SF2 and Dissociation from Mitotic Chromosomes Is Modulated by Histone H3 Serine 10 Phosphorylation

Molecular Cell

Article Chromatin Binding of SRp20 and ASF/SF2 and Dissociation from Mitotic Chromosomes Is Modulated by Histone H3 Serine 10 Phosphorylation Rebecca J. Loomis,1,6 Yoshinori Naoe,1,4,6 J. Brandon Parker,1,2 Velibor Savic,2 Matthew R. Bozovsky,1 Todd Macfarlan,2,5 James L. Manley,3 and Debabrata Chakravarti1,* 1Division of Reproductive Biology Research, Department of Obstetrics and Gynecology and Robert H. Lurie Comprehensive Cancer Center, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA 2Graduate Program in Biological Sciences, University of Pennsylvania, Philadelphia, PA 19104, USA 3Department of Biological Sciences, Columbia University, New York, NY 10027, USA 4Present address: Laboratory for Transcriptional Regulation, Research Center for Allergy and Immunology, RIKEN, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, 230-0045 Japan 5Present address: Gene Expression Laboratory, The Salk Institute, La Jolla, CA 92037, USA 6These authors contributed equally to this work *Correspondence: [email protected] DOI 10.1016/j.molcel.2009.02.003

SUMMARY

Histone H3 serine 10 phosphorylation is a hallmark of mitotic chromosomes, but its full function remains to be elucidated. We report here that two SR protein splicing factors, SRp20 and ASF/SF2, associate with interphase chromatin, are released from hyperphosphorylated mitotic chromosomes, but reassociate with chromatin late in M-phase. Inhibition of Aurora B kinase diminished histone H3 serine 10 phosphorylation and increased SRp20 and ASF/ SF2 retention on mitotic chromosomes. Unexpectedly, we also found that HP1 proteins interact with ASF/SF2 in mitotic cells. Strikingly, siRNA-mediated knockdown of ASF/SF2 caused retention of HP1 proteins on mitotic chromatin. Finally, ASF/SF2depleted cells released from a mitotic block displayed delayed G0/G1 entry, suggesting a functional consequence of these interactions. These findings underscore the evolving role of histone H3 phosphorylation and demonstrate a direct, functional, and histone-modification-regulated association of SRp20 and ASF/SF2 with chromatin. INTRODUCTION Histones play a central role in modulating protein assembly on and release from the chromatin fiber, thereby regulating numerous chromatin-based processes, including transcription, replication, condensation, repair, and segregation (Grewal and Moazed, 2003; Groth et al., 2007; Kouzarides, 2007; Li et al., 2007). The dynamic interaction of proteins on chromatin is regulated primarily by posttranslational modifications of the N-terminal tails of each of the four core histones (Grunstein, 1998; Kouzarides, 2007; Luger and Richmond, 1998; Strahl

and Allis, 2000). The diversity of histone modifications, including acetylation, methylation, phosphorylation, ADP-ribosylation, and ubiquitination, has led to the ‘‘histone code’’ hypothesis and a chromatin signaling network model in which each modification, alone or in combination, serves a distinct function (Schreiber and Bernstein, 2002; Strahl and Allis, 2000). In essence, these modifications direct recruitment or release of proteins that alter chromatin structure and/or regulate DNAbased processes (Kutney et al., 2004). For example, the bromodomain of human p300/CBP-associated factor binds acetylated lysine residues within the tail of histone H3 and H4, and is associated with transcriptionally active genes (Dhalluin et al., 1999). In contrast, chromodomain protein heterochromatin protein 1 (HP1) binding to methylated lysine 9 of histone H3 contributes to the maintenance of large heterochromatic chromosomal domains and transcriptionally repressed genes (Bannister et al., 2001; Lachner et al., 2001; Maison and Almouzni, 2004). Histone H3 serine 10 phosphorylation (H3S10P) plays a critical role in chromosome condensation and chromosome segregation during mitosis and meiosis (Mellone et al., 2003; Nowak and Corces, 2004; Wei et al., 1998, 1999). During M-phase, when H3S10P is high, most HP1 is released from chromatin despite persistence of histone H3 lysine 9 trimethylation. Thus, a methyl-phospho switch is believed to regulate HP1 dissociation from mitotic chromosomes (Fischle et al., 2005; Hirota et al., 2005). Results from another study suggest that phospho-acetylation (S10P-K14Ac) of histone H3 is involved in release of HP1 from chromatin (Mateescu et al., 2004). While these results point to a critical role for H3S10P in chromosome dynamics during mitosis, the full function of this particular histone H3 modification remains to be elucidated. Members of the SR protein family are known to be important for splicing of mRNA precursors (Fu, 1993; Graveley, 2000; Manley and Tacke, 1996). These proteins are modular and comprised of one or more RNA recognition motifs (RRMs) and an arginine-serine rich (RS) domain. The RRM recognizes specific target sequences within the pre-mRNA, while the RS domain, which contains

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multiple phosphorylated serine residues, functions by modulating protein-protein, and perhaps protein-RNA interactions (Fu, 1993; Shen and Green, 2004). SR proteins also participate in other cellular processes (Huang and Steitz, 2005). For example, SR proteins, ASF/SF2 and SC35, have been shown to be critical for maintenance of genome stability and cell-cycle progression (Li and Manley, 2005; Li et al., 2005; Xiao et al., 2007). Genetic depletion of ASF/SF2 led to formation of transcriptional R-loops and DNA rearrangements and caused G2/M cell-cycle arrest, suggesting that SR proteins may have a role in chromatin dynamics and function. Similarly, in mouse embryonic fibroblasts, loss of SC35 resulted in G2/M cell-cycle arrest and genomic instability. To gain more insight into the identity and function of chromatin-associated proteins and the emerging roles of H3S10P, we utilized a protein affinity purification scheme. Unexpectedly, we found that two SR proteins, SRp20 and ASF/SF2, bind to histone tails and their chromatin binding is regulated by H3S10P. We provide several lines of in vitro and in vivo evidence, demonstrating that these SR proteins associate with interphase and late/post-mitotic chromatin but are dissociated from mitotic chromatin. Strikingly, siRNA-mediated knockdown of ASF/SF2 led to retention of HP1 proteins on mitotic chromatin and caused a delay in G0/G1 entry of cells. We propose that these SR proteins have a ‘‘chromatin-sensor’’ activity, which together with HP1, may be necessary for proper cell-cycle progression. RESULTS SRp20 and ASF/SF2 Exhibit H3 Serine 10 Phosphorylation Sensitive Histone Tail Binding Properties To identify modification-sensitive histone-interacting proteins, we incubated HeLa cell nuclear extracts with differentially modified biotinylated histone peptides and analyzed bound proteins by SDS-PAGE and silver stain followed by mass spectrometry of excised bands. The biotinylated histone tail peptides used in the initial experiments were histone H3 tail peptides, methylated at lysine 9 (H3-K9Me) and phosphorylated at serine 10 (H3-S10P). Mass spectrometric analysis of selected isolated bands that bound to H3-K9Me, but not H3-S10P, were identified as HP1a, -b, -g, and SRp20 (Figure 1A). The role of H3-K9Me and H3-S10P in binding and release of HP1 proteins from chromatin has been reported before (Bannister et al., 2001; Fischle et al., 2005; Hirota et al., 2005; Lachner et al., 2001; Maison and Almouzni, 2004). The unexpected binding of the SR protein splicing factor to this histone H3 tail prompted us to further characterize the SRp20-histone H3 interaction in vitro. HeLa cell nuclear extracts were incubated with selected biotinylated histone H3 tail peptides and bound proteins were analyzed by SDS PAGE followed by immunoblot with anti-SRp20 antibody. Endogenous SRp20 bound to unmodified histone H3, H3-K9Ac, H3-K14Ac, and H3-K9Me peptides. However, phosphorylation of serine 10 significantly reduced SRp20 association with histone H3 peptide tails (Figure 1B). We next investigated whether another SR family member, ASF/SF2, also binds histones in a modification-selective manner. To this end, HeLa cell nuclear extracts were incubated with biotinylated histone H3 tail peptides. The histone H3 peptide

