[methods in enzymology] chromatin and chromatin remodeling enzymes, part c volume 377 || analysis of...

[11] signaling to chromatin remodeling 197

[11] Analysis of Histone Phosphorylation: CouplingIntracellular Signaling to Chromatin Remodeling

By Romain Loury and Paolo Sassone-Corsi

The N-terminal domains of histones are subjected to several types ofcovalent modifications, such as acetylation,1,2 phosphorylation,3,4 methyla-tion,5 but also—although not as well investigated—ADP-ribosylation6 andubiquitination.7 A number of these modifications have been associatedwith distinct chromatin-based outputs. In particular, position-specificmodifications of the histone H3 N-terminal tail have been coupled to tran-scriptional regulation (Lys9/Lys14 acetylation, Ser10 phosphorylation),transcriptional silencing (Lys9 methylation), histone deposition (Lys9acetylation), and chromosome condensation/segregation (Ser10/Ser28phosphorylation).8,9 It is generally thought that various combinations ofhistone modifications may elicit differential regulation of the chromatincondensation state which correspond to distinct biological responses.10

Histone phosphorylation seems to be involved in a wide range of cellu-lar processes. One essential feature of phosphorylation versus all other his-tone modifications is that it can be directly linked to the activation ofdistinct signaling pathways, and thereby to unique physiological responses.All core histones contain phosphoacceptor sites in their N-terminaldomains11: H2A is phosphorylated on Ser1, H2B on Ser14 and Ser32, H3on Ser10 and Ser28, while H4 gets phosphorylated on Ser1. A significantnumber of studies on histone phosphorylation have focused on the impor-tance of the Ser10 residue of histone H3. The privileged position of Ser10in H3 is mainly due to its pivotal role in a number of cellular responses andthe availability of a powerful antibody recognizing the phosphorylated

1 P. D. Gregory, K. Wagner, and W. Horz, Exp. Cell Res. 265, 195 (2001).2 S. Y. Roth, J. M. Denu, and C. D. Allis, Annu. Rev. Biochem. 70, 81 (2001).3 L. R. Gurley, R. A. Walters, and R. A. Tobey, Biochem. Biophys. Res. Commun. 50, 744 (1973).4 L. C. Mahadevan, A. C. Willis, and M. J. Barratt, Cell 65, 775 (1991).5 A. J. Bannister, R. Scheider, and T. Kouzarides, Cell 109, 801 (2002).6 A. Huletsky, C. Niedergang, A. Frechette, R. Aubin, A. Gaudreau, and G. G. Poirier, Eur.

J. Biochem. 146, 277 (1985).7 Z. W. Sun and C. D. Allis, Nature 418, 104 (2002).8 T. Jenuwein and C. D. Allis, Science 293, 1074 (2001).9 P. Cheung, C. D. Allis, and P. Sassone-Corsi, Cell 103, 263 (2000).

10 B. D. Strahl and C. D. Allis, Nature 403, 41 (2000).11 S. C. Galasinski, D. F. Louie, K. K. Gloor, K. A. Resing, and N. G. Ahn, J. Biol. Chem. 277,

2579 (2002).

Copyright 2004, Elsevier Inc.All rights reserved.

METHODS IN ENZYMOLOGY, VOL. 377 0076-6879/04 $35.00

TABLE I

Phosphorylation of Histones During Distinct Cellular Processes

Cellular process Histone Residue Kinase Refs.

Gene activation H3 Ser10 RSK2 a

Ser10 MSK1 b

Ser10 IKK-� c

Ser28 MSK1 d

Mitotic chromosome condensation H3 Ser10 Aurora-B e

Ser28 Aurora-B f

Thr11 Dlk g

Apoptosis H2AX Ser139 ? h

H2B Ser14 Mst 1 i

H2B Ser32 ? j

H3 Ser10 ? k

Damage repair H2AX Ser139 ATM l

a P. Sassone-Corsi, C. A. Mizzen, P. Cheung, C. Crosio, L. Monaco, S. Jacquot,

A. Hanauer, and C. D. Allis, Science 285, 886 (1999).b S. Thomson, A. L. Clayton, C. A. Hazzalin, S. Rose, M. J. Barratt, and L. C. Mahadevan,

EMBO J. 18, 4779 (1999).c Y. Yamamoto, U. N. Verma, S. Prajapati, Y. T. Kwak, and R. B. Gaynor, Nature 423, 655

(2003); V. Anest, J. L. Hanson, P. C. Cogswell, K. A. Steinbrecher, B. D. Strahl, and

A. S. Baldwin, Nature 423, 659 (2003).d S. Zhong, C. Jansen, Q. B. She, H. Goto, M. Inagaki, A. M. Bode, W. Y. Ma, and

Z. Dong, J. Biol. Chem. 276, 33213 (2001).e C. Crosio, G. M. Fimia, R. Loury, M. Kimura, Y. Okano, H. Zhou, S. Sen, C. D. Allis,

and P. Sassone-Corsi, Mol. Cell. Biol. 22, 874 (2002).f H. Goto, Y. Yasui, E. A. Nigg, and M. Inagaki, Genes Cells 7, 11 (2002).g U. Preuss, G. Landsberg, and K. H. Scheidtmann, Nucleic Acid Res. 31, 878 (2003).h E. P. Rogakou, W. Nieves-Neira, C. Boon, Y. Pommier, and W. M. Bonner, J. Biol.

