Genomic features underlie the co-option of SVA transposons as cis-regulatory elements in human pluripotent stem cells

2022 ◽  
Author(s):  
Samantha M. Barnada ◽  
Andrew Isopi ◽  
Daniela Tejada-Martinez ◽  
Clement Goubert ◽  
Sruti Patoori ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Gene Regulation ◽  
Stem Cell ◽  
Pluripotent Stem Cells ◽  
Binding Sites ◽  
Human Gene ◽  
Regulatory Elements ◽  
Neighboring Gene ◽  
Genomic Features

Domestication of transposable elements (TEs) into functional cis-regulatory elements is a widespread phenomenon. However, the mechanisms behind why some TEs are co-opted as functional enhancers while others are not are underappreciated. SINE-VNTR-Alus (SVAs) are the youngest group of transposons in the human genome, where ~3,700 copies are annotated, nearly half of which are human-specific. Many studies indicate that SVAs are among the most frequently co-opted TEs in human gene regulation, but the mechanisms underlying such processes have not yet been thoroughly investigated. Here, we leveraged CRISPR-interference (CRISPRi), computational and functional genomics to elucidate the genomic features that underlie SVA domestication into human stem-cell gene regulation. We found that ~750 SVAs are co-opted as functional cis-regulatory elements in human induced pluripotent stem cells. These SVAs are significantly closer to genes and harbor more transcription factor binding sites than non-co-opted SVAs. We show that a long DNA motif composed of flanking YY1/2 and OCT4 binding sites is enriched in the co-opted SVAs and that these two transcription factors bind consecutively on the TE sequence. We used CRISPRi to epigenetically repress active SVAs in stem cell-like NCCIT cells. Epigenetic perturbation of active SVAs strongly attenuated YY1/OCT4 binding and influenced neighboring gene expression. Ultimately, SVA repression resulted in ~3,000 differentially expressed genes, 131 of which were the nearest gene to an annotated SVA. In summary, we demonstrated that SVAs modulate human gene expression, and uncovered that location and sequence composition contribute to SVA domestication into gene regulatory networks.

scholarly journals Regulation of human development by ubiquitin chain editing of chromatin remodelers

2020 ◽  
Author(s):  
David B. Beck ◽  
Mohammed A. Basar ◽  
Anthony J. Asmar ◽  
Joyce Thompson ◽  
Hirotsugu Oda ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Embryonic Development ◽  
Human Development ◽  
Chromatin Remodeling ◽  
Pluripotent Stem Cells ◽  
Regulatory Elements ◽  
Ubiquitin Chain ◽  
Early Differentiation ◽  
Chromatin Regulators

Embryonic development occurs through commitment of pluripotent stem cells to differentiation programs that require highly coordinated changes in gene expression. Chromatin remodeling of gene regulatory elements is a critical component of how such changes are achieved. While many factors controlling chromatin dynamics are known, mechanisms of how different chromatin regulators are orchestrated during development are not well understood. Here, we describe LINKED (LINKage-specific-deubiquitylation-deficiency-induced Embryonic Defects) syndrome, a novel multiple congenital anomaly disorder caused by hypomorphic hemizygous missense variants in the deubiquitylase OTUD5/DUBA. Studying LINKED mutations in vitro, in mouse, and in models of neuroectodermal differentiation of human pluripotent stem cells, we uncover a novel regulatory circuit that coordinates chromatin remodeling pathways during early differentiation. We show that the K48-linkage-specific deubiquitylation activity of OTUD5 is essential for murine and human development and, if reduced, leads to aberrant cell-fate specification. OTUD5 controls differentiation through preventing the degradation of multiple chromatin regulators including ARID1A/B and HDAC2, mutation of which underlie developmental syndromes that exhibit phenotypic overlap with LINKED patients. Accordingly, loss of OTUD5 during early differentiation leads to less accessible chromatin at neural and neural crest enhancers and thus aberrant rewiring of gene expression networks. Our work identifies a novel mechanistic link between phenotypically related developmental disorders and an essential function for linkagespecific ubiquitin editing of substrate groups (i.e. chromatin remodeling complexes) during early cellfate decisions – a regulatory concept, we predict to be a general feature of embryonic development.

scholarly journals An Important Role for DNMT3A-Mediated DNA Methylation in Cardiomyocyte Metabolism and Contractility

Circulation ◽  
2020 ◽  
Vol 142 (16) ◽  
pp. 1562-1578
Author(s):  
Alexandra Madsen ◽  
Grit Höppner ◽  
Julia Krause ◽  
Marc N. Hirt ◽  
Sandra D. Laufer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Stem Cells ◽  
Stem Cell ◽  
Pluripotent Stem Cells ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Heart Tissue ◽  
Engineered Heart Tissue ◽  
Induced Pluripotent

