DNA methylation and histone modifications are essential for regulation of stem cell formation and differentiation in zebrafish development

2021 ◽  
Author(s):  
Alissa D Marchione ◽  
Zanshé Thompson ◽  
Katie L Kathrein
Keyword(s):  
Stem Cell ◽  
Epigenetic Regulation ◽  
Chromatin Structure ◽  
Large Scale ◽  
Genetic Manipulation ◽  
Critical Role ◽  
External Fertilization ◽  
High Fecundity ◽  
Zebrafish Development

AbstractThe complex processes necessary for embryogenesis require a gene regulatory network that is complex and systematic. Gene expression regulates development and organogenesis, but this process is altered and fine-tuned by epigenetic regulators that facilitate changes in the chromatin landscape. Epigenetic regulation of embryogenesis adjusts the chromatin structure by modifying both DNA through methylation and nucleosomes through posttranslational modifications of histone tails. The zebrafish is a well-characterized model organism that is a quintessential tool for studying developmental biology. With external fertilization, low cost and high fecundity, the zebrafish are an efficient tool for studying early developmental stages. Genetic manipulation can be performed in vivo resulting in quick identification of gene function. Large-scale genome analyses including RNA sequencing, chromatin immunoprecipitation and chromatin structure all are feasible in the zebrafish. In this review, we highlight the key events in zebrafish development where epigenetic regulation plays a critical role from the early stem cell stages through differentiation and organogenesis.

scholarly journals Zebrafish Cancer Predisposition Models

2021 ◽  
Vol 9 ◽  
Author(s):  
Kim Kobar ◽  
Keon Collett ◽  
Sergey V. Prykhozhij ◽  
Jason N. Berman
Keyword(s):  
Large Scale ◽  
Genetic Manipulation ◽  
Bone Marrow Failure ◽  
Cancer Predisposition ◽  
Potential Candidate ◽  
Cancer Evolution ◽  
High Fecundity ◽  
Cancer Models ◽  
The Impact

Cancer predisposition syndromes are rare, typically monogenic disorders that result from germline mutations that increase the likelihood of developing cancer. Although these disorders are individually rare, resulting cancers collectively represent 5–10% of all malignancies. In addition to a greater incidence of cancer, affected individuals have an earlier tumor onset and are frequently subjected to long-term multi-modal cancer screening protocols for earlier detection and initiation of treatment. In vivo models are needed to better understand tumor-driving mechanisms, tailor patient screening approaches and develop targeted therapies to improve patient care and disease prognosis. The zebrafish (Danio rerio) has emerged as a robust model for cancer research due to its high fecundity, time- and cost-efficient genetic manipulation and real-time high-resolution imaging. Tumors developing in zebrafish cancer models are histologically and molecularly similar to their human counterparts, confirming the validity of these models. The zebrafish platform supports both large-scale random mutagenesis screens to identify potential candidate/modifier genes and recently optimized genome editing strategies. These techniques have greatly increased our ability to investigate the impact of certain mutations and how these lesions impact tumorigenesis and disease phenotype. These unique characteristics position the zebrafish as a powerful in vivo tool to model cancer predisposition syndromes and as such, several have already been created, including those recapitulating Li-Fraumeni syndrome, familial adenomatous polyposis, RASopathies, inherited bone marrow failure syndromes, and several other pathogenic mutations in cancer predisposition genes. In addition, the zebrafish platform supports medium- to high-throughput preclinical drug screening to identify compounds that may represent novel treatment paradigms or even prevent cancer evolution. This review will highlight and synthesize the findings from zebrafish cancer predisposition models created to date. We will discuss emerging trends in how these zebrafish cancer models can improve our understanding of the genetic mechanisms driving cancer predisposition and their potential to discover therapeutic and/or preventative compounds that change the natural history of disease for these vulnerable children, youth and adults.

scholarly journals Bmi1 Augments Proliferation and Survival of Cortical Bone-Derived Stem Cells after Injury through Novel Epigenetic Signaling via Histone 3 Regulation

2021 ◽  
Vol 22 (15) ◽  
pp. 7813
Author(s):  
Lindsay Kraus ◽  
Chris Bryan ◽  
Marcus Wagner ◽  
Tabito Kino ◽  
Melissa Gunchenko ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Dna Damage ◽  
Stem Cell ◽  
Histone Modification ◽  
Epigenetic Regulation ◽  
Critical Role ◽  
Proliferative Potential ◽  
Therapeutic Effects ◽  
Histone 3