Figure 1. SRp20 and ASF/SF2 Bind Histone H3 in a ModificationSelective Manner (A) Silver stain of a gel showing the proteins that differentially bound to indicated histone H3 peptide tails. The indicated bands were identified by protein microsequencing. (B and C) Endogenous SRp20 (B) or ASF/SF2 (C) does not bind histone H3 phosphorylated at serine 10. HeLa cell nuclear extracts were incubated with indicated biotinylated histone H3 tail peptides. Bound proteins were subject to immunoblot analysis with anti-SRp20 or ASF/SF2 antibody. (D) Domain structure of SRp20 (top panel). SRp20 binds histones through the RS domain (lower panel). Acid-extracted histones isolated from asynchronously growing HeLa cells were separately incubated with equal amounts of indicated GST-fusion proteins and pulled down with glutathione-sepharose beads. Bound proteins were subject to immunoblot analysis with anti-histone H3 antibody.

tails were prebound to streptavidin agarose beads, and the complexes were analyzed by immunoblotting with anti-ASF/ SF2 antibody. Endogenous ASF/SF2 indeed bound strongly to the unmodified, methylated, and acetylated histone H3 tailpeptides, while no detectable binding between ASF/SF2 and either H3-S10P or H3-S10P/K14Ac peptides was observed (Figure 1C). As a control, neither SRp20 (data not shown) nor ASF/SF2 bound to histone H2B tails, further demonstrating the specificity of the histone H3 interaction (Figure 1C).

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Figure 2. Phosphorylation of SRp20 and ASF/SF2 Determines Their Modification-Selective Binding to Histone H3 (A) Bacterially expressed SRp20 bound both unmodified histone H3 and histone H3 phosphorylated at serine 10. GST-SRp20 or GST alone was incubated with acid-extracted HeLa cell histones. Bound proteins were subject to immunoblot analysis with antihistone H3 or anti-phosphoserine 10 histone H3 antibodies. (B) Endogenous SRp20 is phosphorylated. HeLa cell nuclear extracts or GST-SRp20 were treated with or without CIP and run on SDS-PAGE for immunoblot analysis. (C) GST-SRp20 can be phosphorylated in vitro. Recombinant GST-SRp20 was either mock incubated, incubated with buffer or incubated with ATP and HeLa cell S100 fraction, and analyzed by immunoblot with anti-SRp20 and anti-phosphoserine antibodies. (D) Phosphorylated GST-SRp20 no longer binds to histone H3 when phosphorylated at serine 10. GST-SRp20 was preincubated with ATP and HeLa cell S100 fraction prior to GST pull-down assay with acid-extracted histones. (E) Endogenous hyperphosphorylated SR proteins do not bind to histone H3-S10P peptide tail, whereas in vitro hypophosphorylated SR proteins do. Endogenous SRp20 and ASF/SF2 were mock-incubated or incubated with CIP, followed by incubation with phosphoserine 10 histone H3-biotinylated peptide and analyzed by immunoblot with anti-SRp20 and anti-ASF/SF2 antibodies. Input represents 50 mg or 5% of total soluble protein fraction used in the immunoprecipitation reactions. Unbound samples represent 10% of the total volume recovered following immunoprecipitation reactions (approximately equivalent to 100 mg total protein used in the immunoprecipitation). Bound fractions represent all the proteins from the immunoprecipitation reactions that bound to the histone H3S10P peptide.

The RS Domain of SRp20 Is Sufficient for Histone H3 Association Since SRp20 bound to the histone H3 tail in vitro, we wanted to identify the domain of SRp20 necessary for this interaction. SRp20 consists of one RRM and a carboxyl-terminal RS domain (Bourgeois et al., 2004). We expressed and purified these domains as GST fusion-proteins and analyzed their interaction with acid-extracted histones isolated from asynchronously growing HeLa cells. Under conditions where approximately equal amounts of fusion proteins were used, the GST-RS domain, but not the GST-RRM domain, associated with histone H3 (Figure 1D), suggesting that the RS domain of SRp20 is sufficient for the histone H3 interaction. Phosphorylation of SRp20 and ASF/SF2 Is Critical for Their Dissociation from Hyperphosphorylated Histone H3 Tails To confirm the specificity of SRp20 binding to histone H3, GST pull-down assays were performed with recombinant SRp20 and acid-extracted histones isolated from asynchronously growing HeLa cells. Bound proteins were analyzed by immunoblot using anti-histone H3 and anti-phosphoserine 10 histone H3 antibodies. Unlike the endogenous protein, bacterially expressed GST-SRp20 bound both unmodified histone H3 and histone H3 phosphorylated at serine 10 (Figure 2A). SR proteins are known to be phosphorylated at multiple sites in vivo (Bourgeois et al., 2004; Ngo et al., 2005). Therefore, we investigated whether the phosphorylation status of SRp20 accounted for the discrepancy in histone binding between endogenous and recombinant SRp20. To test this hypothesis, we treated HeLa cell nuclear extracts or recombinant GST-SRp20 with calf intestinal phosphatase (CIP). Immunoblot analysis revealed a change in the SDS-PAGE mobility of cellular SRp20, but not of GST-