Background: DNA methylation acts as a mechanism of gene transcription regulation. It has recently gained attention as a possible therapeutic target in cardiac hypertrophy and heart failure. However, its exact role in cardiomyocytes remains controversial. Thus, we knocked out the main de novo DNA methyltransferase in cardiomyocytes, DNMT3A, in human induced pluripotent stem cells. Functional consequences of DNA methylation-deficiency under control and stress conditions were then assessed in human engineered heart tissue from knockout human induced pluripotent stem cell–derived cardiomyocytes. Methods: DNMT3A was knocked out in human induced pluripotent stem cells by CRISPR/Cas9gene editing. Fibrin-based engineered heart tissue was generated from knockout and control human induced pluripotent stem cell–derived cardiomyocytes. Development and baseline contractility were analyzed by video-optical recording. Engineered heart tissue was subjected to different stress protocols, including serum starvation, serum variation, and restrictive feeding. Molecular, histological, and ultrastructural analyses were performed afterward. Results: Knockout of DNMT3A in human cardiomyocytes had three main consequences for cardiomyocyte morphology and function: (1) Gene expression changes of contractile proteins such as higher atrial gene expression and lower MYH7/MYH6 ratio correlated with different contraction kinetics in knockout versus wild-type; (2) Aberrant activation of the glucose/lipid metabolism regulator peroxisome proliferator-activated receptor gamma was associated with accumulation of lipid vacuoles within knockout cardiomyocytes; (3) Hypoxia-inducible factor 1α protein instability was associated with impaired glucose metabolism and lower glycolytic enzyme expression, rendering knockout-engineered heart tissue sensitive to metabolic stress such as serum withdrawal and restrictive feeding. Conclusion: The results suggest an important role of DNA methylation in the normal homeostasis of cardiomyocytes and during cardiac stress, which could make it an interesting target for cardiac therapy.

scholarly journals Human stem cell resources are an inroad to Neandertal DNA functions

2018 ◽  
Author(s):  
Michael Dannemann ◽  
Benjamin Vernot ◽  
Svante Pääbo ◽  
Janet Kelso ◽  
J. Gray Camp
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Stem Cell ◽  
Amino Acid ◽  
Pluripotent Stem Cells ◽  
Human Stem Cell ◽  
Modern Humans ◽  
Functional Variation ◽  
Human Ipscs ◽  
Induced Pluripotent

ABSTRACTPluripotent stem cells from diverse humans offer the potential to study human functional variation in controlled culture environments. A portion of this variation originates from ancient admixture between modern humans and Neandertals, which introduced alleles that left a phenotypic legacy on individual humans today. Here we show that a large repository of human induced pluripotent stem cells (iPSCs) harbors extensive Neandertal DNA, including most known functionally relevant Neandertal alleles present in modern humans. This resource contains Neandertal DNA that contributes to human phenotypes and diseases, encodes hundreds of amino acid changes, and alters gene expression in specific tissues. Human iPSCs thus provide an opportunity to experimentally explore the Neandertal contribution to present-day phenotypes, and potentially study Neandertal traits.

Abstract 498: Combinatorial Extracellular Matrix Microarrays for Probing Endothelial Differentiation of Human Induced Pluripotent Stem Cells

2015 ◽  
Vol 35 (suppl_1) ◽  
Author(s):  
Luqia Hou ◽  
John Coller ◽  
Vanita Natu ◽  
Ngan Huang
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Stem Cell ◽  
Pluripotent Stem Cells ◽  
Pluripotent Stem Cell ◽  
Endothelial Differentiation ◽  
Induced Pluripotent

