Acetylation inactivates the transcriptionalrepressor BCL6Oksana R. Bereshchenko, Wei Gu & Riccardo Dalla-Favera

Published online 28 October 2002; doi:10.1038/ng1018

The proto-oncogene BCL6 encodes a BTB/POZ-zinc finger transcriptional repressor that is necessary for germinal-

center formation and has been implicated in the pathogenesis of B-cell lymphomas. Here we show that the co-

activator p300 binds and acetylates BCL6 in vivo and inhibits its function. Acetylation disrupts the ability of BCL6

to recruit histone deacetylases (HDACs), thereby hindering its capacity to repress transcription and to induce cell

transformation. BCL6 is acetylated under physiologic conditions in normal germinal-center B cells and in germinal

center–derived B-cell tumors. Treatment with specific inhibitors shows that levels of acetylation of BCL6 are con-

trolled by both HDAC-dependent and SIR2-dependent pathways. Pharmacological inhibition of these pathways

leads to the accumulation of the inactive acetylated BCL6 and to cell-cycle arrest and apoptosis in B-cell lymphoma

cells. These results identify a new mechanism of regulation of the proto-oncogene BCL6 with potential for thera-

peutic exploitation. Furthermore, these findings provide a new mechanism by which acetylation can promote

transcription not only by modifying histones and activating transcriptional activators, but also by inhibiting

transcriptional repressors.

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Institute for Cancer Genetics and the Departments of Pathology and Genetics & Development, Columbia University, New York, New York 10032, USA.Correspondence should be addressed to R.D.-F. (e-mail: rd10@columbia.edu).

IntroductionThe proto-oncogene BCL6 was identified by virtue of its involve-ment in chromosomal translocations associated with Bcell–derived non-Hodgkin lymphoma (B-NHL)1–4. The productof BCL6 is a nuclear phosphoprotein belonging to the BTB/POZ(bric-à-brac, tramtrack, broad complex/Pox virus zinc fingers)zinc finger family of transcription factors5. BCL6 can represstranscription from promoters containing its DNA binding site6.This function requires the DNA-binding zinc-finger domain andtwo transcriptional repression domains: the amino-terminalPOZ domain and a second separate domain located in the mid-dle portion of the molecule6,7. In vitro evidence suggests that thetranscriptional repressor function of BCL6 requires interactionof both of these domains with complexes containing HDACs andco-repressor molecules including SMRT and SIN3A8–10.

In the B-cell lineage, BCL6 is selectively expressed in matureB cells within germinal centers11,12, where B cells undergoimmunoglobulin gene hypermutation and isotype switchingand are selected based on affinity maturation13. BCL6 isrequired for the formation of germinal centers14,15. Indepen-dent lines of evidence suggest that the function of BCL6 is torepress genes involved in the control of lymphocyte activation,differentiation and apoptosis within the germinal center16,17.BCL6 can bind the same DNA sequence that is recognized bythe STAT6 transcriptional activator (the main nuclear effectorof signaling induced by interleukin-4) and modulate expression

of those genes that are induced by interleukin-4 and mediatedby STAT6 (refs 15,18).

Expression of BCL6 is regulated by a number of signals thatare critical for germinal-center development. At the proteinlevel, engagement of B-cell receptors by antigens induces MAPkinase–mediated phosphorylation of BCL6, which, in turn, tar-gets BCL6 for degradation by the ubiquitin proteasome path-way19. Signaling by the CD40 receptor, normally activated by Tcells, has been shown to downregulate BCL6 expression at thetranscriptional level20. The normal transcriptional regulationof BCL6 is disrupted by tumor-associated chromosomaltranslocations, which juxtapose heterologous promoters to thecoding exons of BCL6, causing its deregulated expression by amechanism called promoter substitution2. The 5′ non-codingregion of BCL6 is also subjected to somatic hypermutation innormal germinal-center B cells and in germinal center–derivedB-NHL21–23; in some diffuse large B-cell lymphomas (DLBCL),these mutations can deregulate BCL6 expression24). Overall,several lines of evidence suggest that downregulation of BCL6 isnecessary for normal B cells to exit the germinal center, whereasBCL6 remains constitutively expressed in a substantial fractionof B-cell lymphomas11.

Here we report that acetylation regulates the function of BCL6.Acetylation is known to stimulate transcription by modifyinghistones, leading to a transcriptionally active chromatin confor-mation, or by directly targeting transcriptional activators (for

