Genotoxic stress regulates expression of theproto-oncogene Bcl6 in germinal center B cells

Ryan T Phan1,3, Masumichi Saito1, Yukiko Kitagawa1, Anthony R Means2 & Riccardo Dalla-Favera1

Antigen-specific B cells are selected in germinal centers, the structure in which these cells proliferate while accomplishing

genome-remodeling processes such as class-switch recombination and somatic hypermutation. These events are associated

with considerable genotoxic stress, which cells tolerate through suppression of DNA-damage responses by Bcl-6, a transcription

factor required for the formation of germinal centers. Here we show that the expression of Bcl-6 is regulated by DNA damage

through a signaling pathway that promotes Bcl-6 degradation. After DNA damage accumulated, the kinase ATM promoted Bcl-6

phosphorylation, leading to its interaction with the isomerase Pin1 and its degradation by the ubiquitin-proteasome system.

Because Bcl-6 is required for the maintenance of germinal centers, our findings suggest that the extent of genotoxic stress

controls the fate of germinal center B cells by means of Bcl-6.

The germinal center is the structure in secondary lymphoid organswhere B cells stimulated by T cell–dependent antigens undergo clonalexpansion and are selected for high-affinity antibody productionbefore their differentiation into memory B cells and plasma cells1.Germinal center B cells are unique in that they have an extremely highproliferation rate and they accomplish specialized genome-remodelingfunctions such as class-switch recombination (CSR) and somatichypermutation (SHM) of their immunoglobulin genes2. The mechan-isms that regulate the development of germinal centers and control thetransition from the proliferative phase (centroblasts) to the lessproliferative phase (centrocytes) are mostly unknown. In addition,the sustained proliferation and genome-remodeling processes indicatean amount of genotoxic stress that is unique to germinal center B cellsand may require specialized regulatory mechanisms. The identifica-tion of these mechanisms is critical for understanding normalantibody-mediated immunity as well as the pathogenesis of humanB cell lymphomas, most of which derive from germinal center B cells.Indeed, malfunction of CSR and SHM are thought to be the chiefmeans by which proto-oncogenes are activated in tumors derivedfrom germinal centers3.

The development of germinal centers specifically depends on theactivity of Bcl-6, a transcriptional repressor belonging to the BTB-POZzinc finger family4–6. Bcl-6 function requires the carboxy-terminal zincfinger domain that binds to specific DNA sequences7,8 and twotranscriptional repression domains that interact with distinct core-pressor complexes9–11. In the B cell lineage, Bcl-6 protein is abundantlyexpressed only in mature B cells in germinal centers12. The formationof germinal centers and the development of normal T cell–dependent

humoral immune responses require Bcl-6 expression, as Bcl-6-nullmice do not form germinal centers and are therefore unable toproduce high-affinity antibodies5,6. In B cells, Bcl-6 has been shownto modulate many important functions, including activation, differ-entiation, cell cycle arrest and apoptosis13–16. Bcl-6 expression isregulated by signals that are crucial for the development of germinalcenters. Activation of the B cell receptor induces phosphorylation ofBcl-6 activated by mitogen-activated protein kinases, which targetsBcl-6 for rapid degradation by the ubiquitin-proteasome pathway17,whereas stimulation of the CD40 receptor leads to transcriptionaldownregulation of Bcl6 (refs. 14,18). Bcl-6 function is also inactivatedby acetylation, which triggers its dissociation from corepressorcomplexes19. These observations have led to the hypothesis thatdownregulation of Bcl-6 expression is critical for the exit of B cellsfrom the germinal center and their differentiation to memory andplasma cells10,16.BCL6 is also a proto-oncogene involved in chromosomal transloca-

tions in approximately 30% of diffuse large B cell lymphomas(DLBCLs) and in 5–10% of follicular lymphomas20, the two mostcommon forms of B cell non-Hodgkin’s lymphoma. These transloca-tions juxtapose heterologous 5¢ regulatory regions from a variety (over20) of alternative genes to intact BCL6 coding domains, leading to itsderegulated expression by a mechanism called ‘promoter substitu-tion’21,22. In addition, the 5¢ noncoding region of BCL6 is altered bySHM in normal germinal center B cells and in B cell non-Hodgkin’slymphoma23,24, and specific mutations found only in approximately14% of DLBCL cases lead to deregulated expression of Bcl-6 throughdisruption of a negative autoregulatory circuit25,26. It has been shown

