilated cardiomyopathy, which is postulated to result from dysregulated gene expression due to changes in chromatin organization into active and inactive compartments. To test this, we performed genome-wide chromosome conformation analyses in human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs) with a haploinsufficient mutation for lamin A/C. Compared with gene-corrected cells, mutant hiPSC-CMs have marked electrophysiological and contractile alterations, with modest gene expression changes. While large-scale changes in chromosomal topology are evident, differences in chromatin compartmentalization are limited to a few hotspots that escape segregation to the nuclear lamina and inactivation during cardiogenesis. These regions exhibit up-regulation of multiple noncardiac genes including CACNA1A, encoding for neuronal P/Q-type calcium channels. Pharmacological inhibition of the resulting current partially mitigates the electrical alterations. However, chromatin compartment changes do not explain most gene expression alterations in mutant hiPSC-CMs. Thus, global errors in chromosomal compartmentation are not the primary pathogenic mechanism in heart failure due to lamin A/C haploinsufficiency.

scholarly journals Chromatin compartment dynamics in a haploinsufficient model of cardiac laminopathy

2019 ◽  
Author(s):  
Alessandro Bertero ◽  
Paul A. Fields ◽  
Alec S. T. Smith ◽  
Andrea Leonard ◽  
Kevin Beussman ◽  
...  
Keyword(s):  
Gene Expression ◽  
Large Scale ◽  
Induced Pluripotent Stem Cell ◽  
Lamin A ◽  
Chromosome Conformation ◽  
Human Cardiomyocytes ◽  
Nuclear Lamins ◽  
Genome Wide ◽  
Induced Pluripotent ◽  
Gene Expression Alterations

AbstractPathogenic mutations in A-type nuclear lamins cause dilated cardiomyopathy, which is postulated to result from dysregulated gene expression due to changes in chromatin organization into active and inactive compartments. To test this, we performed genome-wide chromosome conformation analyses (Hi-C) in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) with a haploinsufficient mutation for lamin A/C. Compared to gene-corrected cells, mutant hiPSC-CMs have marked electrophysiological and contractile alterations, with modest gene expression changes. While large-scale changes in chromosomal topology are evident, differences in chromatin compartmentalization are limited to a few hotspots that escape inactivation during cardiogenesis. These regions exhibit upregulation of multiple non-cardiac genes including CACNA1A, encoding for neuronal P/Q-type calcium channels. Pharmacological inhibition of the resulting current partially mitigates the electrical alterations. On the other hand, A/B compartment changes do not explain most gene expression alterations in mutant hiPSC-CMs. We conclude that global errors in chromosomal compartmentation are not the primary pathogenic mechanism in heart failure due to lamin A/C haploinsufficiency.SummaryBertero et al. observe that lamin A/C haploinsufficiency in human cardiomyocytes markedly alters electrophysiology, contractility, gene expression, and chromosomal topology. Contrary to expectations, however, changes in chromatin compartments involve just few regions, and most dysregulated genes lie outside these hotspots.Condensed titleGenomic effects of lamin A/C haploinsufficiency

scholarly journals Genome-wide molecular effects of the neuropsychiatric 16p11 CNVs in an iPSC-to-iN neuronal model

2020 ◽  
Author(s):  
Thomas R. Ward ◽  
Xianglong Zhang ◽  
Louis C. Leung ◽  
Bo Zhou ◽  
Kristin Muench ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Cell Model ◽  
Copy Number Variants ◽  
Induced Pluripotent Stem Cell ◽  
Neuronal Model ◽  
Autism Spectrum ◽  
Genome Wide ◽  
Induced Pluripotent ◽  
Methylation Patterns

AbstractCopy number variants (CNVs), either deletions or duplications, at the 16p11.2 locus in the human genome are known to increase the risk for autism spectrum disorders (ASD), schizophrenia, and for several other developmental conditions. Here, we investigate the global effects on gene expression and DNA methylation using a 16p11.2 CNV patient-derived induced pluripotent stem cell (iPSC) to induced neuron (iN) cell model system. This approach revealed genome-wide and cell-type specific alterations to both gene expression and DNA methylation patterns and also yielded specific leads on genes potentially contributing to some of the known 16p11.2 patient phenotypes. PCSK9 is identified as a possible contributing factor to the symptoms seen in carriers of the 16p11.2 CNVs. The protocadherin (PCDH) gene family is found to have altered DNA methylation patterns in the CNV patient samples. The iPSC lines used for this study are available through a repository as a resource for research into the molecular etiology of the clinical phenotypes of 16p11.2 CNVs and into that of neuropsychiatric and neurodevelopmental disorders in general.

