scholarly journals A Novel Chromatin Protein, Distantly Related to Histone H2a, Is Largely Excluded from the Inactive X Chromosome

2001 ◽  
Vol 152 (2) ◽  
pp. 375-384 ◽  
Author(s):  
Brian P. Chadwick ◽  
Huntington F. Willard
Keyword(s):  
X Chromosome ◽  
Fluorescent Protein ◽  
Active Regions ◽  
Micrococcal Nuclease ◽  
Histone H2a ◽  
Histone H4 ◽  
Metaphase Chromosomes ◽  
Double Labeling ◽  
Inactive X Chromosome ◽  
Green Fluorescent

Chromatin on the mammalian inactive X chromosome differs in a number of ways from that on the active X. One protein, macroH2A, whose amino terminus is closely related to histone H2A, is enriched on the heterochromatic inactive X chromosome in female cells. Here, we report the identification and localization of a novel and more distant histone variant, designated H2A-Bbd, that is only 48% identical to histone H2A. In both interphase and metaphase female cells, using either a myc epitope–tagged or green fluorescent protein–tagged H2A-Bbd construct, the inactive X chromosome is markedly deficient in H2A-Bbd staining, while the active X and the autosomes stain throughout. In double-labeling experiments, antibodies to acetylated histone H4 show a pattern of staining indistinguishable from H2A-Bbd in interphase nuclei and on metaphase chromosomes. Chromatin fractionation demonstrates association of H2A-Bbd with the histone proteins. Separation of micrococcal nuclease–digested chromatin by sucrose gradient ultracentrifugation shows cofractionation of H2A-Bbd with nucleosomes, supporting the idea that H2A-Bbd is incorporated into nucleosomes as a substitute for the core histone H2A. This finding, in combination with the overlap with acetylated forms of H4, raises the possibility that H2A-Bbd is enriched in nucleosomes associated with transcriptionally active regions of the genome. The distribution of H2A-Bbd thus distinguishes chromatin on the active and inactive X chromosomes.

scholarly journals Association of BRCA1 with the inactive X chromosome and XIST RNA

2004 ◽  
Vol 359 (1441) ◽  
pp. 123-128 ◽  
Author(s):  
Shridar Ganesan ◽  
Daniel P. Silver ◽  
Ronny Drapkin ◽  
Roger Greenberg ◽  
Jean Feunteun ◽  
...  
Keyword(s):  
X Chromosome ◽  
Fluorescent Protein ◽  
Wild Type ◽  
Cancer Syndrome ◽  
Suppressor Activity ◽  
Inactive X Chromosome ◽  
Xist Rna ◽  
Green Fluorescent ◽  
Cancer Suppression ◽  
Diploid Cells

Breast cancer, early onset 1 (BRCA1) encodes a nuclear protein that participates in breast and ovarian cancer suppression. The molecular basis for the gender and tissue specificity of the BRCA1 cancer syndrome is unknown. Recently, we observed that a fraction of BRCA1 in female cells is localized on the inactive X chromosome (Xi). Chromatin immunoprecipitation (ChIP) experiments have demonstrated that BRCA1 physically associates with Xi–specific transcript (XIST) RNA, a non–coding RNA known to coat Xi and to participate in the initiation of its inactivation during early embryogenesis. Cells lacking wild–type BRCA1 show abnormalities in Xi, including lack of proper XIST RNA localization. Reintroduction of wild–type, but not mutant, BRCA1 can correct this defect in XIST localization in these cells. Depletion of BRCA1 in female diploid cells led to a defect in proper XIST localization on Xi and in the development of normal Xi heterchromatic superstructure. Moreover, depletion of BRCA1 led to an increased likelihood of re–expression of a green fluorescent protein (GFP) reporter gene embedded on Xi. Taken together, these findings are consistent with a model in which BRCA1 function contributes to the maintenance of proper Xi heterochromatin superstructure. Although the data imply a novel gender–specific consequence of BRCA1 loss, the relevance of the BRCA1/Xi function to the tumour suppressor activity of BRCA1 remains unclear and needs to be tested.

