scholarly journals A cohesin cancer mutation reveals a role for the hinge domain in genome organization and gene expression

PLoS Genetics ◽  
2021 ◽  
Vol 17 (3) ◽  
pp. e1009435
Author(s):  
Zachary M. Carico ◽  
Holden C. Stefan ◽  
Megan Justice ◽  
Askar Yimit ◽  
Jill M. Dowen
Keyword(s):  
Gene Expression ◽  
Developmental Disorders ◽  
Three Dimensional ◽  
Embryonic Stem ◽  
Cohesin Complex ◽  
Dna Loops ◽  
Dna Mutation ◽  
Close Physical Proximity ◽  
Pluripotency Gene ◽  
Hinge Domain

The cohesin complex spatially organizes interphase chromatin by bringing distal genomic loci into close physical proximity, looping out the intervening DNA. Mutation of cohesin complex subunits is observed in cancer and developmental disorders, but the mechanisms through which these mutations may contribute to disease remain poorly understood. Here, we investigate a recurrent missense mutation to the hinge domain of the cohesin subunit SMC1A, observed in acute myeloid leukemia. Engineering this mutation into murine embryonic stem cells caused widespread changes in gene expression, including dysregulation of the pluripotency gene expression program. This mutation reduced cohesin levels at promoters and enhancers, decreased DNA loops and interactions across short genomic distances, and weakened insulation at CTCF-mediated DNA loops. These findings provide insight into how altered cohesin function contributes to disease and identify a requirement for the cohesin hinge domain in three-dimensional chromatin structure.

Fluid Flow Modulation of Murine Embryonic Stem Cell Pluripotency Gene Expression in the Absence of LIF

2013 ◽  
Vol 6 (3) ◽  
pp. 335-345 ◽  
Author(s):  
Giovanna G. Lara ◽  
Olesja Hazenbiller ◽  
Tia Gareau ◽  
Robert D. Shepherd ◽  
Michael S. Kallos ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Fluid Flow ◽  
Embryonic Stem Cell ◽  
Embryonic Stem ◽  
Flow Modulation ◽  
Murine Embryonic Stem Cell ◽  
Pluripotency Gene ◽  
Stem Cell Pluripotency ◽  
Embryonic Stem Cell Pluripotency

scholarly journals H4K20me3 methyltransferase SUV420H2 shapes the chromatin landscape of pluripotent embryonic stem cells

Development ◽  
2020 ◽  
Vol 147 (23) ◽  
pp. dev188516
Author(s):  
Jiji T. Kurup ◽  
Zhijun Han ◽  
Wenfei Jin ◽  
Benjamin L. Kidder
Keyword(s):  
Gene Expression ◽  
Cell Fate ◽  
Es Cells ◽  
Three Dimensional ◽  
Embryonic Stem ◽  
Pericentric Heterochromatin ◽  
Es Cell ◽  
Chromatin Interactions ◽  
Repressive Histone Modification ◽  
Dna Elements

ABSTRACTHeterochromatin, a densely packed chromatin state that is transcriptionally silent, is a critical regulator of gene expression. However, it is unclear how the repressive histone modification H4K20me3 or the histone methyltransferase SUV420H2 regulates embryonic stem (ES) cell fate by patterning the epigenetic landscape. Here, we report that depletion of SUV420H2 leads to a near-complete loss of H4K20me3 genome wide, dysregulated gene expression and delayed ES cell differentiation. SUV420H2-bound regions are enriched with repetitive DNA elements, which are de-repressed in SUV420H2 knockout ES cells. Moreover, SUV420H2 regulation of H4K20me3-marked heterochromatin controls chromatin architecture, including fine-scale chromatin interactions in pluripotent ES cells. Our results indicate that SUV420H2 plays a crucial role in stabilizing the three-dimensional chromatin landscape of ES cells, as loss of SUV420H2 resulted in A/B compartment switching, perturbed chromatin insulation, and altered chromatin interactions of pericentric heterochromatin and surrounding regions, indicative of localized decondensation. In addition, depletion of SUV420H2 resulted in compromised interactions between H4K20me3 and gene-regulatory regions. Together, these findings describe a new role for SUV420H2 in regulating the chromatin landscape of ES cells.