Abstract 508: Nuclear Lamins At Enhancers: A New Hypothesis for Laminopathies

2020 ◽  
Vol 127 (Suppl_1) ◽  
Author(s):  
Kohta Ikegami ◽  
Stefano Secchia ◽  
Omar Almakki ◽  
Alexis V Stutzman ◽  
Sachie Ikegami ◽  
...  
Keyword(s):  
Gene Expression ◽  
Histone Acetylation ◽  
Binding Sites ◽  
Nuclear Periphery ◽  
Nuclear Lamina ◽  
Lamin A ◽  
New Paradigm ◽  
Control Of Gene Expression ◽  
Nuclear Lamins ◽  
Nuclear Interior

The segregation of heterochromatin domains (LADs) at the nuclear periphery by the nuclear lamina, composed by polymerized nuclear Lamin A/C, provides a longstanding paradigm for the control of gene expression and for the mechanisms underlying Lamin-A/C-associated disorders, including progeria and cardiomyopathy. Here, we provide evidence supporting a novel paradigm that Lamin A/C functions as a transcription factor in the nuclear interior. We discovered that Ser22-phosphorylated Lamin A/C (pS22-Lamin A/C), required for lamin depolymerization during mitosis, populated the nuclear interior throughout the cell cycle. pS22-Lamin A/C ChIP-deq demonstrated localization at a large subset of putative active enhancers, not LADs. pS22-Lamin A/C-binding sites were co-occupied by the transcriptional activator c-Jun. In progeria patient-derived fibroblasts, a subset of pS22-Lamin A/C-binding sites were lost whereas new pS22-Lamin A/C-binding sites emerged. New pS22-Lamin A/C binding was accompanied by increased histone acetylation and increased c-Jun binding, whereas loss of pS22-Lamin A/C-binding was accompanied by loss of histone acetylation and c-Jun binding. New pS22-Lamin A/C enhancer binding in progeria was associated with upregulated expression of genes implicated in progeria pathophysiology, including cardiovascular disease. In contrast, alteration of LADs in progeria-patient cells could not explain the observed gene expression changes. These results suggest that Lamin A/C regulates gene expression by enhancer binding in the nuclear interior, independent of its function at the nuclear lamina, providing a new paradigm for the pathogenesis of lamin-associated disorders. pS22-Lamin A/C was also present in the nuclear interior of adult mouse cardiomyocytes. Cardiomyocyte-specific deletion of Lmna encoding Lamin A/C in adult mice caused extensive transcriptional changes in the heart and dilated cardiomyopathy, without apparent reduction of nuclear peripheral Lamin A/C. Disruption of the gene regulatory rather than LAD tethering function of Lamin A/C may underlie the pathogenesis of disorders caused by LMNA mutations, including cardiomyopathy.

Abstract 111: Conjoining Gene Expression And Cell Organization With Imaging To Explore Cell States In Hipsc-derived Cardiomyocytes

2021 ◽  
Vol 129 (Suppl_1) ◽  
Author(s):  
Ruwanthi Gunawardane
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Structural Organization ◽  
Large Scale ◽  
Single Cells ◽  
Induced Pluripotent Stem Cell ◽  
Mrna Abundance ◽  
Automated Classification ◽  
Subcellular Organization ◽  
Induced Pluripotent

The Allen Institute for Cell Science is developing a state space of stem cell structural signatures to study changes in cellular organization of human induced pluripotent stem cells (hiPSCs) and other cell states through differentiation. Towards this goal, we have used CRISPR/Cas9 to generate a collection of ~50 endogenous fluorescently tagged hiPSC lines (www.allencell.org), each expressing a monoallelic EGFP-tagged protein that localizes to a particular cellular structure or organelle. In this study, we focuson hiPSC-derived cardiomyocytes and compare the relationship between sarcomeric structural organization and gene expression signatures at large scale. We developed several tools and novel quantitative approaches to achieve this: 1) scarless GFP-tagging of cardiac genes such as ACTN2 to study the organization and morphogenesis of the contractile apparatus; 2) a robust protocol for differentiation of hiPSCs into cardiomyocytes and methods for preparing cells for imaging; and 3) a quantitative, image-based platform for the systematic and automated classification of subcellular organization in single cells. We use these approaches to quantify subcellular organization and gene expression in >30,000 individual human induced pluripotent stem cell-derived cardiomyocytes, producing a publicly available dataset that describes the population distributions of local and global sarcomere organization, mRNA abundance, and correlations between these traits. While the mRNA abundance of some phenotypically important genes correlates with subcellular organization (e.g., MYH7), these two cellular metrics are heterogeneous and often uncorrelated, which suggests that geneexpression alone is not sufficient to classify cell states. Instead, we posit thatcell state should be defined by observing full distributions of quantitative, multidimensional traits in single cells that also account for space, time, and function. This platform provides a multidimensional approach to classify hiPSC-derived cardiomyocytes based on structural organization and gene expression in single cells.

scholarly journals Impaired lamin localization to the nuclear envelope is responsible for nuclear damage in LMNA mutant iPSC-derived cardiomyocytes