Received 22 January; accepted 7 August; published online 9 September 2007; doi:10.1038/ni1508

1Institute for Cancer Genetics, the Departments of Pathology and Genetics & Development, and the Herbert Irving Comprehensive Cancer Center, Columbia University,New York, New York 10032, USA. 2Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.3Present address: CBR Institute for Biomedical Research, Children’s Hospital, Harvard University Medical School, Boston, Massachusetts 02115, USA.Correspondence should be addressed to R.D.-F. (rd10@columbia.edu).

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that Bcl-6 deregulation contributes to lymphomagenesis in vivo, asmice engineered to constitutively express Bcl-6 in B cells developtumors with the critical features of human DLBCL27.

Several observations have identified an important function for Bcl-6in controlling DNA-damage responses in germinal center B cells. Bcl-6antagonizes apoptosis and cell cycle arrest in germinal center B cells bytwo distinct mechanisms. First, Bcl-6 suppresses transcription of thegene encoding the tumor suppressor p53 (TP53) and, consequently,p53-dependent responses by directly binding to the TP53 promoterregion28. Second, Bcl-6 inhibits p53-independent responses by bindingto the transcriptional activator Miz-1 and suppressing Miz-1 targetgenes, including the cell cycle–arrest gene CDKN1A29. Additionalfindings suggest that Bcl-6 may also control the sensing of DNAdamage through transcriptional regulation of the DNA damage–sensing protein ATR30. These results collectively suggest that Bcl-6may prevent normal cell cycle arrest and apoptotic responses ingerminal center B cells to allow rapid cell proliferation and theexecution of DNA-remodeling processes (SHM and CSR) withouteliciting responses to DNA damage.

Such findings raise critical questions regarding how much genotoxicstress germinal center B cells tolerate and how these cells eventuallyresume normal responses to DNA damage. These questions havedirect implications for the control of the normal germinal centerreaction as well as for the phenotype of germinal center–derivedlymphomas. Here we identify a signaling pathway that downregulatesBcl-6 expression in response to increasing DNA damage. This pathwayinvolves the kinase ATM that, in addition to ATR, forms the core ofthe DNA damage–signaling apparatus31. After being activated by itsregulator, the MRN (Mre11-Rad50-NBS1) complex, which sensesdouble-stranded breaks, ATM phosphorylates more than 25 sub-strates, including the checkpoint kinases Chk1 and Chk2 (ref. 32).These kinases, in turn, initiate a secondary wave of phosphorylationevents to extend signaling to DNA damage response effectors, includ-ing p53 and p21. We show here that after DNA damage, an ATM-induced pathway leads to phosphorylation and degradation of Bcl-6.The identification of this pathway suggests that the extent of genotoxic

stress itself may represent, through ATM-induced destruction of Bcl-6,a mechanism for control of the development of germinal centers.

RESULTS

Dose-dependent regulation of Bcl-6 by genotoxic stress

To examine the effect of genotoxic stress on Bcl-6, we used Burkitt’slymphoma cell lines representing transformed germinal center B cells.We treated Ramos Burkitt’s lymphoma cells with increasing doses ofetoposide, a topoisomerase II inhibitor that elicits DNA strand breaksand induces dose-dependent cell cycle arrest and apoptosis in Burkitt’slymphoma cell lines28,29. Immunoblot analysis showed that etoposidetreatment rapidly induced Bcl-6 downregulation in a dose- and time-dependent way (Fig. 1a,b). We noted the same phenomenon aftertreating Ramos cells with other DNA-damaging agents, includingcamptothecin, doxorubicin, cisplatin, hydroxyurea, aphidicolin andionizing radiation. We obtained analogous results with 20 additionalB cell lymphoma lines representative of both the Burkitt’s lymphomaand DLBCL germinal center–derived phenotypes (Fig. 1c and datanot shown), indicating that the phenomenon was not due to tumor-or clone-specific abnormalities. We were unable to use normalgerminal center centroblasts for these experiments, as they rapidlyenter apoptosis during in vitro culture33,34 (Supplementary Fig. 1online) in the absence of CD40 stimulation, which downregulatesBCL6 expression14,18,35.

