Systematic Identification of Methyllysine-Driven Interactions for

Sep 13, 2010 - Department of Biochemistry and the Siebens-Drake Medical Research Institute, Schulich School of Medicine and Dentistry, The University ...
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Systematic Identification of Methyllysine-Driven Interactions for Histone and Nonhistone Targets Huadong Liu,†,‡ Marek Galka,†,‡ Aimee Iberg,§ Zezhou Wang,† Lei Li,† Courtney Voss,† Xinfeng Jiang,† Gilles Lajoie,† Zhiping Huang,| Mark T. Bedford,§ and Shawn S. C. Li*,† Department of Biochemistry and the Siebens-Drake Medical Research Institute, Schulich School of Medicine and Dentistry, The University of Western Ontario, London, Ontario, N6A 5C1, Canada, The University of Texas M.D. Anderson Cancer Center, Science Park-Research Division, P.O. Box 389, Smithville, Texas 78957, United States, and The No.3 Department, Institute of Chemical Defense, P.O. Box 1048, Beijing 102205, China Received June 15, 2010

An important issue in epigenetic research is to understand how the numerous methylation marks associated with histone and certain nonhistone proteins are recognized and interpreted by the hundreds of chromatin-binding modules (CBMs) in a cell to control chromatin state, gene expression, and other cellular functions. We have assembled a peptide chip that represents known and putative lysine methylation marks on histones and p53 and probed the chip for binding to a group of CBMs to obtain a comprehensive interaction network mediated by lysine methylation. Interactions revealed by the peptide array screening were validated by in-solution binding assays. This study not only recapitulated known interactions but also uncovered new ones. A novel heterochromatin protein 1 beta (HP1β) chromodomain-binding site on histone H3, H3K23me, was discovered from the peptide array screen and subsequently verified by mass spectrometry. Data from peptide pull-down and colocalization in cells suggest that, besides the H3K9me mark, H3K23me may play a role in facilitating the recruitment of HP1β to the heterochromatin. Extending the peptide array and mass spectrometric approach presented here to more histone marks and CBMs would eventually afford a comprehensive specificity and interaction map to aid epigenetic studies. Keywords: Lysine methylation • Chromatin-binding modules • Peptide array • Multiple reaction monitoring • Mass spectrometry • Histone modification • Epigenetics

Introduction Histones are subjected to numerous post-translational modifications (PTM) including methylation, acetylation, phosphorylation, ubiquitination, and sumoylation.1 These modifications serve two major functionssmodulation of the physical property of the nucleosome and creation of specific epigenetic marks for the binding of effector modules. Methylation of Lys and Arg and acetylation of Lys residues have been recognized as important epigenetic marks that regulate chromatin structure and gene expression.2,3 Lysine acetylation is associated with euchromatin and therefore facilitates transcription.4 Histone lysine methylation, in contrast, can be either repressive or activating, depending on location. For example, trimethylation of H3K4 promotes transcription whereas trimethylation of H3K9 leads to a transcriptionally repressive heterochromatin structure.5,6 Aberrant histone modifications underpin many human diseases, particularly cancer.7 In addition to histones, nonhistone proteins may also be methylated on Lys and/or Arg residues.8-10 For instance, the * To whom correspondence should be addressed. E-mail: [email protected]. † The University of Western Ontario. ‡ These authors contributed equally to this work. § The University of Texas M.D. Anderson Cancer Center. | Institute of Chemical Defense. 10.1021/pr100597b

 2010 American Chemical Society

tumor suppressor p53 is heavily modified on multiple sites, including acetylation and methylation on lysine residues.11 The numerous methylation and acetylation marks constitute a complex “code” that is read and interpreted by the hundreds of chromatin-binding modules (CBM).12,13 These CBMs have evolved to recognize different types of marks and distinguish between different methylation states of a given methyllysine (meK) mark.14 For instance, the Royal superfamily of CBMs, including chromodomain, tudor, and MBT domains, recognize methyllysine sites whereas the bromodomains seek out acetyllysine marks.14 It was recently shown that the tandem PHD finger of DPF3b could also bind to acetylated histones, thus blurring the boundaries between CBMs that recognize methyllysine and that bind acetyllysine.15 An important question to address is how these methylation and acetylation marks, which can occur on both histone and nonhistone proteins,16,17 are recognized by the hundreds of CBMs encoded in a mammalian genome to regulate the epigenetic program and a host of other cellular functions. Here we apply peptide array and mass spectrometry to systematically uncover meK-CBM interactions and identify novel methylation marks. We started by creating a methyllysine peptide chip not only containing known and but also potential meK marks on histones and p53. This was followed by Journal of Proteome Research 2010, 9, 5827–5836 5827 Published on Web 09/13/2010

research articles screening the chip with several meK-binding modules. Interactions identified from the peptide chip screens were subsequently validated by pull down assays using individual peptides and by measurement of in-solution affinities. Our studies uncovered numerous novel interactions mediated by histone methylation and defined an expanded interaction network mediated by methyllysine. We carried out further analysis on an interaction mediated by H3K23 methylation, a novel HP1β binding site on histone. We showed, using tandem mass spectrometry collected in information-dependent acquisition subsequent to multiple reaction monitoring mass spectrometry,18 that all three methylation states of histone H3 exist in cells. Peptide pull-down and in solution binding experiments suggest that H3K23me is another high-affinity binding site for HP1β besides the known H3K9me mark. Intriguingly, coimmunostaining data suggested that the H3K23me1, but not the H3K23me2 mark, colocalized partially with HP1β to heterochromatin.

