Reconstitution of Nucleosome Demethylation and Catalytic Properties of a Jumonji Histone Demethylase

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Reconstitution of Nucleosome Demethylation and Catalytic Properties ofaJumonjiHistoneDemethylase

Carrie Shiau,1 Michael J. Trnka,2 Alen Bozicevic,3 Idelisse Ortiz Torres,1 Bassem Al-Sady,4 Alma L. Burlingame,2 Geeta J. Narlikar,4 and Danica Galonic Fujimori2,3,* 1Graduate Program in Chemistry and Chemical Biology 2Department of Pharmaceutical Chemistry 3Department of Cellular and Molecular Pharmacology 4Department of Biochemistry and Biophysics University of California, San Francisco, San Francisco, CA 94158, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.chembiol.2013.03.008

SUMMARY dent lysine-specific demethylase family consisting of LSD1 and LSD2 (Karytinos et al., 2009; Shi et al., 2004), as well as the Jumonji histone demethylases catalyze removal of iron- and a-ketoglutarate-dependent Jumonji C domain-con- methyl marks from lysine residues in histone proteins taining demethylases (Klose et al., 2006a; Kooistra and Helin, within nucleosomes. Here, we show that the catalytic 2012). The Jumonji family proteins catalyze a wide set of deme- domain of demethylase JMJD2A (cJMJD2A) utilizes thylation reactions on histone substrates, including removal of a distributive mechanism to remove the histone H3 methyl marks from H3 K4, H3 K9, H3 K27, H3 K36, and H4 lysine 9 trimethyl mark. By developing a method to K20 (Kooistra and Helin, 2012). Common to these proteins is their ability to oxidize methyl groups of methylated lysine sub- assess demethylation of homogeneous, site-specif- strates to form hemiaminal intermediates. Subsequent release ically methylated nucleosomes, we determined that of formaldehyde yields demethylated product (Figure 1A) (Cloos the kinetic parameters for demethylation of nucleo- et al., 2006; Ng et al., 2007; Tsukada et al., 2006). somes by cJMJD2A are comparable to those of The removal of transcriptionally repressive H3 K9me3 marks peptide substrates. These findings imply that other is carried out by the JMJD2 (also known as the KDM4) family domains of the demethylase or its protein partners of demethylases (Cloos et al., 2006; Fodor et al., 2006; Klose may contribute to nucleosome recognition in vivo et al., 2006b; Whetstine et al., 2006). Overexpression of several and, in this way, may further regulate demethylation members of the JMJD2 family have been implicated in various activity and processivity. The quantitative assays diseases (Berry et al., 2012; Cloos et al., 2006; Kawazu et al., of nucleosome demethylation developed in our 2011; Rui et al., 2010), underscoring the importance of precise work provide a platform for future work with complex regulation of demethylation. Members of the JMJD2 family contain JmjN, JmjC, two plant homeodomains (PHD domains), chromatin substrates and full-length demethylases. and a tandem Tudor domain (Figure 1B). While JmjN and JmjC domains form a composite active site (Couture et al., 2007; Ng et al., 2007), the double Tudor domain is thought INTRODUCTION to allow recruitment of demethylases to their target sites by binding H3 K4me3 and H4 K20me3 marks (Lee et al., 2008). Histones, protein components of nucleosomes, are subject to The function of PHD domains in JMJD2 family is yet to be diverse posttranslational modifications. These modifications determined. are predominantly found in the N-terminal tails of histones and The biological significance of this demethylase family has have a profound impact on the regulation of transcription. prompted an investigation into its kinetic and structural features Methylation of lysine 9 in histone H3 (H3 K9) is a conserved eu- (Couture et al., 2007; Hillringhaus et al., 2011; Hopkinson et al., karyotic modification that demarcates heterochromatin, a tran- 2010; Krishnan et al., 2012; Ng et al., 2007). In all of these scriptionally silent chromatin state. Trimethylated H3 K9 (H3 studies, a catalytic construct consisting of JmjN and JmjC K9me3) is required for heritable gene silencing, formation of cen- domain was used (cJMJD2A). To date, there is no published ki- tromeres, and maintenance of telomere stability (Grewal and Jia, netic data on the full-length enzyme. Additionally, no kinetic 2007). H3 K9 methylation has also been observed at genes that studies have evaluated the ability of any demethylase to remove are transcriptionally silenced in cancer (Kondo et al., 2004). The methyl marks from intact and homogenously modified nucleo- extent (mono-, di-, or tri-) and the half-life of methylation are somes, a necessary first step in the analysis of the demethylation controlled by opposing actions of histone methyltransferases of chromatin substrates. and demethylases, as well as histone turnover. Herein, we analyzed the intrinsic processivity and nucleosome Removal of methyl groups from lysine residues in histones is demethylation ability of cJMJD2A. Our study reveals that the carried out by two classes of demethylases: the flavin-depen- cJMJD2A uses a distributive mechanism to affect multiple