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Fig. 1 p300-induced acetylation ofBCL6 and mapping of its acetylationsites. a, Schematic representation ofthe BCL6 protein and its derivativesused in transient transfection experi-ments. POZ, protein–protein interac-tion domain; PEST, region containingthree putative PEST sequences; ZF,zinc finger DNA-binding domain.b, p300 acetylates the N-terminalregion of BCL6. Top panel, vectorsexpressing hemagglutinin-taggedfull-length BCL6 (5 µg) or its N- (∆ZF,2 µg) or C-terminal (ZF, 1 µg) portionswere transiently co-transfected withvectors (7 µg) expressing FLAG-tagged wildtype p300 or a deletionmutant (p300-HAT) lacking acetyl-transferase activity. Whole cellextracts were immunoprecipitated(IP) using an hemagglutinin-specificmonoclonal antibody and analyzedfor BCL6 acetylation by western blot-ting (WB) with an antibody specificfor acetylated lysines (α-AcLys). Bot-tom panel, the same western-blot fil-ter was subsequently probed with anantibody specific for hemagglutininto control for immunoprecipitationand protein loading. c, BCL6 acetyla-tion by p300 maps to its KKYK (376-379) motif. Acetylation of variousBCL6 mutants (a) was analyzed byimmunoprecipitation and westernblotting as described in b. d, The C-terminal lysine (residue 379) of theKKYK motif is the target of p300-mediated acetylation. Acetylation of BCL6-∆ZF mutants with single lysine→arginine substitutions in the KKYK motif wasanalyzed by immunoprecipitation and western blotting as described in b.

example, p53, GATA1, E2F), thereby contributing to theirnuclear localization, association with co-activator complexes andperhaps increased DNA binding25,26. Our results show thatacetylation can directly target and inactivate a transcriptionalrepressor such as BCL6. Acetylation of BCL6 occurs physiologi-cally in germinal-center B cells and leads to its inactivation bypreventing the recruitment of complexes containing HDACs.The results have biological and clinical implications for B-NHLand general implications for the role of acetylation in transcrip-tional regulation.

ResultsThe transcriptional co-activator p300 acetylates BCL6in vivoNoting that BCL6 contained motifs similar to those targeted byp300-mediated acetylation in p53 (ref. 27), we examined whetherBCL6 could also be modified by transcriptional co-activators withhistone acetyltransferase (HAT) activity28. Initial experimentsindicated that BCL6 can be acetylated in vitro by p300, but not bythe p300/CBP-associated acetyltransferase (data not shown). Totest whether BCL6 is also acetylated in vivo, we co-transfectedhuman embryonic kidney 293T cells with vectors expressingBCL6 and p300. The cell lysates were then analyzed by immuno-precipitation using an antibody against BCL6 followed by westernblotting using an antibody previously shown to recognize acety-lated lysines in various transcription factors29,30. Acetylated BCL6was readily detected with co-expression of wildtype p300 but notwith a HAT-deficient mutant of p300 (refs 31,32), indicating thatacetylation of BCL6 required the enzymatic activity of p300.Acetylation of BCL6 was barely detectable in the absence ofexogenous p300, possibly owing to the limiting amounts ofendogenous p300 activity (Fig. 1b).

By co-transfecting 293T cells with vectors expressing p300 andvarious BCL6 mutants (Fig. 1a), we mapped the sequences sus-ceptible to acetylation to the N-terminal region of BCL6 (Fig. 1b).This region contained 21 lysine residues that represented poten-tial acetylation sites; however, we focused on the lysine cluster(KKYK) at position 376–379 because it resembles the site atwhich p53 is acetylated by p300 (ref. 27). We substituted all threelysines with arginines and tested the resulting BCL6 mutant(BCL6-RRYR, Fig. 1a) for acetylation on co-expression withp300 in 293T cells. The BCL6-RRYR mutant, BCL6-∆ZF-RRYRmutant lacking the zinc finger domain but having the RRYR sub-stitutions and a deletion mutant lacking the PEST region thatcontains the KKYK motif (BCL6-∆PEST) were not acetylated byp300 (Fig. 1c), thereby indicating that the KKYK motif wasindeed the primary site of BCL6 acetylation by p300.

To identify the precise target(s) of acetylation among thelysines of the KKYK motif, we generated three point mutants,each of which had one of the lysines substituted with arginine,and tested them for acetylation by p300 in the immunoprecip-itation and western blotting assays. Substitution of the car-boxy-terminal lysine (residue 379) resulted in the completeabrogation of the acetylation signal, whereas substitution ofthe other two lysines did not have any effect on acetylation(Fig. 1d). These results indicate that BCL6 can be acetylated byp300 in vivo, and that Lys379 in the KKYK motif is the primarytarget for acetylation.

BCL6 and p300 interact in vivoTo investigate whether BCL6 can physically interact with p300,we co-transfected 293T cells with vectors expressing the vari-ous forms of BCL6 (Fig 1a) tagged with hemagglutinin andp300 tagged with FLAG, and analyzed the cell lysates by

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immunoprecipitation using an antibody against FLAG (forp300) followed by western blotting with an antibody againsthemagglutinin (for the BCL6 mutants). Full-length BCL6 co-immunoprecipitated with p300 irrespective of the HAT activ-ity of p300; both wildtype p300 and its HAT-deficient mutantbound BCL6 with comparable affinity (Fig. 2a).