Chem. 275, 9390 (2000).i W. L. Cheung, K. Ajiro, K. Samejima, M. Kloc, P. Cheung, C. A. Mizzen, A. Beeser,

L. D. Etkin, J. Chernoff, W. C. Earnshaw, and C. D. Allis, Cell 113, 507 (2003).j K. Ajiro, J. Biol. Chem. 275, 439 (2000).k P. Waring, T. Khan, and A. Sjaarda, J. Biol. Chem. 272, 17929 (1997).l E. P. Rogakou, D. R. Pilch, A. H. Orr, V. S. Ivanova, and W. M. Bonner, J. Biol. Chem.

273, 5858 (1998).

198 histone modifying enzymes [11]

site.12 In more general terms, phosphorylation of histones has been coupledto various cellular processes, which are briefly summarized here andpresented in Table I.

12 M. J. Hendzel, Y. Wei, M. A. Mancini, A. Van Hooser, T. Ranalli, B. R. Brinkley, D. P.

Bazett-Jones, and C. D. Allis, Chromosoma 106, 348 (1997).


Mitosis

Histone phosphorylation during mitosis is a cell cycle-regulated chro-matin modification.3 H3 is phosphorylated at the Ser10 residue duringmitosis, revealing the dual personality of this site.13,14 Indeed, thismodification tightly follows the process of chromosome condensation in aspatio-temporal manner,12 while mutation of the Ser10 residue to Ala inTetrahymena has been shown to lead to aberrant chromatin condensa-tion.13 In mammals, this modification has been shown to be involved inthe initiation of the chromosome condensation process, but not for main-tenance of the condensed state of the chromatin.15 Phosphorylation at H3Ser10 in mammals appears to be mediated by the Aurora-B kinase,16 a pro-tein belonging to the Ipl1/Aurora family of mitotic kinases, strongly impli-cated at diverse levels of chromosome segregation in yeast as well as inmammals.17 Thus, the same phosphorylation event has a dual ‘‘personal-ity.’’ Indeed, histone H3 Ser10 phosphorylation appears to elicit oppositeeffects: chromatin opening in the case of mitogenically-induced gene acti-vation, chromosome condensation during mitosis. This notion highlights ina clear manner the complexity of the histone code.

Additional histone phosphorylation events were reported to take placeduring mitosis: histone H3 Ser28 has also been reported to be phosphory-lated, possibly also by Aurora-B,18 although with slightly different kineticsthan Ser10 phosphorylation.19 The physiological significance of this eventremains to be further clarified. Histone H1, the major linker histone, is alsoknown to be phosphorylated during entry into mitosis. H1 phosphorylationdepends on cdc2 kinase activity.20 However, as recent studies have shownthat chromosome condensation can occur without histone H1 phosphory-lation,21 or even without H1 itself,22 the biological significance of H1phosphorylation remains undefined.

13 Y. Wei, L. Yu, J. Bowen, M. A. Gorovsky, and C. D. Allis, Cell 97, 99 (1999).14 Y. Wei, C. A. Mizzen, R. G. Cook, M. A. Gorovsky, and C. D. Allis, Proc. Natl. Acad. Sci.

USA 95, 7480 (1998).15 A. Van Hooser, D. W. Goodrich, C. D. Allis, B. R. Brinkley, and M. A. Mancini, J. Cell Sci.

111, 3497 (1998).16 C. Crosio, G. M. Fimia, R. Loury, M. Kimura, Y. Okano, H. Zhou, S. Sen, C. D. Allis, and

P. Sassone-Corsi, Mol. Cell. Biol. 22, 874 (2002).17 R. Giet and C. Prigent, J. Cell Sci. 112, 3591 (1999).18 H. Goto, Y. Yasui, E. A. Nigg, and M. Inagaki, Genes Cells 7, 11 (2002).19 H. Goto, Y. Tomono, K. Ajiro, H. Kosako, M. Fujita, M. Sakurai, K. Okawa, A. Iwamatsu,

T. Okigaki, T. Takahashi, and M. Inagaki, J. Biol. Chem. 274, 25543 (1999).20 T. A. Langan, J. Gautier, M. Lohka, R. Hollingsworth, S. Moreno, P. Nurse, J. Maller, and

R. A. Sclafani, Mol. Cell. Biol. 9, 3860 (1989).21 X. W. Guo, J. P. Th’ng, R. A. Swank, H. J. Anderson, C. Tudan, E. M. Bradbury, and