Materials and Methods Peptide Synthesis. Peptides were synthesized on Tentagel resins on an Intavis-AG MultiPep peptide synthesizer using Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry. Peptides were labeled, via a 6-aminohexanoic acid at the N-terminus, either with biotin-NHS for printing and pulldown assays or with fluorescein-NHS for binding studies by fluorescence polarization.19 Purity and identities of the peptides were verified by HPLC and MALDI-MS. Pull-down and Coimmunoprecipitation. For a typical pulldown assay, a methyllysine-containing peptide was labeled at the N-terminus with biotin. Approximately 10 µM of the biotinylated peptide was incubated at 4 °C for an hour with 10 µM purified GST-CBM or 100 µg of HEK293 cell lysate. The complex was then precipitated by incubating with 10 µL of streptavidin sepharose beads (GE Healthcare) for 1 h at 4 °C. After extensive washes in TBST buffer (0.1 M Tris-HCl, pH 7.4, 150 mM NaCl, and 0.1% Tween 20), the pulled-down proteins were separated on SDS-PAGE, transferred onto PVDF membrane and Western blotted with a rabbit anti-GST antibody (Abcam Inc. Cat. #ab3416) or another appropriate antibody (e.g., anti-HP1β, Millipore LV1512441). For pull-down of histone H3 by the HP1β chromodomain, the latter was expressed in E. coli strain BL21 as a (His)6-tagged protein using the expression vector pETM11 and purified on Ni-NTA (Qiagen). The protein was further purified on FPLC and immobilized onto MS300 carboxyl beads (JSR Co., Japan). The immobilized HP1β-CD was incubated with 200 µg of HEK293 cell nuclear lysate for 1 h at 4 °C. After three washes in TBST, the beads were subject to SDS-PAGE analysis and Western blotting with a rabbit anti-H3K23me1 (Active Motif #39387) or anti-H3K9me3 (Active Motif #39285) antibody. For coimmunoprecipitation of H3 and endogenous HP1β, 10 µL of mouse anti-HP1β (Millipore LV1512441) antibody was added to 500 µg of nuclear lysate of HEK293 cells, and the mixture was allowed to mix for 1 h at 4 °C. The immuno-complex was then precipitated by protein G beads (JSR Co.). After three washes in TBST, the complex was subjected to SDS-PAGE and immuno-blotted with either anti-H3K23me1 or anti-H3K9me3 antibodies. Methyllysine Peptide Chip Preparation and Probing. A biotin-labeled peptide was incubated in PBS, pH 7.5 with neutravidin in a 1:1 molar ratio for 1 h at room temperature. The mixture was diluted in PBS to generate three different concentrations of peptide-neutravidin conjugate stocks: 25.0, 5828

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Liu et al. 12.5, and 6.25 µM. SuperAB glass slides (Fisher) were preactivated in 50 mM NaIO4, 0.1 M sodium acetate, pH 5.5 for 0.5 h at room temperature, dried in nitrogen stream, and used immediately. The peptide-neutravidin conjugates were printed onto an activated SuperAB chip using a Bio-Rad VersArray Chipwriter-Pro system. Before probing with a purified protein, the peptide array chip was washed three times in 3% BSA in TBST buffer (0.1 M Tris-HCl, pH 7.4, 150 mM NaCl, and 0.1% Tween 20). For probing, 1.0 µM total GST fusion protein was added directly into the 3%BSA/TBST buffer and incubated with the slide for 1 h at RT. The slide was then washed 3× in TBST and incubated with a rabbit anti-GST antibody (Abcam Inc. Cat. #ab3416). After one hour, the slide was washed 3× in TBST and incubated with a DyLight 649-labeled goat antirabbit IgG antibody (Pierce Co., Cat. #35565) for another hour in the dark. The slide was washed again in TBST, dried in the dark, and scanned with a microarray laser scanner (Tecan Co.). Data processing and quantification were performed using the embedded software of the scanner. The intensity was normalized by a standard score (z-score), which was used to assess whether the signal in a screen is large enough to warrant further attention.20 The z-score was calculated using the equation z ) (χ-µ)/σ for each slide, where χ is a raw score to be standardized; µ is the mean of the population; and σ is the standard deviation of the population. The z-score allowed for easy comparison of screening results obtained for peptide at different concentrations and from using different protein probes. Multiple Reaction Monitoring-Mass Spectrometric Analysis of H3K23 Methylation. Purified histones from HEK293 cells or NIH3T3 were separated on SDS-PAGE and the H3 bands were excised and digested with ArgC. The digests were analyzed by positive ESI-LC-MS/MS on a triple quadrupole (Q3 linear ion trap) mass spectrometer (QTRAP 4000, Applied Biosystems). A nanoAcquity UPLC system (Waters) equipped with C18 analytical column (1.7 µm, BEH130, 75 µm × 200 mm and/or 75 µm × 250 mm) was used to separate the peptides at the flow rate of 300 nL/min and operating pressure of 7000 psi (at 95/5H2O:MeCN). Eluted peptides were directly electrosprayed (Nanosource, ESI voltage +2000 V) into the QTRAP instrument. Peptides were eluted using a 62 min gradient with solvents A (H20, 0.1% formic acid) and B (MeCN, 0.1% formic acid) in 41 min from 5% B to 50% B, 6 min 90% B, 10 min 5% B. Due to close retention times between H3K9 and H3K23 peptides, relative quantification was done using longer, 115 min gradient and 250 mm column (solvents A (H20, 0.1% formic acid) and B (MeCN, 0.1% formic acid) in 100 min from 5% B to 50%B, 5 min 90%B, 10 min 5%B). The instrument was set up to monitor 32 transitions in each sample (100 ms/transition) for a total scan time of 3.2 s. Fluorescence Polarization Measurements. Different concentrations of a purified GST fusion protein were added to a fluorescein-labeled peptide solution which was diluted to give the final concentration of 20 nM in PBS (pH ) 7.6). The mixtures were allowed to stand in the dark for 30 min prior to fluorescent polarization measurements at room temperature on a Perkin-Elmer 2103 multilabel plate reader with excitation set at 480 nm and emission at 520 nm. Binding curves were generated by fitting the isothermal titration data to a hyperbola nonlinear regression model using Prism 3.0 (GraphPad software, Inc., San Diego, CA), which also produced the corresponding dissociation constants (Kd).21 Fluorescence and Confocal Microscopy. NIH3T3 cells were grown on glass coverslips in DMEM + 10% NCS. Cells were

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Figure 1. Systematic mapping of methyllysine (meK) interactions with effector domains using a meK peptide chip. (A) Layout of the meK-peptide chip. A trace amount of fluorescein-labeled neutravidin printed along with the peptide conjugates for quality control of array printing. Image shown represents fluorescent image of a blank peptide chip. The unmodified, mono-, di-, or trimethylated states for each Lys site are printed from top to bottom in four clusters. Within each state cluster, three concentrations of a peptide-neutravidin complex are printed from top to bottom at 25, 12.5, and 6.25 µM, respectively, with duplications in each row. The first and last two columns are GST controls printed in descending concentrations. The cluster at the lower left corner is neutravidin control printed at 25 µM. See Table S1 for sequences of the peptides printed on the array (Supporting Information). (B) Binding of GST to the meK peptide chip. (C) Blow-up image of a region in (B) to show the GST control column and lack of GST binding on the peptide chip. (D) Binding profile of the HP1β chromodomain on a meK-peptide chip. (E) Binding pattern for the Mpp8 chromodomain. Known (green) and novel (yellow) candidate interactions are identified by rectangular circles. For clarity, only part of the chip was shown in D and E. See Figure S5 for the complete domain-chip binding profiles (Supporting Information).

