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1.
EMBO Rep ; 2(10): 920-5, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11571267

ABSTRACT

We have recently shown that heterochromatin protein 1 (HP1) interacts with the nuclear envelope in an acetylation-dependent manner. Using purified components and in vitro assays, we now demonstrate that HP1 forms a quaternary complex with the inner nuclear membrane protein LBR and a sub-set of core histones. This complex involves histone H3/H4 oligomers, which mediate binding of LBR to HP1 and cross-link these two proteins that do not interact directly with each other. Consistent with previous observations, HP1 and LBR binding to core histones is strongly inhibited when H3/H4 are modified by recombinant CREB-binding protein, revealing a new mechanism for anchoring domains of under-acetylated chromatin to the inner nuclear membrane.


Subject(s)
Histones/metabolism , Acetylation , Animals , Binding Sites , Blotting, Western , Cell Nucleus/metabolism , Dose-Response Relationship, Drug , Electrophoresis, Polyacrylamide Gel , Fishes , Glutathione Transferase/metabolism , Heterochromatin/metabolism , Intracellular Membranes/metabolism , Mice , Models, Biological , Plasmids/metabolism , Precipitin Tests , Protein Binding , Protein Structure, Tertiary , Recombinant Fusion Proteins/metabolism , Recombinant Proteins/metabolism , Turkeys
2.
J Biol Chem ; 276(16): 13007-14, 2001 Apr 20.
Article in English | MEDLINE | ID: mdl-11278332

ABSTRACT

We have previously shown that the mouse heterochromatin protein 1 homologue M31 interacts dynamically with the nuclear envelope. Using quantitative in vitro assays, we now demonstrate that this interaction is potently inhibited by soluble factors present in mitotic and interphase cytosol. As indicated by depletion and order-of-addition experiments, the inhibitory activity co-isolates with a 55-kDa protein, which binds avidly to the nuclear envelope and presumably blocks M31-binding sites. Purification of this protein and microsequencing of tryptic peptides identify it as alpha2/6:beta2-tubulin. Consistent with this observation, bona fide tubulin, isolated from rat brain and maintained in a nonpolymerized state, abolishes binding of M31 to the nuclear envelope and aborts M31-mediated nuclear envelope reassembly in an in vitro system. These observations provide a new example of "moonlighting," a process whereby multimeric proteins switch function when their aggregation state or localization is altered.


Subject(s)
Chromosomal Proteins, Non-Histone/metabolism , Heterochromatin/metabolism , Nuclear Envelope/physiology , Tubulin/metabolism , Amino Acid Sequence , Animals , Brain/metabolism , Chromobox Protein Homolog 5 , Chromosomal Proteins, Non-Histone/chemistry , Endometrial Neoplasms , Female , HeLa Cells , Humans , Kinetics , Mice , Molecular Sequence Data , Molecular Weight , Nuclear Envelope/ultrastructure , Peptide Fragments/chemistry , Protein Binding , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Tumor Cells, Cultured
3.
EMBO J ; 19(23): 6558-68, 2000 Dec 01.
Article in English | MEDLINE | ID: mdl-11101528

ABSTRACT

To study the dynamics of mammalian HP1 proteins we have microinjected recombinant forms of mHP1alpha, M31 and M32 into the cytoplasm of living cells. As could be expected from previous studies, the three fusion proteins were efficiently transported into the nucleus and targeted specific chromatin areas. However, before incorporation into these areas the exogenous proteins accumulated in a peripheral zone and associated closely with the nuclear envelope. This transient association did not occur when the cells were treated with deacetylase inhibitors, indicating an acetylation-inhibited interaction. In line with these observations, recombinant HP1 proteins exhibited saturable binding to purified nuclear envelopes and stained the nuclei of detergent-permeabilized cells in a rim-like fashion. Competition experiments with various M31 mutants allowed mapping of the nuclear envelope-binding site within an N-terminal region that includes the chromodomain. A His(6)-tagged peptide representing this region inhibited recruitment of LAP2beta and B-type lamins around the surfaces of condensed chromosomes, suggesting involvement of HP1 proteins in nuclear envelope reassembly.


Subject(s)
Chromosomal Proteins, Non-Histone/metabolism , DNA-Binding Proteins , Nuclear Envelope/metabolism , Acetylation , Animals , Binding Sites , Binding, Competitive , CHO Cells , Cell Line , Cell Nucleus/metabolism , Chromatin/metabolism , Chromobox Protein Homolog 5 , Chromosomal Proteins, Non-Histone/chemistry , Chromosomal Proteins, Non-Histone/genetics , Chromosomes/metabolism , Cricetinae , Cytoplasm/metabolism , Detergents/pharmacology , Dose-Response Relationship, Drug , Electrophoresis, Polyacrylamide Gel , Fluorescent Antibody Technique, Indirect , Glutathione Transferase/metabolism , HeLa Cells , Humans , Immunoblotting , Kinetics , Lamins , Membrane Proteins/metabolism , Mice , Microinjections , Mitosis , Mutation , Nuclear Proteins/metabolism , Octoxynol/pharmacology , Protein Binding , Protein Transport , Recombinant Fusion Proteins/physiology
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