M. Roberge, EMBO J. 14, 976 (1995).


Activation of Gene Expression

The importance of histone N-termini in transcriptional induction is wellrecognized.23,24 The first evidence for a specific kinase involved in the phos-phorylation of a distinct site on a histone, came from the study of theCoffin-Lowry syndrome (CLS), an X-chromosome linked genetic disordercaused by the absence of a functional RSK2 kinase.25 Cells derived fromCLS patients display a strong impairment in the process of mitogenicstimulation. This kind of approach led to the discovery that RSK2 was re-sponsible, after stimulation by EGF and through the Ras/MAPK cascade,for CREB phosphorylation,26 but also histone H3 phosphorylation onSer10,27 in perfect correlation with the induction of immediate-early genes,as c-fos. Thus, RSK2 was the first physiological histone kinase to be directlylinked to the activation of gene expression.

The role for this transduction-coupled modification remains to be fur-ther clarified, but it is tempting to think that histone H3 phosphorylationis restricted to the immediate-early genes, and that by a yet unknown rec-ognition mechanism, the phosphorylated tail becomes a privileged sub-strate for subsequent acetylation by an histone acetyltranferase (HAT)such as CBP (CREB-binding protein) on Lys14, thus leading to promoteropening and transcription of the gene.28 Interestingly, the enzymatic activ-ities eliciting acetylation and phosphorylation are also coupled, as CBP andRSK2 have been found to be associated in a signaling-dependent fashion.29

Several other studies demonstrated the role of histone H3 Ser10 phos-phorylation in response to other stimuli, as UV irradiation for example.4

But in this case, it seems that the kinase responsible for histone H3 phos-phorylation is MSK1, an effector kinase similar in structure to RSK2 thatlies downstream of both the Ras/MAPK and SAPK cascades.30 Moreover,

22 K. Ohsumi, C. Katagiri, and T. Kishimoto, Science 262, 2033 (1993).23 P. Allegra, R. Sterner, D. F. Clayton, and V. G. Allfrey, J. Mol. Biol. 196, 379 (1987).24 L. K. Durrin, R. K. Mann, P. S. Kayne, and M. Grunstein, Cell 65, 1023 (1991).25 E. Trivier, D. De Cesare, S. Jacquot, S. Pannetier, E. Zackai, I. Young, J. L. Mandel, P.

Sassone-Corsi, and A. Hanauer, Nature 384, 567 (1996).26 D. De Cesare, S. Jacquot, A. Hanauer, and P. Sassone-Corsi, Proc. Natl. Acad. Sci. USA 95,

12202 (1998).27 P. Sassone-Corsi, C. A. Mizzen, P. Cheung, C. Crosio, L. Monaco, S. Jacquot, A. Hanauer,

and C. D. Allis, Science 285, 886 (1999).28 P. Cheung, K. G. Tanner, W. L. Cheung, P. Sassone-Corsi, J. M. Denu, and C. D. Allis, Mol.

Cell 5, 905 (2000).29 K. Merienne, S. Pannetier, A. Harel-Bellan, and P. Sassone-Corsi, Mol. Cell. Biol. 21, 7089

(2001).30 S. Thomson, A. L. Clayton, C. A. Hazzalin, S. Rose, M. J. Barratt, and L. C. Mahadevan,

EMBO J. 18, 4779 (1999).


it appears that after UV irradiation Ser28 also becomes phosphorylated,possibly also by MSK1, although this event has not been fully character-ized.31 These studies indicate that various mitogenic or hormonal stimula-tions lead to Ser10 phosphorylation, likely involving a larger number ofsignaling cascades and effector kinases. This notion is confirmed by thefinding that the IKK-� kinase phosphorylates Ser10 in response to cytokinestimulation leading to activation of NF-�B-responsive genes.32,33

Programmed Cell Death

The complex gene expression program that accompanies apoptosis islikely to implicate profound changes in chromatin organization. To date,there is a lack of satisfactory information on the various histone modifica-tions that are likely involved. It seems that the type of modifications impli-cated is highly dependent on the cell type, and on the nature of theapoptotic agent used to trigger the cell death program. This notion furtherhighlights the complexity and variability of the apoptotic processes. Yet,some common hallmarks appear to exist. Phosphorylation of histonesH2A.X (a minor variant of histone H2A) and H2B (respectively onSer139 and Ser32) seems to be induced in response to a wide variety ofapoptotic agents.34,35 The timing of these phosphorylation events seemsto parallel the process of nucleosomal DNA fragmentation occurring atearly stages of apoptosis.