Figure 2. Biochemical characterization of interactions between the chromodomain and the H3K23 methyl peptides. (A) Fluorescence polarization binding curves for the HP1β chromodomain to various H3K23 peptides as labeled on the diagram. (B) Fluorescence polarization binding curves for the Mpp8 chromodomain to the same peptides as in (A). (C) Pull-down of HP1β from HEK293T cell lysate by the biotinylated H3K23me0, -1, -2, or -3 peptide. The H3K9me3 and H3K14me1 peptides were used as positive or negative control, respectively digest of histone 3 (H3). (D) HP1b-CD pull-down (upper panel) and immunopreciptation (lower panel) of histone H3 from nuclear lysates of HEK293 cells. The associated H3K23me1 and H3K9me3 were revealed by Western blotting using the corresponding antibodies. The abundance of the two marks in cells was assessed by separating 10 or 1 µg histones on SDS-PAGE followed by Western blots using anti-H3K23me1 or anti-H3K9me3 antibodies.

fixed for 10 min in 2% formaldehyde and washed with ice-cold methanol. The methanol was rinsed off with two brief PBS washes, and then the blocking solution (3% BSA in PBS) was

applied for 30 min at room temperature. After three washes in staining buffer (PBS + 0.1% NP-40), the primary antibody was added. The antibodies and dilutions used were: H3K23me1 Journal of Proteome Research • Vol. 9, No. 11, 2010 5829

research articles (Active Motif #39387) at 1:50, H3K23me2 (Abcam # ab2370) at 1:50, H3K9me3 (Active Motif #39285) at 1:100 and HP1β (Santa Cruz #SC10212) at 1:50. After a 1-h incubation at room temperature, the primary was removed and the cells were washed three times in staining buffer. Finally, the appropriate secondary (Alexa Fluor-466 antirabbit or Alexa Fluor-647 antimouse or Cy3 antigoat) was added at a 1:500 dilution for 30 min at room temperature. The cells were washed three times with staining solution, DAPI added at a 1:10 000 dilution for one minute, the cells washed again and finally the glass coverslips inverted and placed on a microscope slide with Invitrogen SlowFade mounting medium. Images were acquired on a Zeiss LSM 510 META confocal microscope.

Liu et al. Table 1. In-Solution Binding Affinities for CBM-meK Peptide Interactions Identified from Peptide Array Screensa domain

HP1β chromodomain

Mpp8 chromodomain

Results Visualiztion of Methyllysine Mark-Effector Domain Interactions on a Comprehensive Methyllsyine Peptide Chip. To systematically identify meK-CBM interactions, we produced a peptide array chip that recapitulated established and potential lysine methylation events on histones or p53 (Table S1, Supporting Information) and probed it for binding, respectively, to a set of CBMs that included the chromodomain, tudor domains, PHD fingers, and MBT domains. All peptides were synthesized as 13-mers (except for the H3K4 peptides which were shorter), biotinylated at the N-terminus via a 6-aminohexanoic acid spacer, and verified for purity by HPLC and identity by mass spectrometry (Table S2; Figure S1, Supporting Information). Each Lys mark was represented in four forms of K, Kme1, Kme2, and Kme3, corresponding to the unmodified, mono-, di- and trimethylated states, respectively (Figure 1A). The peptides were then coupled to neutravidin and the resulting conjugates printed on a glass slide by amine-aldehyde condensation. The meK-peptide chip was subsequently incubated with a purified GST-CBM and the bound protein detected with an anti-GST antibody. The binding signals were visualized using a DyLight 649-labeled secondary antibody (Figure 1). The array exhibited no detectable binding to GST (Figure 1 B and C), indicating that signals observed were specific for the fused CBM. To explore the utility of the meK peptide array in determining the specificity of a CBM and identifying meK-CBM interactions, we probed it with some Royal family CBMs,14 including chromodomain, tudor and MBT domains, and with non-Royal family PHD fingers.22 We first tested if the array could recapitulate known interactions by probing it with the HP1β chromodomain (CD) that was previously shown to bind methylated H3K9 and H1K25.23-25 As expected, we detected specific binding of the HP1β CD to all three methylated forms of H3K9 and H1K25, but not to the corresponding unmethylated peptides. Intriguingly, it also bound strongly to the methylated H3K23 peptides (Figure 1D). To explore the peptide chip as a discovery tool, we probed it with the chromodomain of the M-phase phosphoprotein 8 (Mpp8) which, until recently, has few binding partners identified.26,27 The Mpp8 CD exhibited a binding profile that overlapped significantly with that of the HP1β CD. However, besides the H1K25, H3K9 and H3K23 methyl peptides, the Mpp8 CD also recognized the H1K168 and H3K4 methyl marks (Figure 1E). Validation of Interactions Identified from Using the meK Peptide Chip. To ascertain that interactions identified from the peptide array screening occur in solution, we determined the binding affinities (measured as dissociation constant or Kd) of the H3K9 and H3K23 peptides in different methylation 5830

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JMJD2A tudor domains

53BP1 tudor domains

site

Kd (µM)

H3K9 H3K9me1 H3K9me2 H3K9me3 H3K23 H3K23me1 H3K23me2 H3K23me3 H1K25me3 H3K9 H3K9me1 H3K9me2 H3K9me3 H3K23 H3K23me1 H3K23me2 H3K23me3 H1K25 H1K25me1 H1K25me2 H1K25me3 H4K20me3 H1K25me3 H3K23me2 H3K23me3 p53K382me1 p53K382me2 p53K382me3 H4K20me2 p53K381me1 p53K381me2 p53K381me3 p53K382me1 p53K382me2 p53K382me3

NB 12.5 ( 1.6 4.4 ( 0.2 3.2 ( 0.2 NB 16.3 ( 4.3 4.7 ( 0.5 3.0 ( 0.3 7.5 ( 1.4 NB 6.6 ( 0.4 2.3 ( 0.3 1.2 ( 0.1 NB 25.5 ( 3 6.3 ( 0.5 4.7 ( 0.4 N/A 16.6 ( 2.6 7.9 ( 0.5 4.4 ( 0.6 2.6 ( 0.3 6.6 ( 0.8 3.6 ( 0.2 2.2 ( 0.3 20.7 ( 3.8 22.4 ( 2 11 ( 1.1 42.5 ( 24.6 NB 25 ( 3.4 NB NB 27.1 ( 5.4 NB

a Each peptide was synthesized with an N-terminal fluorescein tag via a 6-aminohexanoic acid spacer to facilitate measurement of dissociation constants by fluorescence polarization. Proteins used in the binding assay were expressed in E. coli and purified to homogeneity. NB, no binding.