scholarly journals Cell cycle–dependent localization of macroH2A in chromatin of the inactive X chromosome

2002 ◽  
Vol 157 (7) ◽  
pp. 1113-1123 ◽  
Author(s):  
Brian P. Chadwick ◽  
Huntington F. Willard
Keyword(s):  
Cell Cycle ◽  
X Chromosome ◽  
Degradation Pathway ◽  
S Phase ◽  
Histone H2a ◽  
The Core ◽  
Inactive X Chromosome ◽  
Inactivation Center ◽  
Chromatin Proteins ◽  
Cycle Dependent

One of several features acquired by chromatin of the inactive X chromosome (Xi) is enrichment for the core histone H2A variant macroH2A within a distinct nuclear structure referred to as a macrochromatin body (MCB). In addition to localizing to the MCB, macroH2A accumulates at a perinuclear structure centered at the centrosome. To better understand the association of macroH2A1 with the centrosome and the formation of an MCB, we investigated the distribution of macroH2A1 throughout the somatic cell cycle. Unlike Xi-specific RNA, which associates with the Xi throughout interphase, the appearance of an MCB is predominantly a feature of S phase. Although the MCB dissipates during late S phase and G2 before reforming in late G1, macroH2A1 remains associated during mitosis with specific regions of the Xi, including at the X inactivation center. This association yields a distinct macroH2A banding pattern that overlaps with the site of histone H3 lysine-4 methylation centered at the DXZ4 locus in Xq24. The centrosomal pool of macroH2A1 accumulates in the presence of an inhibitor of the 20S proteasome. Therefore, targeting of macroH2A1 to the centrosome is likely part of a degradation pathway, a mechanism common to a variety of other chromatin proteins.

scholarly journals Polycomb complexes in X chromosome inactivation

2017 ◽  
Vol 372 (1733) ◽  
pp. 20170021 ◽  
Author(s):  
Neil Brockdorff
Keyword(s):  
X Chromosome ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
X Chromosome Inactivation ◽  
Chromosome Inactivation ◽  
Histone H2a ◽  
Post Translational Modifications ◽  
Link Type ◽  
Polycomb Proteins ◽  
Inactive X Chromosome

Identifying the critical RNA binding proteins (RBPs) that elicit Xist mediated silencing has been a key goal in X inactivation research. Early studies implicated the Polycomb proteins, a family of factors linked to one of two major multiprotein complexes, PRC1 and PRC2 (Wang 2001 Nat. Genet. 28 , 371–375 ( doi:10.1038/ng574 ); Silva 2003 Dev. Cell 4 , 481–495 ( doi:10.1016/S1534-5807(03)00068-6 ); de Napoles 2004 Dev. Cell 7 , 663–676 ( doi:10.1016/j.devcel.2004.10.005 ); Plath 2003 Science 300 , 131–135 ( doi:10.1126/science.1084274 )). PRC1 and PRC2 complexes catalyse specific histone post-translational modifications (PTMs), ubiquitylation of histone H2A at position lysine 119 (H2AK119u1) and methylation of histone H3 at position lysine 27 (H3K27me3), respectively, and accordingly, these modifications are highly enriched over the length of the inactive X chromosome (Xi). A key study proposed that PRC2 subunits bind directly to Xist RNA A-repeat element, a region located at the 5′ end of the transcript known to be required for Xist mediated silencing (Zhao 2008 Science 322 , 750–756 ( doi:10.1126/science.1163045 )). Subsequent recruitment of PRC1 was assumed to occur via recognition of PRC2 mediated H3K27me3 by the CBX subunit of PRC1, as has been shown to be the case at other Polycomb target loci (Cao 2002 Science 298 , 1039–1043 ( doi:10.1126/science.1076997 )). More recently, several reports have questioned aspects of the prevailing view, both in relation to the mechanism for Polycomb recruitment by Xist RNA and the contribution of the Polycomb pathway to Xist mediated silencing. In this article I provide an overview of our recent progress towards resolving these discrepancies. This article is part of the themed issue ‘X-chromosome inactivation: a tribute to Mary Lyon’.