more efficiently than ARKme2STGGK. A slow decay of the dimethyl species and concomitant increase in monomethyl species past the initial 4 hr of incubation suggest that the enzyme is mostly inactive by this time. Two consecutive demethylations are required for the formation of monomethylated product from the starting trimethylated peptide. The serial demethylation events can proceed by a processive or distributive mechanism. In a processive demethylation mechanism, the release of the intermediate dimethylated product from the enzyme is substantially slower than its conversion to monomethylated product. In such a scenario, essentially all the dimethylated species exists as bound to the enzyme, and thus this intermediate cannot accumulate to amounts exceeding the concentration of the enzyme. In contrast, in a distributive mechanism, release of dimethylated intermediate is substantially faster than its conversion to monomethylated product. In such a Figure 1. Catalysis and Domain Architecture of JMJD2A scenario, the dimethylated intermediate can accumulate in solu- (A) The abbreviated catalytic mechanism of Jumonji histone demethylases tion to levels that are greater than the concentration of the showing the hemiaminal intermediate. enzyme. The time course of demethylation by MS strongly sug- (B) Domain organization in JMJD2A. gests that the dimethylated species accumulates to amounts greater than that of the enzyme (Figure 2C), implying that demethylation by cJMJD2A proceeds via a distributive mechanism. demethylations. Successful reconstitution of the demethylation We sought to confirm this observation further in an inde- of homogeneous site-specifically methylated nucleosomes has pendent experiment performed with trimethylated peptide allowed us to compare the kinetic parameters for demethylation ARKme3STGGK with the biotin tag attached to the ε-amino of nucleosomes and histone tail peptides. Our findings suggest group of the C-terminal lysine (Figure 3). The kinetic parameters that nucleosome recognition is not intrinsic to cJMJD2A and for demethylation of this peptide (Figure S1) are similar to that of may be mediated by other domains in JMJD2A or its interacting nonbiotinylated peptide (Figure 2). Biotinylated ARKme3STGGK partners. These domains and protein partners could provide a (150 mM) was incubated with the demethylase (5 mM), and the regulatory mechanism for demethylase activity and processivity extent of demethylation was monitored over time by MALDI- in vivo. The ability to measure rates of demethylation of homog- TOF MS. In an analogy to experiments performed with untagged enous, site-specifically modified nucleosomes sets the stage for peptide (Figure 2C), accumulation of dimethylated species in analysis of the demethylation of more complex chromatin sub- excess of enzyme concentration was observed in all time points. strates by JMJD2A and other demethylases. This is consistent with the distributive demethylation, where the formation of monomethylated product observed in later time RESULTS points requires rebinding of dimethylated species. The observation that demethylation of both biotinylated and unlabeled Kinetic Analysis of Peptide Demethylation by cJMJD2A trimethylated peptides results in accumulation of dimethylated To eliminate possible artifacts due to the presence of an affinity intermediate in amounts exceeding the concentration of the tag, we used a tagless catalytic domain construct where the hex- enzyme strongly supports the distributive nature of cJMJD2A. ahistidine tag had been removed from cJMJD2A (amino acids 1– 350 of JMJD2A) after purification (see Supplemental Experi- Demethylation of Nucleosomes mental Procedures available online). Using a fluorescent assay To assess the ability of cJMJD2A to demethylate nucleosomes, that follows the formation of the demethylation byproduct form- we prepared homogeneous, site-specifically modified nucleo- aldehyde (Couture et al., 2007), demethylation of tri- and dime- somes using methyllysine analogs (MLAs) (Simon et al., 2007). thylated H3 K9 peptides corresponding to amino acids 7–14 of In this method, a methyl lysine mimic is incorporated into histone the histone H3 tail (ARKme3STGGK and ARKme2STGGK) by H3 via alkylation of a mutant cysteine residue substituting lysine cJMJD2A was analyzed (Figures 2A and 2B). In these assays, 9 in histone H3, and the modified histone is subsequently incor- ARKme3STGGK is demethylated more efficiently than porated into recombinant nucleosomes (Figure 4A). To compare ARKme2STGGK. The dimethylated peptide exhibited a 6-fold demethylation kinetics quantitatively between analog-modified increase in KM and a 2-fold decrease in turnover number and native substrates, we first determined cJMJD2A activity compared to the trimethylated peptide (KM = 410 mM, kcat = on a trimethyllysine analog peptide ARKCme3STGGK (Fig- 1 1 1.0 min for ARKme2STGGK and KM =67mM, kcat = ure 4B). We observed an 4-fold decrease in kcat (0.48 min ) 1 1.8 min for ARKme3STGGK). and a 5-fold increase in KM (330 mM) as compared to endoge- We used mass spectrometry (MS) to measure the extent of de- nous trimethylated peptide (Figure 2A), consistent with the find- methylation of the trimethylated substrate as a function of time ings of others (Krishnan et al., 2012). (Figure 2C). The rapid disappearance of trimethylated species Demethylation of nucleosomes by cJMJD2A was carried out and accumulation of dimethylated intermediates further support under single-turnover conditions with excess enzyme, a method the observation that cJMJD2A demethylates ARKme3STGGK that better enables the measurement of rates of the catalytic