To map the region of BCL6 that interacts with p300, the sameset of hemagglutinin-tagged BCL6 derivatives was co-expressed

with p300-FLAG in 293T cells, and BCL6–p300 interactionswere analyzed by immunoprecipitation and western blottinganalysis. The BCL6-∆ZF mutant, but not the BCL6-ZFpolypeptide, retained the ability to bind p300 (Fig. 2b), indicat-ing that p300 binds to the N-terminal half of BCL6. The BCL6-∆PEST polypeptide showed considerably weaker binding top300, implicating the PEST region in the interaction withp300. But the BCL6-RRYR and BCL6-∆ZF-RRYR mutants still

Fig. 3 p300 inhibits the tran-scriptional repression func-tion of BCL6. a, Expression ofwildtype p300 but not itsHAT-deficient mutant abro-gates BCL6-mediated tran-scriptional repression. Vectorsexpressing BCL6, p300 andpB6BS-TK-Luc were co-trans-fected into 293T cells. ThepRL-TK-Luc vector (0.3 µg)was included as an internalcontrol for normalization.Cells were collected 48 h post-transfection, and luciferaseactivity was measured as afunction of BCL6-dependentreporter gene transcription6.The experiments were donein duplicate; representativedata depict the mean ± s.d. ofthree independent experi-ments. Below the graph, con-trols are shown for theexpression levels of BCL6,p300-FLAG and p300-HAT-FLAG by western blotting andfor BCL6 acetylation byimmunoprecipitation andwestern blotting (Fig. 1).b, The HA-BCL6-KKYR acety-lation-resistant BCL6 mutantcan repress transcription, butits function cannot be inhib-ited by p300. The BCL6-QQYQ mutant (mimicking the acetylated state of BCL6) had weaker transcriptional repression activity. The pB6BS-TK-Luc reporter wasco-transfected with the indicated amounts of the HA-BCL6, HA-BCL6-KKYR and HA-BCL6-QQYQ expression vectors (color-coded bars), and transcriptionalrepression by the three forms of BCL6 was compared in the luciferase reporter assay, with or without co-transfection of 1 pmol of the p300 expression vector.Below the graph, controls are shown for the expression levels of HA-BCL6, HA-BCL6-KKYR and HA-BCL6-QQYQ, as assessed by immunoprecipitation andwestern blotting analysis after transfection of 0.04 pmol of each vector.

Fig. 2 BCL6 and p300 interactin vivo. a, BCL6 can bind p300independently of its HATfunction. Top panel, Vectorsexpressing FLAG-tagged p300(5 µg) or p300-HAT (7 µg) wereco-transfected with a vectorexpressing full-length BCL6(5 µg) in 293T cells. Immuno-precipitations were done withantibody against FLAGagarose beads, and the pres-ence of BCL6 in each immuno-precipitate was evaluated bywestern blotting with anti-body against BCL6. Middlepanel, the same western-blotfilter was probed with an anti-body against FLAG to controlfor immunoprecipitation effi-ciency and p300-FLAG expres-sion. Bottom panel, westernblotting analysis (with anti-body against BCL6) of the extracts prior to immunoprecipitation showed the expression levels of BCL6. b, p300 binds to the N-terminal domain of BCL6 andrequires the BCL6 PEST region. In vivo mapping of p300 interaction with BCL6 was done with co-immunoprecipitation and western blotting assays. The set ofBCL6 expression vectors (Fig. 1a) was transfected with (+) or without (–) the p300-FLAG expression vector in 293T cells. Middle panel, whole cell extracts wereimmunoprecipitated with M2 beads, and the presence of BCL6 derivatives in each immunoprecipitate was examined by western blotting using antibody againsthemagglutinin. Top panel, the same filter was probed with an antibody against FLAG to control for immunoprecipitation efficiency and p300-FLAG expression.Bottom panel, western blotting analysis (with antibody against hemagglutinin) of the extracts prior to immunoprecipitation showed similar expression levels forall the BCL6 derivatives.

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bound p300 (Fig. 2b), indicating that binding and acetylationrequire distinct domains of BCL6, and that the inability ofp300 to acetylate these mutants is due to the absence of BCL6acetylation sites rather than to the lack of binding to p300.Taken together, these results indicate that BCL6 and p300interact in vivo, and that this interaction requires the PESTregion but not the KKYK acetylation motif of BCL6.

Acetylation inhibits the transcriptional repressorfunction of BCL6To determine the functional consequences of BCL6 acetyla-tion, we analyzed the effects of p300 on BCL6-mediated tran-scriptional repression. To this end, vectors expressing BCL6and p300 were co-transfected into 293T cells along with aluciferase reporter gene (B6BS-TK-Luc) driven by a promoterlinked to the BCL6 DNA binding site. The luciferase activitywas then measured as a function of BCL6-dependent reportergene transcription6. The potent transcriptional repressionactivity of BCL6 was markedly diminished by co-expression ofp300 (Fig. 3a), but the basal reporter gene expression was notaffected, indicating that the effect of p300 on BCL6 was spe-cific and not due to a general enhancement of transcription byp300. The inhibitory effect on BCL6 was clearly dependent onthe HAT activity of p300, as co-expression of the HAT-defi-cient mutant of p300 did not alter BCL6 function. These dataalso indicate that this effect was not due to recruitment of the

PolII machinery to the promoter by p300 (ref. 33), as the HAT-deficient mutant of p300, which binds BCL6 (Fig. 2a) but doesnot acetylate it (Fig. 1b), had no effect on BCL6-mediatedtranscriptional repression.

