Barrier-to-Autointegration Factor influences specific histone ...

RESEARCH PAPER Nucleus 2:6, 1–11; November/December 2011;

G

2011 Landes Bioscience

Barrier-to-Autointegration Factor influences specific histone modifications Rocío Montes de Oca,1 Paul R. Andreassen2 and Katherine L. Wilson1,* 1

Department of Cell Biology; The Johns Hopkins University School of Medicine; Baltimore, MD USA; 2Division of Experimental Hematology & Cancer Biology; Cincinnati Children’s Research Foundation; University of Cincinnati College of Medicine; Cincinnati, OH USA

Keywords: nuclear envelope, Barrier-to-Autointegration Factor, Lamina-Associated Domain, H3-K9 dimethylation, H3-S10 phosphorylation, nucleosome, G9a, SET/I2PP2A, cell cycle, DNA replication, Hutchinson-Gilford Progeria Syndrome, nucleoskeleton Abbreviations: BAF, Barrier to Autointegration Factor; BrdU, 5-Bromo-2’-Deoxyuridine; CBB, Coomassie Brilliant Blue; DMEM, Dulbecco’s modified eagle’s medium; HDAC, histone deacetylase; HGPS, Hutchinson-Gilford progeria syndrome; HP1, heterochromatin protein 1; IP, immunoprecipitation; LADs, Lamina-Associated Domains; LEM, LAP2-Emerin-MAN1; LOCKs, Large Organized Chromatin K9 modifications; NE, nuclear envelope; SET/I2PP2A, protein phosphatase 2A inhibitor; Tet, tetracycline; PARP1, poly(ADP) ribose polymerase 1; PBS, phosphate-buffered saline; PTM, posttranslational modification; RBBP4, retinoblastoma binding protein 4; 2X-SB, 2X-Sample Buffer

Defects in the nuclear envelope or nuclear ‘lamina’ networks cause disease and can perturb histone posttranslational (epigenetic) regulation. Barrier-to-Autointegration Factor (BAF) is an essential but enigmatic lamina component that binds lamins, LEM-domain proteins, DNA and histone H3 directly. We report that BAF copurified with nuclease-digested mononucleosomes and associated with modified histones in vivo. BAF overexpression significantly reduced global histone H3 acetylation by 18%. In cells that stably overexpressed BAF 3-fold, silencing mark H3-K27-Me1/3 and active marks H4-K16-Ac and H4-Ac5 decreased significantly. Significant increases were also seen for silencing mark H3-K9-Me3, active marks H3-K4-Me2, H3-K9/K14-Ac and H4-K5-Ac and a mark (H3-K79-Me2) associated with both active and silent chromatin. Other increases (H3-S10-P, H3-S28-P and silencing mark H3-K9-Me2) did not reach statistical significance. BAF overexpression also significantly influenced cell cycle distribution. Moreover, BAF associated in vivo with SET/I2PP2A (protein phosphatase 2A inhibitor; blocks H3 dephosphorylation) and G9a (H3-K9 methyltransferase), but showed no detectable association with HDAC1 or HATs. These findings reveal BAF as a novel epigenetic regulator and are discussed in relation to BAF deficiency phenotypes, which include a hereditary progeria syndrome and loss of pluripotency in embryonic stem cells.

Introduction Nucleosomes, the basic unit of chromatin structure, are regulated by mobile chromatin-associated proteins1,2 and enzymes that reversibly (‘epigenetically’) modify DNA or specific core histone residues.3 Such modifications generate two general transcriptional states: active, where chromatin is typically more acetylated and silent, where chromatin is more methylated.4 However, the functions and relationships of histone ‘marks’ are complex and dynamic; some marks recruit further regulators (e.g., methyl H3K9 recruits heterochromatin protein 1 [HP1]),5 and some are also important for cell cycle control (e.g., H3-S10-phosphorylation during mitosis).6 Genome organization, gene position and epigenetic control are further influenced by nuclear intermediate filaments formed by A- or B-type lamins (reviewed in refs. 7–9) and by promoter proximity to the nuclear envelope.10-13 Lamin filaments concentrate near the nuclear envelope (NE) and bind most NE inner

membrane proteins directly,14 forming ‘lamina’ networks that support transcription, replication, genome organization, development and DNA repair (reviewed in refs. 7 and 15). Lamina defects cause a myriad of human diseases (laminopathies) including muscular dystrophy, cerebellar disorders and HutchinsonGilford Progeria Syndrome (HGPS).16 Progeria-associated and other mutations in LMNA perturb not only nuclear structure, but also heterochromatin and epigenetic regulation.17-22 How the genome depends on lamin networks is a central question. Lamina networks have three major types of components: lamins, LEM-domain proteins (e.g., LAP2, emerin, MAN1, Lem2/NET25; most located at the NE inner membrane)14 and an enigmatic soluble protein named Barrier-to-Autointegration Factor (BAF).23 These components are mutual interactors that are collectively required to rebuild the nucleus during anaphase and telophase.24-29 The timing of nuclear assembly is regulated, at least in part, by a conserved kinase, Vaccinia-related kinase 1 (VRK1), which directly phosphorylates BAF during mitosis to inhibit its

*Correspondence to: Katherine L. Wilson; Email: [email protected] Submitted: 04/02/11; Revised: 08/31/11; Accepted: 09/02/11 http://dx.doi.org/10.4161/nucl.2.6.17960

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chromatin association and consequently also the chromatin association of LEM-domain proteins and nuclear membranes.28 After assembly, lamins and LEM-domain proteins are in molecular contact with and are proposed to regulate and/or maintain silenced chromatin.8,11,30-32 Whether BAF influenced genome silencing was previously unknown. BAF is abundant in mammalian cells (~9 mM near NE, ~2 mM in nucleoplasm),33 consistent with its role as an essential component of nuclear lamina networks. Perhaps contrary to one’s expectations, BAF also has high diffusional mobility during interphase.34,35 BAF is a functional homodimer with two binding sites for dsDNA36,37 that can condense long DNA by ‘looping’ in vitro.38 BAF profoundly influences higher-order chromatin structure,26,27,29,39 and represses transcription at specific promoters.35,40 BAF also associates in vivo with specific linker histones,41 transcription factors (including Sox2, a master regulator of pluripotency),40,42,43 poly(ADP) ribose polymerase 1 (PARP1) and retinoblastoma binding protein 4 (RBBP4).42 Notably, BAF binds the tail plus helix-aN of histone H3 with high affinity in vitro,41 suggesting it associates with nucleosomes. New findings, presented here, support this model and reveal BAF as an epigenetic regulator. These findings are discussed in relation to the recently discovered roles of BAF in stem cell self-renewal,43 the epigenetic defects caused by progeria-associated lamin A mutations and the