Immunofluorescence analysis of etoposide-treated cells showed thatthe extent of Bcl-6 downregulation was proportional to the magnitudeof DNA damage, as demonstrated by the inverse relationship betweenthe abundance of nuclear Bcl-6 and that of phosphorylated histoneH2AX (g-H2AX), which recognizes DNA breaks (Fig. 1d and Sup-plementary Fig. 2 online). Etoposide-induced downregulation wasspecific for Bcl-6, as the same treatment did not affect the expressionof Oct-2, another nuclear transcription factor expressed in germinalcenter B cells (Fig. 1e). Analysis of freshly isolated normal humangerminal center B cells showed that the amount of DNA damage inthese cells was within the range noted in our experiments with Ramoscells (Fig. 1d). Because loss of Bcl-6 is not compatible with the survival

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Figure 1 DNA damage induces Bcl-6 downregulation in B cells.

(a,b) Immunoblot analysis of the expression of Bcl-6 and b-actin in Ramos

cells treated for various times with 20 mM etoposide (a) or treated for 6 h

with various doses of etoposide (b). (c) Immunoblot analysis of Bcl-6

expression in various B cell lines (left margin) treated for 6 h with various

doses of etoposide. (d) Quantitative analysis of the expression of Bcl-6

and g-H2AX at the single-cell level in etoposide-treated Ramos cells and

native germinal center B cells (GC B) relative to nuclear staining by

DAPI (4,6-diaminobenzidine tetrahydrochloride; n ¼ 118 cells; additional

data, Supplementary Fig. 2). (e) Immunofluorescence analysis of Bcl-6, Oct-2and g-H2AX in etoposide-treated (+Eto) and untreated control (–Eto) Ramos

cells. Original magnification, �40. Data are representative of at least three

independent experiments (error bars (d), s.d.).

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of germinal center B cells in vivo or in vitro5,28, analysis of normalgerminal center B cells may not demonstrate the largest amounts ofg-H2AX with a corresponding low abundance of Bcl-6, as such cellshad probably died. These observations collectively show that theamount of genotoxic stress specifically regulates Bcl-6 expression ingerminal center B cells (Supplementary Fig. 3 online).

DNA damage induces Bcl-6 protein degradation

To investigate the mechanism by which genotoxic stress downregulatesBcl-6 expression, we did immunoblot analysis of Ramos cells treatedwith the proteasome inhibitor MG132 using antibody to Bcl-6 (anti-Bcl-6). Etoposide-induced downregulation of Bcl-6 was completelyblocked by treatment with MG132 (Fig. 2a), which indicated thatDNA damage induces proteasome-dependent degradation of Bcl-6.We confirmed that conclusion by immunoblot analysis of etoposide-treated Ramos cells expressing a Bcl-6 mutant17,28 lacking the PESTmotifs required for protein degradation17. In those cells, etoposidetreatment led to the rapid degradation of endogenous Bcl-6 but not ofthe exogenous Bcl-6 mutant lacking the PEST motifs (Fig. 2b). Theseresults indicate that DNA damage affects Bcl-6 protein stability bytriggering proteasomal degradation of Bcl-6.

ATM-dependent Bcl-6 phosphorylation at Ser/Thr-Pro motifs

It has been shown that B cell receptor signaling also induces Bcl-6degradation by the ubiquitin-proteasome pathway and that degrada-tion is dependent on the phosphorylation of specific serine residues(Ser333 and Ser343) in the PEST domain17. Thus, we investigatedwhether phosphorylation is also involved in Bcl-6 degradationinduced by DNA damage by first examining the effect on Bcl-6expression of various pharmacological inhibitors of known kinases,such as mitogen-activated protein kinase (PD98509), phosphatidyli-nositol 3-OH kinase (wortmannin), p38 kinase (SB203580), proteinkinase C-d and calcium-calmodulin–dependent kinase III (rottlerin),multiple kinases (staurosporin), cyclin-dependent kinases (roscov-itine) and the ATM-ATR kinases (caffeine). Of those, only caffeine,the ATM-ATR inhibitor, blocked Bcl-6 degradation induced by DNAdamage (Fig. 2c), consistent with the function of ATM-ATR kinases assensors of DNA damage36. Further analysis aimed at examining theinvolvement of the ATM and/or ATR pathway showed that a specificATM inhibitor (KU-55933)37 impaired the downregulation of Bcl-6