states for the purified HP1β chromodomain by fluorescence polarization (Figure 2). In agreement with previous work,23 the HP1β CD bound to all three methylated forms of the H3K9 peptides, but not to the unmethylated version. Moreover, diand trimethylation enhanced affinity significantly (Table 1). Intriguingly, the H3K23 and H3K9 peptides of the same methylation state displayed comparable affinities for the HP1β CD despite variations in sequence surrounding the meK site (Table 1; Figure 2A). This data suggests that the HP1β chromodomain possesses a wider specificity than previously appreciated. Similarly, we found that the Mpp8 chromodomain bound to both methylated H3K9 and H3K23 peptides (Figure 2B; Table 1). Moreover, the respective affinities of the H3K9me3 and H3K23me3 peptides for the HP1β and Mpp8 chromodomains are comparable, suggesting that the functions of HP1β and Mpp8 are likely related. To verify that the identified interactions occur for the intact HP1β protein, we employed biotinylated H3K23, H3K23me1, -2, and -3 peptides, respectively, to pull down endogenous HP1β from HEK293 cell lysate. We found that all but the unmethylated H3K23 peptide bound to HP1β. As expected, the H3K9me3 peptide, used as a positive control, bound strongly to HP1β while the H1K14me1 peptide, which exhibited no

Mapping Methyllysine-Driven Interaction Networks

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Figure 3. Identification and relative quantification of in vivo H3K23 methylation by MRM-MS. (A)-(C) Sections of MS/MS spectra showing the key fragment ions indicative of lysine methylation. MS/MS spectra were collected subsequent to MRM signal detection (information dependent acquisition) of different methylation states of H3K23 [KQLATK(Me)AAR]. (D)-(E) MRM peaks showing the approximate relative abundance of methylated vs. non-methylated H3K9 and H3K23. Signal corresponding to non-methylated H3K23 was undetectable under the same conditions.

binding to the chromodomain on the peptide array, failed to precipitate HP1β from the cell lysate (Figure 2C). In a reverse pull-down experiment, we used purified and immoblized HP1β CD to precipitate histone H3 from HEK293 cell nuclear lysate and immublotted for the amount of H3K23me1 or H3K9me3. As shown in Figure 2D, comparable levels of K23me1- and K9me3-containing histone H3 were pulled down by the HP1β chromodomain. Because these two methyl marks coexist on histone H3, it is difficult to ascertain whether the observed binding was due to H3K23me1 or H3K9me3 or both. In an attempt to resolve this uncertainty, we purified histones from the nuclear lysate and immunoblotted for the level of H3Kme1 and H3K9me3, respectively. At the same time, we immunoprecipitated endogenous HP1β from HEK293 cell lysate and assayed for the presence of H3Kme1 or H3K9me3 in the same

Western blot (Figure 2D, lower panel). We found that the level of H3K23me1 was similar to that of H3K9me3, suggesting both are significant modifications. This assertion was also supported by data from mass spec analysis (Figure 3). As expected, HP1β coimmunoprecipitated both H3K23me1 and H3K9me3. Identification of Methylated H3K23 as a Novel Histone Maker Associated with HP1β in vivo. As with other in vitro approaches, candidate interactions identified here using the peptide array approach need to be verified in vivo to fully appreciate their physiological relevance. Data from the meK peptide array screen and peptide pull-down suggest that H3K23 methylation may be involved in recruiting HP1β to heterochromatin in cells. Although H3K23me1 has been shown to exist in cells,28 whether it is a significant PTM or not has not been addressed. In addition, other methylation states of H3K23 Journal of Proteome Research • Vol. 9, No. 11, 2010 5831

research articles have not been established in vivo. We combined multiple reaction monitoring with tandem MS/MS analysis to determine whether different methylation states of H3K23 are present in NIH3T3 cells. We were able to observe by MRM not only the mono-, but also the di- and trimethylated forms of KQLAT[K23]AAR, a peptide drawn from the H3K23 site (Figure S2; Table S3, Supporting Information). Methylation of these peptides was detected by MRM-MS. Signals due to MRM transitions were confirmed by the presence of characteristic fragment ions on the corresponding MS/MS spectra (Figure 3A-C). This analysis provided evidence in support of the notion that the H3K23 site is mono-, di- and tr-methylated in vivo. To examine whether H3K23me methylation is a significant modification, we used MRM to monitor the relative abundance of the monomethylated K23 (or H3K23me1) versus the nonmethylated peptides (or K23) on histone H3 isolated from HEK293 cells. As shown in Figure 3D, while the MRM transitions corresponding to the H3K23me1 peptide produced a large peak, those corresponding to the H3K23 peptide were undetectable under the same condition. This suggests that the H3K23 site is monomethylated in HEK293 cells. In a control experiment, we examined the relative abundance of H3K9 versus H3K9me3 peptides by MRM and found that the level of H3K9me3 peptide was 4-fold over the level of H3K9 peptide (Figure 3E), assuming that the ionization of the two peptides were comparable in the mass spectrometer. To explore the potential function of H3K23 methylation, we examined the colocalization of methylated H3K23 and HP1β in NIH3T3 cells. Because the lack of an antibody specific for H3K23me3, we focused on the H3K23me1 and H3K23me2 marks. To this end, we first determined the specificity of two commercial anti-H3K23me1 and anti-H3K23me2 antibodies by meK peptide chip screening and immunostaining. The antiH3K23me1 antibody bound strongly to the H3K23me1 peptide on the meK peptide chip as expected, but also moderately to H3K14me1 (Figure 4A). In contrast, the anti-H3K23me2 antibody exhibited specific binding to the H3K23me2 peptide on the chip (Figure 4B). The specificity of these antibodies was further verified by the disappearance of the specific immunostaining signal for the H3K23me1 or the H3K23me2 mark in the presence of the corresponding competing peptide antigen (Figure S3A and B, Supporting Information). The H3K14me1 peptide, which showed weaker binding than H3K23me1 to the anti-H3K23me1 antibody on the peptide chip (Figure 3A), was unable to compete off the H3K23me1 immunostaining signals when applied at the same concentration as the H3K23me1 peptide (Figure S3C, Supporting Information). We next immunostained NIH3T3 cells with the H3K23me1 and H3K23me2 antibodies, respectively, and examined their colocalization with H3K9me3, an established mark that is associated with heterochromatin. Visualization by confocal microscopy revealed colocalization between H3K9me3 and H3K23me1 (Figure S4A, Supporting Information). In contrast, the H3K23me2 mark does not appear to be enriched in the H3K9me3 and DAPI foci, at least when using this particular antibody (Figure S4A, Supporting Information). To examine whether H3K23me1 or H3K23me2 colocalized with HP1β, we overexpressed dsRed-HP1β in NIH3T3 cells. Immunostaining of H3K23me1 and H3K23me2 revealed that the former, but not the latter, colocalized with HP1β on dense heterochromatin (Figure S4B, Supporting Information). Again, H3K23me2 was found in the nucleus but did not colocalize with DAPI and HP1β foci. Co-immunostaining of endogenous HP1β with H3K23me1 5832