discovery that BAF insufficiency causes a hereditary progeroid syndrome.44 Results Previous studies using 35S-Met-labeled BAF to probe immobilized core histones41 suggested weak but detectable direct binding to H4, in addition to H3. We confirmed direct binding between recombinant purified Xenopus histone H4 (xH4) and recombinant purified His-tagged human BAF dimers (hereafter, ‘H6BAF’). xH4 pelleted in the presence but not in the absence of H6BAF (Fig. 1A, lanes 4 vs. 2; blot shown is typical of three independent experiments [n = 3]). Direct binding to H4, in addition to H3 and DNA, supported potential association(s) with nucleosomes, non-nucleosomal H3-H4,45 or both. BAF copurifies with isolated mononucleosomes. To test potential BAF association with nucleosomes in vivo, we isolated chromatin from formaldehyde-crosslinked HeLa cells and digested extensively with micrococcal nuclease to generate mononucleosomes, as described by Kim et al.46 Digestion was complete, yielding primarily ~80–180-bp DNA fragments as assayed by agarose gel electrophoresis (Fig. 1B, arrow). The presence of core histones was verified by SDS-PAGE with purified chicken core histones as markers (Fig. 1C, lanes 1 and 2, respectively).

Figure 1. BAF associates with H4 and nucleosomes. (A) Pull-down assays showing BAF binds histone H4 directly in vitro. Untagged recombinant Xenopus H4 (xH4) was incubated with Ni2+beads, plus or minus purified His-tagged recombinant BAF (H6BAF) dimers; bound proteins were resolved by SDS-PAGE and visualized by CBB (Inputs [I] 2%; pellets [P] 70%). One representative gel is shown (n = 3). (B) Ethidium-bromide stained agarose gel of micrococcal nuclease-digested nucleosomes from HeLa cells; arrow indicates , 180 bp DNA. (C) Proteins from microccocal nuclease-digested nucleosomes, resolved by SDS-PAGE and CBB stained (lane 1), with purified chicken core histone markers (lane 2; 4 mg). (D) Protein gel blot of SDS-PAGE-resolved nucleasedigested mononucleosomes before (Input [I] 1%) or after immunoprecipitating with antibodies to BAF (aBAF) or no antibody as control (pellets [P] 50%; supernatants [S] 1%; n = 3). (E) Separately determined BAF dimer (purple) and nucleosome structures overlayed at the same scale by PyMol software (pymol.sourceforge.net), based on BAF (PDB ID: 1CI4) and nucleosome (PDB ID: 1AOI) coordinates from the Protein Data Bank (www.pdb.org). Actual contact(s) are unknown.

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Endogenous BAF co-purified with these isolated mononucleosomes, shown by direct protein gel blotting with antibodies against BAF (Fig. 1D, input (I), lanes 1 and 4; n = 3) and by immunoprecipitating BAF from isolated mononucleosomes (Fig. 1D, lane 5 vs. 2; n = 3). These results supported the hypothesis that BAF associates with nucleosomes in vivo. Strengthening our results, an independent laboratory previously identified BAF by mass spectrometry from purified H3.1nucleosome complexes.47 As ‘food for thought’, the atomic structures of the nucleosome48 and BAF,49 determined separately, are shown at the same scale in Figure 1E. BAF associates with posttranslationally modified histones in vivo. To determine if BAF associated with endogenous posttranslationally modified histones, antibodies specific for each of eight posttranslational modifications on H3 or H4, or control antibodies against the C-terminus of H3 (‘bulk H3'), were used to immunoprecipitate histones from HeLa whole cell Figure 2. BAF co-immunoprecipitates with posttranslationally-modified histones lysates. An example of an original protein gel blot is in vivo. HeLa cell lysates were immunoprecipitated with antibodies specific for each shown in Fig. S1; blots were quantified as described in indicated mark in H3 or H4, or by an antibody that recognizes the C-terminus of H3 Methods. BAF co-precipitated to varying extents with (‘bulk’ H3), then resolved by SDS-PAGE and protein gel blotted simultaneously with antibodies to BAF or bulk H3. Results were quantified by densitometry. The BAF all tested active (dimethylated H3-K4, phosphorylated signal precipitated by each antibody was plotted as a percentage of bulk H3 in the H3-S10, hyperacetylated H4 [H4-Ac5; recognizes trisame precipitate; results for each mark-specific antibody were normalized to the and tetra-acetylated H4], acetylated H4-K5 and BAF-to-bulk-H3 ratio (‘H39), set at 100%. BAF and H3 were not detected in acetylated H4-K16) or silenced (dimethylated H3-K9, no-antibody controls, which were subtracted from all data sets. ‘A’, active chromatin mono/trimethylated H3-K27 and monomethylated mark; ‘S’, silent mark. The mean and SEM of three independent experiments are shown. H4-K20) marks on histones H3 and H4 (Fig. 2; n = 3). Our results revealed no obvious preference for silenced vs. active marks, but supported an association between BAF and endogenously modified chromatin. We Tet-induced BAF expression for 32–52 h and protein gel Decreased H3 acetylation in BAF-overexpressing HeLa blotted whole cell lysates to determine if excess BAF altered the cells. To test if BAF influences histone posttranslational protein levels of specific histone-modifying enzymes. Relative to modifications (PTMs) in vivo, we used metabolic labeling c-tubulin, total BAF (endogenous plus FLAG-BAF) levels were with sodium [14C]-acetate to quantify core histone acetylation ~3-fold higher in the 293:BAF cells (Fig. 3B; n = 4). In contrast, in HeLa cells that transiently overexpressed BAF. Cells that no significant changes were seen in the protein levels of histone overexpressed BAF 2-to-10 fold (data not shown) incorporated deacetylase 2 (HDAC2) or HDAC4 (class I and class IIa HDACs, significantly (~20%) less [14C]-acetate into H3 (Fig. 3A; p = 0.03, respectively)51 (Fig. 3B), emerin or the histone methyltransferase n = 5). Transient BAF overexpression did not detectably alter G9a (Fig. S2A). To quantify potential changes in specific histone histone protein levels (data not shown). The same trend (~20% PTMs, histones were acid-extracted from the same 293:BAF and less acetylation) was seen for H4 and H2A/H2B (which co- 293:CAT samples, resolved by SDS-PAGE and protein gel migrated in our gels), but did not reach statistical significance blotted using antibodies specific for each of eight marks on H3 (Fig. 3A; p = 0.08 each, n = 5), possibly due to variable BAF (Fig. 3C) or six marks on H4 (Fig. 3D). Examples of original expression levels. Reduced bulk H3 acetylation suggested excess protein gel blots are shown in Figure S3 and were quantified as BAF influenced the activity or access of histone-modifying described in Materials and Methods. Several marks were affected enzymes. in BAF-overexpressing cells. Three marks decreased significantly: Stable BAF overexpression alters specific histone ‘marks’ in silencing mark H3-K27-Me1/3 (by ~15%; Fig. 3C; p = 0.02, cells. To achieve consistent BAF overexpression levels, we used n = 4) and active marks H4-K16-Ac (by ~10%; Fig. 3D; previously-described50 stable cell lines, derived from human p = 0.03, n = 4) and H4-Ac5 (by ~30%; Fig. 3D; p = 0.04, HEK293 cells with tetracycline (Tet)-inducible expression of a n = 4). Five marks increased significantly: silencing marks H3-K9single-copy insert of FLAG-tagged BAF (293:BAF cells) and Me3 (by ~20%; Fig. 3C; p = 0.002, n = 4) and H3-K79-Me2 (by corresponding control (293:CAT) cells. Total BAF levels in 293: ~10%; Fig. 3C; p = 0.01, n = 4) and active marks H3-K4-Me2 BAF cells increased ~3 fold within 8.5 h after Tet-induction (by ~15%; Fig. 3C; p = 0.01, n = 4), H3-K9/K14-Ac (by ~15%; (Fig. S2A) and remained near this level for at least three days Fig. 3C; p = 0.02, n = 4) and H4-K5-Ac (by ~15%; Fig. 3D; p = 0.02, n = 4). Other increases that did not reach statistical (Fig. S2B).


