Cbx2 stably associates with mitotic chromosomes via a PRC2- or PRC1-independent mechanism and is needed for recruiting PRC1 complex to mitotic chromosomes

Polycomb group (PcG) proteins are epigenetic transcriptional factors that repress key developmental regulators and maintain cellular identity through mitosis via a poorly understood mechanism. Using quantitative live-cell imaging in mouse ES cells and tumor cells, we demonstrate that, although Polycomb repressive complex (PRC) 1 proteins (Cbx-family proteins, Ring1b, Mel18, and Phc1) exhibit variable capacities of association with mitotic chromosomes, Cbx2 overwhelmingly binds to mitotic chromosomes. The recruitment of Cbx2 to mitotic chromosomes is independent of PRC1 or PRC2, and Cbx2 is needed to recruit PRC1 complex to mitotic chromosomes. Quantitative fluorescence recovery after photobleaching analysis indicates that PRC1 proteins rapidly exchange at interphasic chromatin. On entry into mitosis, Cbx2, Ring1b, Mel18, and Phc1 proteins become immobilized at mitotic chromosomes, whereas other Cbx-family proteins dynamically bind to mitotic chromosomes. Depletion of PRC1 or PRC2 protein has no effect on the immobilization of Cbx2 on mitotic chromosomes. We find that the N-terminus of Cbx2 is needed for its recruitment to mitotic chromosomes, whereas the C-terminus is required for its immobilization. Thus these results provide fundamental insights into the molecular mechanisms of epigenetic inheritance.

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LSD1 Plays an Important Role in GATA Switch during Erythroid Differentiation

Blood ◽

10.1182/blood.v122.21.4846.4846 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4846-4846

Author(s):

Yue Jin ◽

Yidi Guo ◽

Dongxue Liang ◽

Yue Li ◽

Zhe Li ◽

...

Keyword(s):

Molecular Mechanisms ◽

Conflicts Of Interest ◽

Es Cells ◽

Regulatory Element ◽

Erythroid Differentiation ◽

Erythroid Cell ◽

Erythroid Cells ◽

Hematopoietic Stem ◽

Mouse Es Cells ◽

Murine Erythroleukemia

Abstract GATA factors play important role in hematopoiesis. In particular, GATA2 is critical for maintenance of hematopoietic stem and progenitor cells (HS/PCs) and GATA1 is required for erythropoiesis. GATA1 and GATA2 are expressed in reciprocal patterns during erythroid differentiation. It was shown that GATA1 occupied the -2.8Kb regulatory element and mediated repression of the GATA2 promoter in terminally differentiating erythroid cells. However, the detailed molecular mechanisms that control the enhancer/promoter activities of the GATA2 gene remain to be elucidated. In this report, we found that LSD1 and TAL1 co-localize at GATA2 1S promoter through ChIP and double-ChIP assays in murine erythroleukemia (MEL) cells. To further test whether LSD1 and its mediated H3K4 demethylation is important for repression of the GATA2 gene during erythroid differentiation, we silenced LSD1 expression in both MEL cells and mouse ES cells using retrovirus mediated shRNA knockdown and induced them to differentiate into erythroid cells with DMSO and EPO, respectively. GATA2 expression was elevated while the level of GATA1 was repressed by RT-qPCR. Furthermore, consistent with the GATA witch hypothesis, ChIP analysis revealed that the levels of H3K4me2 were increased at the GATA2 1S promoter. In addition, knock-down of LSD1 in MEL cells results in inhibition of erythroid cell differenciation and attenuation of MEL cell proliferation and survival. Thus, our data reveal that LSD1 involved in control of terminal erythroid differentiation by regulating GATA switch. The LSD1 histone demethylase complex may be recruited to the GATA2 1S promoter by interacting with TAL1. The H3K4 demethylation activity of LSD1 leads to downregulation of the active H3K4m2 mark at the GATA2 promoter that alters chromatin structure and represses transcription of the GATA2 genes. Disclosures: No relevant conflicts of interest to declare.

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Zic3 Is Required for Maintenance of Pluripotency in Embryonic Stem Cells

Molecular Biology of the Cell ◽

10.1091/mbc.e06-07-0624 ◽

2007 ◽

Vol 18 (4) ◽

pp. 1348-1358 ◽

Cited By ~ 85

Author(s):

Linda Shushan Lim ◽

Yuin-Han Loh ◽

Weiwei Zhang ◽

Yixun Li ◽

Xi Chen ◽

...