2021 ◽  
Author(s):  
Melanie Maurer ◽  
Shriya Perati ◽  
Lindsey E. Johnson ◽  
Anthony M. Gacita ◽  
Shuping Lai ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
Intracellular Signaling ◽  
Mechanical Stability ◽  
Induced Pluripotent Stem Cell ◽  
Nuclear Lamina ◽  
Lamin A ◽  
Nuclear Abnormalities ◽  
Healthy Control ◽  
Induced Pluripotent ◽  
Cellular Dysfunction

The LMNA gene encodes the nuclear envelope proteins Lamins A and C, which comprise a major part of the nuclear lamina, provide mechanical support to the nucleus, and participate in diverse intracellular signaling. LMNA mutations give rise to a collection of diseases called laminopathies, including dilated cardiomyopathy (LMNA-DCM) and muscular dystrophies. Although nuclear deformities are a hallmark of LMNA-DCM, the role of nuclear abnormalities in the pathogenesis of LMNA-DCM remains incompletely understood. Using induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from LMNA mutant patients and healthy controls, we show that LMNA mutant iPSC-CM nuclei have altered shape or increased size compared to healthy control iPSC-CM nuclei. The LMNA mutation exhibiting the most severe nuclear deformities, R249Q, additionally caused reduced nuclear stiffness and increased nuclear fragility. Importantly, for all cell lines, the degree of nuclear abnormalities corresponded to the degree of Lamin A/C and Lamin B1 mislocalization from the nuclear envelope. The mislocalization was likely due to altered assembly of Lamin A/C. Collectively, these results point to the importance of correct lamin assembly at the nuclear envelope in providing mechanical stability to the nucleus and illustrate that defects in nuclear lamina organization can contribute to the nuclear and cellular dysfunction in LMNA-DCM.

scholarly journals Human Induced Pluripotent Stem Cell-derived Hepatocyte-like Cells Provide Insights on Parenteral Nutrition Associated Cholestasis in the Immature Liver

2021 ◽  
Author(s):  
T. Hang Nghiem-Rao ◽  
Courtney Pfeifer ◽  
Michelle Asuncion ◽  
Joshua Nord ◽  
Daniel Schill ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Bile Acid ◽  
Parenteral Nutrition ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Computational Techniques ◽  
Adult Liver ◽  
Induced Pluripotent ◽  
The Impact

Abstract Parenteral nutrition-associated cholestasis (PNAC) significantly limits the safety of intravenous parenteral nutrition (PN). Critically ill infants are highly vulnerable to PNAC-related morbidity and mortality, however the impact of hepatic immaturity on PNAC is poorly understood. We examined developmental differences between fetal/infant and adult livers, and used human induced pluripotent stem cell-derived hepatocyte-like cells (iHLC) to gain insights into the contribution of development to altered sterol metabolism and PNAC. We used RNA-sequencing and computational techniques to compare gene expression patterns in human fetal/infant livers, adult liver, and iHLC. We identified distinct gene expression profiles between the human feta/infant livers compared to adult liver, and close resemblance of iHLC to human developing livers. Compared to adult, both developing livers and iHLC had significant downregulation of xenobiotic, bile acid, and fatty acid metabolism; and lower expression of the sterol metabolizing gene ABCG8. When challenged with stigmasterol, a plant sterol found in intravenous soy lipids, lipid accumulation was significantly higher in iHLC compared to adult-derived HepG2 cells. Our findings provide insights into altered bile acid and lipid metabolizing processes in the immature human liver, and support the use of iHLC as a relevant model system of developing liver to study lipid metabolism and PNAC.

scholarly journals Batch effects and the effective design of single-cell gene expression studies

2016 ◽  
Author(s):  
Po-Yuan Tung ◽  
John D. Blischak ◽  
Chiaowen Joyce Hsiao ◽  
David A. Knowles ◽  
Jonathan E. Burnett ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Induced Pluripotent Stem Cell ◽  
Expression Levels ◽  
Amplification Bias ◽  
Expression Studies ◽  
Cell Gene Expression ◽  
Gene Expression Studies ◽  
Induced Pluripotent ◽  
Gene Expression Levels

AbstractSingle cell RNA sequencing (scRNA-seq) can be used to characterize variation in gene expression levels at high resolution. However, the sources of experimental noise in scRNA-seq are not yet well understood. We investigated the technical variation associated with sample processing using the single cell Fluidigm C1 platform. To do so, we processed three C1 replicates from three human induced pluripotent stem cell (iPSC) lines. We added unique molecular identifiers (UMIs) to all samples, to account for amplification bias. We found that the major source of variation in the gene expression data was driven by genotype, but we also observed substantial variation between the technical replicates. We observed that the conversion of reads to molecules using the UMIs was impacted by both biological and technical variation, indicating that UMI counts are not an unbiased estimator of gene expression levels. Based on our results, we suggest a framework for effective scRNA-seq studies.

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