Figure 2. Kinetic Analysis of cJMJD2A- Mediated Demethylation of Peptides (A and B) Michaelis-Menten plots of initial velocity as a function of the concentration of ARKme3STGGK (A) and ARKme2STGGK (B). Ex- periments were done in triplicate and are represented as mean ± SEM. (C) Demethylation of ARKme3STGGK (200 mM) by cJMJD2A (1 mM) monitored over time using liquid chromatography-tandem MS. Formation of dimethylated species ([ARKme2STGGK] 150 mMat 15 min) in amounts that largely exceed enzyme concentration supports distributive demethylation. Lines connecting data points are introduced to facilitate visualization and do not represent kinetic ﬁts. For experimental conditions and procedures, see Supplemental Experimental Procedures.

under demethylation conditions, possibly precluding the trimethyl epitope recognition. However, MS analysis of MLA peptide indicates negligible oxidation during the reaction (Figure S3).

DISCUSSION

Histone methylation, a posttranslational modification critical to the regulation of transcription, is controlled by the opposing actions of histone methyltransferases and demethylases. Tight control of the site and extent of methylation (mono-, di-, or trimethylation) is crucial for proper cellular function. Jumonji histone demethylases are critical regulators step (Johnson, 1992). To allow for sufficient assay sensitivity, de- of nucleosomal methylation, and their misregulation is associ- methylation was monitored by quantitative western blotting us- ated with cancer and neurological disorders (Kooistra and Helin, ing antibodies specific for H3 K9me3 and H4. To ensure protein 2012). By antagonizing repressive H3 K9 di- and trimethylation, quantification accuracy, the amounts of nucleosomes used enzymes belonging to JMJD2 family of demethylases have a in these experiments were within the linear detection range crucial impact on transcription, an effect well studied in the of these antibodies (Figure S2A). It is important to note that, context of nuclear hormone-receptor-mediated transcription under these conditions, no cross-reactivity was observed (Wissmann et al., 2007). Herein, we report our findings on the between H3 K9me3-specific antibody and mono- and dimethyl processivity and kinetic analysis of nucleosome demethylation recombinant MLA nucleosomes (Figure S2B). By using H3 by cJMJD2A. Determining the intrinsic behavior of the catalytic K9me3-specific antibody to monitor disappearance of starting domain of JMJD2A provides a better understanding of how material and an antibody against H4 as a loading control, we this demethylase may be regulated. were able to monitor the loss of the trimethylated nucleosome The ability of cJMJD2A to catalyze demethylation was first substrate as a function of enzyme concentration. In these analyzed on methylated histone tail peptides. The kinetic param- experiments, nucleosome concentration was kept constant at eters obtained show high turnover for this family of enzymes— 1 300 nM, while the concentration of cJMJD2A was varied be- kcat(ARKme3STGGK) = 1.8 min ; kcat(ARKme2STGGK) = tween 50 and 400 mM. For each enzyme concentration, the ratio 1.0 min1 (Figure 2)—indicating that enzyme obtained in this of H3 KC9me3 to H4 signal was monitored as a function of time, manner is well behaved and comparable to the most active prep- which is used to determine the first-order rate constant for start- arations published (Krishnan et al., 2012). Our findings also ing material consumption (kobs)(Figure 4C). A catalytic step rate indicate that the trimethylated peptide is a better substrate 1 0 (kmax) of 0.16 min and an apparent KM (K M) of 240 mM were than the dimethylated variant for cJMJD2A, predominantly determined by nonlinear regression analysis of observed first- due to differences in KM: KM(ARKme3STGGK) = 67 mM; order rate constants as a function of enzyme concentration (Fig- KM(ARKme2STGGK) = 410 mM. The preference for trimethylated ure 4D). Due to the presence of thioether moiety, it is possible substrate is consistent with previous reports using His-tagged that the thioether backbone of an MLA can undergo oxidation catalytic constructs of JMJD2A (Couture et al., 2007;

Figure 3. Demethylation of Biotinylated ARKme3STGGK by cJMJD2A Peptide (150 mM) was incubated with the demethylase (5 mM), and the extent of demethylation over time analyzed by MALDI-TOF MS. See also Figure S1.