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Figure 3. BAF overexpression affects specific H3 and H4 marks. (A) HeLa cells were transiently transfected to express H6BAF or empty vector, then metabolically labeled with sodium [14C]-acetate. Histones were acid-extracted, resolved by SDS-PAGE and CBB stained. Incorporated [14C] was detected by fluorography, quantified by densitometry relative to the CBB-signal for each histone and expressed as a percentage of the corresponding 14C-tohistone signal in control cells. The mean and SEM of five independent experiments are shown (p = 0.03; Student T-test). (B) Stable FLAG-BAF expression was induced in 293:BAF (‘BAF’) or control cells (293:CAT; ‘CAT’), then whole cell lysates were resolved by SDS-PAGE and protein gel blotted with antibodies to BAF, HDAC4, HDAC2 or c-tubulin (loading control). Protein levels of BAF and each HDAC were quantified by densitometry, normalized to c-tubulin and plotted as the mean and SEM of four independent experiments (p = 0.01; Student T-test). (C, D) Histones acid-extracted from the same experiment as in (B) were resolved by SDS-PAGE and protein gel blotted with antibodies specific for each indicated mark in H3 (C) or H4 (D). Results were quantified by densitometry and normalized to total H3 or H4 protein levels; the corresponding signal in CAT cells was set to 100%. The mean and SEM of four independent experiments (*p ¡ 0.04, **p ¡ 0.02, ***p = 0.002 by two-tailed Student T-test) are shown. (E) Summary panel categorizing marks as either ‘generally active’, ‘generally silent’ or ‘both’. Note that these designations might change according to the gene studied, genomic location, or as new information becomes available. See also Figure S3.

significance were seen for silencing mark H3-K9-Me2 (increased ~30%; Fig. 3C; n = 4) and phosphorylation at H3-S10 and H3-S28 (increased ~30% and ~20, respectively; Fig. 3C; n = 4). Two factors might have influenced or obscured detection of

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significant BAF-induced changes in H3 phosphorylation: H3 hyperphosphorylation during mitosis and potential effects of BAF overexpression on cell cycle progression. For example BAF-null Drosophila and C. elegans show cell cycle arrest phenotypes,26,27

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and BAF downregulation prolongs S phase in somatic mammalian cells,52 and increases the proportion of cells in G2/M in embryonic stem cells.43 BAF overexpression affects the cell cycle distribution of asynchronous populations. To test for potential cell cycle phenotypes, we examined the cell cycle distribution of unsynchronized 293:CAT and 293:BAF cells Tet-treated for 35 h to induce FLAG-BAF expression. Based on representative histograms of DNA content obtained by flow cytometry (Fig. 4A, ‘DNA content’ panels), we determined the distribution of cells in specific cell cycle phases using ModFit software (Fig. 4A, right). The percentage of G0/G1 cells, averaged from duplicates of three independent experiments, was significantly higher in

Figure 4. Cell cycle analysis of asynchronous 293:BAF cells. Asynchronous 293:CAT and 293:BAF cells were induced for FLAG-BAF expression, then harvested and analyzed by flow cytometry. The percentage of cells in each phase of the cell cycle was determined from histograms of DNA content using ModFit software (A). Percentage of cells in S phase (B) or mitosis (C), identified by co-staining with antibodies to BrdU or MPM-2 (respectively), was calculated from dot plots using CellQuest software. Representative histograms or dot plots are shown at left; the mean and SEM of three independent experiments (run in duplicates in A and B) are plotted at right. (D) Parallel samples of whole cell lysates from asynchronous 293:CAT or 293: BAF cells were protein gel blotted with antibodies to BAF, G9a, SET/I2PP2A, HDAC4, HDAC2, lamins A/C or cyclin E and plotted as the ratio of each signal in 293:BAF vs. 293:CAT cells. The mean and SEM of three independent experiments are shown (p = 0.04; Student T-test).