The best characterized histone modification event that is uniquely asso-ciated with apoptotic chromatin, in species ranging from frogs to humans, isH2B phosphorylation at position Ser14. The kinase involved in this eventwas identified as Mst1 (mammalian sterile twenty), whose function isdirectly regulated by caspase-3.36

Other phosphorylation events have been reported, depending on theapoptotic agent used. These include Ser10 of histone H3, but alsophosphorylation of histones H1 and H4.11,37 However, some of the data

31 S. Zhong, C. Jansen, Q. B. She, H. Goto, M. Inagaki, A. M. Bode, W. Y. Ma, and Z. Dong,

J. Biol. Chem. 276, 33213 (2001).32 Y. Yamamoto, U. N. Verma, S. Prajapati, Y. T. Kwak, and R. B. Gaynor, Nature 423, 655

(2003).33 V. Anest, J. L. Hanson, P. C. Cogswell, K. A. Steinbrecher, B. D. Strahl, and A. S. Baldwin,

Nature 423, 659 (2003).34 E. P. Rogakou, W. Nieves-Neira, C. Boon, Y. Pommier, and W. M. Bonner, J. Biol. Chem.

275, 9390 (2000).35 K. Ajiro, J. Biol. Chem. 275, 439 (2000).36 W. L. Cheung, K. Ajiro, K. Samejima, M. Kloc, P. Cheung, C. A. Mizzen, A. Beeser, L. D.

Etkin, J. Chernoff, W. C. Earnshaw, and C. D. Allis, Cell 113, 507 (2003).


still need confirmation, as these modifications have been observed onlyfollowing a limited range of treatments.

Response to Damage/Repair

An attractive link between histone phosphorylation and the damage/repair mechanisms invokes the fascinating possibility that specific signalingpathways must be implicated in distinct damage/repair events. Followingexposure to various agents that cause double-strand DNA breaks, histoneH2A.X, is rapidly phosphorylated on Ser139 in mammalian cells.38 Thismodification spans megabases of DNA around the lesion.39 The same mod-ification has been implicated in other cellular processes known to occurthrough genomic rearrangements, such as recombination in lymphocytesand in germ cells.40,41 Recent studies have shown the possible implicationof some members of the phosphatidylinositol-3-OH kinase-related kinase(PIKK) family in this process, notably using in vitro kinase assays.42

Methods

Chromatin Immunoprecipitation

This technique is widely used for experiments aiming at the isolation ofspecific subpopulations of nucleosomes, for example nucleosomes contain-ing H3 phosphorylated at Ser10. It constitutes an useful approach to studythe association of chromatin modifications with specific DNA sequences.43

This technique can be applied also to investigate the coupling of differentmodifications (i.e., acetylation, methylation, or even phosphorylation onother residues) on a precise subset of nucleosomes.

1. Harvest cultured confluent cells from a 150-mm (approximately 2� 107 cells) in PBS, pellet the cells by centrifugation (5 min, 2000 rpm, 4

�)

37 P. Waring, T. Khan, and A. Sjaarda, J. Biol. Chem. 272, 17929 (1997).38 E. P. Rogakou, D. R. Pilch, A. H. Orr, V. S. Ivanova, and W. M. Bonner, J. Biol. Chem. 273,

5858 (1998).39 E. P. Rogakou, C. Boon, C. Redon, and W. M. Bonner, J. Cell Biol. 146, 905 (1999).40 H. T. Chen, A. Bhandoola, M. J. Difilippantonio, J. Zhu, M. J. Brown, X. Tai, E. P. Rogakou,

T. M. Brotz, W. M. Bonner, T. Ried, and A. Nussenzweig, Science 290, 1962 (2000).41 S. K. Mahadevaiah, J. M. Turner, F. Baudat, E. P. Rogakou, P. de Boer, J. Blanco-

Rodriguez, M. Jasin, S. Keeney, W. M. Bonner, and P. S. Burgoyne, Nat. Genet. 27, 271

(2001).42 S. Burma, B. P. Chen, M. Murphy, A. Kurimasa, and D. J. Chen, J. Biol. Chem. 276, 42462

(2001).43 A. L. Clayton, S. Rose, M. J. Barratt, and L. C. Mahadevan, EMBO J. 19, 3714 (2000).


and wash in 500 �l of Buffer A (15 mM Tris–HCl, pH 7.4, 0.15 mMspermidine, 0.5 mM spermine, 15 mM NaCl, 60 mM KCl, 2 mM EDTA,0.2 mM EGTA, 0.5 mM PMSF, 20 mM NaF) containing 0.5 M sucrose(same centrifugation parameters).

2. Lyse the cells in 1 ml of Buffer A (containing 0.5 M sucrose and0.5% NP-40), incubate 10 min on ice.

3. Pellet nuclei by centrifugation (15 min, 2000 rpm, 4�) and wash

them twice with Buffer A (containing 0.35 M sucrose) (same centrifuga-tion parameters as in step 1).

4. Resuspend the nuclei in 500 �l of Digestion Buffer (50 mM Tris–HCl, pH 7.4, 0.32 M sucrose, 4 mM MgCl2, 1 mM CaCl2, 0.5 mM PMSF,20 mM NaF), evaluate DNA concentration by measuring the absorbanceat 260 nm of a 1:100 dilution in 2 M NaCl and dilute the nuclei suspensionto a concentration of 100 �g DNA/200 �l.