Ischemic heart disease can lead to myocardial infarction (MI), a major cause of morbidity and mortality worldwide. Multiple stem cell types have been safely transferred into failing human hearts, but the overall clinical cardiovascular benefits have been modest. Therefore, there is a dire need to understand the basic biology of stem cells to enhance therapeutic effects. Bmi1 is part of the polycomb repressive complex 1 (PRC1) that is involved in different processes including proliferation, survival and differentiation of stem cells. We isolated cortical bones stem cells (CBSCs) from bone stroma, and they express significantly high levels of Bmi1 compared to mesenchymal stem cells (MSCs) and cardiac-derived stem cells (CDCs). Using lentiviral transduction, Bmi1 was knocked down in the CBSCs to determine the effect of loss of Bmi1 on proliferation and survival potential with or without Bmi1 in CBSCs. Our data show that with the loss of Bmi1, there is a decrease in CBSC ability to proliferate and survive during stress. This loss of functionality is attributed to changes in histone modification, specifically histone 3 lysine 27 (H3K27). Without the proper epigenetic regulation, due to the loss of the polycomb protein in CBSCs, there is a significant decrease in cell cycle proteins, including Cyclin B, E2F, and WEE as well as an increase in DNA damage genes, including ataxia-telangiectasia mutated (ATM) and ATM and Rad3-related (ATR). In conclusion, in the absence of Bmi1, CBSCs lose their proliferative potential, have increased DNA damage and apoptosis, and more cell cycle arrest due to changes in epigenetic modifications. Consequently, Bmi1 plays a critical role in stem cell proliferation and survival through cell cycle regulation, specifically in the CBSCs. This regulation is associated with the histone modification and regulation of Bmi1, therefore indicating a novel mechanism of Bmi1 and the epigenetic regulation of stem cells.

Stimulation of the Supportive Function of Marrow Stromal Cells by Anticancer Agents: Critical Role of Marrow Macrophages.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4252-4252
Author(s):  
Kensuke Ohta ◽  
Hirohisa Nakamae ◽  
Ki-Ryang Koh ◽  
Kiyotaka Nakaie ◽  
Hong-Zhang Li ◽  
...  
Keyword(s):  
Stem Cell ◽  
Anticancer Drugs ◽  
Anticancer Agents ◽  
Stromal Cells ◽  
Critical Role ◽  
Marrow Stromal Cells ◽  
Gene Expressions ◽  
Gene Response

Abstract Anticancer agents used for the treatment of hematological malignancies are known to cause long-term toxicities to the structure and function of bone marrow microenvironment. These obbservations are based predominantly on the in vivo experiments with anticancer drug-treated mice and also on the clinical features of the patients who survived after inhtensive chemotherapies. In the present study, in order to evaluate immediate gene response of human marrow stromal cells to anticancer drugs, we tested with real-time PCR for the expression of various cytokine mRNAs from the stromal cells treated with anticancer drugs. Contrary to our expectation, we found that treatment of stromal cells with cytarabine (Ara-C; 0.1–100 μmol) for 3 to 14 days significantly and dose-dependently enhances the expression of mRNAs of stem cell factor (SCF) and leukemia inhibitory factor (LIF) while downregulates interleukin-6 (IL-6) mRNA. On the other hand, carboplatin (0.1–100 μmol) up-regulated only SCF mRNA and adriamycin (0.001–1 μmol) did not affect these gene expressions (n=6). In order to determine the responsive cell elements of stromal cells for these novel responses to Ara-C, mRNAs were independently quantified after separating CD14 positive macrophages and CD45 negative mesenchymal cells from Ara-C-treated stromal cells (n=6). In this additional experiments, we found that the above-mentioned changes in gene expression by Ara-C were provoked predominantly in the stromal macrophage fraction. Based on these observations, we treated the stromal cells established from 6 patients with AML and 11 with non-leukemic subjects with Ara-C for 2 weeks, followed by washing 3-times to eliminate Ara-C. Allogenic CD34 positive cells were then recharged and supportive functions of the stromal cells were evaluated with standard 2-stage long-term cultures. Indeed, the results showed that transient treatment with Ara-C significantly and dose-dependently upregulates supporting function of premature hematopoietic cells in non-leukemic and, although to the less extent, in leukemic stromal cells. These novel short-term stimulatory effects of Ara-C to marrow microenvironment may provide new explanations for some clinical events such as mechanism of rapid recovery of normal hematopoiesis and stem cell mobilization observed several days after completion of intensive chemotherapy to acute leukemia. In addfition, the pattern of gene response of stromal cells induced by Ara-C is of biologically great interest since it is quite different from the already known gene response of human stromal cells provoked by IL-1.