Human induced pluripotent stem cell (iPSC)-derived endothelial cells (iPSC-ECs) are a promising cell source for vascular regeneration in patients with peripheral arterial disease. However, a critical bottleneck to their clinical translation is the ability to differentiate the cells reproducibly at high yields. Since endothelial cells interact with the basement membrane extracellular matrix (ECM), we sought to examine the role of ECMs on endothelial differentiation using combinatorial ECM microenvironments. ECM microarrays were developed by covalent conjugation of ECMs (gelatin, fibronectin, laminin, heparin sulfate proteoglycans, collagen IV, matrigel) and the multi-component combinations thereof. The pluripotent stem cells attached to the ECMs and subsequently differentiated over the course of 5 days. Endothelial differentiation was semi-quantitatively scored based on the degree of CD31 staining. Our results demonstrated greater levels of CD31staining when cultured on gelatin + matrigel + laminin (G+M+L) or fibronectin + laminin + heparan sulfate (F+L+H), compared to other combinations across three human pluripotent stem cell lines (iPSC-Huf5, iPSC-CON1, and ESC-H1). This enhancement in endothelial differentiation on the microscale was confirmed at larger cell culture platforms in which a marked increase in CD31+ cells was observed in G+M+L modified-dishes (> 5 fold), and F+L+H combination (> 10 fold), compared to gelatin-modified dishes. RT-PCR further confirmed the transcriptional upregulation in endothelial markers for CD31 (> 2 fold) and VE-cadherin (> 4 fold) on G+M+L, compared to gelatin-modified dishes. To elucidate the role of cell-ECM interactions on endothelial differentiation, gene expression of integrin subunits were examined. Gene expression was markedly upregulated in integrins α1 (>10 fold); α4, α5, and αV (>5 fold); and β1, β3 (>50 fold), and β4, when comparing differentiated cells on day14 to undifferentiated cells. The upregulation of integrin subunits was concomitant with upregulation in endothelial genes. Together, this data suggested that combinatorial ECMs differentially promote endothelial differentiation, in part through integrin-mediated pathways.

scholarly journals CFTR Cooperative Cis-Regulatory Elements in Intestinal Cells

2021 ◽  
Vol 22 (5) ◽  
pp. 2599
Author(s):  
Mégane Collobert ◽  
Ozvan Bocher ◽  
Anaïs Le Nabec ◽  
Emmanuelle Génin ◽  
Claude Férec ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cystic Fibrosis ◽  
Gene Regulation ◽  
Genetic Diseases ◽  
Regulatory Elements ◽  
Cftr Gene ◽  
Intestinal Cells ◽  
Cooperative Effects ◽  
Cis Acting ◽  
Study Techniques

About 8% of the human genome is covered with candidate cis-regulatory elements (cCREs). Disruptions of CREs, described as “cis-ruptions” have been identified as being involved in various genetic diseases. Thanks to the development of chromatin conformation study techniques, several long-range cystic fibrosis transmembrane conductance regulator (CFTR) regulatory elements were identified, but the regulatory mechanisms of the CFTR gene have yet to be fully elucidated. The aim of this work is to improve our knowledge of the CFTR gene regulation, and to identity factors that could impact the CFTR gene expression, and potentially account for the variability of the clinical presentation of cystic fibrosis as well as CFTR-related disorders. Here, we apply the robust GWAS3D score to determine which of the CFTR introns could be involved in gene regulation. This approach highlights four particular CFTR introns of interest. Using reporter gene constructs in intestinal cells, we show that two new introns display strong cooperative effects in intestinal cells. Chromatin immunoprecipitation analyses further demonstrate fixation of transcription factors network. These results provide new insights into our understanding of the CFTR gene regulation and allow us to suggest a 3D CFTR locus structure in intestinal cells. A better understand of regulation mechanisms of the CFTR gene could elucidate cases of patients where the phenotype is not yet explained by the genotype. This would thus help in better diagnosis and therefore better management. These cis-acting regions may be a therapeutic challenge that could lead to the development of specific molecules capable of modulating gene expression in the future.

scholarly journals Strategy to Establish Embryo-Derived Pluripotent Stem Cells in Cattle

2021 ◽  
Vol 22 (9) ◽  
pp. 5011
Author(s):  
Daehwan Kim ◽  
Sangho Roh
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Embryonic Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Stem Cell Research ◽  
Pluripotent Stem Cells ◽  
Embryonic Stem ◽  
Culture Conditions ◽  
Human Diseases ◽  
Induced Pluripotent

Stem cell research is essential not only for the research and treatment of human diseases, but also for the genetic preservation and improvement of animals. Since embryonic stem cells (ESCs) were established in mice, substantial efforts have been made to establish true ESCs in many species. Although various culture conditions were used to establish ESCs in cattle, the capturing of true bovine ESCs (bESCs) has not been achieved. In this review, the difficulty of establishing bESCs with various culture conditions is described, and the characteristics of proprietary induced pluripotent stem cells and extended pluripotent stem cells are introduced. We conclude with a suggestion of a strategy for establishing true bESCs.

scholarly journals T9. EPIGENETIC PROFILING IN SCHIZOPHRENIA DERIVED HUMAN INDUCED PLURIPOTENT STEM CELLS (HIPSCS) AND NEURONS

2020 ◽  
Vol 46 (Supplement_1) ◽  
pp. S234-S234
Author(s):  
Lorna Farrelly ◽  
Shuangping Zhang ◽  
Erin Flaherty ◽  
Aaron Topol ◽  
Nadine Schrode ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mass Spectrometry ◽  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Neural Progenitor Cells ◽  
Gene Expression Pattern ◽  
Early Gene ◽  
Label Free ◽  
Induced Pluripotent

