Selective Recognition of Acetylated Histones by Bromodomain Proteins Visualized in Living Cells

Molecular Cell, Vol. 13, 33–43, January 16, 2004, Copyright 2004 by Cell Press Selective Recognition of Acetylated Histones by Bromodomain Proteins Visualized in Living Cells

Tomohiko Kanno,1,3 Yuka Kanno,1,4 divisions in order to take part in stable transcriptional Richard M. Siegel,2,5 Moon Kyoo Jang,1 memory such as in developmental processes and cellu- Michael J. Lenardo,2 and Keiko Ozato1,* lar differentiation. Evidence suggests that the covalent 1Laboratory of Molecular Growth Regulation modifications of histone tails serve as physical interac- National Institute of Child Health tion surfaces on nucleosome assemblies to recruit chro- and Human Development matin remodeling proteins and transcription regulatory 2 Laboratory of Immunology proteins (Strahl and Allis, 2000; Turner, 2002). National Institute of Allergy Acetylation of histones correlates with gene activation and Infectious Diseases and can be recognized by chromatin-associated pro- National Institutes of Health teins containing bromodomains (Dhalluin et al., 1999; Bethesda, Maryland 20892 Jacobson et al., 2000; Owen et al., 2000). The bromodo- 3 Department of Microbiology and Immunology main is a module of about 110 amino acids that is con- Tohoku University School of Medicine served in many chromatin-associated proteins including Sendai 980-8575 histone acetyltransferases (HATs) such as PCAF and Japan TAFII250, the BET family of nuclear proteins such as Brd2, Brd4, and Bdf1, and ATP-dependent chromatin remodeling factors such as BRG-1 (Jeanmougin et al., Summary 1997). In yeast, bromodomains are involved in gene activation, and in antisilencing of genes at heterochromatin Acetylation and other modifications on histones com- boundaries through chromatin association (Hassan et prise histone codes that govern transcriptional regula- al., 2002; Ladurner et al., 2003; Matangkasombut and tory processes in chromatin. Yet little is known how Buratowski, 2003). Brd4, a mammalian BET protein different histone codes are translated and put into structurally similar to Brd2, has been shown to associate action. Using fluorescence resonance energy transfer, with chromatin (Dey et al., 2000, 2003). Also, an acetyla- we show that bromodomain-containing proteins rec- tion-dependent recruitment of bromodomain-protein ognize different patterns of acetylated histones in in- complexes to chromatin-associated DNA targets has tact nuclei of living cells. The bromodomain protein been demonstrated when nucleosomes were reconsti- Brd2 selectively interacted with acetylated lysine 12 tuted in vitro (Agalioti et al., 2002; Hassan et al., 2002). A variety of HATs exhibit unique substrate specificity, on histone H4, whereas TAFII250 and PCAF recognized H3 and other acetylated histones, indicating fine spec- discriminating between individual lysine residues of his- ificity of histone recognition by different bromodo- tones when assayed in vitro (Roth et al., 2001). Thus the mains. This hierarchy of interactions was also seen in potential exists for creating diverse patterns of acetyla- direct peptide binding assays. Interaction with acet- tion, thereby establishing fine specificity within the his- ylated histone was essential for Brd2 to amplify tran- tone code. This possibility is further strengthened by the scription. Moreover association of Brd2, but not other implication of essential roles of H3 and H4 acetylations in bromodomain proteins, with acetylated chromatin transcription (Howe, et al., 2001; Agalioti et al., 2002; An et al., 2002; Smith et al., 2002). Since bromodomains persisted on chromosomes during mitosis. Thus the contained in a variety of transcriptional regulators and recognition of histone acetylation code by bromodo- chromatin remodeling proteins recognize acetylated mains is selective, is involved in transcription, and histones, they are predicted to possess the ability to potentially conveys transcriptional memory across recognize a diverse array of acetylated histone codes. cell divisions. Although several studies have recently addressed this question (Agalioti et al., 2002; Hassan et al., 2002; La- Introduction durner et al., 2003; Matangkasombut and Buratowski, 2003), the levels of specificity and the mechanisms by Covalent modifications of histone proteins, such as which the bromodomains embedded in the whole pro- acetylation, phosphorylation, methylation, and ubiquiti- tein recognize chromatin have yet to be fully resolved. nation of the N-terminal tails of histones, have been The universal histone code created by acetylation as hypothesized to constitute a histone code that controls well as other modifications is likely to be complex. How- patterns of gene expression (Strahl and Allis, 2000; ever, even a simple rule by which bromodomain proteins Turner, 2002). Some histone modifications are short- recognize specific acetylated histones in living cells has lived and implicated in inducible gene activation or re- not been well established. pression. Others may be inherited down through cell We wished to develop an approach that would allow the delineation of general patterns of the interactions *Correspondence: [email protected] between bromodomains and acetylated histones and 4 Present address: Molecular Immunology and Inflammation Branch, also their persistence during cell division in vivo. To this National Institute of Arthritis, Musculoskeletal and Skin Diseases, end, we employed a flow cytometric adaptation of the National Institutes of Health, Bethesda, MD 20892. 5Present address: Autoimmunity Branch, National Institute of Arthri- fluorescence resonance energy transfer technique (FC- tis, Musculoskeletal and Skin Diseases, National Institutes of Health, FRET) recently developed to study cell surface recep- Bethesda, MD 20892. tors (Siegel et al., 2000a, 2000b; Chan et al., 2001). FRET Molecular Cell 34

is based on the ability of a high-energy donor fluorophore to transfer energy directly to a lower energy acceptor fluorophore. In FC-FRET, spectral variants of the green fluorescent protein (GFP) that carry out energy transfer are quantified by flow cytometry in living cells. A FRET signal between two GFP variants requires prox- imity at the Angstrom level and can identify specific protein-protein interactions. This technique could allow the detection of bimolecular interactions between bromodomain proteins in macromolecular complexes and target histones within nucleosomal histone octamers in the intact nuclei of living cells.