BAF-overexpressing populations (42.6 +/2 4.1%) than in 293: CAT controls (39.0 +/2 4.2%; Fig. 4A, right; p = 0.04). BAFoverexpressing populations appeared to have correspondingly fewer cells in S phase, but this difference was not statistically significant by this assay (Fig. 4A, right; p = 0.08). To study S phase more precisely, we did parallel studies in which asynchronous 293:BAF and 293:CAT cells were Tet-treated 35 h, then pulse-labeled 30 min with 5-Bromo-2’-Deoxyuridine (BrdU). In three independent duplicate experiments, significantly fewer 293:BAF cells (48.55 +/2 3.59%) than 293:CAT controls (54.32 +/2 4.47%) were in S phase, as assessed by BrdU incorporation (Fig. 4B, right; p = 0.02). Thus, G0/G1 phase was significantly longer and S phase was significantly shorter in asynchronous BAF-overexpressing cells. To specifically examine progression to mitosis, asynchronous Tet-induced cells were assayed by flow cytometry using the M-phase antibody MPM-2,53 which recognizes mitosisspecific phospho-epitopes.54 In three independent experiments a significantly higher percentage of BAF-overexpressing 4C cells (G2/M) were MPM-2-positive (2.3 +/2 0.08%) compared with 293:CAT controls (1.44 +/2 0.21%; Fig. 4C, right). This significant (p = 0.02) difference suggested BAFoverexpression reduced G2 phase, or prolonged M phase, or both, in unsynchronized cells. Neither population accumulated polyploid cells (. 4C DNA content; Fig. 4A), suggesting cytokinesis was unaffected. As controls, whole cell lysates were protein gel blotted for BAF and other proteins: G9a, SET/I2PP2A (inhibitor of protein phosphatase 2A), HDAC2, HDAC4, lamins A/C and cyclin E. Tet-induced 293:BAF cells showed a significant ~2.5-fold increase in total BAF (endogenous plus FLAGBAF) relative to 293:CAT controls (Fig. 4D; p = 0.04; n = 3), as expected. Other tested proteins showed no significant changes (Fig. 4D). Overall, asynchronous BAF-overexpressing populations showed a significant (~10%) increase in the proportion of G0/G1 cells, a significant (~10%) decrease in S-phase cells and a significant (~50%) increase in the percentage of M-phase cells. BAF associates with G9a and SET/I2PP2A in vivo. To explain some of the changes in histone posttranslational modification in BAF-overexpressing cells, we hypothesized BAF might interact with and potentially recruit, specific chromatin regulators. Potential interactors from our BAF proteome42 included HDAC1 and/or HDAC2 (distinguishing peptides not found), SET/I2PP2A (blocks H3-S10 dephosphorylation)55 and G9a, an H3-K9 methyltransferase that generates the H3-K9-Me2 mark.56 We therefore tested potential association with HDAC1, SET/I2PP2A or G9a and also tested for potential associations with histone acetyltransferases (HATs) or Aurora B (phosphorylates H3-S10 and H3-S28 during mitosis),6 as controls for the observed changes in histone acetylation and phosphorylation. We transiently expressed in HeLa cells FLAG-tagged versions of three different HATs (PCAF, p300 or Tip60), HDAC1, G9a or SET/I2PP2A, with FLAG-H3.1 and empty FLAG vector as positive and negative controls, respectively.

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Figure 5. BAF associates with G9a and SET/I2PP2A in vivo. (A-C) Protein gel blots of immunoprecipitates from HeLa cells that transiently expressed FLAG-tagged H3.1 (positive control), FLAG-PCAF (n = 5), FLAG-p300 (n = 3), FLAG-Tip60 (n = 3), FLAG-HDAC1 (n . 3), FLAG-G9a (n = 3), FLAG-SET (n = 3), or empty vector (FLAG). Input (I; 1%) and pellet (P; 50%) fractions were resolved by SDS-PAGE and immunoblotted using antibodies against FLAG (aFLAG) or BAF (aBAF). (D, E) FLAG immunoprecipitates from HeLa cells that transiently expressed FLAG, FLAG-BAF (n = 3), FLAG-Aurora B (n = 3) or FLAG-SET (n = 3), protein gel blotted with antibodies to FLAG (aFLAG) or endogenous G9a (aG9a), SET/I2PP2A (aSET), H3 (aH3) or BAF (aBAF serum 3273 sees endogenous BAF [‘Endo’] and FLAG-BAF [F-BAF]).

Cell lysates were immunoprecipitated with FLAG antibodies. Endogenous BAF co-immunoprecipitated with histone H3.1 as expected41 (Fig. 5A, lane 4, aBAF) but showed no detectable association with any tested HAT (Fig. 5A, lanes 6, 8, 10; n ¢ 3) or HDAC1 (Fig. 5A, lane 12; n = 3) in vivo. Endogenous BAF consistently and robustly co-immunoprecipitated with FLAGG9a (Fig. 5B, lane 4, aBAF; n = 3) and FLAG-SET/I2PP2A (Fig. 5C, lane 4, aBAF; n = 3) from cells. Reciprocal experiments gave similar results: FLAG-BAF immunoprecipitated both endogenous G9a (Fig. 5D, lane 4, aG9a; n . 3) and endogenous SET/I2PP2A (Fig. 5D, lane 4, aSET; n = 3). No association with FLAG-Aurora B was detected in HeLa cells, where positive controls verified BAF association with FLAG-SET/I2PP2A (Fig. 5E, lanes 2 vs. 4, aBAF; n = 3). These results independently validated G9a and SET/I2PP2A as BAF-associated (directly or indirectly) in vivo, confirming our previous proteomic results.42 We speculate BAF might favor H3-S10 phosphorylation and H3-K9 methylation in vivo by promoting the association of G9a and SET/I2PP2A with chromatin. On the other hand, despite its significant effects on histone acetylation (decreased global H3 acetylation; increases or decreases in four specific acetylation marks), we found no evidence that BAF associates with any tested

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HDAC or HAT in vivo. Further work is needed to understand the mechanism(s) by which BAF affects histone acetylation. Notably, as an essential-yet-mobile component of the ‘lamina’ network, BAF might influence epigenetic regulation by a mechanism that does not involve the direct recruitment of HDACs, HATs or other chromatin-associated factors to chromatin. Discussion BAF, an essential and highly mobile component of the nuclear lamina network,23 is shown here to associate with nucleosomes and broadly influence the epigenetic landscape. Eight histone marks increased or decreased significantly in cells that overexpressed BAF ~3-fold, with six other marks also potentially affected. BAFoverexpression caused histones to be underacetylated (reduced bulk acetylation and four of six site-specific marks decreased) and hypermethylated (five of six marks increased). These changes are unlikely to reflect a single underlying mechanism, given the complex functional hierarchies and dynamics of histone marks and their regulators,57 and huge gaps in knowledge about how chromatin is influenced by nuclear structure.8,9 Our findings begin to fill this gap, by revealing BAF as a novel epigenetic regulator.