Abstract Background Schizophrenia (SCZ) is a severe psychiatric disorder affecting ~1% of the world’s population. It is largely heritable with genetic risk reflected by a combination of common variants of small effect and highly penetrant rare mutations. Chromatin modifications are known to play critical roles in the mediation of many neurodevelopmental processes, and, when disturbed, may also contribute to the precipitation of psychiatric disorders, such as SCZ. While a handful of candidate-based studies have measured changes in promoter-bound histone modifications, few mechanistic studies have been carried out to explore how these modifications may affect chromatin to precipitate behavioral phenotypes associated with the disease. Methods We applied an unbiased proteomics approach to evaluate the epigenetic landscape of SCZ in human induced pluripotent stem cells (hiPSC), neural progenitor cells (NPCs) and neurons from SCZ patients vs. matched controls. We utilized proteomics-based, label free liquid chromatography mass spectrometry (LC-MS/MS) on purified histones from these cells and confirmed our results by western blotting in postmortem SCZ cortical brain tissues. Furthermore we validated our findings with the application of histone interaction assays and structural and biophysical assessments to identify and confirm novel chromatin ‘readers’. To relate our findings to a SCZ phenotype we used a SCZ rodent model of prepulse inhibition (PPI) to perform pharmacological manipulations and behavioral assessments. Results Using label free mass spectrometry we performed PTM screening of hiPSCs, NPCs and matured neurons derived from SCZ patients and matched controls. We identified, amongst others, altered patterns of hyperacetylation in SCZ neurons. Additionally we identified enhanced binding of particular acetylation ‘reader’ proteins. Pharmacological inhibition of such proteins in an animal model of amphetamine sensitization ameliorated PPI deficits further validating this epigenetic signature in SCZ. Discussion Recent evidence indicates that relevance and patterns of acetylation in epigenetics advances beyond its role in transcription and small molecule inhibitors of these aberrant interactions hold promise as useful therapeutics. This study identifies a role for modulating gene expression changes associated with a SCZ epigenetic signature and warrants further investigation in terms of how this early gene expression pattern perhaps determines susceptibility or severity of the SCZ disease trajectory.

scholarly journals Induced Pluripotent Stem Cells (iPSCs) in Vascular Research: from Two- to Three-Dimensional Organoids

2021 ◽  
Author(s):  
Anja Trillhaase ◽  
Marlon Maertens ◽  
Zouhair Aherrahrou ◽  
Jeanette Erdmann
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Induced Pluripotent Stem Cells ◽  
Stem Cell Research ◽  
Pluripotent Stem Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
3D Models ◽  
Induced Pluripotent

AbstractStem cell technology has been around for almost 30 years and in that time has grown into an enormous field. The stem cell technique progressed from the first successful isolation of mammalian embryonic stem cells (ESCs) in the 1990s, to the production of human induced-pluripotent stem cells (iPSCs) in the early 2000s, to finally culminate in the differentiation of pluripotent cells into highly specialized cell types, such as neurons, endothelial cells (ECs), cardiomyocytes, fibroblasts, and lung and intestinal cells, in the last decades. In recent times, we have attained a new height in stem cell research whereby we can produce 3D organoids derived from stem cells that more accurately mimic the in vivo environment. This review summarizes the development of stem cell research in the context of vascular research ranging from differentiation techniques of ECs and smooth muscle cells (SMCs) to the generation of vascularized 3D organoids. Furthermore, the different techniques are critically reviewed, and future applications of current 3D models are reported. Graphical abstract

scholarly journals Telomere length set point regulation in human pluripotent stem cells critically depends on the shelterin protein TPP1

2020 ◽  
Vol 31 (23) ◽  
pp. 2583-2596
Author(s):  
John M. Boyle ◽  
Kelsey M. Hennick ◽  
Samuel G. Regalado ◽  
Jacob M. Vogan ◽  
Xiaozhu Zhang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Telomere Length ◽  
Pluripotent Stem Cells ◽  
Human Stem Cells ◽  
Set Point ◽  
Hypomorphic Allele ◽  
Point Control ◽  
Set Point Control ◽  
Stem Cell Lines

To better understand telomere length set point control in human stem cells, we generated knockout stem cell lines for TPP1 and contrasted their phenotypes with those of homozygous TPP1 L104A mutant stem cells. This comparison reveals that TPP1 L104A is not a hypomorphic allele but formally establishes TPP1 L104 as a dissociation of function mutant.

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