Results

FRET Reveals Specific Histone-Nuclear Protein Interactions in Living Cells To perform FC-FRET analysis, bromodomain proteins were fused to cyan fluorescent protein (CFP) as a donor, and histones were fused to yellow fluorescent protein (YFP) as an acceptor. We first expressed YFP-histone H1 (YFP-H1) or YFP-histone H4 (YFP-H4) in HeLa cells and found that these histones localized to the nucleus during interphase and to chromosomes during mitosis (Figure 1A). YFP-H4 was in the nucleosomal fraction and acetylated as efficiently as the endogenous H4 (Figure 1B), as in the previous reports (Lever et al., 2000; Kimura and Cook, 2001; Ahmad and Henikoff, 2002). Impor- tantly, lysines K5, K8, K12, and K16 were all acetylated in YFP-H4, as reported for endogenous H4 (Turner et al., 1989). All other YFP-histones, including mutants used in the present study, were also incorporated into chromatin (data not shown). Thus, ectopically expressed YFP- histones behaved identically to the endogenous histones. We first tested a bromodomain protein, Brd2 (formerly Ring3) (Denis and Green, 1996), as an effector molecule that may recognize acetylated histones. Brd2 belongs to the BET family of transcriptional regulators conserved from yeast to humans (Dey et al., 2000; Ladurner et al., 2003; Matangkasombut and Buratowski, 2003). Trans- fected CFP-Brd2 was expressed in reasonable propor- tion to endogenous Brd2 (Figure 1C), localized to the nucleus (Figure 1A), and was associated with an essential component of Mediator, TRAP220 (data not shown) in a similar way to endogenous Brd2 (Jiang et al., 1998). Figure 1. Brd2 Interacts with Histone H4 in Living Cells We then performed FC-FRET between YFP-H4 and CFP- (A) Microscopic images of YFP-H4, YFP-H1, or CFP-Brd2 in HeLa Brd2 in living HeLa cells. Three fluorescent signals were cells. (B) Nucleosomal preparations from parental HeLa cells and those separately measured in each cell: CFP, YFP, and FRET, expressing YFP-H4 were analyzed by immunoblot with antibody where FRET is fluorescence in the YFP emission channel specific for H4, tetra-acetyl H4, or monoacetyl H4. triggered by CFP excitation that was optimally compen- (C) Cells transfected with CFP-Brd2 or untransfected HeLa cells sated as described (Siegel et al., 2000a, 2000b; Chan were blotted with Brd2 antibody. With the 40% transfection effi- et al., 2001). In Figure 1D, a substantial FRET signal ciency observed, the amount of CFP-Brd2 was calculated to be was observed between CFP-Brd2 and YFP-H4, but not approximately 52% of the endogenous Brd2 in transfected cells. (D and E) HeLa cells expressing the indicated fluorescent proteins between CFP-Brd2 and YFP-H1. To compare FC-FRET were analyzed by FC-FRET. In (D), dot plots show FRET versus results among different combinations of proteins, we levels of CFP-Brd2 for all cells. In (E), for each sample, the left panels selected cell populations expressing equivalent levels represent dot plots for levels of CFP- and YFP-fusion proteins. Pink of CFP- and YFP-proteins (Figure 1E). When CFP-Brd2 boxes indicate gates to select for the cells expressing the same and YFP-H4 were cotransfected, 23% of gated CFP/ range of CFP and/or YFP. The right panels show histograms of YFP double-positive cells showed FRET. On the other the cell number versus intensity of FRET signals for the selected populations. The numbers shown are the percentage of FRET-posi- hand, FRET was not detected between CFP-Brd2 and tive cells. Threshold lines are drawn to yield Ͻ1% FRET-positive YFP-H1. We also tested the latency-associated nuclear cells on singly transfected cells. antigen (LANA) of Kaposi’s sarcoma-associated herpes- Histone Acetylation Code Recognition in Live Cells 35