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BAF facilitates S-phase progression. Significantly fewer BAFoverexpressing cells were in S phase, suggesting S phase was shorter. This result was consistent with and strongly supports the proposal by Haraguchi and colleagues that BAF promotes DNA replication,52 based on their finding that S phase is four hours longer in BAF-downregulated HeLa cells. Whether S-phase roles for BAF include lamins15 or LEM-domain proteins58 is still unknown. BAF localizes exclusively in the nucleus during S phase in mortal cells.52 Our findings suggest potential epigenetic mechanism(s) by which BAF might influence replication, since histones are specifically modified at replication origins and during elongation.59,60 For example, H4-K20-Me, which increased without reaching statistical significance in asynchronous BAFoverexpressing cells, has positive roles in replication. Cells downregulated for SET8 (PR-Set7), the methyltransferase responsible for this mark, accumulate in S phase due to slowed replication forks and origin firing, dsDNA breaks and DNA damage response signaling.61,62 Alternatively, BAF might facilitate replicationinduced DNA damage repair,42 or the release of late-replicating heterochromatin, which relocates to the nuclear interior for replication and is subsequently retethered to the NE.63 Interestingly, specific regions (about 1%) of the human genome replicate after canonical S phase, late in G2/M phase.64 Moreover, BAFdownregulated embryonic stem cells (mouse and human) showed significantly fewer cells in S phase and correspondingly more G2/M-phase cells.43 We speculate G2 is prolonged in BAFdeficient embryonic stem cells because BAF is also needed during the final (1%) phase64 of DNA replication. Not surprisingly, BAF-downregulated embryonic stem cells also suffer high rates of apoptosis.43 Proposed mechanisms of BAF epigenetic regulation. BAF showed no detectable association with any tested HAT or HDAC either in vivo or when tested directly in vitro (unpublished observations). However, purified BAF competitively inhibited HAT-mediated acetylation of recombinant purified H3 and H4 in vitro (unpublished observations), consistent with its direct binding to H341 and H4 (reported here). We therefore propose that BAF (like linker histones H1 and H5)65 might sterically and nonspecifically inhibit access of chromatin regulators to nucleosomes. We further propose BAF also influences the epigenome selectively, by recruiting specific regulators to chromatin in vivo. As precedent, we note that HMGN1, a mobile nucleosome-binding protein, promotes H3-K14 acetylation by helping recruit the histone acetylase PCAF.66,67 This ‘recruitment’ model is based on evidence that BAF associates in vivo with several specific regulators: G9a and SET/I2PP2A (shown here) and PARP1, RBBP4, DDB1/2 and CUL4.42 Our proposal that BAF recruits SET/I2PP2A is consistent with a previous study showing H3-S10 phosphorylation decreased in BAF-downregulated cells.52 The 30% increase in the silencing mark H3-K9-Me2 seen in BAF-overexpressing cells did not reach statistical significance, but is noteworthy since we show here BAF associates directly or indirectly with G9a, one of the enzymes responsible for this mark. This mark helps retain G9a68 and further recruits heterochromatin protein 1 to maintain silencing.5 G9a also cooperates


with a specific DNA methyltransferase (Dnmt3) to regulate gene expression during retinal development,69 a process also regulated by BAF.40,70 The developing retina might be an excellent system in which to test for potential BAF-mediated recruitment of G9a as a silencing mechanism. BAF, development and pluripotency. BAF can repress specific developmentally-regulated promoters in C. elegans.35 Since BAF binds DNA nonspecifically, this promoter-specificity is attributed to protein-protein interactions. For example, BAF directly binds Crx and other ‘paired rule’ homeodomain transcription factors and thereby represses Crx-dependent genes.40 Interestingly, BAF also associates with Sox2, one of three master regulators (Oct4, Nanog, Sox2) that maintain pluripotency in embryonic stem cells.43 Downregulating BAF promoted differentiation of mouse embryonic stem cells and appeared to bias differentiation toward mesodermal or trophectodermal fates.43 This would be a fascinating system in which to explore BAF’s proposed roles as an epigenetic regulator and whether these roles depend on B-type lamins, A-type lamins, LEM-domain proteins or other BAFbinding proteins (e.g., NEMP1).70 Lamin B1 and emerin contact large specific regions of silent chromatin (‘Lamina-Associated Domains’ [LADs]).11 These regions of the human genome show ~80% overlap with large blocks of G9a-dependent H3-K9-Me2enriched silent chromatin, known as Large Organized Chromatin K9 modifications (LOCKs).71 Our discovery that BAF associates with nucleosomes and specific chromatin regulators including G9a, raises the hypothesis that BAF may promote chromatin silencing in the context of tethering to the lamina network (lamin filaments, LEM-domain proteins); for example, by inhibiting activation of certain genes, as was observed in mESC where BAF downregulation increased the expression of specific cell lineage genes promoting differentiation.43 BAF, epigenetic regulation and progeria syndromes. Three methyl marks (H3-K4-Me2, H3-K9-Me3, H3-K79-Me2) increased significantly in BAF-overexpressing cells. This ‘promethylation’ activity of BAF is intriguing, but defies simple interpretation. For example, H3-K4-Me2 is generally associated with active chromatin, while H3-K9-Me3 correlates with silent chromatin (i.e., constitutive heterochromatin) and H3-K79-Me2, is enriched in both silent and active chromatin loci.72,73 Conversely, the ‘silent’ mark H3-K27-Me1/3 (i.e., facultative heterochromatin) decreased significantly in BAF-overexpressing cells. We note that lamin-network-disrupted HGPS patient cells also show complex epigenetic changes, which include reduced H3K27-Me3 and increased H4-K20-Me3 (both silent/heterochromatin marks),22 similar to BAF-overexpressing cells. By contrast, silent mark H3-K9-Me3 was reduced in HGPS cells19,20,22 but increased in BAF-overexpressing cells. We suggest epigenetic control in HGPS is perturbed by at least two mechanisms: via loss of BAF epigenetic functions that require intact lamin filament networks and (as lamin networks collapse) via increased levels of ‘free’ BAF that mimic BAF-overexpression. Of note, BAF and lamin A can each associate with the histone chaperone RBBP4,42 which is part of several chromatin remodeling complexes and expression of which is reduced in both premature and normal aging.74