SRp20, suggesting that only endogenous SRp20 was phosphorylated (Figure 2B). We then asked whether in vitro phosphorylated GST-SRp20 fails to associate with histone H3-S10P tails. GST-SRp20 was phosphorylated by preincubation with a HeLa cell S100 fraction (a major source of the SR protein kinase 1, SRPK1, activity) and ATP (Figure 2C) (Gui et al., 1994b). Following incubation, the beads were washed to remove additional proteins and ATP prior to addition of acid-extracted histones. In vitro phosphorylated GST-SRp20 failed to associate with serine 10 phosphorylated histone H3 (Figure 2D, lane 4). In contrast, unphosphorylated GST-SRp20 bound to serine 10 phosphorylated histone H3 (Figure 2D, lane 3). The above data suggest that the phosphorylation state of ASF/ SF2 and SRp20 is an important determinant in their histone-H3binding capabilities. To test this hypothesis, we mock- or CIPtreated endogenous SRp20 and ASF/SF2 and determined their ability to associate with a biotinylated histone H3-S10P peptide tail. A mobility shift was observed for SRp20 and ASF/SF2 treated with CIP, suggesting that endogenous SRp20 and ASF/SF2 were, as expected, phosphorylated (Figure 2E, input). Equal protein amounts were added in both CIP and +CIP inputs as determined by protein quantification, despite the discrepancy in immunoblot signal (Figure 2E, input). Consistent with the above data with purified recombinant proteins, CIP-treated endogenous SRp20 and ASF/SF2 associated with histone H3-S10P peptide, whereas their phosphorylated counterparts did not despite the fact that 1 mg of nuclear extract was used in the binding experiment (Figure 2E, see figure legend for details). These results show that phosphorylated SRp20 and ASF/SF2 fail to bind to histone H3-S10P tails significantly and that association requires dephosphorylation of the histone H3 tail. It remains to be determined whether a single or multiple site phosphorylation event influences the binding properties of SRp20 and ASF/SF2.

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Figure 3. Analysis of Endogenous SRp20 and ASF/SF2-Histone Interactions (A and B) Endogenous SRp20 and ASF/SF2 bound to unmodified, acetylated, and methylated nucleosomes, but neither bound to phosphorylated nucleosomes. Nucleosomes were prepared from control and nocodazole-treated cells as described in the Experimental Procedures and immunoprecipitated with anti-SRp20 or anti-ASF/SF2 antibodies. Bound proteins were subject to immunoblot analysis with (A) anti-SRp20 (left panels) or ASF/SF2 (right panels), (B) anti-histone H3 (first panel), anti-phosphoserine 10 histone H3 (second panel), anti-acetyl lysine 9 histone H3 (third panel), or anti-dimethyl lysine 9 histone H3 (fourth panel) antibodies. Inputs represent 5% of the amount used for immunoprecipitation. (C) Both SRp20 and ASF/SF2 colocalized with total histone H3 and interphase chromatin. HeLa cells were fixed and immunostained for SRp20 (Cb) or ASF/SF2 (Cg) and total histone H3 (Ca and Cf). Hoechst staining showed nuclear staining of DNA (Cc and Ch). Merged images of SRp20/ histone H3 (Cd), SRp20/DNA (Ce), ASF/SF2/ histone H3 (Ci), and ASF/SF2/DNA (Cj) are shown. A section from each field was enlarged to better visualize colocalization (Cd0 , Ce0 , Ci0 , and Cj0 ). Data are representative of three independent experiments for each ASF/SF2 and SRp20 with histone H3 where approximately 30 interphase cells were analyzed.

Analysis of Endogenous SRp20 and ASF/SF2 Interaction with Nucleosomes To confirm the in vitro peptide binding data, we next examined whether SRp20 and ASF/SF2 interact with nucleosomes in intact cells. Previous findings indicate that in mitotic cells, global levels of histone H3 acetylation and methylation do not change, whereas H3 phosphorylation significantly increases (Fischle et al., 2005). To increase histone H3S10P, cells were treated with 50 ng/ml nocodazole for 12 hr. Chromatin fractions were prepared by lysing cells in hypotonic buffer. Nuclei from cells grown under normal conditions or treated with nocodazole were isolated by low speed centrifugation and subjected to micrococcal nuclease (MNase) digestion to prepare nucleosomes for immunoprecipitation with anti-SRp20 or anti-ASF/ SF2 antibodies. Nocodazole treatment did not significantly alter SRp20 and ASF/SF2 levels, and both could be specifically

immunoprecipitated with their respective antibodies from control- and nocodazoletreated cells (Figure 3A). Nucleosomes with acetylated, methylated, and phosphorylated histone H3 are present in nocodazole-treated cells (Figures 3B, input). Endogenous SRp20 and ASF/ SF2 coimmunoprecipitated with nucleosomes containing unmodified (Figure 3B, first panel), acetylated-at-lysine-9 (Figure 3B, third panel), and methylated-at-lysine-9 (Figure 3B, fourth panel) histone H3. Importantly, we failed to detect any measurable interaction of SRp20 or ASF/SF2 with nucleosomes containing H3S10P (Figure 3B, second panel). Additionally, SRp20 coimmunoprecipitated with unmodified histone H3 in control cells (Figure 3B, first panel, left). To support our immunoprecipitation results suggesting that SRp20 and ASF/SF2 associate with endogenous histones, we performed colocalization studies of SRp20 and ASF/SF2 with histone H3 in asynchronously growing HeLa cells using immunocytochemistry. As expected, the level of H3S10P was low to undetectable in interphase cells (data not shown). While the SR proteins stained diffusely throughout the nucleus with characteristic nuclear-speckled patterns (Caceres et al., 1997; Lamond and Spector, 2003), we found that a significant amount of SRp20 and ASF/SF2 colocalized with histone H3 in interphase

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Figure 4. ASF/SF2 Dissociation from Mitotic Chromatin Is Dependent on Serine 10 Phosphorylation ASF/SF2 is dissociated from hyperphosphorylated mitotic chromosomes. (A) ASF/SF2 is primarily excluded from chromatin during prometaphase (Aa–Af), metaphase (Ag–Al), and anaphase (Am–Ar) and reassociates with histone H3 as serine 10 phosphorylation levels decrease during telophase (As–Ax). HeLa cells were synchronized using a double thymidine block, released and blocked in M-phase using a short treatment with nocodazole (2 hr). Cells were released from the M-phase block and collected for microscopy every 30 min for analysis of ASF/SF2-chromatin association in relation to histone H3S10P levels. Data are representative of three independent experiments where 100 mitotic cells were analyzed (approximately 25/ stage). (B) ASF/SF2 dissociation from mitotic chromatin is dependent on H3S10P. Mitotically growing HeLa cells were treated without (Ba–Be0 ) or with (Bf–Bj0 ) Aurora B kinase inhibitor ZM447439. Cells were fixed and immunostained with ASF/SF2 (Bb and Bg) and histone H3-S10P (Ba and Bf) antibodies. Hoechst staining identified condensed chromatin (Bc and Bh). A section from each field was enlarged to better visualize colocalization or a lack thereof of ASF/SF2 and phosphoserine 10-histone H3 (Bd and Bd0 ; Bi and Bi0 ), ASF/SF2 and chromosome (Be and Be0 ; Bj and Bj0 ). Data are representative of three independent experiments, where approximately 40 mitotic cells were analyzed. All stages (prometaphase, metaphase, anaphase, and telophase) demonstrated similar colocalization patterns—dissociation of ASF/SF2 from mitotic chromatin when H3S10P was high and retention when H3S10P was inhibited with the Aurora B kinase inhibitor. (C and D) Inhibition of histone H3S10P increased retention of ASF/SF2 on mitotic chromatin without affecting ASF/SF2 protein levels. Mitotically growing HeLa cells were treated without () or with (+) Aurora B kinase inhibitor ZM447439. Chromatin-enriched fraction (C) or whole-cell extract (D) was subject to immunoblot analysis with indicated antibodies.

