Histone Acetyltransferase hALP and Nuclear Membrane Protein hsSUN1 Function in De-condensation of Mitotic Chromosomes
2007; Elsevier BV; Volume: 282; Issue: 37 Linguagem: Inglês
10.1074/jbc.m703098200
ISSN1083-351X
AutoresYa‐Hui Chi, Kerstin Haller, Jean‐Marie Péloponèse, Kuan‐Teh Jeang,
Tópico(s)RNA Interference and Gene Delivery
ResumoReplicated mammalian chromosomes condense to segregate during anaphase, and they de-condense at the conclusion of mitosis. Currently, it is not understood what the factors and events are that specify de-condensation. Here, we demonstrate that chromosome de-condensation needs the function of an inner nuclear membrane (INM) protein hsSUN1 and a membrane-associated histone acetyltransferase (HAT), hALP. We propose that nascently reforming nuclear envelope employs hsSUN1 and hALP to acetylate histones for de-compacting DNA at the end of mitosis. Replicated mammalian chromosomes condense to segregate during anaphase, and they de-condense at the conclusion of mitosis. Currently, it is not understood what the factors and events are that specify de-condensation. Here, we demonstrate that chromosome de-condensation needs the function of an inner nuclear membrane (INM) protein hsSUN1 and a membrane-associated histone acetyltransferase (HAT), hALP. We propose that nascently reforming nuclear envelope employs hsSUN1 and hALP to acetylate histones for de-compacting DNA at the end of mitosis. The eukaryotic nucleus is separated from other organelles by an envelope containing two membrane layers continuous with the endoplasmic reticulum. Nuclear membrane proteins fall into three categories according to their localization. The first group is the trans-nuclear membrane proteins resident in the nuclear pore complex (NPC). 2The abbreviations used are:NPCnuclear pore complexSUNSad1-UNC84ChIPchromatin immunoprecipitationINMinner nuclear membraneH3pSer10phosphorylated histone H3 at serine 10hALPhuman acetyl-transferase-like proteinLAPlamin-associated polypeptideLBRlamin B receptorHAThistone acetyltransferaseHDAChistone deacetylaseORFopen reading frameRIPAradioimmune precipitation assay bufferPBSphosphate-buffered salineGSTglutathione S-transferaseHAhemagglutininDAPI4′,6-diamidino-2-phenylindoleGFPgreen fluorescent proteinWTwild type 2The abbreviations used are:NPCnuclear pore complexSUNSad1-UNC84ChIPchromatin immunoprecipitationINMinner nuclear membraneH3pSer10phosphorylated histone H3 at serine 10hALPhuman acetyl-transferase-like proteinLAPlamin-associated polypeptideLBRlamin B receptorHAThistone acetyltransferaseHDAChistone deacetylaseORFopen reading frameRIPAradioimmune precipitation assay bufferPBSphosphate-buffered salineGSTglutathione S-transferaseHAhemagglutininDAPI4′,6-diamidino-2-phenylindoleGFPgreen fluorescent proteinWTwild type The second group contains the inner membrane proteins (INM), which include the lamin B receptor (LBR), emerin, and lamin-associated polypeptides (LAPs). The third group includes proteins underlying the nuclear membrane such as nuclear lamina (1Taddei A. Hediger F. Neumann F.R. Gasser S.M. Annu. Rev. Genet. 2004; 38: 305-345Crossref PubMed Scopus (179) Google Scholar). Functionally, the INM provides a physical barrier; the NPC serves for the transport of material between the nucleus and the cytoplasm (2Fahrenkrog B. Koser J. Aebi U. Trends Biochem. Sci. 2004; 29: 175-182Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar); and the nuclear lamina erects a meshwork, which maintains nuclear structure and assists indirectly in DNA replication and RNA processing (3Hutchison C.J. Nat. Rev. Mol. Cell. Biol. 2002; 3: 848-858Crossref PubMed Scopus (247) Google Scholar, 4Mattout-Drubezki A. Gruenbaum Y. Cell Mol. Life Sci. 2003; 60: 2053-2063Crossref PubMed Scopus (113) Google Scholar).Most INM proteins are associated with the nuclear lamina. In a proteomic study of INM proteins, in addition to 13 known proteins, 67 uncharacterized open reading frames (ORFs) were identified (5Schirmer E.C. Florens L. Guan T. Yates J.R. II I Gerace L. Science. 