48 research outputs found
The History And Mystery Of Chromatin
UNE research professors Ada and Don Olins present some of their published and unpublished data on chromatin. The name “Chromatin” was given by W. Flemming (~1880) to the nuclear substance that stains strongly in a light microscope. During the 1940’s to 60’s, it became clear that chromatin is a complex of (anionic) double-stranded DNA with an equal mass of highly basic histone proteins. Following the success of fiber x-ray diffraction in deciphering helical macromolecular structures (e.g., the single peptide alpha-helix, DNA double helix, collagen triple helix, etc.), it became “reasonable” to postulate that chromatin is a helix of nucleohistone. Electron microscopic evidence appeared to support this concept. In 1973-74 the chromatin field witnessed a significant paradigm shift, when our electron micrographs and parallel biochemical data (from other labs) demonstrated that the chromatin polymer is a “string-of-beads”, which were named “nucleosomes”. The “core” nucleosome contains ~160 bp of DNA, wrapped around an inner spool of 8 histones (two each of histones H3, H4, H2A and H2B) related by a dyad axis. Following the discovery of nucleosomes, there was again a profusion of chromatin helical models (e.g., “30 nm fibers”). Unfortunately, there is very little support for such higher-order structures in vivo. Since the discovery of the nucleosome, we have focused on a number of questions related to in situ higher-order chromatin structure: What controls the shape of the interphase nucleus? What are the properties of the chromatin “surface”, adjacent to the nuclear envelope? What happens to chromatin during hyperosmotic dehydration of the live cell?https://dune.une.edu/pharmsci_facpres/1002/thumbnail.jp
Cytoskeletal influences on nuclear shape in granulocytic HL-60 cells
BACKGROUND: During granulopoiesis in the bone marrow, the nucleus differentiates from ovoid to lobulated shape. Addition of retinoic acid (RA) to leukemic HL-60 cells induces development of lobulated nuclei, furnishing a convenient model system for nuclear differentiation during granulopoiesis. Previous studies from our laboratory have implicated nuclear envelope composition as playing important roles in nuclear shape changes. Specifically noted were: 1) a paucity of lamins A/C and B1 in the undifferentiated and RA treated cell forms; 2) an elevation of lamin B receptor (LBR) during induced granulopoiesis. RESULTS: The present study demonstrates that perturbation of cytoskeletal elements influences nuclear differentiation of HL-60 cells. Because of cytotoxicity from prolonged exposure to cytoskeleton-modifying drugs, most studies were performed with a Bcl-2 overexpressing HL-60 subline. We have found that: 1) nocodazole prevents RA induction of lobulation; 2) taxol induces lobulation and micronuclear formation, even in the absence of RA; 3) cytochalasin D does not inhibit RA induced nuclear lobulation, and prolonged exposure induces nuclear shape changes in the absence of RA. CONCLUSIONS: The present results, in the context of earlier data and models, suggest a mechanism for granulocytic nuclear lobulation. Our current hypothesis is that the nuclear shape change involves factors that increase the flexibility of the nuclear envelope (reduced lamin content), augment connections to the underlying heterochromatin (increased levels of LBR) and promote distortions imposed by the cytoskeleton (microtubule motors creating tension in the nuclear envelope)
Chromatin Structure In Situ: The Contribution Of DNA Ultrastructural Cytochemistry
Ultrastructural studies conducted in situ using conventional transmission electron microscopy have had relatively little impact on defining the structural organization of chromatin. This is due to the fact that in routine transmission electron microscopy, together with the deoxyribonucleoprotein, many different intermingled substances are contrasted, masking the ultrastructure of chromatin. By selective staining of DNA in thin sections, using the Feulgen-like osmium-ammine reaction, these drawbacks have been overcome and worthwhile data have been obtained both on the gross morphology and the ultrastructural-functional organization of chromatin in situ. In the present study these results are reviewed and discussed in light of recent achievements in both interphase nuclear chromatin compartmentalization in interphase nuclei and in the structural organization of chromatin fibers in transcriptionally active and inactive chromatin
ELCS In Ice: Cryo-electron Microscopy Of Nuclear Envelope Limited Chromatin Sheets