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Untangling the epigenetic roles of BAF vs. A- and B-type lamins will be challenging, particularly in HGPS cells where the entire nucleoskeleton is disrupted.20,75 Even though BAF is a small and rapidly-diffusing ‘non-structural’ protein, it is also an essential component of the nuclear lamina network.24,27,29 BAF’s importance to the nucleoskeleton is underscored by the discovery that a recessive BAF mutation causes a hereditary progeroid syndrome.44 Individuals homozygous for the BAF missense mutation A12T express BANF1 mRNA at normal levels, but have greatly reduced levels of BAF protein (~5–10% of normal). Patients with this ‘reduced-BAF’ syndrome partially resemble those with typical HGPS, but in contrast present a longer lifespan, profound skeletal abnormalities and lack of cardiovascular problems. This has important medical and therapeutic implications. At the cellular level, BAF A12T fibroblasts showed inefficient emerin localization at the NE and profoundly abnormal nuclear morphology. Both phenotypes were rescued by ectopic expression of wildtype GFP-BAF,44 consistent with evidence that BAF stabilizes binding between emerin and lamin A.33,76 Our new findings suggest BAF is also an epigenetic regulator. This adds an important new piece to the laminopathy and aging ‘puzzles’ that may help to untangle and distinguish how BAF and its nucleoskeletal partners each contribute to nuclear structure and genome function in specific tissues. Materials and Methods Ni2+ pull-down assays for H4 binding to BAF. Purified recombinant His-tagged BAF dimers (10 mg; final concentration 1.78 mM) and purified recombinant Xenopus histone H4 (xH4, 100% identical to human H4; 20 mg, final concentration 7.6 mM; kind gift from J. C. Hansen, Colorado State University) were each centrifuged (14,000 rpm, 10 min, 4°C) to remove aggregates, then mixed and gently rotated (1 h, 25°C) in binding buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 10 mM imidazole, 0.1% Triton X-100, 1 mM PMSF and benzonase (EMD Chemicals Inc.) in a final volume of 200 ml. Reactions were supplemented with 15 ml Ni-NTA agarose beads (Qiagen), incubated overnight (rotating, 4°C) and washed extensively with binding buffer. Bound proteins were eluted with 2X-Sample Buffer (2X-SB), resolved by SDS-PAGE and stained with Coomassie brilliant blue (CBB). Isolation of mononucleosomes from HeLa cells. Mononucleosomes were isolated as described,46 and then immunoprecipitated using antibodies against human BAF (15 ml each of rabbit serum 327324 and rabbit serum 5045,41 per 300 ml reaction) or no antibody as control, overnight at 4°C. Precipitates were resolved by SDS-PAGE (4–12% NuPAGETM gels; Invitrogen), transferred to nitrocellulose (Schleicher and Schuell Bioscience) and protein gel blotted using BAF serum 3273 (1:10,000 dilution). HeLa cell culture and immunoprecipitations with histonespecific antibodies. HeLa cells were cultured in Dulbecco’s modified eagle’s medium (DMEM) containing 10% fetal bovine serum (GIBCO; Invitrogen). Cells were lysed in immunoprecipitation (IP) buffer (20 mM Hepes pH 8, 150 mM NaCl, 0.1% Triton X-100, 10 mM EDTA, 2 mM EGTA, 2 mM DTT,

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1 mM PMSF and benzonase [300 U/ml; EMD Chemicals Inc.]) on ice, 20 min. Lysates were sonicated 3 times (15 sec each) and centrifuged (20 min, 50,000 rpm in a Beckman TL-100 ultracentrifuge, 4°C) to remove insoluble material. Supernatants were incubated (30 min, 4°C) with GammaBind G Sepharose (Amersham Biosciences Corp.), then centrifuged (1,500 rpm, Eppendorf 5415C Microcentrifuge, 4°C) to obtain cleared cell lysates. For each immunoprecipitation, 200 ml cleared lysate was incubated 1 h (4°C) with each indicated antibody: 5–10 ml antihistone H3 #1791, 10 ml anti-H3-K27-Me1/3 #14222, 4 ml antiH4-K20-Me #9051 (all from Abcam); 2–4 ml anti-H3-K4-Me2 #07–030, 5 ml anti-H3-K9-Me2 #07–441, 15 ml anti-H3-S10-P #05–817, 1–2 ml anti-H4-Ac5 #06–946, 1–2 ml anti-H4-K5-Ac #07–327 or 4 ml anti-H4-K16-Ac #07–329 (all from Millipore; Billerica, MA) or no serum as control, then supplemented with 15 ml GammaBind G Sepharose (Amersham Biosciences Corp.) and rotated overnight (4°C), pelleted (1,500 rpm, Eppendorf 5415C Microcentrifuge, 4°C) and washed three times with IP buffer. Nonspecifically-associated proteins were competed by two 15 min (37°C) incubations with non-specific peptide (0.1 mM p53 peptide; C. Wolberger, Johns Hopkins University) and washed once with IP buffer. Bound proteins were eluted using 2X-SB, resolved by SDS-PAGE (4–12% NuPAGETM gels; Invitrogen), transferred and protein gel blotted using rabbit serum 3273 against BAF (1:10,000 dilution) or rabbit antihistone H3 (1:2,500; Abcam #1791). Images were acquired using VersaDoc 5000 (BioRad) and quantified by densitometry using Quantity One 1 Software (BioRad). Transient expression and immunoprecipitation of FLAGtagged proteins. Constructs encoding FLAG-PCAF (from P. Puigserver, Dana Farber Cancer Institute), FLAG-p300 and FLAG-Tip60 (from W. Gu, Columbia University), FLAGHDAC1 (from R. Roeder, Rockefeller University), FLAG-G9a (from M. Stallcup, University of Southern California), FLAGSET/I2PP2A (from T. Papamarcaki, University of Ioannina, Greece), FLAG-Aurora B (from H. Wang, University Alabama, Birmingham), FLAG-BAF (from P. Traktman, Medical College of Wisconsin), FLAG-H3.1 or the empty FLAG vector (described in ref. 41) were transiently transfected into HeLa cells using LT1 (Mirus) per manufacturer instructions and expressed for 24–48 h. Cells were then lysed by incubating 10 min on ice in lysis buffer (20 mM Hepes pH 8, 300 mM NaCl, 0.3% Triton X-100, 0.2 mM EDTA, 0.2 mM EGTA, 1.5 mM MgCl2, 2 mM DTT, 10% glycerol, 1 mM PMSF and 300 U/ml benzonase [EMD Chemicals Inc.]), then diluted 1:1 v/v with lysis buffer to achieve final concentrations of 150 mM NaCl, 0.15% TX-100 and 5% glycerol, incubated 10 more min on ice, sonicated four times (15 sec each) and centrifuged (50,000 rpm, 30 min, 4°C) to recover soluble proteins for subsequent immunoprecipitation. For each immunoprecipitation reaction 250 ml lysate was incubated 3–4 h (4°C) with 3 ml FLAG-M2 agarose beads (Sigma-Aldrich), then pelleted and washed four times with lysis buffer (150 mM NaCl, 0.15% Triton-X). Nonspecifically-associated proteins were competed by two 15 min (37°C) incubations with a non-specific peptide (0.1 mM Sir2 peptide, from C. Wolberger, Johns Hopkins University) and then washed once with lysis buffer.