5. Digest the chromatin by incubating the nuclei 10 min at 37�

withmicrococcal nuclease (4 U/200 �l of nuclei suspension), stop the reaction byadding EDTA to a final concentration of 10 mM and cool 10 min on ice.

6. Centrifuge 10 min 13,000 rpm at 4�

and store the supernatant(which will be further referred as fraction S1) at 4

�.

7. Resuspend the pellet in Lysis Buffer (1 mM Tris–HCl, pH 7.4,0.2 mM EDTA, 0.5 mM PMSF, 20 mM NaF) (same volume as that usedfor Digestion Buffer) and dialyze for 12 h against 2 l of Lysis Buffer.

8. Centrifuge 1000 rpm, 10 min at 4�, keep supernatant (fraction S2)

and resuspend pellet in Lysis Buffer (half volume of Digestion Buffer)(fraction P).

9. In order to determine the concentration of DNA, take 1/10 offractions S1, S2, and P, add SDS to a final concentration of 1% and digestwith proteinase K (final concentration 1 mg/ml) 30 min at 37

�. After

standard phenol/chloroform extraction and precipitation with ethanol,DNA will be analyzed on a 1.5% agarose gel.

10. Pool fractions S1 and S2 together, add NaCl and EDTA to finalconcentrations of 50 mM and 5 mM, respectively and incubate overnight at4�

with rabbit polyclonal antiphospho-Ser10-histone H3 antibody (UpstateBiotechnology Incorporated, Lake Placid, USA) (1:1000 dilution).

11. Add 40 �l of a 50% slurry of Sepharose-protein A and incubate 3 hat room temperature.

12. Pellet the beads by centrifugation (2000 rpm, 5 min at 4�) and wash

three times with 500 �l of Wash Buffer (50 mM Tris–HCl [pH 7.4], 10 mMEDTA, 50 mM NaCl, 0.5 mM PMSF, 20 mM NaF). In the case ofexperiments aiming at studying the coupling of different chromationmodifications by Western blot, boil the beads 10 min in 30 �l of Laemmlibuffer 2�.


13. Elute the DNA fragments by incubating the beads two times15 min at room temperature in 1% SDS, pool the two fractions, performphenol–chloroform extraction and ethanol precipitation.

The presence of modifications associated to phosphorylation on thepurified nucleosomes can be analyzed following a standard Western blotapproach, using antibodies raised against a modified histone (acetylation,methylation, phosphorylation on another residue, . . .).

If the experimenter has an interest in studying the association of thesemodified nucleosomes with the underlying DNA sequences, the fragmentsshould be digested by nuclease S1 in order to remove all possible overhangsgenerated by the micrococcal nuclease digestion step, and subsequentlycloned in linearized plasmids. Sequencing of the clones will thus provide in-formation about the identity of the genes that are affected by these histonemodifications. Once some of these genes are identified, quantitative PCRcan be performed to assess the dynamics of histone phosphorylation. Forexample, in the case of cell stimulation giving rise to gene-specific histonephosphorylation and subsequent gene activation, a comparison between un-stimulated and stimulated samples will give an idea about the modificationstatus of the nucleosomes underlying the sequences of interest.

In Gel Histone Kinase Assay

This technique allows the elucidation of the molecular weights of theproteins bearing a kinase activity towards the substrate of interest. It con-stitutes a good starting point in order to get a clue about the identity of akinase phosphorylating a specific substrate.44

Proteins from a cellular extract are separated on a SDS-PAGE gel,whose resolving gel has been polymerized in the presence of the substrate.After a cycle of denaturation/renaturation of the proteins in the gel, pro-teins are submitted to the kinase reaction by incubation with [�32P]-ATP.The gel is then dried and exposed (see Fig. 1).

To validate the physiological relevance of the results, several controlsshould be performed.

44 M.

—Samples thought to contain an active kinase activity for thesubstrate (e.g., the cellular extract of hormone-stimulated cells)should always be compared with a sample supposed of ‘‘containingno activity’’ (e.g., cellular extract of an untreated cell).

—To exclude the possibility that the radioactive band is due toautophosphorylation of the protein, it is highly recommended to run

J. Monteiro and T. I. Mical, Exp. Cell Res. 223, 443 (1996).

Fig. 1. Overview and interpretation of results of a representative in gel kinase assay. This

technique allows the elucidation of the molecular weights of the proteins bearing a kinase

activity towards the substrate of interest. Proteins from a cellular extract are separated on a

SDS-PAGE gel, whose resolving gel has been polymerized in the presence of the substrate.

After a cycle of denaturation/renaturation of the proteins in the gel, proteins are submitted to

the kinase reaction by incubation with �32P-ATP. The gel is then dried and exposed.

Examples of possible results are shown, along with their interpretation.


the same samples on a gel containing no substrate, or an unrelatedsubstrate (e.g., BSA, . . .).

—If the site of phosphorylation within the substrate has beenpreviously identified, an effective control would be to run thesamples in parallel on a gel which has been polymerized in the


presence of a substrate whose phosphoacceptor residue has beenmutated (serine to alanine substitution).