2003; 301: 1380-1382Crossref PubMed Scopus (517) Google Scholar). 23 of these ORFs map to chromosome regions linked to a variety of dystrophies collectively termed “nuclear envelopathies” (5Schirmer E.C. Florens L. Guan T. Yates J.R. II I Gerace L. Science. 2003; 301: 1380-1382Crossref PubMed Scopus (517) Google Scholar). These diseases have phenotypes ranging from cardiac and skeletal myopathies, lipodystrophy, peripheral neuropathy, and premature aging (6Burke B. Nat. Cell Biol. 2001; 3: E273-E274Crossref PubMed Scopus (25) Google Scholar, 7Burke B. Mounkes L.C. Stewart C.L. Traffic. 2001; 2: 675-683Crossref PubMed Scopus (32) Google Scholar, 8Burke B. Stewart C.L. Nat. Rev. Mol. Cell. Biol. 2002; 3: 575-585Crossref PubMed Scopus (351) Google Scholar, 9Worman H.J. Courvalin J.C. J. Clin. Investig. 2004; 113: 349-351Crossref PubMed Scopus (118) Google Scholar). Genetic studies have associated mutations in emerin, lamin A/C, and lamin B receptor with such pathologies (7Burke B. Mounkes L.C. Stewart C.L. Traffic. 2001; 2: 675-683Crossref PubMed Scopus (32) Google Scholar, 9Worman H.J. Courvalin J.C. J. Clin. Investig. 2004; 113: 349-351Crossref PubMed Scopus (118) Google Scholar). An emerging notion is that the INM proteins are needed to maintain nuclear integrity and guard against mechanical stress (10Holaska J.M. Wilson K.L. Mansharamani M. Curr. Opin. Cell Biol. 2002; 14: 357-364Crossref PubMed Scopus (107) Google Scholar, 11Holmer L. Worman H.J. Cell Mol. Life Sci. 2001; 58: 1741-1747Crossref PubMed Scopus (131) Google Scholar, 12Shumaker D.K. Kuczmarski E.R. Goldman R.D. Curr. Opin. Cell Biol. 2003; 15: 358-366Crossref PubMed Scopus (171) Google Scholar). Plausibly, then, tissues that experience high mechanical stress may have increased sensitivity to the consequence of mutated INM proteins. Nonetheless, a fuller understanding of how abnormalities in nuclear membrane contribute to pathogenesis remains to be elucidated.Some INM proteins have a Sad1-UNC84 (SUN) domain at their C termini (13Starr D.A. Han M. J. Cell Sci. 2003; 116: 211-216Crossref PubMed Scopus (197) Google Scholar). The SUN domain was first identified based on the sequence alignment of Sad1 of Schizosaccharomycespombe and UNC-84 of Caenorhabditis elegans (14Malone C.J. Fixsen W.D. Horvitz H.R. Han M. Development. 1999; 126: 3171-3181Crossref PubMed Google Scholar). All SUN proteins contain putative transmembrane regions, suggesting that they localize to membranes at some periods during the cell cycle. Curiously, steady state S. pombe Sad1 predominates at spindle pole bodies and has been inferred to function in the formation of the mitotic spindle (15Hagan I. Yanagida M. J. Cell Biol. 1995; 129: 1033-1047Crossref PubMed Scopus (342) Google Scholar); on the other hand, UNC-84 localizes in the C. elegans nuclear envelope (16Lee K.K. Starr D. Cohen M. Liu J. Han M. Wilson K.L. Gruenbaum Y. Mol. Biol. Cell. 2002; 13: 892-901Crossref PubMed Scopus (144) Google Scholar). Mammals have four SUN proteins, SUN1 (also called UNC84A), SUN2 (also called UN84B), a sperm-associated antigen 4-like (SPAG4) protein, and a hypothetical protein, MGC33329. To date, other than a described ability to bind nesprin-2 (17Crisp M. Liu Q. Roux K. Rattner J.B. Shanahan C. Burke B. Stahl P.D. Hodzic D. J. Cell Biol. 2006; 172: 41-53Crossref PubMed Scopus (904) Google Scholar, 18Padmakumar V.C. Libotte T. Lu W. Zaim H. Abraham S. Noegel A.A. Gotzmann J. Foisner R. Karakesisoglou I. J. Cell Sci. 2005; 118: 3419-3430Crossref PubMed Scopus (323) Google Scholar), little else is known about the function of mammalian SUN proteins (18Padmakumar V.C. Libotte T. Lu W. Zaim H. Abraham S. Noegel A.A. Gotzmann J. Foisner R. Karakesisoglou I. J. Cell Sci. 2005; 118: 3419-3430Crossref PubMed Scopus (323) Google Scholar, 19Dreger M. Bengtsson L. Schoneberg T. Otto H. Hucho F. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11943-11948Crossref PubMed Scopus (210) Google Scholar, 20Hasan S. Guttinger S. Muhlhausser P. Anderegg F. Burgler S. Kutay U. FEBS Lett. 2006; 580: 1263-1268Crossref PubMed Scopus (59) Google Scholar, 21Hodzic D.M. Yeater D.B. Bengtsson L. Otto H. Stahl P.D. J. Biol. Chem. 2004; 279: 25805-25812Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar).Because the timing of nuclear membrane reformation at the end of mitosis appears to be linked to chromosome de-condensation, we have characterized here the mitotic role for hsSUN1. We find that hsSUN1 is one of the earliest INM factors to associate with segregated daughter chromosomes in anaphase. Knockdown of hsSUN1 leads to hypoacetylated histones and delayed de-condensation of chromosomes at the end of mitosis. A HAT protein, hALP, previously reported to be associated with mammalian inner nuclear membrane (5Schirmer E.C. Florens L. Guan T. Yates J.R. II I Gerace L. Science. 2003; 301: 1380-1382Crossref PubMed Scopus (517) Google Scholar), was found to bind hsSUN1 and to be required for proper mitotic chromosome de-condensation. Our findings broach a mechanism used by nascently enveloped daughter nuclei to de-compact chromosomes, preparing them for gene expression in the impending interphase.EXPERIMENTAL PROCEDURESPlasmid Construction—HsSUN1 (KIAA0810) and hALP cDNA (KIAA1709) were from the Kazusa DNA Research Institute (22Kikuno R. Nagase T. Waki M. Ohara O. Nucleic Acids Res. 2002; 30: 166-168Crossref PubMed Scopus (85) Google Scholar). Full-length hsSUN1 (amino acids 1-785) was amplified from the KIAA0810 (hk05647s1) clone using PCR and ligated into pCDNA3.1+ vector (Invitrogen). HA-tagged full-length hsSUN1 and N- and C-terminal deletion mutants (amino acids 1-581, 1-479, 1-377, 1-238, 40-173, 103-785, 205-785, 307-785, 501-785) were constructed by amplifying the indicated sequences by PCR and cloning into pCDNA3.1+ vector. Full-length hALP (amino acids 1-1025, clone fj18302) was amplified by PCR and tagged with FLAG for detection purposes.Anti-hsSUN1 Antibody Preparation—HsSUN1 amino acids 362-785 were expressed in the pGEX5x-2 vector (Amersham Biosciences). Recombinant GST-fused hsSUN1-(362-785) protein was used for rabbit immunization (Spring Valley Laboratories). Rabbit hsSUN1 antiserum (αhsSUN1-C) was first captured with protein A-agarose (Bio-Rad), and then affinity-purified using GST-hsSUN1-(362-785) fusion protein conjugated to Affi-Gel15 (Bio-Rad).Western Blotting—HeLa cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and supplemented with 2 mml-glutamine and antibiotics. Cells were washed twice with phosphate-buffered saline (PBS), scraped from the culture plate, pelleted, and lysed with RIPA buffer (50 mm HEPES, pH 7.3, 150 mm NaCl, 2 mm EDTA, 20 mm β-glycerophosphate, 0.1 mm Na3VO4, 1 mm NaF, 0.5 mm dithiothreitol, and protease inhibitor mixture (Roche Applied Sciences)) containing 1% SDS. For the peptide competition assay, total cell lysates were analyzed by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene fluoride membrane, and subjected to immunoblotting. Affinity-purified anti-hsSUN1 was either preincubated with GST-agarose, GST-agarose