scholarly journals Dynamics of Centromeres during Metaphase–Anaphase Transition in Fission Yeast: Dis1 Is Implicated in Force Balance in Metaphase Bipolar Spindle

1998 ◽  
Vol 9 (11) ◽  
pp. 3211-3225 ◽  
Author(s):  
Kentaro Nabeshima ◽  
Takashi Nakagawa ◽  
Aaron F. Straight ◽  
Andrew Murray ◽  
Yuji Chikashige ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Topoisomerase Ii ◽  
Fluorescent Protein ◽  
Phase 1 ◽  
Force Balance ◽  
Yeast Cells ◽  
Phase 2 ◽  
Metaphase Chromosomes ◽  
Spindle Elongation ◽  
Green Fluorescent

In higher eukaryotic cells, the spindle forms along with chromosome condensation in mitotic prophase. In metaphase, chromosomes are aligned on the spindle with sister kinetochores facing toward the opposite poles. In anaphase A, sister chromatids separate from each other without spindle extension, whereas spindle elongation takes place during anaphase B. We have critically examined whether such mitotic stages also occur in a lower eukaryote, Schizosaccharomyces pombe. Using the green fluorescent protein tagging technique, early mitotic to late anaphase events were observed in living fission yeast cells. S. pombe has three phases in spindle dynamics, spindle formation (phase 1), constant spindle length (phase 2), and spindle extension (phase 3). Sister centromere separation (anaphase A) rapidly occurred at the end of phase 2. The centromere showed dynamic movements throughout phase 2 as it moved back and forth and was transiently split in two before its separation, suggesting that the centromere was positioned in a bioriented manner toward the poles at metaphase. Microtubule-associating Dis1 was required for the occurrence of constant spindle length and centromere movement in phase 2. Normal transition from phase 2 to 3 needed DNA topoisomerase II and Cut1 but not Cut14. The duration of each phase was highly dependent on temperature.

scholarly journals A Novel Dynamin-like Protein Associates with Cytoplasmic Vesicles and Tubules of the Endoplasmic Reticulum in Mammalian Cells

1998 ◽  
Vol 140 (4) ◽  
pp. 779-793 ◽  
Author(s):  
Yisang Yoon ◽  
Kelly R. Pitts ◽  
Sophie Dahan ◽  
Mark A. McNiven
Keyword(s):  
Endoplasmic Reticulum ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Cultured Cells ◽  
Double Labeling ◽  
Subcellular Membrane ◽  
Morphological Studies ◽  
Cytoplasmic Vesicles ◽  
Membrane Compartment ◽  
Green Fluorescent

Abstract. Dynamins are 100-kilodalton guanosine triphosphatases that participate in the formation of nascent vesicles during endocytosis. Here, we have tested if novel dynamin-like proteins are expressed in mammalian cells to support vesicle trafficking processes at cytoplasmic sites distinct from the plasma membrane. Immunological and molecular biological methods were used to isolate a cDNA clone encoding an 80-kilodalton novel dynamin-like protein, DLP1, that shares up to 42% homology with other dynamin-related proteins. DLP1 is expressed in all tissues examined and contains two alternatively spliced regions that are differentially expressed in a tissue-specific manner. DLP1 is enriched in subcellular membrane fractions of cytoplasmic vesicles and endoplasmic reticulum. Morphological studies of DLP1 in cultured cells using either a specific antibody or an expressed green fluorescent protein (GFP)- DLP1 fusion protein revealed that DLP1 associates with punctate cytoplasmic vesicles that do not colocalize with conventional dynamin, clathrin, or endocytic ligands. Remarkably, DLP1-positive structures coalign with microtubules and, most strikingly, with endoplasmic reticulum tubules as verified by double labeling with antibodies to calnexin and Rab1 as well as by immunoelectron microscopy. These observations provide the first evidence that a novel dynamin-like protein is expressed in mammalian cells where it associates with a secretory, rather than endocytic membrane compartment.