scholarly journals In Vivo HP1 Targeting Causes Large-Scale Chromatin Condensation and Enhanced Histone Lysine Methylation

2005 ◽  
Vol 25 (11) ◽  
pp. 4552-4564 ◽  
Author(s):  
Pernette J. Verschure ◽  
Ineke van der Kraan ◽  
Wim de Leeuw ◽  
Johan van der Vlag ◽  
Anne E. Carpenter ◽  
...  
Keyword(s):  
Chromatin Structure ◽  
Mammalian Cells ◽  
Large Scale ◽  
Chromosome Region ◽  
Chromatin Condensation ◽  
Regulation Of Gene Expression ◽  
Lac Repressor ◽  
Homologous Proteins ◽  
Lac Operator

ABSTRACT Changes in chromatin structure are a key aspect in the epigenetic regulation of gene expression. We have used a lac operator array system to visualize by light microscopy the effect of heterochromatin protein 1 (HP1) α (HP1α) and HP1β on large-scale chromatin structure in living mammalian cells. The structure of HP1, containing a chromodomain, a chromoshadow domain, and a hinge domain, allows it to bind to a variety of proteins. In vivo targeting of an enhanced green fluorescent protein-tagged HP1-lac repressor fusion to a lac operator-containing, gene-amplified chromosome region causes local condensation of the higher-order chromatin structure, recruitment of the histone methyltransferase SETDB1, and enhanced trimethylation of histone H3 lysine 9. Polycomb group proteins of both the HPC/HPH and the EED/EZH2 complexes, which are involved in the heritable repression of gene activity, are not recruited to the amplified chromosome region by HP1α and HP1β in vivo targeting. HP1α targeting causes the recruitment of endogenous HP1β to the chromatin region and vice versa, indicating a direct interaction between the two HP1 homologous proteins. Our findings indicate that HP1α and HP1β targeting is sufficient to induce heterochromatin formation.

scholarly journals Large-scale chromatin structure of inducible genes: transcription on a condensed, linear template

2009 ◽  
Vol 185 (1) ◽  
pp. 87-100 ◽  
Author(s):  
Yan Hu ◽  
Igor Kireev ◽  
Matt Plutz ◽  
Nazanin Ashourian ◽  
Andrew S. Belmont
Keyword(s):  
Chromatin Structure ◽  
Transcriptional Activation ◽  
Large Scale ◽  
Nuclear Speckles ◽  
Interphase Chromosome ◽  
Chromosome Conformation ◽  
Dynamic Events ◽  
Classical Models ◽  
Chromatin Fibers

The structure of interphase chromosomes, and in particular the changes in large-scale chromatin structure accompanying transcriptional activation, remain poorly characterized. Here we use light microscopy and in vivo immunogold labeling to directly visualize the interphase chromosome conformation of 1–2 Mbp chromatin domains formed by multi-copy BAC transgenes containing 130–220 kb of genomic DNA surrounding the DHFR, Hsp70, or MT gene loci. We demonstrate near-endogenous transcription levels in the context of large-scale chromatin fibers compacted nonuniformly well above the 30-nm chromatin fiber. An approximately 1.5–3-fold extension of these large-scale chromatin fibers accompanies transcriptional induction and active genes remain mobile. Heat shock–induced Hsp70 transgenes associate with the exterior of nuclear speckles, with Hsp70 transcripts accumulating within the speckle. Live-cell imaging reveals distinct dynamic events, with Hsp70 transgenes associating with adjacent speckles, nucleating new speckles, or moving to preexisting speckles. Our results call for reexamination of classical models of interphase chromosome organization.

scholarly journals Conservation of epigenetic regulation by the MLL3/4 tumour suppressor in planarian pluripotent stem cells

2017 ◽  
Author(s):  
Yuliana Mihaylova ◽  
Prasad Abnave ◽  
Damian Kao ◽  
Samantha Hughes ◽  
Alvina Lai ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Epigenetic Regulation ◽  
Cell System ◽  
Stem Cell Population ◽  
Loss Of Function ◽  
Tumour Suppressors ◽  
A Genome ◽  
Regulatory Effects

AbstractCurrently, little is known about the evolution of epigenetic regulation in animal stem cells. Using the planarian stem cell system to investigate the role of the COMPASS family of MLL3/4 histone methyltransferases, we demonstrate that their role as tumour suppressors in stem cells is conserved over a large evolutionary distance in animals. This also suggested the potential conservation of a genome wide epigenetic regulation program in animal stem cells, so we assessed the regulatory effects of Mll3/4 loss of function by performing RNA-seq and ChIP-seq on the G2/M planarian stem cell population, part of which contributes to the formation of outgrowths. We find many oncogenes and tumour suppressors among the affected genes that are therefore likely candidates for mediating MLL3/4 tumour suppression function in mammals, where little is known about in vivo regulatory targets. Our work demonstrates conservation of an important epigenetic regulatory program in animals and highlights the utility of the planarian model system for studying epigenetic regulation.