212 Increased expression of YAP/TAZ encourages outgrowth establishment and three-dimensional colony formation and boosts plasticity of parthenogenetic stem cells

2020 ◽  
Vol 32 (2) ◽  
pp. 234
Author(s):  
S. Arcuri ◽  
G. Pennarossa ◽  
F. Gandolfi ◽  
T. Brevini
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Three Dimensional ◽  
Embryonic Stem ◽  
Direct Interaction ◽  
High Plasticity ◽  
Dna Hypomethylation ◽  
Regulatory Pathways ◽  
Pluripotency Gene ◽  
The Cost

Cell proliferation, apoptosis, and differentiation are essential processes from the early phases of embryogenesis to adult tissue formation and maintenance. These mechanisms also play a key role in embryonic stem cells (ESCs), which are able to proliferate maintaining pluripotency and, at the same time, give rise to all populations belonging to the three germ layers in response to specific stimuli. ESCs are, therefore, considered a well-established invitro model to study the complexity of these processes. We previously generated porcine and human diploid parthenogenetic embryonic stem cells (ParthESC) that showed many features and regulatory pathways common to biparental ESCs. However, we observed that monoparental cells demonstrated a greater ability to form outgrowths and generate stable three-dimensional (3D) spheroid colonies, which are distinctive signs of high plasticity (Brevini et al. 2010 STCR 6; https://doi.org/10.1007/s12015-010-9153-2). In the present study, we compared porcine and human biparental ESCs and ParthESC. Pluripotency gene expression was analysed and ELISA tests for global DNA methylation and total YAP levels were performed. Our results showed that ParthESCs showed significantly higher expression levels of YAP and TAZ than biparental ESCs. In contrast, the analysis of the upstream genes involved in the Hippo signalling pathway revealed no differences. We demonstrated that YAP remains in a dephosphorylated form, encouraging its nuclear compartmentalization and direct interaction with the nuclear transcription factors TEADs and SMADs, that are also upregulated and maintain pluripotency, repressing differentiation processes (Beyer et al. 2013 Cell Rep. 5, 1611-1624; https://doi.org/10.1016/j.celrep.2013.11.021). These complex regulatory interactions accompanied the overexpression of the pluripotency-related genes OCT4, NANOG, REX1, SOX2, UTF1, and TERT. In parallel, we observed a decrease of DNMT3 and DNMT4 activity that resulted in a global DNA hypomethylation and a chromatin high-permissive state. Altogether, our results demonstrate a significant upregulation of YAP/TAZ activity in monoparental cell lines. This may account for their greater ability to form outgrowths and generate 3D spheroid colonies and increased plasticity compared with biparental counterparts. It is interesting to note that YAP mRNA is supplied and expressed by the oocyte and is maternally inherited (Yu et al. 2016 Cell Res. 26, 275-287; https://doi.org/10.1038/cr.2016.20). Although further clarifications are needed, we hypothesise that its higher expression in ParthESC may be related to the strictly maternal origin of these cells. This study was supported by the Carraresi Foundation. Authors are members of the COST Actions CA16119.

scholarly journals Functional impact of cancer-associated cohesin variants on gene expression and cellular identity

Genetics ◽  
2021 ◽  
Author(s):  
Natalie L Rittenhouse ◽  
Zachary M Carico ◽  
Ying Frances Liu ◽  
Holden C Stefan ◽  
Nicole L Arruda ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Chromosome Structure ◽  
Embryonic Stem ◽  
Sequence Variants ◽  
Dna Loops ◽  
Murine Embryonic Stem Cells ◽  
Recurrent Mutations ◽  
Cellular Identity

Abstract Cohesin is a ring-shaped protein complex that controls dynamic chromosome structure. Cohesin activity is important for a variety of biological processes, including formation of DNA loops that regulate gene expression. The precise mechanisms by which cohesin shapes local chromosome structure and gene expression are not fully understood. Recurrent mutations in cohesin complex members have been reported in various cancers, though it is not clear whether many cohesin sequence variants have phenotypes and contribute to disease. Here, we utilized CRISPR/Cas9 genome editing to introduce a variety of cohesin sequence variants into murine embryonic stem cells and investigate their molecular and cellular consequences. Some of the cohesin variants tested caused changes to transcription, including altered expression of gene encoding lineage-specifying developmental regulators. Altered gene expression was also observed at insulated neighborhoods, where cohesin-mediated DNA loops constrain potential interactions between genes and enhancers. Furthermore, some cohesin variants altered the proliferation rate and differentiation potential of murine embryonic stem cells. This study provides a functional comparison of cohesin variants found in cancer within an isogenic system, revealing the relative roles of various cohesin perturbations on gene expression and maintenance of cellular identity.

scholarly journals Laminin 521 Stabilizes the Pluripotency Expression Pattern of Human Embryonic Stem Cells Initially Derived on Feeder Cells