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Figure 4. Colocalization of H3K23me1 or -2 with H3K9me3 or HP1β in cells. (A) Binding profiles of an anti-H3K23me1 antibody on the meK peptide chip. (B) Binding profile of an anti-H3K23me2 antibody. Note that each peptide was printed in three concentrations and with replicates for each concentration on the peptide chip, therefore each rectangular box identifies a single peptide. The H3K23me2 antibody is specific, however the H3K23me1 antibody recognizes both H3K23me1 and H3K14me1, albeit with lower affinity for the latter. (C) Confocal microscopic images of coimmunostained H3K23me1 (red) and HP1β (green) in NIH3T3 cells. Nuclei were stained blue with DAPI. White arrowheads indicate sites of colocalization. (D) Coimmunstaining of H3K23me2 (red) and HP1β (green) in NIH3T3 cells.

(Figure 4C) or H3K23me2 (Figure 4D) in NIH3T3 cells revealed the colocalization of HP1β with the former but not the latter mark. Collectively, data from the confocal microscopic analysis suggest that, like H3K9me3, H3K23me1 may facilitate the association of HP1β with heterochromatin. It is unlikely that the observed cross-reactivity of the anti-H3K23me1 antibody to the H3K14me1 mark on the Kme peptide chip would contribute to the collocalization of HP1β and H3K23me1 because the H3K14me1 peptide was unable to bind HP1β (Figure 2C) and block H3K23me1 immunofluorescence (Figure S3C, Supporting Information). While additional experiments are needed to address the in vivo function of H3K23 methylation, our data on the H3K23me1-HP1β interaction and colocalization in cells provide evidence for the expansion of the histone code to include H3K23 methylation. Systematic Identification of meK-CBM Interactions. To explore broad utility of the meK peptide chip, we used it to identify binding sites for the tudor domains of 53BP1, JMJD2A, and PHF20, respectively. We found that the 53BP1 and PHF20 tudor domains only recognized dimethyl-Lys marks whereas JMJD2A read both di- and trimethylated marks, but with a greater proclivity for the latter (Table 2; Figures S5, Supporting Information). We compared the affinities of the JMJD2A and 53BP1 tudor domains for their target peptides using fluorescence polarization (Table 2, Figure S6A, Supporting Informa-

research articles

Mapping Methyllysine-Driven Interaction Networks a

Table 2. CBM-meK Interactions Identified from Screening a Methyllysine Peptide Array

a Only interactions with a binding z score greater than 2.0 are listed in the table. Known interactions are marked by “+”, missed known interactions by “-”, and novel candidate interactions by a checkmark.

tion) and peptide pull-down assays (Figure S6B, Supporting Information) and found that the former exhibited significantly greater affinities than the latter for the H1K25me3, H3K18me1, H3K23me2/3, H3K4me3, and H4K20me3 peptides. Except for H3K4me3 and H4K20me3,29-31 the remaining docking sites are novel, suggesting that JMJD2A is a demethylase with a broad spectrum of targets. We also probed the meK peptide array with the PHD finger from PHF2 and the MBT domain of PHF20L1, which, like the other CBMs, displayed unique binding profiles (Figure S5, Supporting Information). Because the peptides are immobilized through their N-termini, which are thus not free, it is likely that not all potential interactions may be observed from such screens. Indeed, interactions of the 53BP1 tudor domains with K3K79me2 and p53K370 and an interaction between the JMJD2A tudor domains and H3K4me3 were not detected on the peptide chip for unknown reasons. Nevertheless, the meK peptide array screens recapitulated 85% (or 17/20) of the interactions reported in the literature and led to the identification of 41 novel candidate interactions (Table 2), allowing us to generate a putative, expanded histone meK-CBM interaction network (Figure 5). Although the physiological functions of these interactions await further investigation, our results suggest that there exists an extensive histone methyllysine markeffector domain interaction network and that the dynamics of this network may play a pivotal role in regulating the epigenetic program.

Discussion In an attempt to systematically identify interactions mediated by methyllysine and their binding domains, we combined peptide array screening, multiple reaction monitoring mass spectrometry, and in-solution binding assays to define an expanded epigenetic regulatory network. Our strategy is distinct from SPOT arrays32,33 in that all peptides were purified and identity-verified before they were printed onto a slide as neutravadin conjugates. This ensures quality of the array and minimizes the potential loss of binding due to direct immobilization. Another advantage of the meK peptide chip over traditional SPOT array is that multiple copies of an array may be produced from the same batch of peptide stocks, thus allowing direct comparison of binding profiles between different CBMs. Compared to previous work that focused on identifying meK-CBM interactions for known histone meK marks26 our peptide array contains not only the established sites but also potential methylation/binding sites (Table S1, Supporting Information), which enabled us to combine peptide array and mass spectrometry to identify interactions mediated by either known or potential methylation sites. Importantly, these candidate binding events identified from the meK peptide array screens were subsequently validated by in-solution binding assays. Our meK-peptide array exhibited superior sensitivity and low background signal and allowed for the identification of a significantly greater number of known and Journal of Proteome Research • Vol. 9, No. 11, 2010 5833

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Figure 5. Systematic mapping of methyllysine-CBM interactions. (A) Heat map representation of candidate interactions between CBMs and meK peptides based on results from the meK peptide chip screens. The z-scores corresponding to interactions detected on the peptide chip were used to generate the heat map using the MeV Multi Experiment Viewer. (B) In vitro CBM-histone methyllysine mark interaction network identified from the meK peptide chip screens (using a z score cutoff of 2.0). The histone methylation sites are identified with small circles, binding domains with larger circles, and histones with barrels.