plus 100 μl of purified GST (400 μg/ml), or GST-agarose plus GST-hsSUN1-(362-785) fusion protein (300 μg/ml, 50 and 200 μl, respectively). Antibodies after incubation with/without GST-agarose plus GST or GST-hsSUN1 fusion proteins were then added to polyvinylidene difluoride membranes blocked with 0.2% I-Block (Tropix) in PBS and 0.1% Tween-20 (Bio-Rad). Alkaline phosphatase-conjugated anti-rabbit secondary antibody was added, and the blots were developed by chemiluminescence following the manufacturer's protocol (Tropix).Chromatin Association Assay—The chromatin association assay was performed by modifying the chromatin immunoprecipitation (ChIP) protocol described by Upstate. Briefly, cells were transfected with plasmids using Lipofectamine (Invitrogen). 24 h later, cells were cross-linked by adding 1% formaldehyde to the medium and incubated for 10 min at room temperature. The cross-linking reaction was quenched by addition of 0.125 m glycine and incubation at room temperature for another 10 min. Cells were washed with cold PBS, scraped, and pelleted by centrifugation. To extract soluble chromatin and its associated proteins, cells were lysed in SDS-lysis buffer (1% SDS, 50 mm Tris-HCl, pH 8.0, 10 mm EDTA, and protease inhibitor mixture) and sonicated for 5 times for 10-s pulses (Branson, Sonifier, Model 450) and incubated on ice inbetween. Lysates were centrifuged at 12,000 × g at 4 °C for 10 min. Soluble fractions of cell lysates were diluted 50 times in RIPA buffer (as described in Western blotting) and incubated with monoclonal anti-HA or anti-FLAG agarose (Sigma-Aldrich) for 16 h at 4 °C. The agarose beads were washed five times with RIPA buffer. Before analyzing the samples with SDS-PAGE, samples were boiled in one volume of 2× Laemmli loading buffer (2% SDS, 20% glycerol, 120 mm Tris-HCl, pH 6.8, 200 mm dithiothreitol, bromphenol blue) for 30 min to reverse the cross-linking.Immunofluorescence and Confocal Microscopy—Cells were fixed in 4% paraformaldehyde for 20 min at room temperature and permeabilized with 0.1% Triton X-100 in PBS for 5 min at room temperature. To block nonspecific binding, cells were incubated with 1% bovine serum albumin in PBS for 30 min. Antibodies against hsSUN1, emerin (Santa Cruz Biotechnology), lamin B (Santa Cruz Biotechnology), nuclear pore complex (mab414, Covance), α-tubulin (Sigma-Aldrich), CENP-A (MBL) and anti-LAP2 (Sigma-Aldrich), anti-LBR (Epitomics) were added to cells at dilutions of 1:200 to 1:2000 and incubated for 1 h at room temperature. Cells were washed three times with PBS and then probed with fluorescent (Alexa-488, Alexa-594, or Alexa-647)-conjugated secondary antibodies. Cell nuclei were stained with DAPI (Molecular Probes). Cells on the coverslips were mounted on glass slides with antifade reagents (Molecular Probes). Slides were monitored using a Leica TCS-NP/SP confocal microscope. For time-lapse confocal microscopy, live cells were incubated at 37 °C in a humidified Pe-Con environmental chamber supplied with 5% CO2.RNAi—Synthetic siRNA duplexes targeting hsSUN1 (5′-CCAUCCUGAGUAUACCUGUCUGUAU-3′) and hALP (5′-CGGCCUUCAGUGCUGUGGUGUUAUA-3′) were from Invitrogen. HeLa cells were transfected with hsSUN1 RNAi using TransMessenger transfection reagent (Qiagen). A GFP-expressing plasmid (Clontech) was co-transfected with hsSUN1 RNAi to monitor transfection efficiency. We also employed unrelated siRNAs with the same GC content as controls. 