JMJD2A predominantly recognizes residues immediately surrounding the H3 K9 and does not recognize additional features on the nucleosome. Recognition of other chromatin features or other modifications by the double Tudor or PHD domains of the full-length demethylase may result in tighter association and an increase in demethylase activity. Such additional interactions may also increase the processivity of the catalytic domain. By analogy, protein-interacting partners Hillringhaus et al., 2011). Additionally, MS monitoring of deme- may also contribute to the regulation of activity and processivity. thylation of the trimethylated peptide over time showed the rapid In vivo, it is plausible that, in the presence of the relevant chro- disappearance of trimethylated species and the accumulation of matin marks and/or protein partners, full-length JMJD2A may the intermediate dimethylated peptide, which slowly decays to a demethylate in a processive manner. Such regulation of pro- monomethyl product (Figure 2C). These findings are consistent cessivity would have significant implications for controlling with our kinetic measurements indicating strong preference for the specific output of JMJD2A proteins in a context-dependent trimethylated peptide. manner (Bua et al., 2009; Collins et al., 2008; Iwase et al., Mechanistically, multiple methyl marks can be demethylated 2007; Kim et al., 2006; Rottach et al., 2010). either distributively or processively, depending on whether Overall, our ability to quantitatively assess the removal of peptide substrates dissociate or remain enzyme-bound be- methyl marks from site-specifically methylated nucleosomes tween consecutive demethylations. Our findings indicate that by a demethylase is the necessary first step in further detailed in- the catalytic domain of JMJD2A by itself is distributive in its vestigations of chromatin modification by demethylases and de- action. The release of the dimethyl intermediate may have a methylase-containing protein complexes. These studies will also significant impact on the regulation of transcription. For address the importance of the crosstalk between chromatin example, HP1 is recruited to heterochromatic loci by specific marks in the regulation of histone methylation. association with H3 K9me2/3, while sites containing H3 K9me1/0 generally lack HP1 and remain euchromatic (Bannis- SIGNIFICANCE ter et al., 2001; Canzio et al., 2011; Fischle et al., 2008; Grewal and Jia, 2007; Lachner et al., 2001). Thus, in one possible Histone lysine methylation within nucleosomes is a revers- scenario, the release of the dimethyl peptide may serve as ible process catalyzed by opposing actions of histone an additional checkpoint before full commitment to the func- methyltransferases and histone demethylases. As precise tional output signaled by the monomethylated state. Moreover, control of the methylation extent of nucleosomes is the observation that the dimethylated peptide is a 10-fold required for proper gene expression, nucleosome methyl- poorer substrate than the trimethylated peptide raises the ation must be tightly regulated. Jumonji histone demethy- possibility that demethylation of dimethylated substrates may, lases are a class of Fe(II) and a-ketoglutarate-dependent in some instances, be carried out by other demethylases. For dioxygenases that catalyze methyl removal from a number example, in the context of androgen receptor-mediated of methylated lysine residues, and their misregulation is transcription, JMJD2C associates with a flavoprotein demethy- correlated with diseases. By quantitatively analyzing the lase LSD1, and in this complex, LSD1 acts on mono- and demethylation activity of a catalytic construct of JMJD2A, dimethyl H3 K9 marks (Metzger et al., 2005; Wissmann et al., a member of the human Jumonji demethylases, we provide 2007). Like JMJD2C, JMJD2A is also known to associate insight into the ability of a prototypic member of the family with LSD1 in an androgen-receptor-dependent fashion (Kauff- to remove a methyl mark. The results point to two important man et al., 2011). features of the cJMJD2A: an intrinsic distributive nature and Our data further imply that cJMJD2A does not significantly its predominant recognition of the histone tail segment sur- discriminate between histone tail peptide and nucleosomal sub- rounding the methyl lysine residue. Our studies directly strates. The kcat/KM values for these two substrates are within raise the possibility that the activity and processivity of

2.5-fold of one another: kcat/KM (ARKC9me3STGGK) = 1.5 3 the catalytic domain may be regulated in a context-speciﬁc 3 1 1 0 3 10 mM min and kmax/K M (H3 KC9me3 Nuc) = 0.67 3 10 manner by domains outside the catalytic domain. The mM1min1. This result implies that the catalytic domain of method developed here will enable future quantitative