virus, which is known to interact with H1 (Cotter and of coprecipitation assays, since all core histones are Robertson, 1999). A positive FRET signal was observed precipitated as a complex. Therefore, to identify the between CFP-LANA and YFP-H1, but not between CFP- residues on the H4 tail that actually contact Brd2, we LANA and YFP-H4. Also, cells transfected with CFP- performed in vitro peptide-precipitation assays. HeLa proteins alone or YFP-histone alone were negative for nuclear extracts were incubated with biotinylated H4 tail FRET. Thus, Brd2 interacts with the core histone H4, peptides, either unacetylated or acetylated at various but not with the linker histone H1, while LANA interacts lysines, and bound complexes were precipitated by bio- with H1, but not with H4. These data show that FC-FRET tin-avidin interactions. Endogenous Brd2 selectively allows detection of specific bimolecular interactions be- bound to tetra-acetylated H4 (Figure 2D) and H4 pep- tween histones and nuclear proteins in the native chro- tides di-acetylated at K5/K12 but not at K8/16 (Figure matin. These interactions could not be attributed to 2E). In blocking experiments, unbiotinylated H4 peptides overexpression of the transfected proteins driving spuri- di-acetylated at K5/12 but not K8/16 blocked Brd2 bind- ous interactions, since the amount of YFP-H4 was about ing to tetra-acetylated H4 peptides (Figure 2E). Further 3% of endogenous H4 whereas the amount of CFP-Brd2 analysis showed that CFP-Brd2 bound strongly to H4 was about 50% of endogenous Brd2 (see Figure 1C peptides acetylated at K12, but very weakly to those legend). Competition between the CFP-labeled Brd2 as acetylated at K8 or K16 (revealed only after long expo- well as YFP-labeled histones with large amounts of their sure), and not at all to those acetylated at K5 (Figure endogenous counterparts most likely resulted in the ob- 2F). In contrast, Brd2 did not bind to H3 peptides unmod- served population of cells that exhibited FRET of less ified or acetylated at K9 and/or K14 (Figure 2F). These than 100%. In all the FRET analyses performed in the results provide strong biochemical validation of our present study, gated cell populations expressing equiv- FRET data and show that Brd2 specifically recognizes alent levels of CFP- and YFP-proteins were compared, acetylated H4 through K12. Indeed, the single amino and threshold lines were drawn to yield Ͻ1% FRET- acid substitution of YFP-H4 at K12, but not at K5, with positive cells on singly transfected cells in the same set arginine effectively abolished FRET with CFP-Brd2 (Fig- of experiments. ure 2G). Also, the weak FRET with H2B represented a weak intreraction of Brd2 with acetylated H2B, since Selectivity of Brd2-Histone Interactions removing the N-terminal tail (H2B⌬N) significantly re- We next determined whether Brd2 interacts with other duced FRET (Figure 2H), and in peptide binding experi- core histones (Figure 2A). H2B produced a significant ments Brd2 bound to acetyl-lysine at K5 and K12 but FRET signal, although consistently less than H4. In con- not K15 or K20 of H2B (Figure 2I). trast, H3 and H2A yielded little or no FRET signals. Thus Brd2 selectively interacts with H4 and, to a lesser de- HATs Bromodomains Interact with H3 as Well as H4 gree, H2B. Given the short range over which energy Given that H3 is a preferred substrate for many HATs transfer can occur, it is highly unlikely that the Brd2- in in vitro assays (Roth et al., 2001), the absence of FRET H4 interaction is indirect (Siegel et al., 2000b). Further between H3 and Brd2 was striking. This could not be supporting a direct, specific recognition of H4 acetyla- explained by a lack of H3 acetylation, since HeLa cells tions by Brd2, we found that removing the N-terminal contained significant amounts of acetylated H3 (Figure tail (H4⌬N) abolished FRET, indicating that the N-ter- 3A). This led us to test another bromodomain protein, minal tail of H4 is essential for interaction (Figure 2A). TAFII250, for interaction with H3 (Figure 3B). TAFII250 is In the H4 tail, mutations in lysines K5 and K12, but a component of the basal transcription factor TFIID, and not K8 and K16, effectively abolished FRET (Figure 2A), its double bromodomain module binds to an acetylated indicating that K5 and/or K12 are the main residues H4 peptide in vitro (Jacobson et al., 2000) and acetylated involved in the interaction with Brd2 in vivo. Importantly, H3 in a reconstituted chromatin target (Agalioti et al., the levels of acetylation at unaltered lysines in these 2002). Transfected CFP-TAFII250 associated with other mutants were similar to those of wild-type (Figure 2B; components of TFIID in HeLa cells, indicating that it and data not shown), indicating that the selective aboli- was incorporated into a stable TFIID complex (data not tion of acetylation at mutated residues accounted for shown). We found that TAFII250 produced a significant the loss of FRET signal. FRET signal with H3 as well as H4 and to a lesser extent

To establish independent evidence for the interaction H2B but not with H2A (Figure 3C). Because TAFII250 has between Brd2 and acetylated H4 observed with FC- a HAT activity and contains a histone recognition site FRET, we examined the biochemical association of Brd2 outside the bromodomains (Mizzen et al., 1996), we with nucleosomes (Figure 2C). Mononucleosomes were tested deletions that removed the HAT region, but re- prepared from HeLa cells expressing FLAG-tagged Brd2 tained the bromodomains (Figure 3B). The deletions pro- or control FLAG constructs by digesting isolated nuclei duced FRET with H3 and H4 as well as did full length with micrococcal nuclease. Anti-FLAG antibody copre- TAFII250 (Figure 3C; and data not shown). It is of note that cipitated nucleosomes containing acetylated H4 from these deletions also lacked the TBP binding surface, and cells transfected with FLAG-Brd2, but not FLAG control TAFII250-BD is further devoid of the putative HMG-like cells (Figure 2C, right panel). Similar results were ob- region, indicating that DNA binding of TAFII250 is dis- tained for endogenous Brd2-nucleosome complexes pensable for its histone association. Thus, H3 is recog- immunoprecipitated by anti-Brd2 antibody (data not nized by the bromodomains of TAFII250 but not of Brd2. shown). Thus, Brd2 is associated with nucleosomes in Moreover, in contrast to FRET with Brd2, both mutants vivo. However, the nucleosomal histones that directly H4-K(5,12)G and H4-K(8,16)G partially reduced FRET contact with Brd2 cannot be determined by this type with TAFII250, indicating that K5/K12 and K8/K16 of H4 Molecular Cell 36