2018 ◽  
Vol 2018 ◽  
pp. 1-9 ◽  
Author(s):  
Halima Albalushi ◽  
Magdalena Kurek ◽  
Leif Karlsson ◽  
Luise Landreh ◽  
Kristín Rós Kjartansdóttir ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Embryonic Stem ◽  
Cell Types ◽  
Culture Conditions ◽  
Pluripotency Marker ◽  
Human Foreskin Fibroblasts ◽  
Pluripotency Gene ◽  
Hes Cells ◽  
Cells Cultured

Human embryonic stem (hES) cells represent an important tool to study early cell development. The previously described use of human recombinant laminin (LN) 521 represented a step forward in generating clinically safe culture conditions. To test the short-term effect of LN521 on cultured hES cells, five male hES cell lines were cultured on human foreskin fibroblasts (hFFs), Matrigel, LN521, and LN121 and characterized by qPCR, immunofluorescence analysis, as well as their potential for three-germ layer differentiation. Variations in gene expression related to pluripotency, stemness, and testicular cells at different passages and culture conditions were evaluated by qPCR. All cell lines expressed pluripotency markers at protein and RNA level and were able to differentiate into cell types of the three germ layers after being cultured on LN521 for nine passages. Reduction in variation of pluripotency marker expression could be observed after culturing the cells on LN521 for nine passages. hES cells cultured on LN521 exhibited less differentiation, faster cell growth, and attachment when compared to hES cells cultured on LN121 or Matrigel. Our results indicate a positive effect of LN521 in stabilizing pluripotency gene expression and might be the first step towards more controllable and robust culture conditions for hES cells.

scholarly journals Chronic Loss of Stag2 Leads to Altered Chromatin Structure Contributing to De-Regulated Transcription in Aml.

2020 ◽  
Author(s):  
James Smith ◽  
Stephanie G Craig ◽  
Fabio G Liberante ◽  
Katrina M Lappin ◽  
Clare M Crean ◽  
...  
Keyword(s):  
Gene Expression ◽  
Genome Structure ◽  
Three Dimensional ◽  
Cohesin Complex ◽  
Mapk Signalling Pathway ◽  
Altered Gene Expression ◽  
Increased Sensitivity ◽  
Altered Gene ◽  
Structure Lead ◽  
The Impact

Abstract Background The cohesin complex plays a major role in folding the human genome into 3D structural domains. Mutations in members of the cohesin complex are known early drivers of myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML), with STAG2 the most frequently mutated complex member.Methods Here we use functional genomics (RNA-seq, ChIP-seq and HiChiIP) to investigate the impact of chronic STAG2 loss on three-dimensional genome structure and transcriptional programming in a clinically relevant model of chronic STAG2 loss.Results The chronic loss of STAG2 led to loss of smaller loop domains and the maintenance/formation of large domains that in turn led to altered genome compartmentalisation. These changes in genome structure lead to altered gene expression, including deregulation of the HOXA locus and the MAPK signalling pathway resulting in increased sensitivity to MEK inhibition.Conclusions The altered genomic architecture driven by the chronic loss of STAG2 results in altered gene expression that may contribute to leukaemogenesis which may be therapeutically targeted.

scholarly journals Modeling gene expression networks to predict interchromosomal organization during human embryonic stem cell differentiation

2018 ◽  
Author(s):  
Kyle V. Laster ◽  
Arturo G. Garza-Gongora ◽  
Elizabeth Daley ◽  
Alexey Terskikh ◽  
Evangelos Kiskinis ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
In Silico ◽  
Regulatory Networks ◽  
Cellular Differentiation ◽  
Three Dimensional ◽  
Embryonic Stem ◽  
Chromosome Organization ◽  
Neuronal Precursor Cells