1. Prepare SDS-PAGE gels (8–10% acrylamide), containing in theresolving parts the substrate of interest (for histone H3 [calf thymushistone H3, Boehringer Ingelheim GmbH, Germany]: 0.1 mg/ml; BSA:0.5 mg/ml), or even no substrate at all. For each sample, load 50 �gof protein on the gel and perform migration the same way as a standardprotein electrophoresis.

2. Remove SDS from the gel by washing it twice 30 min in WashingBuffer (20% 2-propanol, 50 mM Tris–HCl, pH 8.0) at room temperatureand denature the proteins by incubating the gel 20 min at 20

�in

Denaturing Buffer (6 M guanidine–HCl, 50 mM Tris–HCl, pH 8.0,5 mM 2-mercaptoethanol).

3. Incubate the gel for at least 16 h at 4�

in Renaturing Buffer (50 mMTris–HCl, pH 8.0, 5 mM 2-mercaptoethanol, 0.04% Tween 40 [v/v],100 mM NaCl, 5 mM MgCl2, 1 mM DTT). Several changes of buffer willallow a better efficiency of renaturation.

4. Wash the gel three times 20 min at 20�

in Kinase Buffer (40 mMHEPES, pH 7.4, 2 mM MnCl2, 5 mM MgCl2, 1 mM DTT, 0.2 mM EGTA).

5. Incubate the gel 4 h at room temperature in 3 ml Kinase Buffercontaining 50 �M ATP and 70 �Ci of [�32P]-ATP (3000 Ci/mol). In orderto eliminate the unincorporated radioactivity, wash the gel four times5 min at room temperature in 1% tetrasodium pyrophosphate-5%trichloroacetic acid.

6. Stain the gel with Coomassie blue R-250, dry the gel and expose.

As the kinase activity of a protein may undergo various regulatory stepswhich would not be necessarily reproduced in gel kinase assay (i.e., inter-action with regulatory proteins), this approach would be useful for proteinkinases whose activity is regulated by an increase in their protein amountor by a change in their modification status (e.g., phosphorylation eventaltering activity). Hence, the experimenter will not always be able to estab-lish the physiological relevance of the results obtained. For example, asshown with band 3 on Fig. 1, a kinase can give no signal if it requires inter-action with other proteins to be activated. On the contrary, a physiologicalkinase for the substrate can also give a signal in the negative control, asexamplified by band 4 on Fig. 1: this situation occurs in the case of kinasesactivated by the release from an inhibitory protein, or in the case ofkinases requiring translocation to the same cellular compartment as thesubstrate.


In Vitro Kinase Assay

This technique allows one to test the kinase activity of a given proteinfor a putative substrate. Histone proteins as substrate can derive from dif-ferent sources.

Nucleosomes: Given the expected complexity of the ‘‘histone code,’’it is predictable that cells contain a wide range of different nucleosomesubpopulations, all defined by unique modification states. Some prior his-tone modifications might affect the ability of enzymes to execute their sub-sequent modification. In the case of histone H3, phosphorylation at Ser10has been shown to enhance the activity of HATs for Lys14,28,45 but to inter-fere with methylation at Lys9 by Suv39h1.46 The use of nucleosomes recon-stituted in vitro, and thus supposedly devoided of modifications, assubstrate for kinase assays will not allow the experimenter to take intoaccount the complexity of the histone code. On the other hand, it is alsopossible to perform the assay on nucleosomes directly extracted from thecell, bearing in mind that possibly only a few specific subpopulations of nu-cleosomes remained phosphorylated. In any rate, the use of nucleosomes inthe kinase assay remains the best approach that takes into account thestructural requirements for the enzymatic activity.

A mix of free histones: In which all four core histones are present inequimolar amounts, but not folded as a nucleosome.

A recombinant N-terminal tail of a histone: Although reductive, the useof a recombinant protein will enable the experimenter to easily identify themodified residue, by targeted mutagenesis (e.g., Ser to Ala mutation).

The kinase to be used in the assay can either be isolated from the cell byimmunoprecipitation, or generated as recombinant protein in bacteria.

1. Incubate the kinase with 15 �l 2� Kinase Buffer (40 mM HEPES,pH 7.4, 300 mM KCl, 10 mM MnCl2), 5 mM NaF, 1 mM DTT, 50 �M coldATP, 20 �M [�32P]-ATP (3000 Ci/mol) and the substrate (nucleosomes:1–5 �g; mix of free histones: 15 �g; an N-terminal tail of the histone: 8 �g).Complete to 30 �l with distilled water, and incubate 20 min at 37

�.

2. Stop the reaction by adding 30 �l of Laemmli Buffer 2�, and boil10 min.

3. Run 5 to 10 �l on a 15% acrylamide SDS-PAGE gel and rinse thegel briefly several times (8 times should be enough) with distilled water, inorder to eliminate all unincorporated radioactivity.