To confirm that acetylation was responsible for BCL6 inactiva-tion, we studied the transcriptional activity of the acetylation-resistant BCL6-KKYR mutant (see Fig. 1d) and a second mutant(BCL6-QQYQ) in which the three lysine residues of the KKYKmotif were each substituted by glutamine, a residue that stericallymimics the effect of acetylated lysines29,34. The BCL6-KKYRmutant showed no difference in transcriptional repression activ-ity compared with the wildtype BCL6 (Fig. 3b), but unlike wild-type BCL6, its activity was not inhibited by p300. This indicatesthat the inhibition of BCL6 function is dependent on p300-mediated acetylation. Consistent with this, the acetylation-mim-icking mutant (BCL6-QQYQ) showed considerably lesstranscriptionally repressive activity than wildtype BCL6, compa-rable to that of BCL6 after acetylation by p300. Co-expression ofp300 did not impair transcriptional repression by BCL6-QQYQfurther, suggesting that physical interaction with p300 does notcontribute to BCL6 inactivation in the absence of acetylation(data not shown). Analogous results were obtained by transfect-ing the same vectors in H1299 small lung cell carcinoma cells(data not shown). Taken together, these results indicate that p300negatively regulates BCL6 transcriptional repression functionand that this regulatory activity requires BCL6 acetylation.

Fig. 4 Acetylation of BCL6 leads to itsdissociation from HDACs. a, BCL6-mediated transcriptional repression isinhibited by TSA. Luciferase reporterassays were done as in Fig. 3 in thepresence of the indicated concentra-tions of TSA (color-coded bars) ordimethyl sulfoxide (DMSO), added12 h before cell harvesting. BCL6 pro-tein expression levels (for 0.4 pmol oftransfected pMT2T-HA-BCL6) areshown by western blotting analysisbelow the graph. b, BCL6 acetylationand dose-dependent dissociationfrom co-repressors. 293T cells wereco-transfected with vectors express-ing FLAG-HDAC2 (7.5 µg), HA-BCL6(1 µg) and GAL4-p300 (1, 2.5 and12.5 µg). Cells were harvested 48 hafter transfection and divided in twoparts for analysis of BCL6 interactionswith HDAC2 and BCL6 acetylation sta-tus. For the analysis of BCL6–HDAC2interactions, whole-cell extracts wereimmunoprecipitated with M2 beads(antibody against FLAG), and theamount of BCL6 in each immunopre-cipitate was evaluated by westernblotting (panel 1). The same western-blot filter was then probed with anantibody against FLAG to control forHDAC2 immunoprecipitation effi-ciency and protein loading (panel 5).BCL6 acetylation was evaluated asdescribed in Fig. 1 (panels 2,3). Cellextracts prior to immunoprecipitationwere analyzed for the expression lev-els of GAL4-p300 (panel 4). c, Amutant polypeptide (HA-BCL6-QQYQ)that mimics the acetylated isoform ofBCL6 cannot bind HDAC2. Equalamounts (10 µg) of pMT2T-HA-BCL6, pMT2T-HA-BCL6-KKYR and pMT2T-HA-BCL6-QQYQ were co-transfected with an expression vector for FLAG-HDAC2 (7.5 µg)in 293T cells. Top panel, the HDAC2–BCL6 interactions were analyzed as in b by immunoprecipitation with M2 beads (antibody against FLAG) and western blot-ting with antibody against BCL6. Middle panel, the expression levels and immunoprecipitation efficiency for FLAG-HDAC2 were monitored by analyzing thesame western-blot filter with antibody against FLAG. Bottom panel, western blotting (with antibody against BCL6) of the extracts prior to immunoprecipitationshowed similar expression levels of the three BCL6 isoforms.