AbstractCellular differentiation occurs through the regulation of lineage-specific gene expression networks that are facilitated by the spatial organization of the genome. Although techniques based on the chromatin conformation capture (3C) approach have yielded intrachromosomal genome-wide interaction maps, strategies to identify non-random interchromosomal associations is lacking. Therefore, we modeled the genomic organization of chromosomes based on the regulatory networks involved in the differentiation of pluripotent human embryonic stem cells (hESCs) to committed neuronal precursor cells (cNPCs). Importantly, transcriptional regulation has been demonstrated to be a driving force in non-random genome organization. Thus, we constructed coarse-grained in silico networks using gene expression data to identify potential physical associations among chromosomes occurring in situ and then analyzed the three-dimensional (3D) distribution of these chromosomes, assessing how their associations contribute to nuclear organization. Our analysis suggests that coordinate regulation of differentially expressed genes is correlated with the 3D organization of chromosomes in hESC nuclei induced to differentiate to cNPCs.Author SummaryThe cellular commitment and differentiation of stem cells is a hallmark of metazoan development. The ultimate fate of a stem cell is defined by the synergistic modulation of key gene regulatory networks within the nucleus. In our work, we formulate an in silico model describing how the similarity in the expression profile of differentially regulated gene networks is correlated with the higher-order organization of chromosomes during differentiation from human embryonic stem cells (hESCs) to committed neuronal precursor cells (cNPCs). Using graph statistics, we observe that the genome networks generated using the in silico model exhibit properties similar to real-world networks. In addition to modeling how gene expression relates to dynamic changes in chromosome organization, we test the model by calculating the relative proximity of multiple chromosome pairs using 3D fluorescence in situ hybridization (FISH). While various chromosomal properties, including gene density and overall length, have been attributed to chromosome organization, our previous work has identified the emergence of cell-type specific chromosomal topologies related to coordinate gene regulation during cellular differentiation. Here we extend these findings by determining whether our in silico model can predict chromosome association based upon coordinate gene expression. Our work supports the idea that gene co-regulation, in addition to inherent organizational constraints of the nucleus, influences three-dimensional chromosome organization.

scholarly journals Chronic Loss of STAG2 Leads to Altered Chromatin Structure Contributing to DE-Regulated Transcription in AML.

2020 ◽  
Author(s):  
James Smith ◽  
Katrina M Lappin ◽  
Stephanie G Craig ◽  
Fabio G Liberante ◽  
Clare M Crean ◽  
...  
Keyword(s):  
Gene Expression ◽  
Genome Structure ◽  
Three Dimensional ◽  
Cohesin Complex ◽  
Mapk Signalling Pathway ◽  
Altered Gene Expression ◽  
Mapk Signalling ◽  
Increased Sensitivity ◽  
Altered Gene ◽  
The Impact

Abstract Background The cohesin complex plays a major role in folding the human genome into 3D structural domains. Mutations in members of the cohesin complex are known early drivers of myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML), with STAG2 the most frequently mutated complex member. Methods Here we use functional genomics (RNA-seq, ChIP-seq and HiChIP) to investigate the impact of chronic STAG2 loss on three-dimensional genome structure and transcriptional programming in a clinically relevant model of chronic STAG2 loss. Results The chronic loss of STAG2 led to loss of smaller loop domains and the maintenance/formation of large domains that, in turn, led to altered genome compartmentalisation. These changes in genome structure resulted in altered gene expression, including deregulation of the HOXA locus and the MAPK signalling pathway, resulting in increased sensitivity to MEK inhibition. Conclusions: The altered genomic architecture driven by the chronic loss of STAG2 results in altered gene expression that may contribute to leukaemogenesis and may be therapeutically targeted.

scholarly journals Subnuclear localisation is associated with gene activation not repression or parental origin at the imprinted Dlk1-Dio3 locus

2021 ◽  
Author(s):  
Rahia Mashoodh ◽  
Lisa C Hulsmann ◽  
Frances L Dearden ◽  
Nozomi Takahashi ◽  
Anne C Ferguson-Smith
Keyword(s):  
Gene Expression ◽  
Transcriptional Repression ◽  
Es Cells ◽  
In Situ Hybridisation ◽  
Nuclear Periphery ◽  
Gene Activation ◽  
Three Dimensional ◽  
Embryonic Stem ◽  
Parental Origin ◽  
Fluorescence In Situ Hybridisation

At interphase, de-condensed chromosomes have a non-random three-dimensional architecture within the nucleus, however, little is known about the extent to which nuclear organisation might influence expression or vice versa. Here, using imprinting as a model, we use 3D RNA- and DNA-fluorescence-in-situ-hybridisation in normal and mutant mouse embryonic stem cells to assess the relationship between imprinting control, gene expression and allelic distance from the nuclear periphery. We compared the two parentally inherited imprinted domains at the Dlk1-Dio3 domain and find a small but reproducible trend for the maternally inherited domain to be further away from the periphery if the maternally expressed gene Gtl2/Meg3 is active compared to when it is silenced. Using Zfp57KO ES cells, which harbour a paternal to maternal epigenotype switch, we observe active alleles significantly further away from the nuclear periphery with the distance from the periphery being proportional to the number of alleles active within the cell. This distribution of alleles suggests an activating effect of the nuclear interior rather than a repressive association with the nuclear periphery. Although we see a trend for the paternally inherited copy of the locus to be closer to the nuclear periphery, this appears to be linked to stochastic gene expression differences rather than parental origin. Our results suggest that transcriptional activity, rather than transcriptional repression or parental origin, defines sub-nuclear localisation at an endogenous imprinted domain.

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