4. Stain the gel with Coomassie blue R-250, dry it, and expose.

45 W. S. Lo, R. C. Trievel, J. R. Rojas, L. Duggan, J. Y. Hsu, C. D. Allis, R. Marmorstein, and

S. L. Berger, Mol. Cell 5, 917 (2000).46 S. Rea, F. Eisenhaber, D. O’Carroll, B. D. Strahl, Z. W. Sun, M. Schmid, S. Opravil,

K. Mechtler, C. P. Ponting, C. D. Allis, and T. Jenuwein, Nature 406, 593 (2000).


GST-Pulldown Assays on Cellular Extracts

The protocol we provide below is the one we used to demonstrate theinteraction between histone H3 N-terminal tail and Aurora-B, the kinase in-volved in mitotic H3 phosphorylation.16 In this experiment, a cellular extractwas incubated with beads previously coupled to a GST-fusion protein encom-passing the first 30 aminoacids of histone H3 and the precipitate, containinghistone H3 interacting partners, was analyzed for the presence of Aurora-B by Western blot. The protocol is slightly different from the standard tech-nique due to the high insolubility of chromatin. Thus, some parametershad to be modified, especially concerning the preparation of the extract.

Although most of the standard protocols for GST-pulldown assaysadvise to use a physiological concentration of NaCl (between 150 and200 mM) both during preparation of the extract and during the incubationwith the GST-fusion protein, the fact that Aurora-B was tightly associatedto the chromatin in vivo, thus displaying a high-level of insolubility,prompted us to prepare the extract using a high concentration in NaCl(600 mM) before finally lowering it to 170 mM during the incubation withthe fusion protein. We therefore advise the experimenter pay special careto the NaCl concentration required to dissociate the protein from thechromatin to fully solubilize it.

Histone tails contain a high number of basic residues, which confer ahigh electrostatic charge and thereby high propension to nonspecificin vitro interactions. We advise the experimenter to use as a negative con-trol the tail of another histone, for example H4, to discriminate betweennonspecific and specific interactions. The optimal NaCl concentration forincubation and washing steps should be set up by comparing resultsobtained with the two tails.

1. Wash twice the cells with PBS, once with EBC buffer (50 mM Tris–HCl,pH 8.0, 50 mM NaF, 0.5 mM PMSF) containing 170 mM NaCl directly on theplate (diameter 100 mm). Lyse the cells with 1 ml of EBC buffer (containing600 mM NaCl and 0.5% NP-40), and collect them in an Eppendorff tube.

2. Pass through a syringe several times (around 10 times), in order tosolubilize the chromatin and incubate 30 min at 4

�with constant stirring.

3. Centrifuge 10 min at 13,000 rpm in a microfuge, keep the supernatant(soluble fraction) and the pellet (insoluble fraction), in order to checkcomplete solubilization of the protein.

4. Dilute the supernatant with EBC buffer (without NaCl, 0.5% NP-40)in order to bring NaCl concentration to 170 mM and measure proteinconcentration of the extract by Bradford assay. Dilute the extract withEBC buffer (170 mM NaCl, 0.5% NP-40) to a final concentration of1 mg/ml.


5. For each binding reaction, preclear 1 mg of extract by incubating 1 hat 4

�with 30 �l of beads preequilibrated in EBC buffer (170 mM NaCl,

0.5% NP-40). Centrifuge 1 min at full speed, keep the precleared extracton ice.

6. Couple the beads to the GST-fusion protein by washing 30 �l ofbeads/binding reaction with 500 �l BCO buffer (20 mM Tris–HCl, pH 8.0,0.5 mM EDTA, 20% glycerol, 500 mM KCl, 1% NP-40, 1 mM DTT,0.5 mM PMSF) four times by centrifuging 5 s at 7000 rpm in a microfuge.Resuspend the beads in 500 �l BCO buffer and 10 �g GST-fusion protein,incubate 1 h at 4

�with constant stirring. Wash the beads four times with

500 �l BCO buffer and then twice with EBC buffer (170 mM NaCl, 0.5%NP-40).

7. Incubate the coupled beads with the precleared extract for 12 h at4�. Centrifuge 5 s at 7000 rpm, discard the supernatant (an aliquot of the

supernatant should be kept in order to compare it with the initial extract).8. Wash the beads three times with EBC buffer (170 mM NaCl, 0.5%

NP-40) and boil the beads in 30 �l Laemmli buffer 2�.

The results of the interaction can then be visualized by Western analy-sis, using a specific antibody to the protein of interest. Due to possible non-specific interactions, it is also advised to include a negative control, that is aprotein that is not supposed to interact with the fusion protein.

Depending on the protein to be studied, some parameters (NaCl con-centration, amount of protein in the interaction assay, time of incuba-tion, etc.) of the above protocol may be modified to suit the specificexperimenter’s goal.