scholarly journals Novel Histone-Based DNA Carrier Targeting Cancer-Associated Fibroblasts

Polymers ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 1695
Author(s):  
Alexey Kuzmich ◽  
Olga Rakitina ◽  
Dmitry Didych ◽  
Victor Potapov ◽  
Marina Zinovyeva ◽  
...  
Keyword(s):  
Gene Delivery ◽  
Fluorescent Protein ◽  
Transfection Efficiency ◽  
Growth Factor Receptor ◽  
Surface Protein ◽  
Firefly Luciferase ◽  
Luciferase Assay ◽  
Cancer Associated Fibroblasts ◽  
Histone H2a ◽  
Green Fluorescent

Nuclear proteins, like histone H2A, are promising non-viral carriers for gene delivery since they are biocompatible, biodegradable, bear intrinsic nuclear localization signal, and are easy to modify. The addition of surface-protein-binding ligand to histone H2A may increase its DNA delivery efficiency. Tumor microenvironment (TME) is a promising target for gene therapy since its surface protein repertoire is more stable than that of cancer cells. Cancer-associated fibroblasts (CAFs) are important components of TME, and one of their surface markers is beta-type platelet-derived growth factor receptor (PDGFRβ). In this study, we fused histone H2A with PDGFRβ-binding peptide, YG2, to create a novel non-viral fibroblast-targeting DNA carrier, H2A-YG2. The transfection efficiency of histone complexes with pDNA encoding a bicistronic reporter (enhanced green fluorescent protein, EGFP, and firefly luciferase) in PDGFRβ-positive and PDGFRβ-negative cells was estimated by luciferase assay and flow cytometry. The luciferase activity, percentage of transfected cells, and overall EGFP fluorescence were increased due to histone modification with YG2 only in PDGFRβ-positive cells. We also estimated the internalization efficiency of DNA-carrier complexes using tetramethyl-rhodamine-labeled pDNA. The ligand fusion increased DNA internalization only in the PDGFRβ-positive cells. In conclusion, we demonstrated that the H2A-YG2 carrier targeted gene delivery to PDGFRβ-positive tumor stromal cells.

scholarly journals Binding of Drosophila Orc Proteins to Anaphase Chromosomes Requires Cessation of Mitotic Cyclin-Dependent Kinase Activity

2008 ◽  
Vol 29 (1) ◽  
pp. 140-149 ◽  
Author(s):  
Tina Baldinger ◽  
Manfred Gossen
Keyword(s):  
Origin Recognition Complex ◽  
Kinase Activity ◽  
Fluorescent Protein ◽  
Initial Step ◽  
Dynamic Imaging ◽  
Cyclin Dependent Kinase ◽  
Metaphase Chromosomes ◽  
Mitotic Cyclin ◽  
Green Fluorescent

ABSTRACT The initial step in the acquisition of replication competence by eukaryotic chromosomes is the binding of the multisubunit origin recognition complex, ORC. We describe a transgenic Drosophila model which enables dynamic imaging of a green fluorescent protein (GFP)-tagged Drosophila melanogaster ORC subunit, DmOrc2-GFP. It is functional in genetic complementation, expressed at physiological levels, and participates quantitatively in complex formation. This fusion protein is therefore able to depict both the holocomplex DmOrc1-6 and the core complex DmOrc2-6 formed by the Drosophila initiator proteins. Its localization can be monitored in vivo along the cell cycle and development. DmOrc2-GFP is not detected on metaphase chromosomes but binds rapidly to anaphase chromatin in Drosophila embryos. Expression of either stable cyclin A, B, or B3 prevents this reassociation, suggesting that cessation of mitotic cyclin-dependent kinase activity is essential for binding of the DmOrc proteins to chromosomes.