Nuclear Envelope-Limited Chromatin Sheets (ELCS) form during excessive interphase nuclear envelope growth in a variety of cells. ELCS appear as extended sheets within the cytoplasm connecting distant nuclear lobes. Cross-section stained images of ELCS, viewed by transmission electron microscopy, resemble a sandwich of apposed nuclear envelopes separated by ~30 nm, containing a layer of ordered chromatin fibers. EM Procedures: The ultrastructure of ELCS was compared by three different methods: 1) aldehyde fixation/dehydration/plastic embedding/sectioning and staining; 2) high-pressure freezing/freeze substitution into plastic/sectioning and staining; 3) high-pressure freezing/cryo-sectioning/cryo-electron microscopy. Human leukemic (HL-60/S4) cells were treated with retinoic acid (4 days) to induce granulopoiesis, growth of nuclear envelope membranes, formation of lobulated nuclei and ELCS. Conclusions: ELCS exist in vivo; they are not an artifact of fixation. ELCS in ice are thicker than after dehydration and embedding in plastic. EM tomography of aldehyde fixed cells supports that the putative “30 nm fiber” in ELCS are composed of two overlapping layers of parallel “10 nm fibers”.https://dune.une.edu/pharmsci_facpost/1005/thumbnail.jp
Retrotransposon Alu Is Enriched In The Epichromatin Of HL-60 Cells
Epichromatin, the surface of chromatin facing the nuclear envelope in an interphase nucleus, reveals a “rim” staining pattern with specific mouse monoclonal antibodies against histone H2A/H2B/DNA and phosphatidylserine epitopes. Employing a modified ChIP-Seq procedure on undifferentiated and differentiated human leukemic (HL-60/S4) cells, \u3e95% of assembled epichromatin regions overlapped with Alu retrotransposons. They also exhibited enrichment of the AluS subfamily and of Alu oligomers. Furthermore, mapping epichromatin regions to the human chromosomes revealed highly similar localization patterns in the various cell states and with the different antibodies. Comparisons with available epigenetic databases suggested that epichromatin is neither “classical” heterochromatin nor highly expressing genes, implying another function at the surface of interphase chromatin. A modified chromatin immunoprecipitation procedure (xxChIP) was developed because the studied antibodies react generally with mononucleosomes and lysed chromatin. A second fixation is necessary to securely attach the antibodies to the epichromatin epitopes of the intact nucleus
Phosphatidylserine colocalizes with epichromatin in interphase nuclei and mitotic chromosomes
Cycling eukaryotic cells rapidly re-establish the nuclear envelope and internal architecture following mitosis. Studies with a specific anti-nucleosome antibody recently demonstrated that the surface (“epichromatin”) of interphase and mitotic chromatin possesses a unique and conserved conformation, suggesting a role in postmitotic nuclear reformation. Here we present evidence showing that the anionic glycerophospholipid phosphatidylserine is specifically located in epichromatin throughout the cell cycle and is associated with nucleosome core histones. This suggests that chromatin bound phosphatidylserine may function as a nucleation site for the binding of ER and re-establishment of the nuclear envelope
Transcriptomes reflect the phenotypes of undifferentiated, granulocyte and macrophage forms of HL-60/S4 cells
Author Posting. © The Author(s), 2017. This is the author's version of the work. It is posted here by permission of Taylor & Francis for personal use, not for redistribution. The definitive version was published in Nucleus 8 (2017): 222-237, doi:10.1080/19491034.2017.1285989.In order to understand the chromatin changes underlying differential gene expression during
induced differentiation of human leukemic HL-60/S4 cells, we conducted RNA-Seq analysis
on quadruplicate cultures of undifferentiated, granulocytic- and macrophage-differentiated
cell forms. More than half of mapped genes exhibited altered transcript levels in the
differentiated cell forms. In general, more genes showed increased mRNA levels in the
granulocytic form and in the macrophage form, than showed decreased levels. The majority
of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were significantly enriched in genes that exhibited differential transcript levels after either RA or TPA
treatment. Changes in transcript levels for groups of genes with characteristic protein
phenotypes, such as genes encoding cytoplasmic granular proteins, nuclear envelope and
cytoskeletal proteins, cell adhesion proteins, and proteins involved in the cell cycle and
apoptosis illustrate the profound differences among the various cell states. In addition to the
transcriptome analyses, companion karyotyping by M-FISH of undifferentiated HL-60/S4
cells revealed a plethora of chromosome alterations, compared to normal human cells. The
present mRNA profiling provides important information related to nuclear shape changes
(e.g., granulocyte lobulation), deformability of the nuclear envelope and linkage between the
nuclear envelope and cytoskeleton during induced myeloid chromatin differentiation.DMW
thanks the Bay and Paul Foundations for support. ALO and DEO thank the College of
Pharmacy at UNE for their support. ALO and DEO are recipients of a 2015 UNE Mini-
Grant from the Vice President for Research and Scholarship. ALO and DEO thank the
German Cancer Research Center (Heidelberg) for the awards of Guest Scientist fellowships.2018-02-0