24-72 h after transfection, cells were analyzed by Western blotting or confocal microscopy.RESULTSDeterminants of hsSUN1 Localization to the Nuclear Envelope—HsSUN1 was initially described to contain 824 amino acids (13Starr D.A. Han M. J. Cell Sci. 2003; 116: 211-216Crossref PubMed Scopus (197) Google Scholar). However, various lengths (ranging from 909 to 974 amino acids) for SUN1 (UNC84A) have been suggested (17Crisp M. Liu Q. Roux K. Rattner J.B. Shanahan C. Burke B. Stahl P.D. Hodzic D. J. Cell Biol. 2006; 172: 41-53Crossref PubMed Scopus (904) Google Scholar, 18Padmakumar V.C. Libotte T. Lu W. Zaim H. Abraham S. Noegel A.A. Gotzmann J. Foisner R. Karakesisoglou I. J. Cell Sci. 2005; 118: 3419-3430Crossref PubMed Scopus (323) Google Scholar, 20Hasan S. Guttinger S. Muhlhausser P. Anderegg F. Burgler S. Kutay U. FEBS Lett. 2006; 580: 1263-1268Crossref PubMed Scopus (59) Google Scholar, 21Hodzic D.M. Yeater D.B. Bengtsson L. Otto H. Stahl P.D. J. Biol. Chem. 2004; 279: 25805-25812Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 23Haque F. Lloyd D.J. Smallwood D.T. Dent C.L. Shanahan C.M. Fry A.M. Trembath R.C. Shackleton S. Mol. Cell. Biol. 2006; 26: 3738-3751Crossref PubMed Scopus (367) Google Scholar, 24Wang Q. Du X. Cai Z. Greene M.I. DNA Cell Biol. 2006; 25: 554-562Crossref PubMed Scopus (48) Google Scholar). Updated sequencings (KIAA0810, clone hk05647s1) from Kazusa DNA Research Institute and from our own cloning of cDNAs (data not shown) from several human cells (Jurkat, HeLa, and primary human lymphoblastoid cells) reveal that hsSUN1 is 785 amino acids in length. At its C terminus, hsSUN1 conserves with C. elegans UNC-84 a 190-amino acid SUN domain (Fig. 1A). In silico analyses indicate that hsSUN1 has three transmembrane regions located at amino acids 239-256, 263-282, and 289-306; and two coiled-coils at positions 377-402 and 428-466 (Fig. 1A).Evidence supports that hsSUN1 is an INM protein. However, published reports do not agree on which hsSUN1 domain specifies nuclear location (17Crisp M. Liu Q. Roux K. Rattner J.B. Shanahan C. Burke B. Stahl P.D. Hodzic D. J. Cell Biol. 2006; 172: 41-53Crossref PubMed Scopus (904) Google Scholar, 18Padmakumar V.C. Libotte T. Lu W. Zaim H. Abraham S. Noegel A.A. Gotzmann J. Foisner R. Karakesisoglou I. J. Cell Sci. 2005; 118: 3419-3430Crossref PubMed Scopus (323) Google Scholar, 20Hasan S. Guttinger S. Muhlhausser P. Anderegg F. Burgler S. Kutay U. FEBS Lett. 2006; 580: 1263-1268Crossref PubMed Scopus (59) Google Scholar, 21Hodzic D.M. Yeater D.B. Bengtsson L. Otto H. Stahl P.D. J. Biol. Chem. 2004; 279: 25805-25812Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 23Haque F. Lloyd D.J. Smallwood D.T. Dent C.L. Shanahan C.M. Fry A.M. Trembath R.C. Shackleton S. Mol. Cell. Biol. 2006; 26: 3738-3751Crossref PubMed Scopus (367) Google Scholar, 24Wang Q. Du X. Cai Z. Greene M.I. DNA Cell Biol. 2006; 25: 554-562Crossref PubMed Scopus (48) Google Scholar). To clarify structure-function relationship, we constructed several hsSUN1 deletion mutants (Fig. 1B) and expressed each in HeLa cells. We observed that hsSUN1 despite removal of amino acids 480-785 (Fig. 1C, panels 1-3, see WT, ΔC1, and ΔC2 proteins) still retained a nuclear envelope pattern indicating that hsSUN1 C terminus, including its SUN domain, is dispensable for nuclear membrane localization. When we deleted into hsSUN1 coiled-coils, as in hsSUN1 ΔC3, ∼10% of the protein partitioned from the nuclear envelope into the cytoplasm (Fig. 1C, panel 4). Further removal of all three transmembrane regions (amino acids 1-238, ΔC4; Fig. 1C, panel 5) dispersed increased amounts of hsSUN1.The above analyses were complemented with deletions starting from the N terminus. Removing the first 102 N-terminal amino acids from hsSUN1 (amino acids 103-785 ΔN1, Fig. 1C, panel 6) shifted more than 60% of the protein from the envelope into the ER. Removing the next 102 amino acids (amino acids 205-785 ΔN2, Fig. 1C, panel 