Keyword(s):

Stem Cells ◽

Transcription Factors ◽

Embryonic Stem Cells ◽

Regulatory Networks ◽

Molecular Mechanisms ◽

Es Cells ◽

Embryonic Stem ◽

Es Cell ◽

Lineage Specification ◽

Mouse Es Cells

Embryonic stem (ES) cell pluripotency is dependent upon sustained expression of the key transcriptional regulators Oct4, Nanog, and Sox2. Dissection of the regulatory networks downstream of these transcription factors has provided critical insight into the molecular mechanisms that regulate ES cell pluripotency and early differentiation. Here we describe a role for Zic3, a member of the Gli family of zinc finger transcription factors, in the maintenance of pluripotency in ES cells. We show that Zic3 is expressed in ES cells and that this expression is repressed upon differentiation. The expression of Zic3 in pluripotent ES cells is also directly regulated by Oct4, Sox2, and Nanog. Targeted repression of Zic3 in human and mouse ES cells by RNA interference–induced expression of several markers of the endodermal lineage. Notably, the expression of Nanog, a key pluripotency regulator and repressor of extraembryonic endoderm specification in ES cells, was significantly reduced in Zic3 knockdown cells. This suggests that Zic3 may prevent endodermal marker expression through Nanog-regulated pathways. Thus our results extend the ES cell transcriptional network beyond Oct4, Nanog, and Sox2, and further establish that Zic3 plays an important role in the maintenance of pluripotency by preventing endodermal lineage specification in embryonic stem cells.

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Kif18A promotes Hec1 dephosphorylation to coordinate chromosome alignment with kinetochore microtubule attachment

10.1101/304147 ◽

2018 ◽

Cited By ~ 2

Author(s):

Haein Kim ◽

Jason Stumpff

Keyword(s):

Molecular Mechanisms ◽

Spindle Assembly Checkpoint ◽

Primordial Germ Cells ◽

Direct Interaction ◽

Mitotic Arrest ◽

Microtubule Dynamics ◽

Mitotic Chromosomes ◽

Microtubule Attachment ◽

C Terminus ◽

Chromosome Alignment

SUMMARYMitotic chromosomes are spatially confined at the spindle equator just prior to chromosome segregation through a process called chromosome alignment. Alignment requires temporal coordination of kinetochore microtubule attachment and dynamics. However, the molecular mechanisms that couple these activities are not understood. Kif18A (kinesin-8) suppresses the dynamics of kinetochore microtubules to promote chromosome alignment during metaphase. Loss of Kif18A function in HeLa and primordial germ cells leads to alignment defects accompanied by a spindle assembly checkpoint (SAC)-dependent mitotic arrest, suggesting the motor also plays a role in regulating kinetochore-microtubule attachments. We show here that Kif18A increases attachment by promoting dephosphorylation of the kinetochore protein Hec1, which provides the primary linkage between kinetochores and microtubules. This function requires a direct interaction between the Kif18A C-terminus and protein phosphatase-1 (PP1). However, the Kif18A-PP1 interaction is not required for chromosome alignment, indicating that regulation of kinetochore microtubule dynamics and attachments are separable Kif18A functions. Mitotic arrest in Kif18A-depleted cells is rescued by expression of a Hec1 variant that mimics a low-phosphorylation state, indicating that Kif18A-dependent Hec1 dephosphorylation is a key step for silencing the checkpoint and promoting mitotic progression. Our data support a model in which Kif18A provides positive feedback for kinetochore microtubule attachment by directly recruiting PP1 to dephosphorylate Hec1. We propose that this function works synergistically with Kif18A’s direct control of kinetochore microtubule dynamics to temporally coordinate chromosome alignment and attachment.

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How do histone modifications contribute to transgenerational epigenetic inheritance in C. elegans?

Biochemical Society Transactions ◽

10.1042/bst20190944 ◽

2020 ◽

Vol 48 (3) ◽

pp. 1019-1034 ◽

Cited By ~ 1

Author(s):

Rachel M. Woodhouse ◽

Alyson Ashe

Keyword(s):

Histone Modifications ◽

Molecular Mechanisms ◽

Epigenetic Inheritance ◽

Nematode Caenorhabditis Elegans ◽

Histone Methyltransferases ◽

Transgenerational Epigenetic Inheritance ◽

C Elegans ◽

Recent Developments ◽

Gene Regulatory

Gene regulatory information can be inherited between generations in a phenomenon termed transgenerational epigenetic inheritance (TEI). While examples of TEI in many animals accumulate, the nematode Caenorhabditis elegans has proven particularly useful in investigating the underlying molecular mechanisms of this phenomenon. In C. elegans and other animals, the modification of histone proteins has emerged as a potential carrier and effector of transgenerational epigenetic information. In this review, we explore the contribution of histone modifications to TEI in C. elegans. We describe the role of repressive histone marks, histone methyltransferases, and associated chromatin factors in heritable gene silencing, and discuss recent developments and unanswered questions in how these factors integrate with other known TEI mechanisms. We also review the transgenerational effects of the manipulation of histone modifications on germline health and longevity.