Figure 2. Brd2-Histone Interactions Require K12 of H4 or K5/K12 of H2B (A) FRET analysis of Brd2 interaction with H2A, H2B, H3, or H4 N-terminal tail mutants. Histograms shown are gated on CFP- and YFP-fusion protein-expressing cells as in Figure 1. (B) YFP-H4, YFP-H4-K(5,12)G, YFP-H4-K(8,16)G, or YFP was acid-extracted from transfected HeLa cells and blotted with the indicated antibodies. (C) Mononucleosome preparations from HeLa cells expressing FLAG-Brd2 or control FLAG were immunoprecipitated with FLAG antibody followed by elution with FLAG peptide. Eluted nucleosomes were analyzed by immunoblot with tetra-acetyl H4 antibody (the right panel). For input samples, nucleosomal DNA staining in agarose gel after proteinase K digestion, and immunoblots of FLAG-Brd2 and acetyl H4 are shown. An asterisk indicates nonspecific bands. (D) HeLa nuclear extract was incubated with biotinylated H4 peptides, either unmodified (H4) or tetra-acetylated (AcH4), and precipitated with streptavidin beads followed by immunoblot with Brd2 antibody. (E) Peptide precipitation assays as in (D) with biotinylated H4 peptides di-acetylated at K8/K16 or K5/K12 (the left two lanes). In the right two lanes, extracts were incubated with unconjugated H4 peptides di-acetylated at K8/K16 or K5/K12 followed by incubation with biotin-conjugated tetra-acetylated H4. (F) Peptide precipitation assays with extracts from CFP-Brd2 transfected HeLa cells and mono-acetylated H4, or mono- or di-acetylated H3 peptides. CFP-Brd2 was detected with GFP antibody. (G and H) FRET analysis of Brd2 interaction with H4 mutants (G) or a H2B mutant (H). (I) Peptide precipitation assays with extracts from CFP-Brd2-transfected HeLa cells and mono-acetylated H2B peptides. Histone Acetylation Code Recognition in Live Cells 37

Figure 4. Specificity of Histone Recognition Is Intrinsic to Bromodo- mains (A) Diagram of chimeric proteins. BD, bromodomain; NLS, nuclear localization signal; ET, the ET domain; B1 and B2, the bromodomains #1 and #2 of Brd2, respectively; T1 and T2, the bromodomains #1

and #2 of TAFII250, respectively; LB, the linker region of Brd2; LT,

the linker region of TAFII250. (B) CFP-tagged bromodomain chimeric proteins were tested for FRET with YFP-histones and analyzed as in Figure 1.

tides acetylated at K14 (Figure 3E). We conclude that specific lysine residues of both H3 and H4 are recog-

nized by the TAFII250 bromodomains in vivo and that

TAFII250 has broader recognition specificity than Brd2. As the third bromodomain protein, we tested PCAF, a HAT that has a single bromodomain. PCAF produced FRET signals with both H3 and H4 but not H2A or H2B (Figure 3F). Peptide-precipitation assays revealed PCAF Figure 3. TAFII250 Bromodomains Interact with H3 as Well as H4 (A) Nucleosomal preparations from HeLa cells and those expressing binding to both acetylated H3 and H4 (data not shown), YFP-H3 were analyzed by immunoblot with di-acetyl H3 antibody. consistent with the previous study (Dhalluin et al., 1999).

(B) Diagram of wild-type TAFII250 and truncated constructs. HAT, These results collectively indicate that there is fine spec- histone acetyltransferase; NLS, nuclear localization signal; BD, bro- ificity among bromodomains in recognizing differentially modomain. acetylated histones and that bromodomains of HAT pro- (C) FRET analysis of the interaction between the indicated core teins constitute a discrete recognition group different histone and TAF 250 or TAF 250BD. II II from Brd2. (D) FRET analysis for TAFII250 and H4 N-terminal tail mutants.

(E) Peptide precipitation assays using extracts from CFP-TAFII250- transfected HeLa cells and the indicated H4 or H3 peptides. CFP- Histone Recognition Specificity Is an Intrinsic TAFII250 was detected with GFP antibody. (F) FRET analysis of the interaction between PCAF and the indicated Property of Bromodomains core histone. Histograms in (C), (D), and (F) were generated as in To gain insight into the basis of distinct recognition Figure 1. of H3 and H4 by different bromodomain proteins, we constructed two chimeric proteins in which both of the bromodomains in Brd2 were replaced with those from both critically contribute to the interaction with TAFII250 TAFII250 in the Brd2 backbone (Figure 4A). In Brd2- (compare Figures 2A and 3D). Peptide precipitation TAFBD1/BD2, the linker region between the original analysis showed that TAFII250 bound H4 peptides Brd2 bromodomains was preserved, while in Brd2- mono-acetylated at K8, K12, or K16 as well as H3 pep- TAFBD-unit, the whole double bromodomain unit from Molecular Cell 38