novel interactions than previous studies.26 It is likely that the combination of different factors such as the purity of the peptides, the extra linker (i.e., 6-amino hexanoic acid) at the N-terminus, the preconjugation of biotinylated peptide with neutravidin, the multiple concentrations of a peptide (ranging from 6.25 to 25 µM) printed on a meK peptide chip, and the sensitive DyLight 649 fluorescence detection, contributed to the difference in sensitivity between this study and others. Indeed, our meK peptide array allowed for a wide dynamic range of binding signals to be observed (Figure S7, Supporting Information). Despite the high sensitivity of the meK peptide chip, some known interactions, notably a well-characterized interaction between the JMJD2A tudor domains and H3K4me3,30 were missed from our screens when a stringent z score cutoff of 2.0 was applied (Table 2). Because a peptide is biotinylated at the N-terminus and immobilized through neutravidin, it is possible that the short N-terminus of the H3K4me3 peptide (4 residues compared to 6 in all other peptides) constrained the peptide conformation upon immobilization and compromised its binding to the JMJD2A tudor domain. Our screen also missed the interactions of the 53BP1 tudor domains with H3K79me2 and p53K370me2 due likely to 5834

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the generally weak binding between the 53BP1 tudors with its methyllysine marks. This comprehensive interaction analysis led to several observations. First, although different histone marks are recognized by different CBMs, cross-binds often occur. This is particularly true for CBMs from the same family such as the HP1β and Mpp8 chromodomains and the JMJD2A and 53BP1 tudor domains. Recent work suggested that many other domains are capable of read out histone methyllysine marks besides those studied herein, making the histone meK-CBM interaction network even more complex to comprehend than previously thought. For example, the repressive methyl H3K27 mark is recognized by chromobox 7 (CBX7) within the polycomb repressive complex 1 (PRC1)34 and by EED within PRC2, an interaction that plays a critical role in the spreading of the H3K27me3 mark.35 The methylated histone H3 tail is also the site for binding by PELP1, an oncogenic, proline, glutamic acid and leucine-rich protein36 and by the DNA methyltranferase DNMT3A. Interestingly, while the DNMT3A PWWP domain seeks out H3K36me3 and thereby directs DNA methylation,37 its ADD domain binds to H3 histone 1-19 tail. Intriguingly the latter interaction is disrupted by H3K4 trimethylation or Ser/

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Mapping Methyllysine-Driven Interaction Networks 38

Thr phosphorylation. Second, although a given CBM may have a preference for certain methylation states, this bias is not absolute, but rather it varies with the methylation site.14 Similar observations have been made on the PHD finger and other CBMs. For instance, although PHD finger domains from different proteins recognize the H3K4 methylation mark, different domains selectively bind to mono-, di- or trimethylated H3K4.39-43 Third, there exist extensive overlaps in specificity between different families of CBMs. For instance, H3K23me2 or -3 is recognized by both the chromodomains and PHD fingers whereas H4K20me2 or -3 is capable of binding to either the tudor domains or PHD fingers. Therefore the specificity of a CBM cannot be properly gauged by affinity for a single mark. A more appropriate measurement of CBM specificity is therefore by its association with a group of specific marks (Figure 5). It should also be recognized that CBM may function in a combinatorial manner. Besides tudor domains that are often present in tandem in a protein to read out methyllysine site, the tandem PHD finger-bromodomain of human KAP1 has been shown to function as a unit to facilitate lysine SUMOylation.30 Besides recapitulating known interactions, our meK peptide array analysis led to the identification of numerous candidate interactions (Table 2). While the biological relevance of these latter interactions await further investigation, we interrogated the physiological significance of a potential interaction between the HP1β chromodomain and the methyl H3K23 mark. We verified by mass spectrometry that H3K23 is mono-, di-, or trimethylated and that the abundance of H3K23me1 is comparable to that of H3K9me3 in cells. Moreover, we showed that the H3K23me1 mark partially colocalizes with HP1β to heterochromatin, suggesting that H3K23 monomethylation may play a role in regulating chromatin structure. It is also possible that the two marks function together to regulate HP1β function and/ or chromatin state. This assertion is supported by our observation that the K23me1 and K9me3 marks coexist on histone H3, hinting at the possibility that the functions of the two sites may be related. In this regard, interdependence of H3K23 acetylation and other histone marks have been described in previous work.44,45 Apparently, the identification of the methyl transferase and demethylase responsible for writing and erasing the H3K23 methylation, respectively, would help define the role of this novel modification in chromatin biology. Intriguingly, we found that the H3K23me and H3K9me peptides of the same methylation state bind with comparable affinities to the HP1β chromodomain despite limited identity in sequence flanking the methyllysine residue. The structure of the HP1β chromodomain in complex with an H3K9me3 peptide revealed a specific binding pocket enriched in aromatic residues for the trimethyl moiety, explaining why Kme3 is favored over Kme2 or Kme1. In contrast, the residues surrounding the Kme3 bound in an induced-fit manner and formed a β-strand that was packed against the β-sheet the chromodomain.23 This mode of ligand recognition suggests that considerable flexibility and variability may exist for the residues surrounding the Kme3. Promiscuity in peptide sequence recognition was also observed for other CBMs, including the Mpp8 chromodomain and the JMJD2A tudor domains. In the latter case, the JMJD2A tudor domains were found to bind with comparable affinities to the H3K4me3, H3K20me3, H3K23me2/3 and H1K25me3 marks, respectively (Figure 5, Table 2). Understanding lysine methylation and methyllysine-driven protein-protein interactions at a global scale is essential for a

systematic understanding of the molecular basis of epigenetic regulation of cellular functions. We have presented here a method that combines peptide array, mass spectroscopy, biochemical and cellular assays to identify novel methylation events and to systematically map candidate interactions enabled by methylation. Our data on lysine methylation and methyllsine-binding modules provided the proof-of-principle for this approach, which, at the same time, generated a wealth of new information (e.g., novel methylation sites and candidate interactions) for further exploration. HP1β is a versatile protein capable of interacting with both histone and nonhistone targets (data not shown), and a full understanding of the function of this heterochromatin-associated protein would require characterization of these diverse interactions. We anticipate that this approach, when extended to other types of histone modifications such as acetylation and phosphorylation and to additional chromatin-binding modules, would afford a comprehensive interaction map enabled by histone modifications that would help decipher the histone code.46