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Bound proteins were eluted with 2X-SB, resolved by SDS-PAGE (4–12% NuPAGETM gels; Invitrogen) and protein gel blotted with rabbit anti-FLAG (1:7,000; Sigma #F7425), rabbit anti-G9a (1:500; Cell Signaling #3306), rabbit anti-SET (1:300; Abcam #1183), rabbit anti-histone H3 (1:2,500; Abcam #1791) or rabbit serum 3273 against BAF (1:10,000). Transient transfection of HeLa cells for metabolic labeling with [14C]-acetate. An N-terminally His-tagged human BAF (H6BAF) construct76 or corresponding control empty vector, were transiently transfected into HeLa cells using LT1 reagent (Mirus) per manufacturer instructions and expressed 24–48 h. Then new media containing [14C]-sodium acetate (~90–150 mCi final concentration; 57.5 mCi/mmol; Sigma) or an equal volume of solvent (water), plus deacetylase inhibitor (1 mM TSA)77 were added to cells for 4 h. Cells were then harvested, pelleted and 20% of each pellet was lysed and protein gel blotted to quantify BAF expression levels. The remaining cell pellet (80%) was fractionated to generate nuclei; histones were isolated by acidextraction as described,78 resolved by SDS-PAGE (4–12% NuPAGETM gels; Invitrogen) and stained with CBB to quantify protein levels of H3, H4 and H2A/H2B (which co-migrated in these gels). Incorporated [ C]-acetate was measured by fluorography, quantified by densitometry (Quantity One 1 Software; BioRad) and plotted as the percentage of [14C]-incorporated per total histone, with control cells set to 100%. Significance was determined using a two-tailed Student T-test. Analysis of histone posttranslational modifications in 293: BAF and 293:CAT cells. HEK293 cell lines with stable Tetinducible expression of FLAG-BAF (293:BAF cells) and corresponding control 293:CAT cells were from P. Traktman.50 Cells were cultured in DMEM containing 10% Fetal Bovine Serum (GIBCO; Invitrogen), 100 mg/ml hygromycin (Invitrogen) and 15 mg/ml blasticidin (Invitrogen). Cells were treated 32–52 h with Tet (1.5 mg/ml; Sigma) to induce FLAG-BAF expression and harvested: 20% was used to generate whole cell lysates and 80% was used to acid-extract histones (described above). Whole cell extracts were resolved by SDS-PAGE and protein gel blotted with antibodies specific for BAF (serum 3273; 1:10,000), c-tubulin (loading control; 1:500,000; Sigma #T6557), a-actin (loading control; 1:10,000; Millipore), HDAC4 (1:1,000; BioLegend), HDAC2 (1:2,000; sc-7899, Santa Cruz Biotechnology, Inc.), G9a (1:500; Cell Signaling #3306), SET (1:300; Abcam #1183), lamins A/C (1:1,000; Millipore #MAB3211), emerin (1:2,000; Santa Cruz #sc-15378) or cyclin E (1:500; Santa Cruz #sc-248). Protein levels were quantified by densitometry (described above) and normalized to c-tubulin or a-actin. Acid-extracted histones were resolved by SDS-PAGE and protein gel blotted using antibodies specific for the following marks: H3-K4-Me2 (1:10,000; Millipore #07–030), H3-K9-Me2 (1:30,000; Millipore #07–441), H3-K9-Me3 (1:20,000; Abcam #8898), H3-K9/K14Ac (1:30,000; Millipore #06–599), H3-S10-P (1:5,000; Millipore #05–817), H3-K27-Me1/Me3 (1:20,000; Abcam #14222), H3S28-P (1:5,000; Millipore #07–145), H3-K79-Me2 (1:30,000; Abcam #3594), H4-K5-Ac (1:10,000; Millipore #06–759), H4K8-Ac (1:10,000; Millipore #06–760), H4-K12-Ac (1:10,000; Millipore #06–761), H4-K16-Ac (1:10,000; Millipore #06–762), 14


H4-K20-Me (1:30,000; Abcam #9051) and H4-Ac5 (1:30,000; Millipore #06–946). Results for each mark specific antibody were quantified by densitometry as described above, normalized to each Ponceau-stained H3 or H4 band and plotted as a percentage of the corresponding 293:CAT signal. Significance was determined using a two-tailed Student T-test. BrdU incorporation and analysis of asynchronous cells by flow cytometry. Asynchronous 293:BAF or 293:CAT cells were treated with Tet (1.5 mg/ml) to induce FLAG-BAF expression for 35 h. The distribution of cells in G0/G1, S and G2/M phases was estimated for each sample from histograms of DNA content using the ModFit LT software (Becton Dickinson and Co.). This program fit a binomial curve to the G0/G1 and G2/M peaks and determined the percentage of cells in S phase by subtracting the percentage of cells in G0/G1 and G2/M. To analyze DNA content, cells were stained with 50 mg/ml propidium iodide (Sigma #P4170) plus 30 U/ml RNase A. Data was collected using a FACScan or FACScalibur instrument (Becton Dickinson and Co.). For each sample, cell aggregates were gated out and 10,000 cell events were analyzed. To specifically measure the percentage of cells in S phase, samples were pulsed with 30 mM 5-Bromo-2’-Deoxyuridine (Sigma #B5002) for 30 min, then washed, harvested, fixed, labeled with anti-BrdU and analyzed by flow cytometry. Briefly, we resuspended 106 cells in denaturating buffer (2 M HCl in PBS/0.5% TritonX-100) for 30 min (25°C), then pelleted and resuspended in neutralizing buffer (0.1 M sodium tetraborate in PBS, pH 8.5), washed once with PBS and incubated ~1 h (37°C) with FITC-conjugated mouse anti-BrdU (#556028; BD Biosciences) in PBS/3% BSA/0.05% Tween. DNA was propidium iodide stained as above. Harvest of cells for incubation with mouse anti-MPM-2 (1:200; #05–368) primary antibody from Millipore and analysis by flow cytometry was as previously described.79 FITC-conjugated goat anti-mouse (1:1,000; #115–095–146) secondary antibody was from Jackson Laboratories. The percentage of MPM-2 positive cells was determined using CellQuest version 3.3 software (Becton Dickinson and Co.). Acknowledgments