TAFII250 was transplanted onto the Brd2 backbone, domain mutant BD-m8 enhanced the promoter activity eliminating the Brd2 linker region. The FRET analysis of in response to constitutively active Ras in a Brd2 dose- the chimeric constructs in Figure 4B revealed that both dependent manner (Figure 6A). Interestingly, Ras ex- constructs interacted with H3 as well as H4, demonstra- pression led to a marked increase in H4 acetylation at ting that the broad specificity of histone recognition is K12 in these cells, the specific residue that promotes an intrinsic property of the TAFII250 bromodomains. This Brd2-H4 association (Figure 6C). The effect of Ras was also indicates that the lack of H3 recognition by Brd2 selective for K12 of H4, in that acetylation levels of K8 was not due to a potential steric inhibition by the Brd2 on H4 and K9/K14 on H3 remained unchanged. There- structure surrounding the bromodomains. Furthermore, fore, we conclude that the interaction of its bromodo- substitution of only one bromodomain in Brd2 with one mains with acetylated histone H4, especially K12, is bromodomain from TAFII250 was found sufficient to con- essential for the role of Brd2 in amplifying Ras-mediated fer the ability to recognize H3 upon Brd2 (data not cyclin E transcription. shown). Brd2-Histone Interaction during Mitosis Bromodomain Residues Critical Some BET family proteins persist on mitotic chromo- for Acetyl-Histone Recognition somes (Chua and Roeder, 1995; Dey et al., 2000, 2003), The bromodomains are generally composed of four ␣ while other bromodomain proteins leave chromosomes helices with two loops: one between ␣Z and ␣A, and during mitosis (Muchardt et al., 1996; Kruhlak et al., the other between ␣B and ␣C (Figure 5A) (Zeng and 2001). YFP-tagged Brd2 mostly localized on mitotic Zhou, 2002). Conserved residues V, Y, Y, and Y (boxed chromosomes, whereas the double-bromodomain mu- in Figure 5A) in the loops in PCAF are critical for the tants, BD(1ϩ2)-Y/F and BD-m8, which did not interact formation of a hydrophobic pocket that accepts an ace- with acetylated H4 in FRET analysis, were dispersed into tyl moiety (Dhalluin et al., 1999), and the first tyrosine the extrachromosomal space (Figure 7A). The single- residue in the corresponding residues of Gcn5p makes bromodomain mutant BD2-Y/F that interacted with H4 a strong hydrogen bond with acetyl-lysine (Owen et al., retained the ability to associate with mitotic chromo- 2000). We therefore assessed which amino acid resi- somes (Figure 7A). These data indicate that bromodo- dues of the two Brd2 bromodomains were important for main-mediated recognition of acetylated H4 is crucial the specific interactions with acetylated histones. First, for the retention of Brd2 on mitotic chromosomes. In mutations were introduced into the four conserved contrast, we found that YFP-TAFII250 and YFP-PCAF amino acids in each of the bromodomains (BD1-m4, were dispersed into the nonchromosomal space during BD2-m4), or all eight in both domains (BD-m8) (Figure mitosis (Figure 7B; and data not shown), consistent with 5B). BD-m8 failed to produce FRET with YFP-H4, the previous study for the endogenous counterparts whereas BD1-m4 and BD2-m4 showed reduced, but (Kruhlak et al., 2001). To further assess the ability of significant, levels of FRET (Figure 5C). The histone tail the TAFII250 bromodomains to associate with mitotic deleted control (H4⌬N) showed the background level of chromosomes, we tested the two chimeric Brd2 con- FRET. Thus, one intact Brd2 bromodomain is sufficient structs that have their bromodomains substituted with for a partial interaction with H4, and mutations in both the TAFII250 bromodomains. Both of them no longer bromodomains abrogate the interaction. Peptide pre- associated with mitotic chromosomes and were dis- cipitation assays confirmed the complete loss of acetyl- persed into the nonchromosomal space (Figure 7B). H4 binding activity by BD-m8 and that the association These results indicate that it is the bromodomain struc- of H4 with Brd2 depended on the acetylation (Figure tures unique to Brd2 that govern the retention on mi- 5D). We then tested Brd2 bromodomain mutants with totic chromosomes. fewer substitutions (Figures 5B and 5E) and found that To gain further insight into the basis of the bromodo- single substitutions of the critical tyrosine to phenylala- main-mediated mitotic chromosomal retention of Brd2, nine in both bromodomains were sufficient to abolish we wished to visualize interactions under microscope. FRET with H4, which is dependent on H4 K12. These To do so, we used bimolecular fluorescence comple- results reveal the importance of the conserved residues mentation (BiFC) (Hu et al., 2002). In this technique, of the bromodomains for interaction with acetyl-H4 and the full length YFP is split into two fragments, amino- show that the observed FRET signal is mediated by a terminal YFP [Y(N)] and carboxy-terminal YFP [Y(C)], direct interaction between the hydrophobic pockets of both of which are nonfluorescent. When these two non- the Brd2 bromodomains and the acetylated K12 of H4 fluorescent YFP fragments closely associate, they com- tail. The Brd2 mutants with the substitutions in both plement each other and emit YFP fluorescence, which bromodomains also failed to produce FRET with YFP- can be visualized in living cells. Complementation of H2B (data not shown), indicating that the interaction YFP signal is achieved only when the two fragments are with H2B is also mediated by the hydrophobic pocket individually fused to a pair of directly interacting protein of the Brd2 bromodomains. partners. We therefore constructed Y(N)-Brd2 and Y(C)- H4 to attempt to visualize the Brd2-H4 interaction. When Brd2-Histone Interaction and Transcription transfected alone, neither Y(N)-Brd2 nor Y(C)-H4 pro- We next explored a link between the Brd2-acetylated duced a YFP signal, as expected (Figure 7C). However, histone interaction and transcriptional regulation. Pre- coexpression of the two proteins generated a clear YFP viously, it was reported that Brd2 regulates Ras-medi- (BiFC-YFP) signal seen throughout the interphase nu- ated cyclin E promoter activity (Denis et al., 2000). We cleus with the exception of nucleoli (Figure 7C). Control found that the wild-type Brd2 but not its double bromo- experiments using Y(N)-Brd2-BDm8, Y(C)-H4⌬N, or Histone Acetylation Code Recognition in Live Cells 39