cells (Figure 3C, top panel, Figures Cd and Cd0 , and bottom panel, Figures Ci and Ci0 , respectively). Similarly, SR protein/ DNA merged images show that a significant amount of endogenous SRp20 and ASF/SF2 associated with chromosomes in interphase cells (Figures 3Ce, Ce0 , Cj, and Cj0 ). Together, these results strongly suggest association of SRp20 and ASF/SF2 with interphase chromatin and H3S10P-sensitive interaction of SRp20 and ASF/SF2 with nucleosomes/chromatin. SRp20 and ASF/SF2 Are Excluded from Histone H3 Serine 10 Phosphorylated Mitotic Chromatin and Reassociate with Postmitotic Hypophosphorylated Chromatin In light of our observations that SRp20 and ASF/SF2 associated with interphase chromatin but failed to interact with histone H3

phosphorylated at serine 10 in vitro and with hyperphosphorylated histones/nucleosomes isolated from cultured cells, we examined association of the SR proteins with chromatin as a consequence of histone phosphorylation as cells progress through mitosis. HeLa cells were synchronized by a double thymidine block and mitotically arrested with a short nocodazole treatment 9 hr after release from the second thymidine block to enhance the number of cells in mitosis. Mitotic cells were then harvested, fixed with paraformaldehyde, permeabilized with methanol, and probed with anti-ASF/SF2 and anti-histone H3-S10P antibodies followed by Hoechst staining for DNA (Figure 4B). Consistent with previous reports, mitotic cells showed extensive histone H3 hyperphosphorylation (Figure 4Ba) (Hendzel et al., 1997; McManus and Hendzel, 2006). In contrast to its extensive association with interphase chromatin (Figure 3C),

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Figure 5. SRp20 Dissociation from Mitotic Chromatin Is Dependent on Serine 10 Phosphorylation SRp20 is dissociated from hyperphosphorylated mitotic chromosomes. (A) SRp20 is primarily excluded from chromatin during prometaphase (Aa–Af), metaphase (Ag–Al), and anaphase (Am–Ar) and reassociates with histone H3 as serine 10 phosphorylation levels decrease during telophase (As–Ax). Experimental procedures and data analysis are similar to those described under Figure 4. (B) SRp20 dissociation from mitotic chromatin is dependent on H3S10P. Mitotically growing HeLa cells were treated without (Ba–Be0 ) or with (Bf–Bj0 ) Aurora B kinase inhibitor ZM447439. Cells were fixed and immunostained with SRp20 (Bb and Bg) and histone H3-S10P (Ba and Bf) antibodies. Hoechst staining identified condensed chromatin (Bc and Bh). A section from each field was enlarged to better visualize colocalization or a lack thereof of SRp20 and phosphoserine 10histone H3 (Bd and Bd0 ; Bi and Bi0 ) and SRp20 and chromosome (Be and Be0 ; Bj and Bj0 ). Data are representative of three independent experiments where approximately 40 mitotic cells were analyzed. (C and D) Inhibition of histone H3S10P increased retention of SRp20 on mitotic chromatin without affecting SRp20 protein levels. Mitotically growing HeLa cells were treated without () or with (+) Aurora B kinase inhibitor ZM447439. Chromatinenriched fraction (C) or whole-cell extract (D) was subject to immunoblot analysis with indicated antibodies.

a significant reduction in ASF/SF2 association with mitotic chromosomes (Figures 4Be and 4Be0 ) became evident. Similar results were also obtained with nocodazole-arrested mitotic cells (data not shown) and with SRp20 (Figures 5Be and 5Be0 ). This finding suggests that H3S10P is an important determinant for release of SR proteins from mitotic chromosomes. Residual ASF/SF2 and SRp20 association with mitotic chromatin (Figure 4Be0 and Figure 5Be0 , respectively) might represent their interaction with unmodified, acetylated, or methylated nucleosomal domains (Figure 3B). We next wanted to determine if H3S10P is indeed a critical regulator of SR protein-chromatin interactions and sufficient to cause dissociation of the SR proteins from mitotic chromatin. To investigate this, HeLa cells were synchronized by a double thymidine block and released for 9 hr, followed by a short nocodazole treatment to enhance the number of cells in mitosis. We then performed a time-course analysis of the disappearance of H3S10P and reassociation of SRp20 and ASF/SF2 following

release from the M-phase block. Cells were harvested every 30 min after release, fixed with paraformaldehyde, permeabilized with methanol, and probed with anti-ASF/SF2 (Figure 4A) or anti-SRp20 (Figure 5A) and anti-histone H3-S10P antibodies to analyze SR protein association/dissociation from chromatin in relation to the presence of H3S10P. Cells in prometaphase, metaphase, and anaphase displayed high levels of H3S10P (Figures 4Aa, 4Ag, and 4Am and Figures 5Aa, 5Ag, 5Am) and largely showed ASF/SF2 and SRp20 to be dissociated (excluded) from mitotic chromatin (Figures 4Af, 4Al, and 4Ar and Figures 5Af, 5Al, and 5Ar, respectively). As cells progressed through telophase, a late stage of mitosis, the levels of H3S10P decreased (Figure 4As and Figure 5As) and were accompanied by a dramatic reassociation of both ASF/SF2 and SRp20 with chromatin (Figure 4Ax and Figure 5Ax, respectively), even before chromatin decondensation was complete. These data suggest that SR protein dissociation from chromatin during M-phase and their reassociation during/following telophase is dependent on H3S10P. To confirm that H3S10P is indeed required for release of SRp20 and ASF/SF2 from mitotic chromosomes, we next analyzed association of the SR proteins with mitotic chromatin