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Acetylation of BCL6 leads to its dissociation fromhistone deacetylasesTo investigate the mechanism by which p300-mediated acetyla-tion impairs BCL6 function, we tested whether acetylation inter-fered with the ability of BCL6 to recruit complexes containingHDACs, which contribute to transcriptional repression bydeacetylating histones in the proximity of promoter regions35. Tothis end, we first confirmed that BCL6-mediated repression wasdependent on HDAC function8,9,36,37 by showing that additionof trichostatin A (TSA), a potent and specific inhibitor ofHDACs38, led to a dose-dependent inhibition of BCL6-mediatedrepression (Fig. 4a). On the basis of experiments indicating thatBCL6 interacts preferentially with HDAC2 and, to a lesser extent,with HDAC1 (data not shown), we then examined the effect ofBCL6 acetylation on the stability of the HDAC2–BCL6 interac-tion in 293T cells after transient co-transfection with vectorsexpressing BCL6, FLAG-tagged HDAC2 and p300 fused withGAL4. The results of co-immunoprecipitation and western blot-ting showed that BCL6 interacted with HDAC2 (Fig. 4b), andthat this interaction was abolished when BCL6 was acetylated byp300. Consistent with its ability to functionally mimic acetylatedBCL6 in the reporter assay (Fig. 3b), the BCL6-QQYQ mutantalso showed much weaker binding to HDAC2 (Fig. 4c), indicat-ing that the KKYK motif is required for BCL6 to bind HDACcomplexes. Conversely, the KKYR mutant retained the ability tobind HDAC2, suggesting that acetylation (or glutamine substitu-tion) has a specific effect in preventing HDAC2 binding. In anal-ogous experiments, we observed that neither p300-mediatedacetylation nor glutamine substitutions affected the ability of

BCL6 to bind DNA or localize to the nucleus (data not shown).Taken together, these results suggest that p300-mediated acetyla-tion regulates BCL6 function principally by interfering with itsability to bind HDAC-containing complexes.

BCL6 acetylation occurs in B cells and is controlled byHDAC- and SIR2-dependent pathwaysTo verify that acetylation and deacetylation of BCL6 occurs innative cells and does not result from protein overexpression typi-cal of transient transfection assays, we analyzed the presence ofacetylated BCL6 in normal germinal-center B cells isolated fromhuman tonsils and in various B-cell lymphoma lines represent-ing transformed counterparts of germinal-center B cells contain-ing normal (Ramos), mutated (P3HR1 and Ly-1) or translocated(Val) copies of BCL6 (refs 2,22,23). Acetylated BCL6 wasdetected in both normal and transformed germinal-center B cells(Fig. 5a). Inhibition of HDAC activity by addition of TSA led toan increase in the levels of acetylated BCL6 (see Fig. 5b for repre-sentative results in Ramos cells), indicating that TSA-sensitivedeacetylases are involved in deacetylating BCL6. An analogouseffect was obtained after addition of niacinamide (NIA; Fig. 5b),which interferes with a different deacetylation pathway involvingSIR2, recently shown to deacetylate p53 (refs 39,40). Inhibitionof both the HDAC- and SIR2-dependent pathways by co-treat-ment with TSA and NIA led to an additive effect on the accumu-lation of acetylated BCL6 in both normal and transformed B cells(Fig. 5b). These results demonstrate that BCL6 acetylation anddeacetylation processes occur physiologically in B cells and arecontrolled by both HDAC- and SIR2-dependent pathways.

Fig. 5 Protein interactionsand acetylation of endoge-nous BCL6 in B cells. a, BCL6is acetylated in normalhuman germinal-center Bcells and in germinal-cen-ter–derived lymphoma cells.Germinal center–centroblasts(CB) were isolated and pooledfrom human tonsils of fourindividuals. Top panel, cellpellets from CB33 lym-phoblastoid cells (lackingBCL6 expression; negativecontrol; lane 1), Burkitt lym-phoma cells (Ramos, lane 2,and P3HR1, lane 4), DLBCLcells (Val, lane 3, and Ly-1,lane 5) and normal germinal-center B cells (GC, lane 6)were analyzed for BCL6 acety-lation by immunoprecipita-tion and western blottingusing antibodies against BCL6and acetyl-lysine (AcLys). Mid-dle panel, the same western-blot filters were subsequentlyprobed with antibody againstBCL6 to analyze the total lev-els of BCL6. Bottom panel,western blotting with antibody against β-actin was done for cell extracts used in immunoprecipitation. b, BCL6 acetylation in B cells is controlled by HDAC- andSir2-dependent deacetylation pathways. Ramos, Val, P3HR1 and Ly-1 cells were grown in the presence of 1 µM TSA and 5 mM NIA, added together or individu-ally, as indicated, for 3 h. Cell extracts were analyzed for BCL6 acetylation (top panel) and total BCL6 levels (lower panel) as in a. Human tonsillar lymphocyteswere isolated and kept in culture for 6 h in the presence or absence of TSA and NIA. BCL6 acetylation status was evaluated as in a (lanes 11,12). c, BCL6 associateswith endogenous HDAC2-containing complexes in Ramos cells, and BCL6 acetylation leads to its dissociation from HDAC2. Nuclear extracts from Ramos cellswere immunoprecipitated with rabbit polyclonal antibody against BCL6. The antibody against Notch was used as a negative control in immunoprecipitation.Presence of HDAC2 in each immunoprecipitate was evaluated by western blotting with an antibody against HDAC2 (panel 1). The amounts of BCL6 in theimmunoprecipitates were controlled by western blotting using an antibody against BCL6 (panel 2). Ramos cells were treated with 0.5 µM TSA and 5 mM NIA dur-ing the indicated time. Cells were divided in two aliquots: one for analysis of BCL6-HDAC2 interactions (panels 1,2) as in b, and one for analysis of BCL6 acetyla-tion (panels 3,4) as in a and b. Panel 5 shows the expression levels of HDAC2 in the nuclear extracts used in the BCL6–HDAC2 interaction analysis, which werequantified by densitometry for normalization of the amount of HDAC2 bound to BCL6 in panel 1.