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Analysis of the Zic3, Mbd3, Chd4 and Zic2 cistromes in mouse ES cells

Signaling Pathways Project Datasets ◽

10.1621/o7pnnlgjxf ◽

2019 ◽

Author(s):

Z Luo

Keyword(s):

Es Cells ◽

Mouse Es Cells

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Gene Targeting

10.1093/oso/9780199637928.001.0001 ◽

1999 ◽

Cited By ~ 2

Keyword(s):

Homologous Recombination ◽

Gene Targeting ◽

Mammalian Cells ◽

Es Cells ◽

Practical Approach ◽

Simple Method ◽

Mouse Es Cells ◽

Cell Clones ◽

Previous Edition ◽

Pros And Cons

Since the publication of the first edition of Gene Targeting: A Practical Approach in 1993 there have been many advances in gene targeting and this new edition has been thoroughly updated and rewritten to include all the major new techniques. It provides not only tried-and-tested practical protocols but detailed guidance on their use and applications. As with the previous edition Gene Targeting: A Practical Approach 2e concentrates on gene targeting in mouse ES cells, but the techniques described can be easily adapted to applications in tissue culture including those for human cells. The first chapter covers the design of gene targeting vectors for mammalian cells and describes how to distinguish random integrations from homologous recombination. It is followed by a chapter on extending conventional gene targeting manipulations by using site-specific recombination using the Cre-loxP and Flp-FRT systems to produce 'clean' germline mutations and conditionally (in)activating genes. Chapter 3 describes methods for introducing DNA into ES cells for homologous recombination, selection and screening procedures for identifying and recovering targeted cell clones, and a simple method for establishing new ES cell lines. Chapter 4 discusses the pros and cons or aggregation versus blastocyst injection to create chimeras, focusing on the technical aspects of generating aggregation chimeras and then describes some of the uses of chimeras. The next topic covered is gene trap strategies; the structure, components, design, and modification of GT vectors, the various types of GT screens, and the molecular analysis of GT integrations. The final chapter explains the use of classical genetics in gene targeting and phenotype interpretation to create mutations and elucidate gene functions. Gene Targeting: A Practical Approach 2e will therefore be of great value to all researchers studying gene function.

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Palmitoylation of Sindbis Virus TF Protein Regulates Its Plasma Membrane Localization and Subsequent Incorporation into Virions

Journal of Virology ◽

10.1128/jvi.02000-16 ◽

2016 ◽

Vol 91 (3) ◽

Cited By ~ 20

Author(s):

Jolene Ramsey ◽

Emily C. Renzi ◽

Randy J. Arnold ◽

Jonathan C. Trinidad ◽

Suchetana Mukhopadhyay

Keyword(s):

Plasma Membrane ◽

Sindbis Virus ◽

Molecular Mechanisms ◽

Wild Type Virus ◽

Membrane Localization ◽

Clear Understanding ◽

Wild Type ◽

Plasma Membrane Localization ◽

C Terminus ◽

N Terminus

ABSTRACT Palmitoylation is a reversible, posttranslational modification that helps target proteins to cellular membranes. The alphavirus small membrane proteins 6K and TF have been reported to be palmitoylated and to positively regulate budding. 6K and TF are isoforms that are identical in their N termini but unique in their C termini due to a −1 ribosomal frameshift during translation. In this study, we used cysteine (Cys) mutants to test differential palmitoylation of the Sindbis virus 6K and TF proteins. We modularly mutated the five Cys residues in the identical N termini of 6K and TF, the four additional Cys residues in TF's unique C terminus, or all nine Cys residues in TF. Using these mutants, we determined that TF palmitoylation occurs primarily in the N terminus. In contrast, 6K is not palmitoylated, even on these shared residues. In the C-terminal Cys mutant, TF protein levels increase both in the cell and in the released virion compared to the wild type. In viruses with the N-terminal Cys residues mutated, TF is much less efficiently localized to the plasma membrane, and it is not incorporated into the virion. The three Cys mutants have minor defects in cell culture growth but a high incidence of abnormal particle morphologies compared to the wild-type virus as determined by transmission electron microscopy. We propose a model where the C terminus of TF modulates the palmitoylation of TF at the N terminus, and palmitoylated TF is preferentially trafficked to the plasma membrane for virus budding. IMPORTANCE Alphaviruses are a reemerging viral cause of arthritogenic disease. Recently, the small 6K and TF proteins of alphaviruses were shown to contribute to virulence in vivo. Nevertheless, a clear understanding of the molecular mechanisms by which either protein acts to promote virus infection is missing. The TF protein is a component of budded virions, and optimal levels of TF correlate positively with wild-type-like particle morphology. In this study, we show that the palmitoylation of TF regulates its localization to the plasma membrane, which is the site of alphavirus budding. Mutants in which TF is not palmitoylated display drastically reduced plasma membrane localization, which effectively prevents TF from participating in budding or being incorporated into virus particles. Investigation of the regulation of TF will aid current efforts in the alphavirus field searching for approaches to mitigate alphaviral disease in humans.