Acknowledgment. This work with supported by grants (to S.S.C.L.) from the Canadian Cancer Society and Genome Canada through the Ontario Genomics Institute. M.T.B. is supported by a grant from NIDA. S.S.C.L. holds a Canada Research Chair in Functional Genomics and Cellular Proteomics. Supporting Information Available: Supplementary tables and figures. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Smith, B. C.; Denu, J. M. Chemical mechanisms of histone lysine and arginine modifications. Biochim. Biophys. Acta 2009, 1789 (1), 45–57. (2) Neff, T.; Armstrong, S. A. Chromatin maps, histone modifications and leukemia. Leukemia 2009, 23 (7), 1243–51. (3) Litt, M.; Qiu, Y.; Huang, S. Histone arginine methylations: their roles in chromatin dynamics and transcriptional regulation. Biosci. Rep. 2009, 29 (2), 131–41. (4) Allfrey, V. G.; Faulkner, R.; Mirsky, A. E. Acetylation and Methylation of Histones and Their Possible Role in the Regulation of Rna Synthesis. Proc. Natl. Acad. Sci. U.S.A. 1964, 51, 786–94. (5) Eissenberg, J. C.; Shilatifard, A. Histone H3 lysine 4 (H3K4) methylation in development and differentiation. Dev. Biol. 2009. (6) Shinkai, Y. Regulation and function of H3K9 methylation. Subcell Biochem. 2007, 41, 337–50. (7) Esteller, M. Cancer epigenomics: DNA methylomes and histonemodification maps. Nat. Rev. Genet. 2007, 8 (4), 286–98. (8) Pradhan, S.; Chin, H. G.; Esteve, P. O.; Jacobsen, S. E. SET7/9 mediated methylation of non-histone proteins in mammalian cells. Epigenetics 2009, 4 (6), 383–7. (9) Huang, J.; Berger, S. L. The emerging field of dynamic lysine methylation of non-histone proteins. Curr. Opin. Genet. Dev. 2008, 18 (2), 152–8. (10) Teyssier, C.; Le Romancer, M.; Sentis, S.; Jalaguier, S.; Corbo, L.; Cavailles, V. Protein arginine methylation in estrogen signaling and estrogen-related cancers. Trends Endocrinol. Metab. 2010, 21 (3), 181–9. (11) Murray-Zmijewski, F.; Slee, E. A.; Lu, X. A complex barcode underlies the heterogeneous response of p53 to stress. Nat. Rev. Mol. Cell. Biol. 2008, 9 (9), 702–12. (12) de la Cruz, X.; Lois, S.; Sanchez-Molina, S.; Martinez-Balbas, M. A. Do protein motifs read the histone code. Bioessays 2005, 27 (2), 164–75. (13) Berger, S. L. The complex language of chromatin regulation during transcription. Nature 2007, 447 (7143), 407–12. (14) Taverna, S. D.; Li, H.; Ruthenburg, A. J.; Allis, C. D.; Patel, D. J. How chromatin-binding modules interpret histone modifications: lessons from professional pocket pickers. Nat. Struct. Mol. Biol. 2007, 14 (11), 1025–40.

Journal of Proteome Research • Vol. 9, No. 11, 2010 5835

research articles (15) Zeng, L.; Zhang, Q.; Li, S.; Plotnikov, A. N.; Walsh, M. J.; Zhou, M. M. Mechanism and regulation of acetylated histone binding by the tandem PHD finger of DPF3b. Nature 2010, 466 (7303), 258– 62. (16) Trojer, P.; Reinberg, D. A gateway to study protein lysine methylation. Nat. Chem. Biol. 2008, 4 (6), 332–4. (17) Glozak, M. A.; Sengupta, N.; Zhang, X.; Seto, E. Acetylation and deacetylation of non-histone proteins. Gene 2005, 363, 15–23. (18) Kondrat, R. W.; Mcclusky, G. A.; Cooks, R. G. Multiple Reaction Monitoring in Mass Spectrometry Mass Spectrometry for Direct Analysis of Complex-Mixtures. Anal. Chem. 1978, 50 (14), 2017– 21. (19) Jia, C. Y.; Nie, J.; Wu, C.; Li, C.; Li, S. S. Novel Src homology 3 domain-binding motifs identified from proteomic screen of a Prorich region. Mol. Cell. Proteomics 2005, 4 (8), 1155–66. (20) Darby, S. C.; Reissland, J. A. Low-levels of ionizing-radiation and cancer - are we underestimating the risk. J. R. Stat. Soc., Ser. A: Stat. Soc. 1981, 144, 298–331. (21) Hwang, P. M.; Li, C.; Morra, M.; Lillywhite, J.; Muhandiram, D. R.; Gertler, F.; Terhorst, C.; Kay, L. E.; Pawson, T.; Forman-Kay, J. D.; Li, S. C. A “three-pronged” binding mechanism for the SAP/ SH2D1A SH2 domain: structural basis and relevance to the XLP syndrome. EMBO J. 2002, 21 (3), 314–23. (22) Bienz, M. The PHD finger, a nuclear protein-interaction domain. Trends Biochem. Sci. 2006, 31 (1), 35–40. (23) Jacobs, S. A.; Khorasanizadeh, S. Structure of HP1 chromodomain bound to a lysine 9-methylated histone H3 tail. Science 2002, 295 (5562), 2080–3. (24) Daujat, S.; Zeissler, U.; Waldmann, T.; Happel, N.; Schneider, R. HP1 binds specifically to Lys26-methylated histone H1.4, whereas simultaneous Ser27 phosphorylation blocks HP1 binding. J. Biol. Chem. 2005, 280 (45), 38090–5. (25) Bannister, A. J.; Zegerman, P.; Partridge, J. F.; Miska, E. A.; Thomas, J. O.; Allshire, R. C.; Kouzarides, T. Selective recognition of methylated lysine 9 on histone H3 by the HP1 chromo domain. Nature 2001, 410 (6824), 120–4. (26) Bua, D. J.; Kuo, A. J.; Cheung, P.; Liu, C. L.; Migliori, V.; Espejo, A.; Casadio, F.; Bassi, C.; Amati, B.; Bedford, M. T.; Guccione, E.; Gozani, O. Epigenome microarray platform for proteome-wide dissection of chromatin-signaling networks. PLoS One 2009, 4 (8), e6789. (27) Quinn, A. M.; Bedford, M. T.; Espejo, A.; Spannhoff, A.; Austin, C. P.; Oppermann, U.; Simeonov, A. A homogeneous method for investigation of methylation-dependent protein-protein interactions in epigenetics. Nucleic Acids Res. 2009. (28) Garcia, B. A.; Hake, S. B.; Diaz, R. L.; Kauer, M.; Morris, S. A.; Recht, J.; Shabanowitz, J.; Mishra, N.; Strahl, B. D.; Allis, C. D.; Hunt, D. F. Organismal differences in post-translational modifications in histones H3 and H4. J. Biol. Chem. 2007, 282 (10), 7641–55. (29) Huang, Y.; Fang, J.; Bedford, M. T.; Zhang, Y.; Xu, R. M. Recognition of histone H3 lysine-4 methylation by the double tudor domain of JMJD2A. Science 2006, 312 (5774), 748–51. (30) Lee, J.; Thompson, J. R.; Botuyan, M. V.; Mer, G. Distinct binding modes specify the recognition of methylated histones H3K4 and H4K20 by JMJD2A-tudor. Nat. Struct. Mol. Biol. 2008, 15 (1), 109– 11. (31) Kim, J.; Daniel, J.; Espejo, A.; Lake, A.; Krishna, M.; Xia, L.; Zhang, Y.; Bedford, M. T. Tudor, MBT and chromo domains gauge the degree of lysine methylation. EMBO Rep. 2006, 7 (4), 397–403. (32) Rathert, P.; Dhayalan, A.; Murakami, M.; Zhang, X.; Tamas, R.; Jurkowska, R.; Komatsu, Y.; Shinkai, Y.; Cheng, X.; Jeltsch, A.