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when H3S10P was inhibited. It has been established that H3 serine 10 is phosphorylated by Aurora B kinase in mitotic cells (Giet and Glover, 2001; Hsu et al., 2000). Mitotic cells were obtained following a double thymidine block, a 10 hr release, and a 1 hr treatment with an Aurora B kinase inhibitor, ZM447439 (Ditchfield et al., 2003, 2005). As expected, the Aurora B kinase inhibitor significantly reduced H3S10P on condensed mitotic chromatin (Figures 4Bf and 4C, middle panel, and Figures 5Bf and 5C, middle panel). Importantly, we observed a concomitant increase in the retention of ASF/SF2 on the condensed mitotic chromatin when H3S10P was blocked (compare Figures 4Be0 with 4Bj0 ; Figure 4C, top panel). As with ASF/SF2, we found that SRp20 associated with interphase chromatin (Figure 3C, upper panel) and was largely dissociated from mitotic chromosomes (Figure 5A). In the presence of the Aurora B kinase inhibitor, however, an increase in retention of SRp20 on condensed mitotic chromatin was observed (compare Figures 5Be0 with 5Bj0 ; Figure 5C, top panel). Next, we sought to corroborate our immunocytochemistry results (Figures 4B and 5B) by biochemical fractionation and analysis. To this end, we collected mitotic cells following double thymidine block, a 9 hr release, and a 2 hr treatment with nocodazole and ZM447439. These cells were either fractionated to obtain a chromatin-enriched fraction as previously described (Fischle et al., 2005; Mendez and Stillman, 2000) (Figures 4C and 5C) or lysed to obtain whole-cell lysates (Figures 4D and 5D). In both cases, lysates were analyzed for ASF/SF2 (Figures 4C and 4D, top panels), SRp20 (Figures 5C and 5D, top panels), H3S10P (Figures 4C and 4D and 5C and 5D, second panels), and loading control, either histone H1 (Figures 4C and 5C, bottom panels) or actin (Figures 4D and 5D, bottom panels), using the appropriate antibodies. Total levels of ASF/SF2 (Figure 4D, top panel) or SRp20 (Figure 5D, top panel) in whole-cell extracts were unchanged following inhibition of H3S10P by Aurora B kinase inhibitor treatment. As expected, cells treated with the Aurora B kinase inhibitor significantly decreased H3S10P (Figures 4C and 4D and 5C and 5D, second panels). Importantly, consistent with our immunocytochemistry data (Figures 4B and 5B), increased retention of both ASF/SF2 (Figure 4C, top panel) and SRp20 (Figure 5C, top panel) on mitotic chromatin in the absence of H3S10P was evident. As a positive control, and in agreement with the results of Fischle et al. (2005), we also found that HP1b was retained on mitotic chromatin in the absence of H3S10P (Fischle et al., 2005) (Figure 4C, third panel). Together with the results of Figure 3, these immunocytochemical and biochemical analyses demonstrate association of SRp20 and ASF/SF2 with interphase chromatin, their dissociation from mitotic chromatin, and reassociation with late/post-mitotic chromatin in a H3S10P-dependent manner. Knockdown of ASF/SF2 Affects HP1-Chromatin Association Our data so far have shown that SRp20 and ASF/SF2 associate with interphase chromatin; are largely dissociated from mitotic chromatin in a H3S10P-dependent manner; and subsequently reassociate with chromatin late in M-phase when H3S10P diminishes. These observations raise the interesting question as to the function of SRp20 and ASF/SF2 dissociation from chromatin

during M-phase and reassociation with postmitotic chromatin. To address this issue, we considered the possibility that ASF/ SF2 may be necessary to remove other chromatin-associated protein(s) to facilitate M-phase progression. HP1 proteins have been found to be recruited to discrete regions of the genome by histone H3 lysine 9 trimethylation where they are involved in controlling chromatin structure and dynamics—predominantly regulation of gene expression and heterochromatin formation (Fischle et al., 2005; Hirota et al., 2005). HP1 dissociation from chromatin is dependent on H3S10P, and treatment with the Aurora B kinase inhibitor leads to retention of HP1 proteins on mitotic chromatin (Fischle et al., 2005), similar to what we observed with SRp20 and ASF/SF2. Since ASF/SF2 and HP1 proteins are all significantly released from hyperphosphorylated mitotic chromatin, we reasoned that they may interact with each other and that such an interaction may be necessary for their release. To determine if ASF/SF2 and HP1b interact, we analyzed the association between ASF/ SF2 and HP1b in control siRNA treated mitotic cells by immunocytochemistry. Although not extensive, we nonetheless observed colocalization of ASF/SF2 and HP1b in control siRNA treated mitotic cells in which both proteins were primarily excluded from chromatin (Figures 6Ae and 6Ae0 and Figure 6D where the same cell is shown with different overlapping images). To provide direct support for an interaction, we made mitotic soluble protein extracts, immunoprecipitated with either control IgG or anti-ASF/SF2 antibody, and analyzed bound proteins by immunoblotting for HP1b. In agreement with the results of Figure 6A, we found that a fraction of ASF/SF2 and HP1b (Figure 6B) associated in mitotic cell extracts. In the future, it will be important to determine whether binding of SRp20 and ASF/ SF2 to histone H3K9Me plays any functional role in the abovementioned process. To address the possibility that the ASF/SF2-HP1b association in mitotic cells is important for HP1 release from mitotic chromatin, we determined the effect of ASF/SF2 knockdown on HP1 protein association with mitotic chromatin. We transfected HeLa cells with either control or ASF/SF2 siRNAs, fixed cells with paraformaldehyde, permeabilized with methanol and analyzed HP1b association by immunocytochemistry (Figure 6D). ASF/ SF2 protein levels were significantly diminished after 48 hr of RNAi treatment (Figure 6C). Consistent with previous findings, we observed HP1b remained nuclear but was predominantly excluded from chromatin in control siRNA treated mitotic cells (Figure 6Da, 6De, and 6De0 ) (Fischle et al., 2005). Importantly, more HP1b (compare Figures 6De0 with 6Dj0 ) was retained on mitotic chromatin in ASF/SF2-knockdown cells despite the presence of H3S10P (Figure S1 available online). We next wished to confirm the immunocytochemistry results with biochemical fractionation and analysis. To this end, we collected mitotic cells after ASF/SF2 knockdown and nocodazole treatment, and they were either fractionated to obtain a chromatin-enriched fraction as previously described (Fischle et al., 2005; Mendez and Stillman, 2000) (Figure 6E) or lysed to obtain whole-cell lysates (Figure 6F). Lysates were analyzed for HP1b, ASF/SF2, and a loading control, either histone H1 or actin using appropriate antibodies. Our findings demonstrate that increased amounts of HP1b were indeed retained on mitotic