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To establish whether acetylation prevents BCL6–HDAC inter-action in native cells, we examined the association of endogenousBCL6, p300 and HDAC2 in Ramos cells. Indirect immunofluo-rescence staining experiments indicated that all three proteinsresided in discrete nuclear subdomains outside of the nucleoli,consistent with the possibility of physical interaction (data notshown). Co-immunoprecipitation and western blotting experi-ments showed that BCL6 was associated with HDAC2 underbasal conditions (Fig. 5c). To confirm that acetylation regulatesthe binding of BCL6 to HDAC2, we analyzed BCL6 acetylationand its binding to HDAC2 in parallel in Ramos cells treated withTSA and NIA. Drug-induced accumulation of acetylated BCL6resulted in its progressive dissociation from HDAC2 (Fig. 5c).This phenomenon was reversible, as shown by the fact that drugremoval by washing led to BCL6 deacetylation and restoredbinding to HDAC2. Taken together, these findings indicate thatacetylation prevents stable association of BCL6 with HDACs, andthat the function of BCL6 as a transcriptional repressor is regu-lated by the balance of the acetylation and deacetylation path-ways in B cells.

Acetylation sites are critical for BCL6 transformingactivityTo investigate whether acetylation affected the biological func-tion of BCL6, we tested its effects on the ability of BCL6 to act asan oncogene. Although no assay is available to test its transform-ing potential in B cells, BCL6, like other oncogenes, can conferanchorage-independent growth to immortalized rodent fibrob-lasts, a classic trait of malignant transformation. Thus, we stablytransfected Rat-1 cells with vectors expressing either wildtypeBCL6 (pMT2T-HA-BCL6) or the mimetic of acetylated BCL6(pMT2T-HA-BCL6-QQYQ). After antibiotic selection, trans-fected clones (>100) were pooled and compared for their abilityto form colonies in soft agar41. The ability of the BCL6-QQYQmutant to induce in vitro clonogenicity in Rat-1 cells was consid-erably impaired (Fig. 6). Both the number (Fig. 6a) and the size(data not shown) of the colonies induced by BCL6-QQYQ weremarkedly smaller than those induced by wildtype BCL6, despitethe fact that both proteins were expressed at similar levels(Fig. 6b). These results indicate that the KKYK acetylation site iscritical for BCL6-mediated transformation.

DiscussionBCL6 is expressed in germinal-center centroblasts and centro-cytes where it inhibits the expression of genes controlling cellcycle, apoptosis and differentiation16,17. Downregulation ofBCL6 expression is required for B-cell differentiation towardsmemory and plasma cells17, and is achieved at the transcriptional

level by T cell–dependent CD40 signaling20 and at the proteinlevel by antigen-dependent B-cell receptor activation19. Thus,acetylation-mediated downregulation of BCL6 activity repre-sents a new pathway for the rapid and possibly signal-inducedinhibition of BCL6 function. The signals and effectors inducingBCL6 acetylation in B cells are not known, but do not includeDNA damage (data not shown), which has been shown to induceacetylation of p53 (ref. 42) or signaling from CD40 and B-cellreceptors, which have been shown to regulate BCL6 (refs 19,20).Under experimental conditions, p300, but not p300/CBP-associ-ated factor, efficiently acetylates BCL6, strongly suggesting thatp300 is the physiologic effector of BCL6 acetylation in B cells.Identifying the pathways controlling BCL6 acetylation will becritical to understand the mechanisms that regulate germinal-center formation and lymphomagenesis.

Both acetylation and substitution of critical lysines withamino-acid residues that mimic acetylation markedly impair theability of BCL6 to repress transcription. In both cases, loss offunction is associated with loss of the ability of BCL6 to bindHDAC2, suggesting that recruitment of HDAC is an essentialcomponent of the transcription-repressing function of BCL6.The C-terminal lysine (residue 379) within the KKYK motiflocated in the central portion of BCL6 seems to be critical bothfor BCL6 function and for its interaction with HDACs. Thisobservation explains previous findings that the 100-amino-acidregion encompassing the KKYK motif is essential for full tran-scriptional repression by BCL6 (ref. 6) and is included in theregion required for binding to the SIN3A co-repressor in experi-mental ‘two-hybrid’ systems8, and that a 17-amino-acid regionencompassing the acetylation site is required for BCL6 transcrip-tional repression and can function as an autonomous repressionmodule43. Overall, these observations indicate that acetylationmay inhibit BCL6 function by preventing the recruitment of co-repressor complexes containing HDAC.