We are grateful to P. Traktman (Medical College of Wisconsin) for the 293:BAF and 293:CAT cell lines; R. Stolle for purified BAF; M. J. Eddins and K. Fahie (Johns Hopkins School of Medicine) for chromatography help and M. J. Eddins for Figure 1E. We thank C. Lerin and P. Puigserver (Dana Farber Cancer Institute), I. Celic and J. Boeke (Johns Hopkins School ofMedicine), W. L. Kraus (Cornell), J. Th’ng (Northwestern Ontario Regional Cancer Centre) and C. Slawson and L. Blosser (Johns Hopkins Flow Cytometry Facility) for reagents and advice. We thank W. Gu (Columbia), R. Roeder (Rockefeller), M. R. Stallcup (Univ. Southern California), T. Papamarcaki (Univ. Ioannina) and H. Wang (Univ. Alabama) for constructs and S. Taverna and the Wilson lab for insightful discussions. This work was funded by American Heart Association Predoctoral fellowship 0615601U (R.M.) and National Institutes of Health RO1 GM48646 (K.L.W.).

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Disclosure of Potential Conflicts of Interest

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41. Montes de Oca R, Lee KK, Wilson KL. Binding of barrier to autointegration factor (BAF) to histone H3 and selected linker histones including H1.1. J Biol Chem 2005; 280:42252-62; PMID:16203725; http:// dx.doi.org/10.1074/jbc.M509917200 42. Montes de Oca R, Shoemaker CJ, Gucek M, Cole RN, Wilson KL. Barrier-to-autointegration factor proteome reveals chromatin-regulatory partners. PLoS ONE 2009; 4:e7050; PMID:19759913; http://dx.doi.org/ 10.1371/journal.pone.0007050 43. Cox JL, Mallanna SK, Ormsbee BD, Desler M, Wiebe MS, Rizzino A. Banf1 is required to maintain the selfrenewal of both mouse and human embryonic stem cells. J Cell Sci 2011; 124:2654-65; PMID:21750191; http://dx.doi.org/10.1242/jcs.083238 44. Puente XS, Quesada V, Osorio FG, Cabanillas R, Cadinanos J, Fraile JM, et al. Exome sequencing and functional analysis identifies BANF1 mutation as the cause of a hereditary progeroid syndrome. Am J Hum Genet 2011; 88:650-6; PMID:21549337; http://dx. doi.org/10.1016/j.ajhg.2011.04.010 45. Park YJ, Luger K. Histone chaperones in nucleosome eviction and histone exchange. Curr Opin Struct Biol 2008; 18:282-9; PMID:18534842; http://dx.doi.org/ 10.1016/j.sbi.2008.04.003 46. Kim MY, Mauro S, Gevry N, Lis JT, Kraus WL. NAD +-dependent modulation of chromatin structure and transcription by nucleosome binding properties of PARP-1. Cell 2004; 119:803-14; PMID:15607977; http://dx.doi.org/10.1016/j.cell.2004.11.002 47. Foltz DR, Jansen LE, Black BE, Bailey AO, Yates JR, 3rd, Cleveland DW. The human CENP-A centromeric nucleosome-associated complex. Nat Cell Biol 2006; 8:458-69; PMID:16622419; http://dx.doi.org/10. 1038/ncb1397 48. Luger K, Möder AW, Richmond RK, Sargent DF, Richmond TJ. Crystal structure of the nucleosome core particle at 2.8 A resolution. Nature 1997; 389:251-60; PMID:9305837; http://dx.doi.org/10.1038/38444 49. Umland TC, Wei S-Q, Craigie R, Davies DR. Structural Basis of DNA Bridging by Barrier-toAutointegration Factor. Biochemistry 2000; 39:9130-8; PMID:10924106; http://dx.doi.org/10.1021/bi000572w 50. Wiebe MS, Traktman P. Poxviral B1 kinase overcomes barrier to autointegration factor, a host defense against virus replication. Cell Host Microbe 2007; 1:187-97; PMID:18005698; http://dx.doi.org/10.1016/j.chom. 2007.03.007 51. Bolden JE, Peart MJ, Johnstone RW. Anticancer activities of histone deacetylase inhibitors. Nat Rev Drug Discov 2006; 5:769-84; PMID:16955068; http://dx. doi.org/10.1038/nrd2133 52. Haraguchi T, Koujin T, Osakada H, Kojidani T, Mori C, Masuda H, et al. Nuclear localization of barrier-toautointegration factor is correlated with progression of S phase in human cells. J Cell Sci 2007; 120:1967-77; PMID:17519288; http://dx.doi.org/10.1242/jcs.03461 53. Andreassen PR, Margolis RL. Microtubule dependency of p34cdc2 inactivation and mitotic exit in mammalian cells. J Cell Biol 1994; 127:789-802; PMID:7962060; http://dx.doi.org/10.1083/jcb.127.3.789 54. Davis FM, Tsao TY, Fowler SK, Rao PN. Monoclonal antibodies to mitotic cells. Proc Natl Acad Sci USA 1983; 80:2926-30; PMID:6574461; http://dx.doi.org/ 10.1073/pnas.80.10.2926 55. Nowak SJ, Pai CY, Corces VG. Protein phosphatase 2A activity affects histone H3 phosphorylation and transcription in Drosophila melanogaster. Mol Cell Biol 2003; 23:6129-38; PMID:12917335; http://dx.doi. org/10.1128/MCB.23.17.6129-6138.2003


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