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Figure 6. ASF/SF2 Associates with HP1b in Mitotic Cells and ASF/SF2 Knockdown Leads to Retention of HP1b on Mitotic Chromatin (A) ASF/SF2 and HP1b colocalize in mitotic cells (Aa–Ae0 ). (B) ASF/SF2 and HP1b associate in HeLa mitotic cells. HeLa cells were mitotically arrested using 100 ng/ml nocodazole. The soluble protein fraction was used for immunoprecipitation with either control IgG or anti-ASF/SF2 antibody. Bound proteins were subject to immunoblot analysis with anti-HP1b antibody. Input represents 10 mg of total lysate used in the experiment. (C) RNAi treatment of HeLa cells significantly reduces ASF/ SF2 expression. HeLa cells were treated with control or ASF/SF2 siRNA two times, 24 hr apart, using Lipofectamine 2000. Cells were collected for immunoblot analysis 48 hr after the first transfection and probed with anti-ASF/SF2 and antiactin antibodies. (D–F) ASF/SF2 knockdown led to an increased retention of HP1b on mitotic chromatin despite histone H3S10P and without affecting HP1b protein levels. (D) Top panel in (D) is the same cell as that shown in (A) except with different overlapping images. Cells were fixed with paraformaldehyde, permeabilized with methanol, and stained with anti-HP1b and anti-ASF/SF2 antibodies at 48 hr post-first transfection. ASF/SF2 knockdown led to increased association of HP1b with chromatin (compare De and De0 with Dj and Dj0 ) despite the presence of H3S10P in metaphase cells. HP1b data are representative of 3 independent experiments and approximately 50 mitotic cells were analyzed. (E and F) HeLa cells were treated with control or ASF/SF2 siRNA two times, 24 hr apart, and then with nocodazole for 12 hr before harvesting to enrich for mitotic cells. Chromatin-enriched fraction (E) or whole-cell extract (F) was subject to immunoblot analysis with indicated antibodies.

chromatin upon ASF/SF2 knockdown (Figure 6E, top panel) despite no significant change in the levels of HP1b (Figure 6F, top panel). Additional immunoprecipitation, immunocytochemical and biochemical fractionation analyses were conducted for HP1a and HP1g in the ASF/SF2 knockdown cells and similar results were obtained (Figure S2). Collectively, our findings strongly suggest that along with histone H3 phosphorylation, ASF/SF2 also plays a potentially important role in mediating HP1 protein dissociation from mitotic chromatin. Since SRp20 and ASF/SF2 associated with interphase and postmitotic, but not mitotic, chromatin, we next analyzed whether ASF/SF2 plays a role in affecting cell-cycle progression. We tested this possibility by utilizing DT40-ASF cells, which are a derivative of chicken DT40 cells where the only copy of the ASF/SF2 gene is a human cDNA under the control of a tetracycline (tet)-repressible promoter (Wang et al., 1996). DT40-ASF cells were treated with 1 mg/ml doxycycline for 16 hr before adding 100 ng/ml nocodazole for 8 hr to prevent cell-cycle progression past prometaphase (Figure 7A). ASF/SF2 protein expression levels were significantly diminished after 24 hr of doxycycline treatment, which represents hour 0 of the schematic shown in Figure 7A (Figure 7C, inset). After the nocodazole treatment, we released the cells and analyzed the effect of ASF/SF2 depletion on cell-cycle progression via flow cytometry using propidium iodide to stain the DNA. Cell-cycle analysis indicated that ASF/SF2 depletion inhibited G2/M progression (Figure 7B) and subsequently delayed entry of cells into G0/G1 (Figure 7C).

Subsequently, as expected, and consistent with our overall hypothesis, inhibition of histone H3S10P by treatment with the Aurora B kinase inhibitor essentially prevented all cells from entry into G0/G1 in both ASF/SF2 depleted and normal cells (Figure S3). Although our experimental design does not directly demonstrate a causal relationship between ASF/SF2 chromatin reassociation at the end of telophase and cell entry into G0/G1 phase, our findings together demonstrate that depletion of endogenous ASF/SF2 alters retention of HP1 proteins on mitotic chromatin and prevents G0/G1 entry after release from an M-phase block. DISCUSSION This study has provided the first demonstration that two members of the human SR protein family, SRp20 and ASF/ SF2, associate with interphase chromatin, dissociate from mitotic chromosomes following H3S10P, and reassociate with postmitotic chromatin. In the future it will be important to determine whether this is a common property of all members of the SR family of proteins. It appears that the release of SR proteins from chromatin is determined by SR protein phosphorylation by SRPKs and histone H3 serine 10 phosphorylation by Aurora B kinase. Consistent with this view, SRPK1 has been shown to translocate to the nucleus at the onset of M-phase, and some SR proteins have been shown to be hyperphosphorylated in M-phase (Ding et al., 2006; Gui et al., 1994a). We propose that

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Figure 7. ASF/SF2 Depletion Delays G0/G1 Entry Depletion of ASF/SF2, followed by an M-phase block and release, caused cells to be delayed entering G0/G1. DT40-ASF cells were treated with doxycycline for 16 hr, followed by cotreatment with doxycycline and nocodazole for 8 hr, and then released and analyzed for cell-cycle progression by propidium iodide staining and subsequent flow cytometry analysis. (A) Schematic of experimental design shown in (B) and (C). (B) DT40 ASF cells depleted of ASF/SF2 demonstrated a significant increase in the percentage of G2/M cells compared to ASF/SF2-expressing cells. (C) ASF/SF2 depletion in DT40-ASF cells demonstrated a significant decrease in the percentage of cells entering G0/G1 compared to cells expressing ASF/SF2. ([C], inset) DT40-ASF chicken cells, treated with 1 mg/ml of doxycycline for 24 hr, showed depletion of ASF/SF2. Cells were collected for immunoblot analysis and probed with anti-ASF/SF2 and anti-actin antibodies. Data shown in (B) and (C) are representative of four independent experiments.

a dual phosphorylation event involving SRPK-mediated phosphorylation of SR proteins and Aurora B kinase-mediated H3 phosphorylation probably regulates the dynamics of SR protein interactions with chromatin. Although primarily nuclear, shuttling of SRp20 and ASF/SF2 to the cytoplasm when transcription is blocked or Clk/Sty kinase is exogenously expressed has been observed (Caceres et al., 1998). It remains to be seen whether these events are linked and, thus, establish a regulatory circuit. Another interesting feature of the SR protein interaction with chromatin is that while a dual modification of histone H3 appears necessary for HP1 release (Fischle et al., 2005; Hirota et al., 2005; Mateescu et al., 2004), H3S10P alone is sufficient for release of SRp20 and ASF/SF2 from mitotic chromatin. While the interaction of SRp20 and ASF/SF2 with chromatin might appear unexpected, previous work established that inactivation of ASF/SF2 causes transcription-related and cell-cycle progression defects, suggesting a role for ASF/SF2 in modulation of chromosome dynamics. Li et al. (2005) suggested that a mechanism other than an alteration in mRNA splicing, specifically the generation of DNA double-strand breaks resulting from cotranscriptional R-loop formation (Li and Manley, 2005; Li et al., 2005), could be responsible for the G2/M transition defects in cells depleted of ASF/SF2. Similarly, roles for another splicing factor, SC35, beyond its function in RNA processing, have recently been reported in chromatin based processes: SC35 regulates transcription elongation of specific genes and also plays a critical role in regulating genomic stability and cell-cycle progression (Lin et al., 2008; Xiao et al., 2007). Based on these and our current findings, we propose that chromatin association/dissociation properties of the SR proteins also contributes to proper cell-cycle progression, and that they may contribute

