Histone Acetyltransferase hALP and Nuclear Membrane Protein hsSUN1 Function in De-condensation of Mitotic Chromosomes*

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.

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). 2 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 (1). Functionally, the INM provides a physical barrier; the NPC serves for the transport of material between the nucleus and the cytoplasm (2); and the nuclear lamina erects a meshwork, which maintains nuclear structure and assists indirectly in DNA replication and RNA processing (3,4).
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 (5). 23 of these ORFs map to chromosome regions linked to a variety of dystrophies collectively termed "nuclear envelopathies" (5). These diseases have phenotypes ranging from cardiac and skeletal myopathies, lipodystrophy, peripheral neuropathy, and premature aging (6 -9). Genetic studies have associated mutations in emerin, lamin A/C, and lamin B receptor with such pathologies (7,9). An emerging notion is that the INM proteins are needed to maintain nuclear integrity and guard against mechanical stress (10 -12). 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 (13). The SUN domain was first identified based on the sequence alignment of Sad1 of Schizosaccharomycespombe and UNC-84 of Caenorhabditis elegans (14). 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 (15); on the other hand, UNC-84 localizes in the C. elegans nuclear envelope (16). 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 (17,18), little else is known about the function of mammalian SUN proteins (18 -21).
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 (5), 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.
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. □ S The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1
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 ϫ 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 2ϫ 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), ␣-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% CO 2 .
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 ⌬N1, 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 ⌬N2, Fig. 1C, panel 7) did not cause further changes. However, when the deletion was extended to amino acid 306, hsSUN1 ⌬N3 (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.
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  3 and 4). B-G, fixed HeLa cells were immunostained with ␣hsSUN1-C (green) antibody and antibodies, as indicated, to lamin B1, emerin, NPC (mab414), ␣-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.  6 and 7) agarose beads. Histone H2B co-immunoprecipitated by HA-hsSUN1-BD or FLAG-BAF was detected by immunoblotting.
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 chromatinbinding domains (31). 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 precip-itation 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.
geted 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.
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. 32, 33). 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, arrowheads). 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 DAPIstained 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 ␣-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).
Acetylation of Histone H2B and H4 Is Decreased in hsSUN1depleted Cells-We sought to understand what accounted for delayed chromosome de-condensation in hsSUN siRNA cells. Condensed chromosomes are wrapped by histones whose function is regulated by post-translational acetylation and phosphorylation among other events (34 -36). Phosphorylation of histone H3 at serine 10 (H3pSer10) was previously proposed to initiate chromatin condensation when cells enter mitosis (37). On the other hand, what event specifies chromatin de-condensation as cells exit mitosis is unknown. In our experiments, H3pSer10 phosphorylation in anaphase and telophase did not differ between hsSUN1 and control RNAi cells (supplemental Fig. S1), suggesting that this event does not explain results in Fig. 5B.
Histone acetylation modulates compacted chromatin to allow transcription factors to access DNA (38,39). We wondered whether histone acetylation might also regulate mitotic DNA de-condensation. To investigate this notion, the acetylation status of histones in control and hsSUN1 RNAi cells was characterized by Western blotting (Fig. 5C). Total acetylated H2B (AcH2B; Fig. 5C), determined using a mixture of antibodies individually specific for acetyl-Lys 5 , -Lys 12 , -Lys 15 , and -Lys 20 , and acetylated H4 (AcH4; Fig. 5C), verified with an antibody mix specific for acetyl-Lys 5 , -Lys 8 , -Lys 12 , and -Lys 16 , were reduced in hsSUN1-RNAi versus control RNAi samples. On the other hand, acetylated H3 (AcH3; Fig. 5C) was insignificantly changed. We next analyzed several individual lysine acetylation sites in H2B and H4. HsSUN1-RNAi cells were significantly reduced for acetylation at Lys 12 and Lys 15 , but not at Lys 5 , of H2B; and for acetylation at Lys 8 , Lys 12 , and Lys 16 of H4 (Fig.  5D). These results show that depletion of hsSUN1 not only affected nuclear envelope integrity (Fig. 4) and mitotic chromosome de-condensation (Fig. 5, A and B), but also the acetylation of H2B and H4 (Fig. 5, C and D).