Figure 5. Point Mutations in the Bromodomain Abolish H4 Interactions (A) Diagram of bromodomains. Dashed boxes indicate ␣ helices. (B) Diagram showing wild-type Brd2 and its mutants. BD, bromodomain. Positions of point mutations are marked as “A” (alanine substitutions) or “F” (phenylalanine substitutions). (C and E) Brd2 bromodomain mutants were tested for FRET. (D) Peptide precipitation assays using H4 peptides (unmodified or tetra-acetylated) and extracts from HeLa cells expressing CFP-Brd2 or CFP-Brd2-BDm8.

Y(C)-H3 showed negligible reconstitution of YFP signals. duced BiFC-YFP signals on mitotic chromosomes, indi- To quantitate the specific complementation by Y(N)- cating that K8 and K16 of H4 are not critically required Brd2 and Y(C)-H4 in comparison with these controls, for the interaction between Brd2 and H4 on mitotic chro- the complemented YFP signals were measured by flow mosomes (Figure 7E). cytometry. Only the combination of Y(N)-Brd2 and Y(C)- H4 produced significant YFP signals over background Discussion levels (Figure 7D). Thus it is the specific Brd2-H4 interaction that was visualized in living cells by BiFC. In cells In the present study, we found highly selective recogni- undergoing mitosis, coexpression of Y(N)-Brd2 and tion of acetylated histones by different bromodomain Y(C)-H4 showed clear Brd2-H4 interaction on mitotic proteins in the physiological context of living cells. The chromosomes (Figure 7E). Moreover, Y(N)-Brd2 and concept of a histone code raises the question of the Y(C)-H4-K(8,16)G complemented each other and pro- generality of the information contained in various his- Molecular Cell 40

tone modifications and how it is interpreted by the nuclear transcription machinery. Although the histone code is likely to be very complex and difficult to fully unravel, our approaches with FC-FRET and BiFC al- lowed us to observe patterns of selective recognition of acetylated histones by different bromodomains taking place in living cell nuclei. The fact that these recognition patterns were assayed independently of any particular gene or promoter, and that they were fully consistent with in vitro peptide binding assays, suggests that they may represent general patterns that guide bromodomain-histone interactions involving many genes in vivo. To detect interactions between bromodomains and nucleosomal histones in living cells, we utilized a recently developed FC-FRET technique. The advantages of this technique include (1) avoiding fixation or destruction of cells and (2) allowing analyses of individual interactions occurring in or between protein complexes in the physiological environment. The N-terminal tail of each core histone protrudes from the nucleosome core in a similar Figure 6. Point Mutations in the Bromodomain Abolish the Tran- manner, allowing direct comparisons between them for scriptional Function of Brd2 the interaction with bromodomain proteins. We ob- (A) Luciferase assays were performed in triplicate in NIH 3T3 cells served specific binding patterns with selected histone using a cyclin E promoter reporter, expression vectors for active Ras (rasV12), and increasing amounts of wild-type Brd2 or Brd2-BDm8. tails by each of the bromodomain proteins analyzed. (B) Comparable expression of Brd2 and Brd2-BDm8 was confirmed Because of the tight packing of nucleosomes and the by immunoblot. potential flexibility of N-terminal histone tails, however, (C) NIH 3T3 cells and those expressing rasV12 were acid extracted it may be theoretically possible that binding to one his- and analyzed by immunoblot with the indicated antibodies. tone tail or even other types of nucleosome binding such as direct DNA binding might juxtapose a bromodomain

Figure 7. Brd2-H4 Interactions Persist during Mitosis (A and B) Microscopic images of YFP-tagged constructs on mitotic P19 cells and the corresponding DNA staining. (C) BiFC between Y(N)-Brd2 and Y(C)-H4 in living HeLa cells visualized by a confocal microscope tuned for YFP signals. (D) BiFC signals (fluorescence in the YFP emission channel) in living HeLa cells transfected with indicated constructs were analyzed by flow cytometry. Threshold lines are drawn to yield Ͻ0.5% YFP-positive cells on singly transfected cells. (E) BiFC analysis was performed on living P19 cells undergoing mitosis. Histone Acetylation Code Recognition in Live Cells 41

protein to noninteracting histone tails in the same or binding of yeast SWI/SNF and SAGA complexes to re- a neighboring nucleosome and produce “false” FRET. constituted nucleosome arrays, and they reported that However, our domain-swap and mutagenesis experi- there was no difference between acetylated H3 and H4 ments argue against this possibility. For Brd2, point mu- in their ability to retain the entire protein complexes tations introduced in either H4 or the Brd2 bromodo- containing bromodomain proteins. However, one of the mains abrogated FRET signals, indicating a specific bromodomain proteins in the SAGA complex failed to interaction with H4. The absence of FRET by Brd2 with interact with acetylated histones despite its intrinsic H1, H2A, H3, and the H4 and H2B mutants indicates ability, indicating that specificity of bromodomain func- that the potential juxtaposition of these histones to Brd2 tion is context dependent. Agalioti et al. (2002) demon- when it was bound to native H4 was not sufficient to strated a differential requirement of distinct acetyl- produce FRET. For TAFII250, although it binds to DNA lysines by bromodomain proteins in their nucleosomal in the TFIID complex, the truncated versions devoid of association in the interferon-␤ promoter reconstituted in DNA binding still produced specific and comparable vitro. The recruitment of BRG-1 in the SWI/SNF complex