7) did not cause further changes. However, when the deletion was extended to amino acid 306, hsSUN1 ΔN3 (amino acids 307-785) became wholly cytoplasmic (Fig. 1C, panel 8). Collectively, the results show hsSUN1 three putative transmembrane motifs and its first 102 N-terminal amino acids are needed for retention in the nuclear envelope.HsSUN1 Nucleates Daughter Nuclear Envelope Formation—Because antibodies are not available, we generated and affinity-purified rabbit antisera (αhsSUN1-C) to hsSUN1 C-terminal 362-785 amino acids (Fig. 2A). Using αhsSUN1-C, we first studied the distribution of cell endogenous hsSUN1. Interphase hsSUN1 stained with lamin B1 around the nucleus (Fig. 2B). In early mitosis even as the envelope commences breakdown, hsSUN1, along with lamin B1 and emerin, is found at the nuclear membrane, (Fig. 2C, panels 1-6). During this period, hsSUN1 and the nuclear pore complex (NPC, detected with mab414, which recognizes the conserved FXFG repeats in nucleoporins) are partially overlapping (Fig. 2C, panels 7-9). As the cell moves into metaphase, hsSUN1, lamin B1, and emerin disperse into the mitotic cytosol (Fig. 2D, panels 1-6) while NPC-staining with mab414 is extinguished (Fig. 2D, panels 7-9). By anaphase, hsSUN1 reorganizes around nascently separated daughter DNAs (Fig. 2E) at the peripheral edges of condensed chromosomes (see Fig. 2F; compare the locations of hsSUN1 and CENP-A). We note that as hsSUN1 reforms structurally from metaphase to anaphase NPC-staining follows co-incidentally (Fig. 2E, panels 1-3). By contrast, re-organization of anaphase lamin B1 (Fig. 2E, panels 4-6) lags initially (compare NPC and lamin B1 staining relative to α-tubulin-staining; Fig. 2E, panels 2, 5, and 8); but by telophase, lamin B1 too converges with hsSUN1 at newly reformed daughter nuclear envelopes (Fig. 2G). While other interpretations are possible, these sequential views suggest that hsSUN1 leads NPC and lamin B1 in nucleating daughter envelopes.FIGURE 2Cell cycle localization of hsSUN1. A, characterization of the specificity of affinity-purified αhsSUN1-C in Western blotting experiments using competition with either an excess of GST (lane 2) or GST-hsSUN1-(362-785) proteins (lanes 3 and 4). B-G, fixed HeLa cells were immunostained with αhsSUN1-C (green) antibody and antibodies, as indicated, to lamin B1, emerin, NPC (mab414),α-tubulin, or CENP-A. Interphase B, prophase C, metaphase D, anaphase E and F, and telophase G cells are shown. DNA was stained with DAPI (blue). F, two views (panels 1 and 2) of anaphase cells stained with hsSUN1 (green) and CENP-A (red). Panel 2 shows an enlarged view of hsSUN1 at the edge of segregated chromosomes in anaphase. Arrows in E point to hsSUN1 at the lateral margins of anaphase chromosomes.View Large Image Figure ViewerDownload Hi-res image Download (PPT)HsSUN1 Congresses to Newly Segregated Chromosomes before LAP—Above, anaphase hsSUN1 precedes lamin B1 in reorganizing around newly segregated chromosomes (Fig. 2E). Previously, LAP2 and LBR were reported as INM proteins (25Chaudhary N. Courvalin J.C. J. Cell Biol. 1993; 122: 295-306Crossref PubMed Scopus (200) Google Scholar, 26Haraguchi T. Koujin T. Hayakawa T. Kaneda T. Tsutsumi C. Imamoto N. Akazawa C. Sukegawa J. Yoneda Y. Hiraoka Y. J. Cell Sci. 2000; 113: 779-794Crossref PubMed Google Scholar, 27Dechat T. Gajewski A. Korbei B. Gerlich D. Daigle N. Haraguchi T. Furukawa K. Ellenberg J. Foisner R. J. Cell Sci. 2004; 117: 6117-6128Crossref PubMed Scopus (149) Google Scholar) with congruent timing in their association with partitioning mitotic chromosomes (25Chaudhary N. Courvalin J.C. J. Cell Biol. 1993; 122: 295-306Crossref PubMed Scopus (200) Google