Transgenic expression of the jellyfish green fluorescent protein in the cone photoreceptors of the mouse

2001 ◽  
Vol 18 (4) ◽  
pp. 615-623 ◽  
Author(s):  
YIJIAN FEI ◽  
THOMAS E. HUGHES
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Pcr Analysis ◽  
Regulatory Sequence ◽  
Cone Photoreceptors ◽  
Isolated Cells ◽  
Double Labeling ◽  
Transgenic Expression ◽  
Entire Cell ◽  
Green Fluorescent

The goal of this study was to determine whether the jellyfish green fluorescent protein (GFP) could be used in transgenic mice to label and purify cone photoreceptors from the living retina. We created a transgene containing the 5′ regulatory sequence of the human red pigment gene (pR6.5 lacZ clone; kindly provided by J. Nathans & Y. Wang), fused to the GFP coding sequence. This transgene was used to generate seven lines of PCR-positive founders. Three of the lines had bright green fluorescent cone photoreceptors. The GFP fills the entire cell. Two mouse lines had only a few (∼10–100) fluorescent cells per retina, and one line (R6.85933) had many thousands. In the latter, double labeling of the cones with RITC-conjugated peanut agglutinin reveals that in the ventral retina a small proportion of the cones express GFP, while in the dorsal retina the majority do. Cells dissociated from the retinae of line R6.85933 continue to fluoresce and can be readily detected and enriched with flow cytometry. The signal provides a log unit of separation between the fluorescent cone soma and the remaining retinal cells. Roughly 3% of the cells are this fluorescent, and it is possible to purify up to 30,000 cells from one mouse. RT-PCR analysis of the mRNA from these isolated cells detects both the middle and short wavelength opsins with little if any contamination from rhodopsin.

scholarly journals Epstein–Barr virus nuclear antigen-1 is highly colocalized with interphase chromatin and its newly replicated regions in particular

2002 ◽  
Vol 83 (10) ◽  
pp. 2377-2383 ◽  
Author(s):  
Sayuri Ito ◽  
Eisuke Gotoh ◽  
Shigeru Ozawa ◽  
Kazuo Yanagi
Keyword(s):  
Epstein Barr Virus ◽  
Fluorescent Protein ◽  
Chromosomal Localization ◽  
Nuclear Antigen ◽  
Interphase Chromatin ◽  
Premature Chromosome Condensation ◽  
Metaphase Chromosomes ◽  
Barr Virus ◽  
Epstein Barr ◽  
Green Fluorescent

Epstein–Barr virus (EBV) nuclear antigen-1 (EBNA-1), which binds to both the EBV origin of replication (oriP) and metaphase chromosomes, is essential for the replication/retention and segregation/partition of oriP-containing plasmids. Here the chromosomal localization of EBNA-1 fused to green fluorescent protein (GFP–EBNA-1) is examined by confocal microscopy combined with a ‘premature chromosome condensation’ (PCC) procedure. Analyses show that GFP–EBNA-1 expressed in living cells that lack oriP plasmids is associated with cellular chromatin that has been condensed rapidly by the PCC procedure into identifiable forms that are unique to each phase of interphase as well as metaphase chromosomes. Studies of cellular chromosomal DNAs labelled with BrdU or Cy3-dUTP indicate that GFP–EBNA-1 colocalizes highly with the labelled, newly replicated regions of interphase chromatin in cells. These results suggest that EBNA-1 is associated not only with cellular metaphase chromosomes but also with condensing chromatin/chromosomes and probably with interphase chromatin, especially with its newly replicated regions.

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