FRET signals as the full length TAFII250, indicating that specifically required acetylated K8 of H4, while the sub-

DNA binding was unlikely to account for FRET signals. sequent recruitment of TAFII250 required acetylated K9 These observations lend credence to the proposition and K14 of H3. However, the highly specific interaction that only direct interactions between bromodomains of TAFII250 with H3 was observed only in the enhancer/ and histone tails produced significant FRET in the pres- promoter and chromatin context, and when tested in ent study. Another important consideration is that FRET isolation, the TAFII250 bromodomains interacted with depends on both the distance between fluorophores both H3 and H4. It is not clear to what extent the require- and their relative orientation so that the conformation ment of acetylated histones by bromodomain proteins as well as the size of a fluorophore-tagged protein may observed for a model gene applies to the general behav- affect the FRET efficiency. Therefore it was important ior of bromodomains in the cell or to those on different to confirm that the absence of FRET between YFP-H3 genes/promoters. and CFP-Brd2 was not due to an unfavorable three- Our FC-FRET analysis allows measurement of global dimensional conformation of Brd2. The chimeric pro- bromodomain-histone interactions in a steady state in teins in which bromodomains of Brd2 were exchanged living cells. Although individual interactions may take with those of TAFII250 exhibited FRET with H3 in the place in a variety of contexts, average signals in each Brd2 backbone, eliminating the possibility that a unique cell were acquired in this study. The present results structure of Brd2 prevented FRET with H3. Together, clearly indicate the distinct recognition of acetyl-lysines the present study demonstrates that the FRET technique on H3 and H4 by different bromodomains. As revealed can be applied to the study of protein-protein interac- by bromodomain swap experiments, recognition of spe- tions in the native chromatin environment. In addition, cific acetyl-lysine on histone is an intrinsic property of the flow cytometric method employed in this work allows the bromodomain. Thus, the bromodomains present in an analysis of thousands of cells simultaneously. The different proteins must harbor sufficient structural diver- data collected by FC-FRET represent statistical values sity to support specific recognition of acetylation codes, of in vivo interactions and allow quantitative compari- most likely located in the loop structures which form a sons among different combinations of interactions. Im- hydrophobic pocket. What allows the bromodomains of portantly, the same specificity of acetyl-histone recogni- TAFII250 and PCAF, but not those of Brd2, to interact tion by bromodomains was found using peptide with H3 is a question for future study. It is possible precipitation assays. that amino acids near the critical acetyl-lysine of H3 In the present study, we found that bromodomains contribute to the interaction specificity, as is the case from Brd2 specifically recognized acetylated K12 of H4, for the PCAF-HIV Tat interaction (Mujtaba et al., 2002) whereas those in TAFII250 and PCAF recognized both or the Gcn5-H4 interaction (Owen et al., 2000; reviewed acetylated H3 and H4 with broader acetyl-lysine speci- in Zeng and Zhou, 2002). ficities. The results suggest that each acetyl-lysine resi- While the state of histone acetylation is shown to due carries a distinct code that is recognized by distinct influence inducible gene expression in various organ- downstream transcriptional regulatory proteins con- isms, what role they play in the heritable memory pro- taining bromodomains. Although several recent studies cesses maintaining gene expression patterns across have addressed similar questions, these studies have cell division is not fully understood (Turner, 2002; Smith not provided general principles governing bromodomain et al., 2002; Ladurner et al., 2003). In yeast, the memory recognition of acetylated histones. A yeast bromodo- of the transcriptionally active state of telomeric genes main protein Bdf1 is reported to interact with both acet- correlates with acetylation of H4 K12 (Smith et al., 2002). ylated H3 and H4 in vitro (Ladurner et al., 2003; Matang- In higher eukaryotes, transcription factors generally dis- kasombut and Buratowski, 2003). While Bdf1 and the sociate from chromatin during mitosis, and histones be- closely related protein Bdf2 both interacted with unacet- come hypoacetylated, coinciding with the general re- ylated and acetylated H4, only Bdf1 exhibited prefer- pression of transcription (Gottesfeld and Forbes, 1997). ence for hyperacetylated forms of H4, and this H4 inter- Nevertheless, significant levels of acetylation remain for action was crucial for chromatin association and the K5 and K12 of H4 at metaphase (Turner and Fellows, support of viability by Bdf1 (Matangkasombut and Bura- 1989) but not for H2B (Kruhlak et al., 2001). We found towski, 2003). Ladurner et al. (2003) demonstrated that that the selective interaction of Brd2 with acetylated the interaction with H4 but not H3 by Bdf1 was tight K12 of H4 was stable enough to retain Brd2 on mitotic enough to protect acetyl-lysines from Sir2-mediated chromosomes. BiFC revealed that this association was deacetylation. Hassan et al. (2002) measured in vitro distributed broadly throughout the length of mitotic Molecular Cell 42