Our results (Fig. 5) indicate that BCL6 acetylation is controlledby two deacetylation pathways. On the basis of its inhibition byTSA and the observation that BCL6 binds HDACs, the first path-way conceivably involves HDACs belonging to conserved fami-lies of Class I and/or II histone deacetylases, including HDAC1and HDAC2, but possibly involving other members44. The sec-ond pathway is inhibited by NIA (vitamin B3), strongly suggest-ing the involvement of the NAD-dependent TSA-resistant SIR2deacetylase, which has been shown to deacetylate histones45, aswell as a transcription factor such as p53 (refs 39,40). Theinvolvement of SIR2 in inducing the non-acetylated active stateof BCL6 is consistent with its established role as a transcriptionalsilencer46. On the other hand, the observation that the NAD-dependent deacetylase activity of SIR2 is closely linked to cellular

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metabolism in both yeast and mammalian cells46 suggests thatBCL6 acetylation, and, therefore, the regulation of its function,may also be influenced by the cell metabolic rate.

A notable aspect of our results is that the same two pathways(HDAC and SIR2) that regulate acetylation-mediated activationof p53 (refs 40,47) also control acetylation-mediated inactivationof BCL6. We have recently shown that BCL6 inhibits cell-cyclearrest and apoptosis and directly represses the transcription of anumber of p53-induced genes, including the cell-cycle regulatorWAF1/p21 and the putative anti-apoptotic protein PIG-7(encoded by p53-induced gene 7; ref. 48 and Niu and R.D.-F., inpreparation). Thus, it is possible that specific acetylation anddeacetylation pathways may control cell-cycle arrest and apopto-sis by activating p53 and inactivating BCL6 in germinal-center Bcells. Consistent with this hypothesis, treatment with TSAorNIA, which induces p53 activation and BCL6 inactivation,induces apoptosis and expression of WAF1/p21 and PIG-7 inmultiple B-NHL lines (data not shown). These responses are notentirely dependent on p53, because they can be observed in p53-mutant cell lines49, consistent with a p53-independent, possiblyBCL6-mediated, role of acetylation in controlling cell cycle andapoptosis in germinal-center B cells.

The fact that induction of BCL6 acetylation, and thereforeinhibition of its function, can be achieved pharmacologically byinhibiting HDAC- and SIR2-dependent deacetylation hasimportant therapeutic implications. Combined treatment withTSA and NIA leads to complete cell death within 28–48 hours forB-NHL cell lines derived from Burkitt lymphoma with eithernormal or mutant p53 and DLBCL carrying either normal ortranslocated BCL6 genes (data not shown). As both HDAC-tar-geting deacetylase inhibitors such as TSA, SAHA and analogs38

and SIR2-targeting deacetylase inhibitors such as NIA representpharmacologically well-characterized substances usable inhumans, further research on their use for the therapy of B-NHLis warranted. This therapeutic approach would be generallyapplicable to most types of B-NHL (>90%) because all, exceptfor mantle cell lymphoma, express BCL6 (ref. 11). On the basis ofthe data we obtained from the Ramos cell line, this approachcould also be useful for p53-null chemotherapy-resistant cases50.

Finally, these results have general implications for the mecha-nisms controlling gene expression in that they point to a new rolefor acetylation in transcriptional regulation. Multiple lines ofevidence show that acetylation promotes gene transcription byrendering chromatin structure permissive for transcriptionthrough modification of histones and by directly targeting tran-scriptional activators so as to increase their transactivating activ-ity, nuclear localization or stability25. The results herein indicatethat acetylation can also promote transcription by inactivatingtranscriptional repressors.

MethodsPlasmid constructs and antibodies. We used pMT2T-HA and pB6BS-TK-Luc constructs as previously described6. The pMT2T-HA-based BCL6mutants were generated by point mutagenesis using the QuickChange Site-Directed Mutagenesis Kit (Stratagene). The pCI-p300-FLAG and pCI-p300-HAT-FLAG constructs were as previously described32. The pBJ-HDAC1-FLAG and pME18SFLAG-HDAC2 were obtained from R. Eisen-man. We used antibody against acetyl-lysine (Cell Signaling Technology),antibody against FLAG (M2, Sigma) and antibody against hemagglutinin(a gift from J. Kitajewsky). Antibodies against BCL6 (N3 and C19), againstGAL4 (DBD), against Notch2 (25-255) and against HDAC2 (H54) werepurchased from Santa Cruz Biotechnology. PGB6 antibody was receivedfrom B. Falini.

Transfections and immunoprecipitations. We transfected 293T cellsusing the calcium phosphate precipitation method as described6. For

luciferase reporter assays, we seeded 7 × 105 cells per 60-mm dish 1 dbefore transfection. At 48 h post-transfection, we analyzed cell lysates forluciferase activity using the Dual-Luciferase Reporter Assay Kit (Promega)and for protein expression by western blotting as described40, using nitro-cellulose membrane for immobilizing proteins and ECL reagent for detect-ing proteins (Amersham Pharmacia Biotech).