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Deletion of the Actin-Associated Tropomyosin Tpm3 Leads to Reduced Cell Complexity in Cultured Hippocampal Neurons—New Insights into the Role of the C-Terminal Region of Tpm3.1

Cells ◽

10.3390/cells10030715 ◽

2021 ◽

Vol 10 (3) ◽

pp. 715

Author(s):

Tamara Tomanić ◽

Claire Martin ◽

Holly Stefen ◽

Esmeralda Parić ◽

Peter Gunning ◽

...

Keyword(s):

Amino Acid ◽

Hippocampal Neurons ◽

Molecular Mechanisms ◽

Growth Cones ◽

Amino Acid Residues ◽

Terminal Amino Acid ◽

C Terminus ◽

Primary Mouse ◽

Terminal Amino

Tropomyosins (Tpms) have been described as master regulators of actin, with Tpm3 products shown to be involved in early developmental processes, and the Tpm3 isoform Tpm3.1 controlling changes in the size of neuronal growth cones and neurite growth. Here, we used primary mouse hippocampal neurons of C57/Bl6 wild type and Bl6Tpm3flox transgenic mice to carry out morphometric analyses in response to the absence of Tpm3 products, as well as to investigate the effect of C-terminal truncation on the ability of Tpm3.1 to modulate neuronal morphogenesis. We found that the knock-out of Tpm3 leads to decreased neurite length and complexity, and that the deletion of two amino acid residues at the C-terminus of Tpm3.1 leads to more detrimental changes in neurite morphology than the deletion of six amino acid residues. We also found that Tpm3.1 that lacks the 6 C-terminal amino acid residues does not associate with stress fibres, does not segregate to the tips of neurites, and does not impact the amount of the filamentous actin pool at the axonal growth cones, as opposed to Tpm3.1, which lacks the two C-terminal amino acid residues. Our study provides further insight into the role of both Tpm3 products and the C-terminus of Tpm3.1, and it forms the basis for future studies that aim to identify the molecular mechanisms underlying Tpm3.1 targeting to different subcellular compartments.

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Epigenetic memory effects in forest trees: a victory of “Michurinian biology”?

Central European Forestry Journal ◽

10.1515/forj-2017-0024 ◽

2017 ◽

Vol 63 (4) ◽

pp. 173-179 ◽

Cited By ~ 5

Author(s):

Dušan Gömöry ◽

Matúš Hrivnák ◽

Diana Krajmerová ◽

Roman Longauer

Keyword(s):

Molecular Mechanisms ◽

Cytosine Methylation ◽

Epigenetic Inheritance ◽

Memory Effects ◽

Forest Trees ◽

Small Interfering Rnas ◽

Adaptation To Climate Change ◽

Growth Environment ◽

Micro Rnas ◽

Covalent Modifications

AbstractThe study reviews trait inheritance, which is in contradiction with the rules of Mendelian genetics, and which was object of controversies among biologists (sometimes with grave political consequences) in the USSR and Sovietcontrolled countries in the 1930s-1960s. “Carryover” or “memory” effects of the climate, to which maternal trees are exposed during seed development, on phenological behavior and other adaptively relevant traits of their offspring in conifers are mentioned; similar effects are associated with the germination and early growth environment. Molecular mechanisms underlying these effects include covalent modifications of DNA or DNA-associated proteins (cytosine methylation, various types of histone modifications), micro-RNAs and small interfering RNAs. Tools for the identification of these modifications are reviewed with a focus on cytosine methylation, along with an overview of the hitherto knowledge on the occurrence of DNA modifications in forest trees. The practical implications of epigenetic inheritance in forest trees are discussed with the focus on the adaptation to climate change and legislation on forest reproductive materials.

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