Scholar, 28Buendia B. Courvalin J.C. Exp. Cell Res. 1997; 230: 133-144Crossref PubMed Scopus (77) Google Scholar, 29Ellenberg J. Siggia E.D. Moreira J.E. Smith C.L. Presley J.F. Worman H.J. Lippincott-Schwartz J. J. Cell Biol. 1997; 138: 1193-1206Crossref PubMed Scopus (625) Google Scholar, 30Yang L. Guan T. Gerace L. J. Cell Biol. 1997; 137: 1199-1210Crossref PubMed Scopus (194) Google Scholar). We next queried whether the hsSUN1 association with segregated chromatids (Fig. 2E) occurs prior to or after LAP2/LBR.To assess the relative ordering of hsSUN1 and LAP2, we immunostained simultaneously cell-endogenous hsSUN1 and LAP2. HsSUN1 and LAP2 are together in interphase (Fig. 3A, panels 1-3). By metaphase, both hsSUN1 and LAP2 become dispersed (Fig. 3A, panels 4-6). In early anaphase, hsSUN1 reorganizes at the peripheral edges of chromosomes (Fig. 3A, panels 7-9) with LAP2 following later to chromosome proximal locales (Fig. 3A, panels 10-15 and Ref. (27Dechat T. Gajewski A. Korbei B. Gerlich D. Daigle N. Haraguchi T. Furukawa K. Ellenberg J. Foisner R. J. Cell Sci. 2004; 117: 6117-6128Crossref PubMed Scopus (149) Google Scholar)). These comparisons place hsSUN1 interaction with newly segregated chromosomes before LAP2.FIGURE 3HsSUN1 binds chromatin prior to LAP2. A, localization of hsSUN1 (green) and LAP2 (red) were compared in interphase (panels 1-3), metaphase (panels 4-6), anaphase (panels 7-12), and telophase (panels 13-15) cells. HsSUN1 appears first at the peripheral rim of separated sister chromatids, as denoted by arrows with LAP2 appearing later (indicated by arrowheads). DNA was stained with DAPI (blue). Bar, 10 μm. B, alignment of N-terminal amino acids of human and mouse SUN1. Identities are shaded in gray; conserved basic amino acids are shaded in black. The basic domains of human and mouse SUN1 are underlined. C, chromatin binding assay was performed using HeLa cells expressing transfected full-length HA-hsSUN1-WT (lane 2), HA-hsSUN1-BD (lane 4), or HA-hsSUN1-SUN (lane 5), or FLAG-BAF (lanes 7). Lanes 1, 3, and 4 are mock-transfected samples. Cell lysates were immunoprecipitated with monoclonal anti-HA- (lanes 1-5) or monoclonal anti-FLAG- (lanes 6 and 7) agarose beads. Histone H2B co-immunoprecipitated by HA-hsSUN1-BD or FLAG-BAF was detected by immunoblotting.View Large Image Figure ViewerDownload Hi-res image Download (PPT)A current notion is that LAP2/LBR forms a scaffold onto which other NE proteins coalesce to assemble a new nuclear envelope. LAP2 and LBR contain basic amino acid chromatin-binding domains (31Ulbert S. Platani M. Boue S. Mattaj I.W. J. Cell Biol. 2006; 173: 469-476Crossref PubMed Scopus (84) Google Scholar). Because SUN1 associates with segregated chromosomes before LAP2, we wondered if SUN1 also has a chromatin-binding domain. We compared human and mouse SUN1 sequences and noted that both conserved a basic N-terminal amino acid region (hsSUN1 amino acids 40-109; musSUN1 amino acids 40-111; both pIs are 11.5, Fig. 3B). To check if this N-terminal fragment can bind chromatin, we overexpressed HA-tagged wild type full-length hsSUN1 (hsSUN1-WT) and the hsSUN1 basic domain (hsSUN1-BD, amino acids 40-173, Fig. 3C) and performed a modified chromatin precipitation assay (as described under “Experimental Procedures”). As a positive control, the known chromatin-binding protein, BAF (barrier-to-autointegration factor), was used in a parallel assay (Fig. 3C, lane 7). Indeed, full-length hsSUN1-WT and hsSUN1-BD co-precipitated histone H2B (Fig. 3C, lanes 2 and 4) like BAF (Fig. 3C, lane 7); by contrast, a protein containing only the hsSUN1 SUN domain (hsSUN1-SUN, amino acids 501-785) did not (Fig. 3C, lane 5). These results identify