hALP Contributes to Chromosome De-condensation-The above results suggest that mitotic chromosome de-condensation is linked to a histone acetyltransferase (HAT) activity. To ask which HAT contributes this activity, we reasoned that such a HAT must be a nuclear membrane-associated moiety. An in silico search revealed that the human genome encodes a minimum of sixteen HATs (40,41); however, only one, KIAA1709/hALP (41), is a nuclear membrane-associated protein (5).
We investigated whether hALP would interact with mitotic DNA. In mitotic cells, hALP was stained with condensed chromosome in a sheath-like array ( Fig. 6A; Ref. 42). Such interaction is compatible with hALP providing a HAT activity for de-condensing mitotic chromosomes. Indeed, consistent with this interpretation, when we used siRNA to deplete hALP (Fig. 6, B and C) and followed in time lapse GFP-H2B-marked DNA de-condensation, prolonged chromosome condensation was seen in hALP-siRNA cells compared with control cells (Fig. 6D).
hsSUN1 Targets hALP Activity to Chromosomes-A plausible model from our results is that condensed mitotic chromosomes as they become wrapped by newly forming daughter nuclear envelope contact the chromatin-binding domain of hsSUN1, which brings membrane-associated hALP to facilitate DNA decondensation. This model which suggests that hsSUN1 targets hALP to condensed chromosome can be tested by constructing a chimeric protein with the chromatin binding domain of hsSUN1 (Fig. 3B) fused to hALP. A prediction is that an N terminus hsSUN-hALP fusion would directly target chromatin and would enhance DNA de-condensation.
The findings from the artificial hsSUN1⌬C4-hALP fusion protein are consistent with hsSUN1 bridging hALP interaction with DNA. To ask if an intracellular bridging interaction could be explained by protein-protein binding between hsSUN1 and hALP, we assayed whether overexpressed hsSUN1⌬C4A coimmunoprecipitates hALP. As a control, we also used a deleted version of hsSUN1, which contains only its SUN domain (i.e. hsSUN1-SUN, Fig. 3C). Cell lysates from respectively transfected cells were prepared and co-immunoprecipitations were performed. Fig. 7C shows that hALP indeed co-precipitated with hsSUN1⌬C4 (Fig. 7C, lane 5) but not hsSUN1-SUN (Fig.  7C, lane 6).

DISCUSSION
Except for a shared SUN motif, hsSUN1 is unrelated in sequence to other SUN proteins. HsSUN1 has three predicted transmembrane domains. Here, we show that the hsSUN1 three transmembrane domains are needed for nuclear membrane retention and that its N terminus is needed for chromatin binding. Additional evidence shows that hsSUN1 serves an early role in the proper reformation of daughter nuclear envelopes and in targeting hALP to chromosomes.