scholarly journals Nucleosomes impede Cas9 access to DNA in vivo and in vitro

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Max A Horlbeck ◽  
Lea B Witkowsky ◽  
Benjamin Guglielmi ◽  
Joseph M Replogle ◽  
Luke A Gilbert ◽  
...  
Keyword(s):  
Large Scale ◽  
Nucleosome Occupancy ◽  
Critical Role ◽  
Genetic Screens ◽  
Guide Rnas ◽  
Highly Active ◽  
Bacterial Enzyme ◽  
Eukaryotic Chromatin

The prokaryotic CRISPR (clustered regularly interspaced palindromic repeats)-associated protein, Cas9, has been widely adopted as a tool for editing, imaging, and regulating eukaryotic genomes. However, our understanding of how to select single-guide RNAs (sgRNAs) that mediate efficient Cas9 activity is incomplete, as we lack insight into how chromatin impacts Cas9 targeting. To address this gap, we analyzed large-scale genetic screens performed in human cell lines using either nuclease-active or nuclease-dead Cas9 (dCas9). We observed that highly active sgRNAs for Cas9 and dCas9 were found almost exclusively in regions of low nucleosome occupancy. In vitro experiments demonstrated that nucleosomes in fact directly impede Cas9 binding and cleavage, while chromatin remodeling can restore Cas9 access. Our results reveal a critical role of eukaryotic chromatin in dictating the targeting specificity of this transplanted bacterial enzyme, and provide rules for selecting Cas9 target sites distinct from and complementary to those based on sequence properties.

Abstract 206: Stem Cell-derived Exosomes Induce Cardiac Repair via mRNA Modification

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Prabhu Mathiyalagan ◽  
Yaxuan Liang ◽  
Adriano S Martins ◽  
Douglas W Losordo ◽  
Roger J Hajjar ◽  
...  
Keyword(s):  
Stem Cell ◽  
Myocardial Ischemia ◽  
Target Genes ◽  
Critical Role ◽  
Cell Communication ◽  
Target Cells ◽  
Mouse Heart ◽  
Loss Of Function

Exosomes are cell-derived nanovesicles that carry and shuttle microRNAs (miRNAs) to mediate cell-cell communication. Vast majority of cell types including cardiac myocytes and progenitors actively secrete exosomes, whose miRNA contents are altered after physiological or pathological changes such as myocardial ischemia (MI). In this new study, we have discovered that chemical modification to mRNAs is a novel regulator of ischemia-induced gene expression changes in the heart. We hypothesized that the benefits of human CD34 + stem cell-derived exosomes (CD34exo) are mediated by mRNA modifications in the target cells via miRNA delivery. MiRNA profiling and bioinformatic analysis identified that CD34exo is selectively enriched with a number of miRNAs that directly target genes implicated in regulation of mRNA modifications. Interestingly, under myocardial ischemia, there was a significant increase in mRNA modifications in the mouse heart, which was decreased by about 70% with CD34exo-treatment. In line with the in vivo MI data, in vitro hypoxic stimulation in neonatal / adult rodent myocytes and non-myocytes increased mRNA modifications and controls known regulators of those mRNA modifications. Loss-of-function studies for regulators of mRNA modifications attenuated hypoxia-induced changes to epitranscriptome indicating important roles for these molecules under stress conditions. Finally, using gain-of-function and loss-of-function studies, we demonstrate that miR-126, one of the most enriched miRNAs in CD34exo, plays a critical role in regulating the mRNA modifications. We conclude that miRNAs enriched in CD34exo mediate their cardioprotective effect at least in part, by regulating the mRNA epitranscriptome of the target cell. Our new data suggests hypoxia as a novel regulator of the mRNA epitranscriptome and provides novel insights to post-transcriptional gene regulation in the heart.