chromosomes, suggesting that it affected many differ- cells were transfected on a chambered coverglass (Lab-Tek) and ent genes. Hence, K12 acetylation and the association observed with a Leica TCS SP2 confocal microscope. of Brd2 may be a plausible molecular mechanism for Luciferase Reporter Assays epigenetic “marking” in mammalian cells. It is of note NIH 3T3 cells on 24-well plates were transfected using FuGene 6 that mitotic retention is a property of a subfamily of (Roche) with a cyclin E promoter-driven luciferase reporter (from K. bromodomains that includes Brd2, Bdf-1 (Chua and Ohtani) (0.2 ␮g), an internal control vector coding for Renilla lucifer- Roeder, 1995), and Brd4 (Dey et al., 2000, 2003) but ase (0.002 ␮g), and a combination of expression vectors, including does not include BRG-1 (Muchardt et al., 1996), p300, RasV12 (0.2 ␮g), and Brd2 or Brd2-BD-m8 (0.2, 0.4, or 0.6 ␮g). PCAF, or TAF 250 (Kruhlak et al., 2001). The inefficiency Luciferase/Renilla luciferase ratios measured using a dual-luciferase II reporter assay system (Promega) were expressed as relative lucifer- of the TAFII250 bromodomains in the mitotic chromo- ase activities. some retention was confirmed by bromodomain swap experiments. It should be noted that although the major- Peptide Precipitation Assays ity of TFIID including TBP, TAFII15, TAFII20, TAFII100, Biotinylated H4 peptides, unmodified or tetra-acetylated, and H3 peptides were obtained from Upstate Biotechnology. Other H4 pep- and TAFII250 are displaced from mitotic chromosomes, a small subpopulation remains associated with mitotic tides and H2B peptides were obtained from United States Biological. Extracts were incubated with a biotinylated peptide in a buffer con- chromosomes, possibly required for rapid reassembly taining 20 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM EDTA, 0.2% NP- of preinitiation complexes in the newly divided cells 40, and protease inhibitors at 4ЊC for 2–4 hr. Streptavidin-coupled (Segil et al., 1996; Christova and Oelgeschlager, 2001; magnetic beads (Dynal Biotech) were then added for 1 hr. Beads Chen et al., 2002). were washed three times and subjected to SDS-PAGE and immu- In summary, bromodomains demonstrate diverse fine noblot. specificity in recognition of acetylated histones and abil- Nucleosomal Preparations and FLAG Immunoprecipitation ity to remain on mitotic chromosomes. These properties For analysis of YFP-histone incorporation in the nucleosome (Fig- may be critical in understanding how histone acetylation ures 1B and 3A), polynucleosome fractions were prepared as de- regulates the initiation and maintenance of gene expres- scribed (Schnitzler, 2000). To obtain the mononucleosome fraction sion patterns. in Figure 2, HeLa nuclei isolated as described (Schnitzler, 2000) were digested with micrococcal nuclease and extracted in a low- salt buffer according to the method by Mizzen and Allis (Mizzen et Experimental Procedures al., 1999). Immunoprecipitation with FLAG antibody and elution with FLAG peptide were performed using ANTI-FLAG M2 affinity gel sys- Constructs tem (Sigma). CFP- and YFP constructs were cloned in pECFP-C1 and pEYFP- C1 (Clontech), respectively, positioning the fluorescent tags at N Antibodies termini. Inserted cDNAs were: mouse Brd2 (identical to gi 3041763, Rabbit polyclonal antibody was produced against a baculovirus cloned from F9 ␭ZAP library), LANA (from J. Jung), human TAF 250 II recombinant Brd2. The following antibodies were purchased from (from R.G. Roeder), human PCAF (from Y. Nakatani), human H1 commercial sources: H4 tetra-acetylated (K5/K8/K12/K16), H4 acet- (IMAGE:376009), human H2A (IMAGE:418370), human H2B (IM- ylated at K8 or K12, and H3 di-acetylated (K9/K14) were from Up- AGE:2339049, the 4th residue T therein was corrected to A), mouse state Biotechnology; H4 acetylated at K5 or K16 from Serotec; and H3 (gi 51299, from ATCC), and human H4 (IMAGE:2130477). IMAGE GFP from Santa Cruz and Roche. clones were purchased from ResGen, and nucleotide sequences were confirmed. Mutagenesis was performed using a Quick change Acknowledgments site-directed mutagenesis kit (Stratagene). Y(N)-Brd2 and Y(C)-H4 were produced by deleting amino acids 155–238 and 2–154 from We thank Y. Goto, J. Jung, Y. Nakatani, K. Ohtani, R.G. Roeder, and YFP-Brd2 and YFP-H4, respectively. In YFP-H4⌬N, amino acids A. Vassilev for reagents; K. Holmes and R. Swofford of the NIAID flow 1–16 were deleted from YFP-H4. In H4-K(5,12)G and H4-K(8,16)G, cytometry facility, L. He and D. Olson of the NIAMS flow cytometry lysines K5 and K12, and lysines K8 and K12 were replaced with facility, and J. Muppidi, F. Chitsaz, and L. Balsam for excellent glycines, respectively. In H4-K5R and H4-K12R, lysines K5 and K12 technical assistance. of H4 were replaced with arginine, respectively. In YFP-H2B⌬N, amino acids 1–22 were deleted from YFP-H2B. TAF 250⌬N and II Received: July 18, 2003 TAF 250BD contain amino acids 1207–1872 and 1351–1608 of II Revised: October 20, 2003 TAF 250, respectively. 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