Chromosomes are structurally organized and occupy discrete nuclear territories (43). The nuclear envelope provides a scaffold for anchoring chromatin and for maintaining nuclear integrity (32,44). Nuclear envelope and associated proteins such as nuclear lamina, NPC, LAP2, LBR, and emerin directly or indirectly interact with chromatin to regulate DNA replication and transcription (27,43,45). A pivotal event in the mammalian cell cycle is nuclear membrane dissolution as a cell enters mitosis. Much about nuclear envelope breakdown and reassembly remain incompletely understood (33, 46 -48). Recent findings suggest that nuclear envelope proteins first reassembles via tethering to discrete regions on segregated chromatids (27,29,30,49,50). Which protein sets the stage for others to follow has not been fully defined. Here, we report that hsSUN1 precedes LAP2 and lamin B1 in interacting with segregated chromosomes in anaphase. Whether hsSUN1 is the first INM or follows a yet earlier protein is unclear. However, findings that hsSUN1 has a chromatin binding domain in its N terminus and that its depletion leads to failed NPC formation Mitotic chromosomes are thought to be highly compacted for physical reasons required for segregation. After separating sister chromatids, the completion of mitosis mandates that new envelopes reform around DNA to consummate two daughter nuclei. Transcription is silenced in mitosis as expected for highly condensed chromatin (51). However, the start of the next cell cycle (i.e. G1) needs de novo mRNA synthesis from de-condensed genes (52,53). While it is accepted that as a cell begins mitosis DNA condensation correlates with phosphorylation of Ser 10 on H3 (37), what dictates DNA de-condensation at the end of mitosis is less clear. Our data now suggest that the signal to trigger de-condensation is not a change at phosphorylated H3Ser10 (supplemental Fig. S1). Instead, our findings indicate that de-condensation is marked by several acetylated lysines in H2B and H4 (Fig. 5, C and D), including Lys 15 of H2B and Lys 8 , Lys 12 , and Lys 16 of H4. Acetylation of specific lysine(s) in histones has been extensively reported to regulate transcriptional activation, histone deposition, DNA repair, and chromatin structure (35,54). Increasing evidence has led to the idea of a histone modification code, which might be recognized by various cellular machineries. We suggest hsSUN1 is responsible for inducing the acetylation a subset of histones at the end of mitosis and generating a code for the initiation of chromosome de-condensation. An unexpected result from our work is that chromosome segregation does not appear to be obligatory for DNA de-condensation. Hence, a chromatin-targeted HAT is sufficient to initiate DNA de-condensation of duplicated sister chromatids that have yet to separate fully (Fig. 7B, see supplemental movie 1).
Our results suggest three ways to view the link between hsSUN1 and chromatin de-condensation/histone acetylation. The first view is that hsSUN1 modulates histone acetyltransferases (HATs), or histone deacetylases (HDACs), which modifies H2B and H4. Perturbation of this HAT (HDAC)-activity leads to (de)acetylated H2B and H4 (Fig. 5C), which promotes chromatin (de)compaction. A second view is that DNA de-condensation requires the proper completion of a daughter nuclear envelope. Here, loss of hsSUN1 interrupts nuclear membrane reassembly thereby interfering with de-condensation. That defects in BAF (55) and nuclear lamina (56) also affect nuclear envelope formation and retard chromatin de-condensation are consistent with this latter perspective. A third view is that both of the above two processes are important. Accordingly, in the context of a reforming daughter envelope an INM protein is used to recruit a HAT (HDAC) for purposes of regulating DNA de-condensation. Indeed, reports that LAP2␤ can interact with HDAC3 and contribute to histone H4 deacetylation (57) are compatible with a mechanistic model in which interplay between HATs and HDACs at the termination of mitosis tips the DNA condensation/de-condensation balance.
If a HAT is needed, then which HAT works with hsSUN1? While many HATs exist in the human genome, only one, hALP, based on proteomic data (5), is nuclear membrane-associated. Moreover, hALP was detected in mitotic chromosome scaffold fraction by proteomics, supporting its role in mitosis (42).
Three pieces of evidence support the relevance of hALP for chromosome de-condensation: 1) hALP congresses to mitotic DNA (Fig. 6A); 2) knockdown of hALP prolongs DNA condensation (Fig. 6D); and 3) direct targeting of hsSUN1⌬C4-hALP to chromatin accelerates DNA de-condensation (Fig. 7B). Hence, while we cannot exclude the involvement of other HATs and/or HDACs, our results are consistent with requirements for hALP and hsSUN1 in mitotic DNA de-condensation. What remains possible is that hsSUN1 may interact with other nuclear or nuclear matrix-associated HATs in addition to nuclear-membrane associated hALP. Such additional interactions, if identified, could also contribute to mitotic DNA de-condensation. Indeed, understanding how condensed mitotic chromatin is de-condensed complements insights on how heterochromatin is transformed into a transcriptionally active state (58). These complementary studies add to the richness of our appreciation for the regulatory roles played by histones in chromosome biology (59).