Inactivation of p38 MAPK Extends Self-Renewal Capacity of Haematopoietic Stem Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 265-265
Author(s):  
Keisuke Ito ◽  
Atsushi Hirao ◽  
Fumio Arai ◽  
Sahoko Matsuoka ◽  
Keiyo Takubo ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
P38 Mapk ◽  
Cell Population ◽  
Critical Role ◽  
Dependent Manner ◽  
Self Renewal ◽  
Haematopoietic Stem Cells ◽  
P38 Inhibitor

Abstract Haematopoietic stem cells (HSCs) undergo self-renewing cell divisions and maintain blood production for their lifetime. Appropriate control of HSC self-renewal is critical for maintenance of haematopoietic homeostasis. Here we show that activation of p38 MAPK limits lifespan of HSCs in response to increasing levels of reactive oxygen species (ROS) in vivo. Although normal quiescent HSCs maintain a low level of oxidative stress, an increase in ROS was observed in HSCs after transplantation as well as in aged mice. In vitro treatment with BSO (Buthionine sulfoximine), which depletes intra-cellular glutathion, increased ROS (H2O2) level in immature hematopoietic cell population, c-kit+Sca1+Lin- (KSL) cells, in a dose-dependent manner. Low dose concentration of BSO suppressed reconstitution capacity of HSCs, whereas higher concentration did not affect progenitors. These data indicate that HSCs are much more sensitive to increased ROS than progenitors and are consistent with our previous results from Atm−/− mice in which ROS level is elevated in vivo. Here we focused on MAPKs for the stem cell dysfunction since it has been shown that several MAPKs are activated in response to ROS. We evaluated effects of MAPK inhibitors for p38, JNK or ERK in incubation of KSL cell with BSO. p38 inhibitor (SB203580), neither JNK nor ERK inhibitor, restored reconstitution capacity of HSCs after transplantation, suggesting that activation of p38 may contributes to defect of stem cell function in vivo. To address the question, we evaluated p38 activation in Atm−/− BM cells by immunohistochemistry. Surprisingly, p38 protein was phosphorylated only in KSL cells, but not other more differentiated cell populations, despite that the ROS levels were comparable among the cell population of mice. In response to activation of p38, p16INK4a was up-regulated only in KSL cells. The data indicates a possibility that stem cell-specific p38 activation negatively regulates self-renewal of HSCs. We then investigated a role of p38 activation on self-renewal of HSCs in vivo. When p38 inhibitor was intraperitoneally administered both before and after BMT, the level of repopulation achieved was comparable to that of the wild-type. Furthermore, Atm−/− mice that received long-term p38 inhibitor treatment did not show either anemia, a decrease in progenitor colony-forming capacity, or reduced frequencies of stem cell subsets. These data demonstrate that the activation of p38 present in HSCs promotes the exhaustion of stem cell pool in response to elevation of ROS. It has been proposed that aging is driven in part by a gradual depletion of stem cell functional capacity. There are evidences that inappropriate production of oxidants is connected to aging and life span. We propose a possibility that p38 activation in response to ROS plays a critical role for limit of stem cell capacity.

In Vivo Fate Tracing Studies Using the SCL Stem Cell Enhancer: Embryonic Hematopoietic Stem Cells Significantly Contribute to Adult Hematopoiesis.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 559-559
Author(s):  
Joachim R. Gothert ◽  
Sonja E. Gustin ◽  
Mark A. Hall ◽  
Anthony R. Green ◽  
Berthold Gottgens ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
De Novo ◽  
Genetic Manipulation ◽  
Fetal Liver ◽  
Hematopoietic Stem ◽  
Reporter Mice ◽  
First Time

Abstract Evidence for the direct lineage relationship between embryonic and adult hematopoietic stem cells (HSCs) in the mouse is primarily indirect. In order to study this relationship in a direct manner we expressed the tamoxifen-inducible Cre-ERT-recombinase under the control of the SCL-stem-cell-enhancer in transgenic mice (HSC-SCL-Cre-ERT). To determine functionality, HSC-SCL-Cre-ERT transgenics were bred with the Cre-reporter-mice ROSA26R and R26R-EYFP. Flow-cytometric and transplantation studies revealed tamoxifen-dependent recombination occurring in more than 90% of adult long-term HSCs, whereas the targeted proportion within mature progenitor populations was significantly lower. Moreover, the transgene was able to irreversibly tag embryonic HSCs on days 10 and 11 of gestation. These cells contributed to bone marrow hematopoiesis five months later. In order to investigate whether the de novo HSC-generation is completed during embryogenesis, HSC-SCL-Cre-ERT marked fetal liver cells were transplanted into adult recipients. Strikingly, the proportion of marked cells within the transplanted and the in vivo-remaining HSC-compartment was not different, implying that no further HSC-generation occurred during late fetal and neonatal stages of development. These data demonstrate for the first time the direct lineage relationship between mid-gestation embryonic and adult HSCs in the mouse. Additionally, the HSC-SCL-Cre-ERT mice will provide a valuable tool to achieve temporally controlled genetic manipulation of HSCs.

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