Figure 4. Kinetic Analysis of cJMJD2A- Mediated Demethylation of MLA-Contain- ing Peptides and Nucleosomes (A) Preparation of recombinant homogeneous H3

KC9me3 nucleosomes. Recombinant histone H3 K9C was alkylated with (2-bromoethyl)trimethy-

lammonium bromide to form H3 KC9me3 histones. Subsequent assembly with histone H2A, H2B, H4, and 601 sequence DNA provided homogeneous

H3 KC9me3 nucleosomes. (B) Michaelis-Menten plot for demethylation of MLA-containing peptide. (C) Western blot analysis of an experiment performed at 350 mM cJMJD2A. Data analysis and

extrapolation of kobs is described in Experimental Procedures. (D) Kinetics of demethylation of MLA-containing nucleosomes. Data were obtained from three in- dependent experiments and represented as mean ± SEM. See also Figures S2 and S3.

kobst k studies on mechanistic roles of the noncatalytic regions as [H3 KC9me3] = [H3 KC9me3]t=0 e . Obtained obs values were then 0 well as interacting proteins of demethylases in regulating graphed against cJMJD2A concentrations and ﬁt to kobs =(kmaxX)/(X+K M) k K0 demethylation of nucleosomes. with nonlinear regression to determine max and M parameters, where X is the concentration of cJMJD2A. EXPERIMENTAL PROCEDURES SUPPLEMENTAL INFORMATION Demethylation of Nucleosomes Demethylations of nucleosomes were performed by incubation of nucleo- Supplemental Information includes three ﬁgures and Supplemental Experi- a somes containing H3 KC9me3 (300 nM) with -ketoglutarate (1 mM), ascorbate mental Procedures and can be found with this article online at http://dx.doi. m (1 mM), varying concentrations of cJMJD2A (50–400 M), and Fe(NH4)2(SO4)2 org/10.1016/j.chembiol.2013.03.008. (twice the concentration of cJMJD2A) in 50 mM HEPES (pH 7.5) at room temperature. Reactions were initiated by the addition of methylated nucleosomes. Time points were quenched using a 3:1 mixture of 63 SDS sample loading ACKNOWLEDGMENTS buffer and 0.5 M EDTA, pH 8.0, and boiled at 100C for 2 min. Samples were run on 15% Criterion gels at 200 V for 40 min and transferred onto Im- We thank the Lim, Shokat, and Wells laboratories for use of their instru- mun-blot PVDF membranes (Bio-Rad) at 4C for 2 hr at 600 mA. Membranes ments. We thank Lindsey Pack and the past and present Fujimori lab mem- were blocked at room temperature in 10 ml Odyssey Blocking Buffer (LI-COR bers for helpful discussions. We also thank Drs. Lisa Racki and Daniele Biosciences) for 75 min and then incubated overnight with H3 K9me3 antibody Canzio for their help with nucleosome reconstitutions. This work is sup- (Upstate 07-442, Lot: 2017310) and H4 antibody (Active Motif 39269, Lot: ported by the American Heart Association Predoctoral Fellowship (to 11908001) at 1:800 and 1:1,000 dilutions, respectively, in a 1:1 mixture of C.S.), the University of California, San Francisco (UCSF) Program for Break- 13 PBS and Odyssey Blocking Buffer containing 0.02% Tween-20 at 4C. through Biomedical Research, the Sidney Kimmel Foundation for Cancer Membranes were subsequently washed with 13 TBS and 0.05% Tween-20 Research, a Basil O’Connor Starter Scholar Research Award, and an award (four times, 4 min each), and then incubated with IRDye 680LT goat antirabbit from the Searle Scholars Program (to D.G.F.). MS was provided by the Bio- secondary antibody (LI-COR 926-68021, Lot: C10628-01) at 1:20,000 dilutions Organic Biomedical Mass Spectrometry Resource at UCSF, supported by in a 1:1 mixture of 13 PBS and Odyssey Blocking Buffer containing 0.02% National Institute of General Medical Sciences Grants P41GM103481 and Tween-20 for 40 min at room temperature. Following this, membranes were 1S10RR026662. washed as described earlier and analyzed using Odyssey Application Soft- ware (LI-COR Biosciences). Received: December 13, 2012

Data were analyzed by dividing ﬂuorescent antibody signal of H3 KC9me3 Revised: March 2, 2013 by that of H4. These ratios were normalized to the H3 KC9me3/H4 ratio Accepted: March 19, 2013 at time 0, graphed as a function of time, and ﬁtted to the equation Published: April 18, 2013

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