Immunofluorescence

The visualization of the intracellular localization of a kinase with re-spect to its substrate is an essential step towards the validation of its naturalphysiological function. A number of antibodies that recognize modifiedhistone tails have been used successfully in immunofluorescence andimmunostaining analyses. The outcome depends greatly on the qualityof the antibody, for some their effectiveness in other experimentalapproaches is not a guarantee of success for immunofluorescence studies.

Mitotic phosphorylation of histone H3 at Ser-10 results in a strikingvisualization of condensing and condensed chromatin. The experimentalsteps described below can be applied also to other antibodies. The anti-phospho-H3 is a very efficient marker of the different phases of mitosisand shows a distinct colocalization with the kinase involved in mitotic H3phosphorylation, the passenger protein Aurora-B. Indeed, mitoticphosphorylation of histone H3 is a highly dynamic process (see Fig. 2).

Fig. 2. The Ser-10 P-H3 signal allows an easy discrimination between the different phases

of mitosis. NIH3T3 cells are fixed in paraformaldehyde 4%, then hybridized with a mouse

monoclonal antibody raised against the Ser-10-phosphorylated form of histone H3 along with

a rabbit polyclonal antibody raised against the Aurora-B kinase, and then stained with DAPI.


1. Grow the cells on 35 mm diameter plates.2. Discard the medium and wash the cells twice with ice-cold PBS.3. Fix the cells by incubating them with ice-cold PBS (4% paraform-

aldehyde) 20 min on ice and wash them four times with PBS.4. Permeabilize the cells by incubation with PBS (0.2% Triton X-100) 10 min

at room temperature and wash them four times with PBS (0.05% Tween-20).5. Incubate the cells with PBS (5% BSA) in order to avoid aspecific

interactions.


6. Hybridize the cells overnight with the antibody in PBS (5% BSA) at4�, using the appropriate dilution (rabbit polyclonal anti-phospho-Ser10-

histone H3 antibody: 1:1000; mouse monoclonal anti-phospho-Ser10-histone H3 antibody: 1:200) and wash them four times with PBS(0.05% Tween-20).

7. Incubate the cells 1 h at room temperature in the dark with theappropriate secondary antibodies conjugated to fluorophores diluted inPBS (5% BSA) and wash them twice with PBS (0.05% Tween-20).

8. Incubate the cells for 1 min in a 1:50,000 dilution of DAPI in PBS(0.05% Tween-20) and wash them with PBS.

9. Cover the cells with one drop of Vectashield and put a coverslip ontop. Slides can be stored at 4

�in the dark.

Fig. 3. Rapid induction of H3 phosphorylation in mouse hippocampal neurons in response

to in vivo injection of pharmacological agonists to specific receptors. This is an example of

immunohistochemistry with an antibody that recognizes the phosphorylated Ser-10 site on

hippocampi of mice injected with 35 mg/kg of kainic acid (an agonist of glutamaergic

receptors) or with a saline solution (0.9% NaCl). H3 phosphorylation is rapid and transient,

following the kinetics of the immediate early response.


Analogous protocols can also be applied on tissue sections, either toidentify the mitotic cells or to visualize other nonmitotic phosphorylationevents. For example, a rapid and transient phosphorylation of histone H3in hippocampal cells in response to several neurotransmitter receptoragonists can be revealed on mouse brain sections (paraffin or bouin). Thisevent is coupled to the transcriptional induction of the immediate-earlygenes (e.g., c-fos) (see Fig. 3). As example we show phosphorylation of his-tone H3 on serine 10 that, differently from mitotic phosphorylation, iscoupled to localized chromatin decondensation and transcriptional acti-vation. This is elicited by injection of the mouse with kainic acid, an agonistof the glutamaergic receptors, and this event can also be easily visualizedby immunocyto- or immunohistochemistry. Similar results have beenobtained by studying neurons of the suprachiasmatic nucleus, a small struc-ture in the lower hypothalamus of the mammalian brain, where the en-dogenous circadian clock resides. A simple pulse of light induces a rapidand transient phosphorylation at Ser10 of H3, underscoring the great plas-ticity of these neurons in responding to physiological stimuli that reset theendogenous clock system.47 This event is mediated by signaling throughGABAB receptors and is tightly coupled to activation of both clock andearly response genes.

Concluding Remarks

Among all histone modifications, phosphorylation has the unique fea-ture of constituting a direct link with intracellular signaling pathways. Thus,its analysis will provide further information into how epigenetic modifica-tions occur in response to physiology, in cellular differentiation and duringdevelopment. The techniques described here will allow the experimenterto study different aspects of histone phosphorylation. The in gel kinaseassay and the in vitro kinase assay will be of great help for the identificationof specific kinases phosphorylating a given residue, an aspect that could becomplementary to the GST-pulldown assay. Functional information canbe gathered by chromatin immunoprecipitation and immunofluorescencestudies using specific antibodies directed to phosphorylated residues. Theseexperimental approaches will shed further light on the physiologicalimportance of the ‘‘histone code.’’

47 C. Crosio, N. Cermakian, C. D. Allis, and P. Sassone-Corsi, Nat. Neurosci. 3, 1241 (2000).

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