a chromatin-association domain in the N terminus of hsSUN1.Cells Depleted for hsSUN1 Have Defective Nuclear Envelope—Next, using RNAi-mediated depletion, we characterized the requirement for hsSUN1 in nuclear envelope integrity (Fig. 4A). HsSUN1-siRNA or control-siRNAi was introduced separately into cells with a nuclear-targeted green fluorescent protein (GFP). Green cells from hsSUN1-siRNA or control-siRNA transfections were compared, and hsSUN1 protein was found to be depleted from the former but not the latter (Fig. 4B, compare panel 7 to panel 2). Interestingly, whereas nuclear-targeted GFP was wholly circumscribed in the nucleus in control-RNAi cells (compare GFP to DAPI, Fig. 4B, panels 1 and 3), the GFP protein showed a whole-cell distribution in hsSUN1-RNAi cells (Fig. 4B, panels 6 and 8). This latter profile suggests a nuclear envelope defect in hsSUN1-depleted cells, which fail to retain nuclear-targeted GFP.FIGURE 4Depletion of hsSUN1 impairs nuclear envelope integrity and NPC formation. A, immunoblotting of lysates from cells treated with 0, 1, or 4 μg (lanes 1-3) of hsSUN1 siRNA and probed with αhsSUN1-C. Actin was used as a loading control. B, HeLa cells were co-transfected with a GFP-expressing plasmid and control- (panels 1-5) or hsSUN1- (panels 6-10) siRNA and immunostained with αhsSUN1-C. GFP is green; hsSUN1 is red; and DNA (DAPI) is blue. GFP-expressing cells (arrows) indicate cells transfected with siRNA. Bar, 10 μm. C, control- (panels 1-3) or hsSUN1- (panels 4-6) siRNA transfected cells were stained using αhsSUN1-C (green, panels 1 and 4) and mab414 (NPC, red, panels 2 and 5). DAPI (blue) was used to visualize DNA. Bar, 10 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To independently check nuclear envelope integrity, we stained for NPC. In control cells, NPC staining was seen appropriately in anaphase (Fig. 4C, panels 1-3; Refs. 32Hetzer M.W. Walther T.C. Mattaj I.W. Annu. Rev. Cell Dev. Biol. 2005; 21: 347-380Crossref PubMed Scopus (247) Google Scholar, 33Margalit A. Vlcek S. Gruenbaum Y. Foisner R. J. Cell. Biochem. 2005; 95: 454-465Crossref PubMed Scopus (82) Google Scholar). By contrast, hsSUN1-RNAi cells were absent for hsSUN1 and showed failed NPC staining/reorganization at the edges of segregated DNA masses in anaphase (Fig. 4C, panels 4-6, arrow-heads). Hence, hsSUN1 appears to be required in anaphase for NPC formation; failed NPC assembly may explain the inability of nuclear envelope to retain nuclear-GFP (Fig. 4B, panels 6 and 9).hsSUN1-depleted Cells Have Delayed Chromosome Decondensation—Two events occur at the end of mitosis: daughter nuclei form and chromosomes de-condense. Currently, it is unclear whether these two events are linked. To ask if the daughter nuclear envelope reassembly influences DNA de-condensation, we visualized chromosome segregation in control and hsSUN1 RNAi cells. We digitized signals from DAPI-stained chromosomes using heightened colored intensities to reflect increased DNA compaction (Fig. 5A, panels 4 and 8). By this measure, control-RNAi cells compared with hsSUN1-RNAi cells at the same juncture during cell division (as monitored by α-tubulin staining) had consistently lower DAPI intensity (see Fig. 5A, panels 4 and 8; the averaged fluorescent intensity is 2.7 times lower in panel 4 than panel 8). Thus, hsSUN1 depletion affects nuclear envelope integrity (Fig. 4B) and results in an apparent increase in DNA compaction (Fig. 5A).FIGURE 5Knockdown of hsSUN1 prolonged chromatin condensation with hypoacetylated histones. A, cells transfected with control- or hsSUN1-siRNA were immunostained to visualize hsSUN1 (gray, panels 1 and
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