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(33)

(34)

(35)

(36)

(37)

(38)

(39)

(40)

(41)

(42) (43)

(44)

(45) (46)

Protein lysine methyltransferase G9a acts on non-histone targets. Nat. Chem. Biol. 2008, 4 (6), 344–6. Nady, N.; Min, J.; Kareta, M. S.; Chedin, F.; Arrowsmith, C. H. A SPOT on the chromatin landscape? Histone peptide arrays as a tool for epigenetic research. Trends Biochem. Sci. 2008, 33 (7), 305– 13. Yap, K. L.; Li, S.; Munoz-Cabello, A. M.; Raguz, S.; Zeng, L.; Mujtaba, S.; Gil, J.; Walsh, M. J.; Zhou, M. M. Molecular interplay of the noncoding RNA ANRIL and methylated histone H3 lysine 27 by polycomb CBX7 in transcriptional silencing of INK4a. Mol. Cell 2010, 38 (5), 662–74. Margueron, R.; Justin, N.; Ohno, K.; Sharpe, M. L.; Son, J.; Drury, W. J., 3rd; Voigt, P.; Martin, S. R.; Taylor, W. R.; De Marco, V.; Pirrotta, V.; Reinberg, D.; Gamblin, S. J. Role of the polycomb protein EED in the propagation of repressive histone marks. Nature 2009, 461 (7265), 762–7. Nair, S. S.; Nair, B. C.; Cortez, V.; Chakravarty, D.; Metzger, E.; Schule, R.; Brann, D. W.; Tekmal, R. R.; Vadlamudi, R. K. PELP1 is a reader of histone H3 methylation that facilitates oestrogen receptor-alpha target gene activation by regulating lysine demethylase 1 specificity. EMBO Rep. 2010, 11 (6), 438–44. Dhayalan, A.; Rajavelu, A.; Rathert, P.; Tamas, R.; Jurkowska, R. Z.; Ragozin, S.; Jeltsch, A. THE DNMT3A PWWP domain reads histone 3 lysine 36 trimethylation and guides DNA methylation. J. Biol. Chem. 2010. Zhang, Y.; Jurkowska, R.; Soeroes, S.; Rajavelu, A.; Dhayalan, A.; Bock, I.; Rathert, P.; Brandt, O.; Reinhardt, R.; Fischle, W.; Jeltsch, A. Chromatin methylation activity of Dnmt3a and Dnmt3a/3L is guided by interaction of the ADD domain with the histone H3 tail. Nucleic Acids Res. 2010, 38 (13), 4246–53. Feng, W.; Yonezawa, M.; Ye, J.; Jenuwein, T.; Grummt, I. PHF8 activates transcription of rRNA genes through H3K4me3 binding and H3K9me1/2 demethylation. Nat. Struct. Mol. Biol. 2010, 17 (4), 445–50. Wen, H.; Li, J.; Song, T.; Lu, M.; Kan, P. Y.; Lee, M. G.; Sha, B.; Shi, X. Recognition of histone H3K4 trimethylation by the plant homeodomain of PHF2 modulates histone demethylation. J. Biol. Chem. 2010, 285 (13), 9322–6. Matthews, A. G.; Kuo, A. J.; Ramon-Maiques, S.; Han, S.; Champagne, K. S.; Ivanov, D.; Gallardo, M.; Carney, D.; Cheung, P.; Ciccone, D. N.; Walter, K. L.; Utz, P. J.; Shi, Y.; Kutateladze, T. G.; Yang, W.; Gozani, O.; Oettinger, M. A. RAG2 PHD finger couples histone H3 lysine 4 trimethylation with V(D)J recombination. Nature 2007, 450 (7172), 1106–10. Chakravarty, S.; Zeng, L.; Zhou, M. M. Structure and site-specific recognition of histone H3 by the PHD finger of human autoimmune regulator. Structure 2009, 17 (5), 670–9. Chang, P. Y.; Hom, R. A.; Musselman, C. A.; Zhu, L.; Kuo, A.; Gozani, O.; Kutateladze, T. G.; Cleary, M. L. Binding of the MLL PHD3 finger to histone H3K4me3 is required for MLL-dependent gene transcription. J. Mol. Biol. 2010, 400 (2), 137–44. Taverna, S. D.; Ueberheide, B. M.; Liu, Y.; Tackett, A. J.; Diaz, R. L.; Shabanowitz, J.; Chait, B. T.; Hunt, D. F.; Allis, C. D. Long-distance combinatorial linkage between methylation and acetylation on histone H3 N termini. Proc. Natl. Acad. Sci. U.S.A. 2007, 104 (7), 2086–91. Daujat, S.; Bauer, U. M.; Shah, V.; Turner, B.; Berger, S.; Kouzarides, T. Crosstalk between CARM1 methylation and CBP acetylation on histone H3. Curr. Biol. 2002, 12 (24), 2090–7. Goldberg, A. D.; Allis, C. D.; Bernstein, E. Epigenetics: a landscape takes shape. Cell 2007, 128 (4), 635–8.

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