For detection of protein–protein interactions in transfected 293T cells,we prepared whole-cell extracts in co-immunoprecipitation lysis buffer(50 mM Tris, 150 mM NaCl, 0.1% Nonidet P-40, 10% glycerol, 5 mMMgCl2) supplemented with 1 mM dithiothreitol, 10 mM β-glycerophos-phate, 1 mM NaF, 1 mM sodium orthovanadate, 10 µM TSA (Sigma) andprotease inhibitors cocktail (Calbiochem). Immunoprecipitations weredone using the M2 beads (Sigma) for 6 h at 4 °C. We washed immobilizedcomplexes five times with co-immunoprecipitation lysis buffer, elutedbound proteins from the beads using the FLAG peptide (Sigma) andanalyzed eluates by western blotting using the indicated antibodies.Unless otherwise specified, western blots for BCL6 were done using theN3 antibody.

For detection of BCL6-interacting proteins in Ramos cells, we preparednuclear extracts from 1 × 108 cells and immunoprecipitated them with 2 µgof rabbit polyclonal antibody against BCL6 (C19) or with antibody againstNotch as a negative control. Immune complexes were immobilized on Pro-tein G beads (Amersham Pharmacia Biotech), washed five times with coldco-immunoprecipitation lysis buffer, and bound proteins were eluted fromthe beads using BC1000 buffer (20 mM Tris, pH 7.6, 1000 mM NaCl, 10%glycerol, 0.1 mM dithiothreitol, 10 µM TSA) to avoid contamination withimmunoglobulin proteins. We analyzed eluates for the presence of HDAC2and boiled and analyzed beads for BCL6 immunoprecipitation efficiencyby western blotting using antibody against HDAC2 and antibody againstBCL6, respectively.

For detection of the acetylated endogenous BCL6, we washed 4 × 107

Ramos cells with cold phosphate-buffered saline containing 0.1 µM TSAand 1 mM NIA (Sigma) and lysed them in RIPA buffer (50 mM Tris,250 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholic acid, 0.1% SDS) con-taining 50 µM TSA, 10 mM NIA and the protease inhibitors cocktail. Priorto immunoprecipitations, cell extracts were diluted 1:3 with co-immuno-precipitation buffer containing fresh TSA and NIA and cleared twice bycentrifugation at 14,000 r.p.m. We immunoprecipitated whole-cell extractsusing a mouse monoclonal antibody against BCL6 (PGB6) overnight at4 °C. Immune complexes were immobilized on Protein G beads, washedfour times with cold RIPA buffer containing TSA and NIA and eluted fromthe beads by boiling. Immunoprecipitated proteins were resolved bySDS–PAGE (4–15% gels, BIORAD) and analyzed by western blotting withantibody against acetyl-lysine.

B-cell purification. Tonsils were obtained from routine tonsillectomiesperformed at the Babies and Children’s Hospital of Columbia–Presbyter-ian Medical Center. The specimens were kept on ice immediately after sur-gical removal. Separations were carried out in a cold room using ice coldphosphate-buffered saline with 0.5% bovine serum albumin. We isolatedtonsillar mononuclear cells by mincing tonsillar tissue followed by Ficoll-Isopaque density centrifugation. Germinal-center centroblasts (defined asCD77+CD38+ B cells) were purified from tonsils of four individuals bymagnetic cell separation using the MidiMACS system (Miltenyi Biotech).We isolated centroblasts by staining tonsillar mononuclear cells with anti-body against CD77 (Coulter/Immunotech), incubating with mouse anti-body against rat IgM (Pharmingen) and then antibody against IgG1MicroBeads (Miltenyi Biotech) and subsequently passing the stained cellsuspension over an LS column (Miltenyi Biotech). The purity of the isolat-ed centroblasts (95% in all cases) was determined on a Calibur fluores-cence-activated cell sorter (Becton Dickinson) by staining for CD38, CD77and CD3.

Treatment with deacetylase inhibitors. We treated Ramos, Val andP3HR1 cells grown at 0.75 × 106 cells ml–1 in RPMI (Life Technologies)and Ly-1 B cells grown in Iscove’s modified Dulbecco’s medium supple-mented with 10% fetal bovine serum (Life Technologies) with 1 µM TSA(dissolved in dimethyl sulfoxide) and 5 mM NIA (dissolved in water) forthe indicated time. We placed normal tonsillar cells at a density of 5 × 106

cells ml–1 after isolation in RPMI medium supplemented with 20% fetalbovine serum and treated them the same way.

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AcknowledgmentsWe thank R. Baer and L. Pasqualucci for critical reading of the manuscript;U. Klein and G. Cattoretti for the isolation of human B cells; and J.C. Luofor the preliminary experiments on in vivo and in vitro interaction of p300and BCL6. This work was supported by the US National Institutes of Health(to R.D.-F.).

Competing interests statementThe authors declare that they have no competing financial interests.

Received 26 March; accepted 28 August 2002.

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