to the G2/M defect, as well as explain the delayed G0/G1 entry we observed upon ASF/SF2 depletion. SRp20 and ASF/SF2’s chromatin association/dissociation properties provide insight into how these proteins may work to regulate proper chromatin function. We hypothesize that the release of SR proteins from hyperphosphorylated chromatin and the subsequent reassociation of SR proteins with chromatin once histone H3S10P has diminished are both important events for proper chromatin function. Knockdown of ASF/SF2 by siRNA led to retention of HP1 proteins on histone H3 despite serine 10 phosphorylation, suggesting that dissociation of ASF/SF2 from hyperphosphorylated histone H3 also influences HP1 dissociation from mitotic chromatin. Indeed, Fischle et al. (2005) suggested that in addition to H3S10P, other mechanisms may be involved in mitotic release of HP1 from chromatin, such as further modification of HP1 proteins and/or their interaction partners (Fischle et al., 2005). Our findings implicate ASF/SF2 as one such interacting partner responsible for HP1 release from mitotic chromatin. HP1 and ASF/SF2 are both associated with interphase chromatin, are dissociated from chromatin upon H3S10P, and are associated with each other in mitotic cells. Due to the inherent limitations of mammalian cells for mutational analysis, neither this study nor previous ones (Fischle et al., 2005; Hirota et al., 2005) provided a direct causal relationship linking the chromatin association/ dissociation properties of SRp20, ASF/SF2, and HP1 proteins with M-phase progression and chromosome segregation. However, M-phase progression and chromosome segregation defects have been noted in S. pombe and Tetrahymena mutants defective in H3S10P (Mellone et al., 2003; Wei et al., 1999). Additionally in S. pombe, the HP1 homolog, Swi6, is required for proper chromatin segregation (Pidoux and Allshire, 2004).

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Our studies in conjunction with the above results provide a strong correlation between SR and HP1 proteins and their release from chromatin with proper chromosome segregation and M-phase progression. It is, therefore, reasonable to suggest that the coordinated removal of HP1 and ASF/SF2 during M-phase is probably necessary to allow access by factors required for mediating proper chromatin condensation and faithful chromosome segregation. In this context we note that although 14-3-3 proteins were shown to bind H3S10P, they did not significantly associate with hyperphosphorylated condensed chromosomes in mitotic cells. Since H3S10P is also implicated in transcriptional activation, it appears that 14-3-3 and H3S10P association may be restricted to transcription of inducible genes (Macdonald et al., 2005). We also found that removal of an M-phase block in cells depleted of ASF/SF2 significantly delayed both exit from G2/M and entry into G0/G1. There are several possibilities for why ASF/SF2 depleted cells, blocked in M-phase, would delay G0/ G1 entry. The first is that ASF/SF2 depletion blocks the cells at G2, as suggested by Li et al. (2005). These authors demonstrated that DT40-ASF cells depleted of ASF/SF2 induced a G2-block and an increase in apoptotic cell death by 72 hr post-tet treatment. However, it is important to note that our experimental time frame was significantly shorter (28.5 hr) than theirs, and we did not see increased apoptotic cell death as indicated by the absence of a sub-G0/G1 peak in all time points analyzed within the experimental time frame (28.5 hr). This, by no means, eliminates the possibility that part of the G2/M increase we see in our cell-cycle analysis is due to a G2 block and not an M-phase block. The second, more intriguing possibility for the delay of G0/G1 entry upon ASF/SF2 depletion and release from an M-phase block is because ASF/SF2 is not present to reassociate with chromatin in telophase once H3S10P has diminished. ASF/SF2 reassociation with chromatin at the end of mitosis may be a trigger for M-phase completion, and it may allow additional factors, such as HP1 proteins, to reassociate with chromatin as the cell enters G0/G1. We propose that inhibition of H3S10P in ZM447439treated cells, ASF/SF2, and SRp20, as well as HP1 proteins, are prevented from dissociating from mitotic chromatin and, thus, exacerbating the effect we see with ASF/SF2 depletion alone. In summary, this work has established the association of two SR proteins, SRp20 and ASF/SF2, with interphase chromosomes; demonstrates their release from hyperphosphorylated mitotic chromosomes and reassociation with postmitotic chromatin; and provides insight into our evolving understanding of the function of H3S10P. The ability of the SR proteins to associate with and dissociate from chromosomes in a histone H3 modification-selective manner may account for G2/M cell-cycle arrest and defects in G0/G1 accumulation. Significantly, the SR proteins have similar association/dissociation characteristics as HP1 proteins. ASF/SF2 and HP1 proteins associate in mitotic cells and knockdown of ASF/SF2 led to retention of HP1 proteins on mitotic chromatin, suggesting a mechanistic link between the release of ASF/SF2 and HP1 proteins from mitotic chromatin. Future studies will investigate the underlying mechanisms of SR protein association/dissociation from chromatin and the potential role of SR proteins in directly regulating chromatin function and cell-cycle progression.

EXPERIMENTAL PROCEDURES Plasmids Expression constructs were comprised of PCR-amplified fragments of SRp20 and its derivatives cloned into pGEX-4T1 (CLONTECH) vectors. Cell Culture HeLa cells were grown in monolayers in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum, penicillin/streptomycin, and L-glutamine and incubated in a 5% CO2/95% humidified atmosphere at 37 C. To induce M-phase arrest, 50 ng/ml nocodazole was used for 12 hr where indicated. Synchronization of HeLa cells was performed using a double thymidine block. Cells were released from the second block by washing and incubating in fresh culture media. After approximately 10 hr, as the cells neared mitosis, they were treated with 2 mM ZM447439 (an Aurora B kinase inhibitor, Tocris Bioscience) (Ditchfield et al., 2003, 2005) or DMSO control. After 1 hr treatment, mitotic cells were shaken off the plates, washed with 13 PBS, and spun onto coated cytospin slides using Cytospin 2 (Shandon) at 1000 rpm for 4 min. For analysis of cell-cycle progression after mitotic block, HeLa cells were synchronized using double thymidine block, 8 hr after the second release, 50 ng/ml nocodazole was added for 2.5 hr before cells were released from the mitotic block and analyzed by immunocytostaining. Immunocytostaining was performed using the method as described under Immunocytochemistry. DT40-ASF Chicken Cells DT40-ASF chicken cells are cultured at 1 3 105 cells/ml in RPMI medium supplemented with 10% fetal calf serum, 1% chicken serum, penicillin/streptomycin with antimycotics, and L-glutamine and incubated in a 5% CO2/95% humidified atmosphere at 37 C (Li et al., 2005; Wang et al., 1996). ASF depletion was induced using 1 mg/ml doxycycline for indicated times. To block cells in mitosis, they were cultured in 100 ng/ml of nocodazole for 8 hr. Histone Peptide Binding Assay and Protein Microsequencing See details in the Supplemental Data. In Vitro Binding Assay See details in the Supplemental Data. In Vitro Phosphorylation Assay See details in the Supplemental Data. CIP Treatment of HeLa Mitotic Cells HeLa cells are treated with 50 ng/ml nocodazole for 12 hr to induce M-phase arrest. Mitotic cells were collected and lysed in lysis buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.5% NP-40, 50 mM NaF, 1 mM EDTA, 1mM DTT, and protease inhibitor cocktail). Cell lysates (1 mg) were treated with 100 units of calf intestinal phosphatase (CIP) (NEB) or mock-treated at 37 C for 1 hr. After CIP inactivation by addition of 10 mM sodium vanadate and 10 mM EDTA, lysate was incubated with histone H3S10P-biotinylated peptide (AbCam) or no peptide for 45 min at room temperature. The peptide and associated proteins were pulled down using streptavidin-agarose beads (Roche) at 4 C for 1 hr. The beads and associated proteins were washed, and bound proteins were separated by SDS-PAGE and analyzed by immunoblot. Nucleosome Extraction and Immunoprecipitation HeLa cells were suspended in nuclei isolation buffer (NIB; 15 mM Tris-HCl [pH 7.5], 60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 1 mM CaCl2, 2 mM sodium vanadate, 10 mM NaF, 250 mM sucrose, protease inhibitor cocktail, and 200 mg/ml RNase A). An equal volume of NIB containing 0.6% NP-40 was added to the cells, and the suspension was gently mixed and incubated on ice for 5 min. Nuclei were pelleted by centrifugation at 20003 g for 5 min at 4 C and digested at 37 C for 5 min with 15 U of MNase (GE Healthcare) in 300 ml of NIB buffer. Following centrifugation for 10 min at 10,0003 g at 4 C, the supernatant was removed by aspiration and the pellet was resuspended by pipetting in 300 ml of ice-cold 2 mM EDTA (pH 8.0). The nucleosome preparation was incubated with protein A-beads (Invitrogen) and either 10 ml of anti-SRp20 antibody

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(Invitrogen) or anti-ASF/SF2 antibody (Invitrogen) for 16 hr at 4 C. The immunoprecipitates were washed 5 times with 0.1% NP-40 containing wash buffer. Bound proteins were separated by SDS-PAGE and analyzed by immunoblot. For HP1 protein-ASF/SF2 association experiments, 1 mg (for HP1b) or 2.5 mg (for HP1g) of soluble protein isolated from mitotically arrested HeLa cells was used for the immunoprecipitations with control IgG or mouse anti-ASF/SF2 antibody (Invitrogen). Bound proteins were separated by SDS-PAGE and analyzed by immunoblot. Chromatin Fractionation Mitotic HeLa cells were harvested and whole cell extracts were prepared by lysing cells in lysis buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.5% NP-40, 50 mM NaF, 1 mM EDTA, 1 mM DTT and protease inhibitor cocktail) for 1 hr at 4 C. For chromatin fractionation, we followed the protocol previously described (Fischle et al., 2005; Mendez and Stillman, 2000). Briefly, mitotic HeLa cells were resuspended in 10 mM HEPES (pH 7.9), 10 mM KCl, 1.5 mM MgCl2, 0.34 M sucrose, 10% glycerol, 1 mM DTT, 1 mM PMSF, 0.1% Triton X-100, and protease inhibitor cocktail and incubated on ice for 5 min followed by centrifugation at 13003 g for 5 min at 4 C. The supernatant represents the soluble protein fraction. The remaining pellet is washed two times with HEPES buffer described above and resuspended in 3 mM EDTA, 0.2 mM EGTA, 1 mM DTT, and protease inhibitor cocktail and incubated on ice for 30 min, followed by centrifugation at 17003 g for 5 min at 4 C. The pellet is then resuspended in the original HEPES buffer, sonicated to release chromatin-associated proteins, and centrifuged to remove residual insoluble proteins and nuclear matrix. The resulting supernatant is designated as the chromatin-enriched fraction and was probed with anti-ASF/SF2 (Invitrogen), anti-SRp20 (Invitrogen), anti-HP1a (Upstate), anti-HP1b (Upstate), anti-HP1g (AbCam), anti-H3S10P (Cell Signaling), and/or anti-histone H1 (Upstate) antibodies. RNAi Knockdown in HeLa Cells HeLa cells were plated on coverslips in a 12-well plate at 2.5 3 104 cells/well the day before the first transfection. Thirty nM control RNAi or ASF/SF2 RNAi (Invitrogen) were transfected using Lipofectamine 2000 (Invitrogen). The RNAi transfection was repeated 24 hr later. Cells were immunocytostained according to method described under Immunocytochemistry 48 hr after the first transfection. RNAi treated HeLa cells used for biochemical fractionation analysis or whole-cell lysate analysis by SDS-PAGE were treated with 100 ng/ml nocodazole for 12 hr before harvesting to enrich for mitotic cells. Immunocytochemistry HeLa cells grown in a monolayer on coverslips were fixed in 4% paraformaldehyde for 15 min at 4 C and permeabilized for 30 s with ice-cold methanol. Coverslips were washed three times with 1% milk/150 mM sodium acetate in 13 PBS and 3 times with 1% milk in 13 PBS and incubated with anti-SRp20 (Invitrogen) or anti-ASF/SF2 (Invitrogen) antibodies with either anti-H3 S10P (Upstate), anti-H3 K9Me2 (Upstate), anti-H3 acetylated lysine (AbCam), antiH3 (Upstate), anti-HP1b (Upstate), anti-HP1a (Upstate), or anti-HP1g (Upstate) antibodies overnight at 4 C. After washing three times in 1% milk in 13 PBS, coverslips were subsequently incubated with both anti-mouse-Alexa 488 and anti-rabbit-Alexa 563 (Invitrogen) for 1 hr at room temperature, followed by Hoechst 33342 (Invitrogen) staining to visualize DNA. Cells were visualized under 1003 magnification on a LSM 510 Meta microscope (Carl Zeiss), and the midplane of each cell was imaged. Flow Cytometry For cell cycle analysis, DT40-ASF chicken cells were harvested at desired times, washed twice in cold 1 ml 13 PBS, resuspended to single-cell suspension in 50 ml 13 PBS prior to fixation with ethanol (950 ml ice-cold 70% ethanol). Cells were stored at 20 C in fixative for at least 2 hr. Cells were centrifuged for 5 min at 2003 g and ethanol was aspirated. The cell pellet was resuspended in 1 ml 13 PBS, centrifuged for 5 min at 2003 g, and decanted, and wash was repeated two more times. The cell pellet was resuspended in 500 ml of PI staining solution (50 mg/ml PI, 0.1% Triton X-100, 2 mg RNase A in 13 PBS) and incubated at 37 C for 20 min. Flow cytometry analysis was performed at the Robert H. Lurie Comprehensive Cancer Center Flow Cytometry Core Facility.

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