after treatment of Ramos cells with etoposide (Fig. 2d). In the samecells, this inhibitor also prevented ATM autophosphorylation, asexpected, but also impaired phosphorylation of Chk1, usually targetedby ATR, but not of Chk2, usually targeted by ATM38. Although thisobservation suggests a distinct ‘downstream wiring’ of the ATM-ATRpathways in germinal center B cells, the results presented abovecollectively indicate that the pathway leading to Bcl-6 degradationby DNA damage involves ATM. This pathway is distinct from thatinduced by B cell receptor signaling, which involves phosphorylationof Bcl-6 mediated by mitogen-activated protein kinase17.

While determining which Bcl-6 residues could be targeted byphosphorylation, we noted that after DNA damage, Bcl-6 was recog-nized by the MPM-2 antibody (Fig. 2e), which specifically detectsphosphorylated ‘S/T-P motifs’ (serine or threonine residue followedby a proline residue)39. As the entire Bcl-6 molecule contains 14 S/T-Pmotifs, it remains to be determined how many of these sites can bephosphorylated after DNA damage. These findings collectivelydemonstrate that DNA damage–induced degradation of Bcl-6 involvesits phosphorylation at S/T-P motifs mediated by the ATM pathway.

Phosphorylated Bcl-6 interacts with Pin1

The finding that DNA damage induced Bcl-6 phosphorylation atS/T-P motifs prompted us to assess the involvement of Pin1, a highlyconserved peptidyl-prolyl isomerase that specifically recognizes phos-phorylated S/T-P peptide bonds and catalyzes cis-trans interconversionof its substrates39,40. Bcl-6 immunoprecipitated together with Pin1 inRamos cells (Fig. 3a). Notably, we also detected the Bcl-6–Pin1complex by coimmunoprecipitation in purified normal germinalcenter B cells (Fig. 3b), thus confirming its physiological function.

The coimmunoprecipitation assays showed that Bcl-6 bound to thefull-length Pin1 polypeptide, and further analysis of specific Bcl-6deletion mutants suggested that a domain in the amino-terminal halfof Bcl-6 (amino acids 120–300) was required for the interaction withPin1 (Fig. 3c,d). Conversely, the full-length Pin1, but not a derivativelacking the WW domain (characterized by the presence of twoconserved tryptophan residues), was able to interact with Bcl-6(Fig. 3e), consistent with published reports showing that the WWdomain is important for the interaction of Pin1 with its substrates39,40.These results collectively indicate that Bcl-6 can physically interactwith Pin1.

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Figure 2 DNA damage–induced phosphorylation of Bcl-6 and protein degradation. (a,b) Immunoblot analysis of the

expression of Bcl-6 and b-actin in normal Ramos cells (a) and in Ramos cells expressing a hemagglutinin-tagged

Bcl-6 mutant lacking the PEST motifs required for protein degradation (Ramos–HA–Bcl-6(DPEST); b); cells wereleft untreated (–) or were treated for 6 h with 20 mM etoposide (+), in the presence (+) or absence (–) of 50 mM

MG132. (c) Immunoblot analysis of the expression of Bcl-6 and b-actin in etoposide-treated Ramos cells in the

presence of increasing doses of caffeine (0 mM (–), 2 mM (+) and 5 mM (++)). (d) Immunoblot of lysates of

Ramos cells left untreated or treated for 6 h with 20 mM etoposide, in the presence of ATM kinase inhibitor (ATMi;

0, 2 and 10 mM (wedges)), analyzed with anti-Bcl-6, anti-ATM, antibody to ATM phosphorylated at Ser1981 (p-ATM

(S1981)), anti-Chk1, antibody to Chk1 phosphorylated at Ser345 (p-Chk1 (S345)), anti-Chk2, antibody to Chk2

phosphorylated at Thr68 (p-Chk2 (T68)) and anti-b-actin. (e) Immunoassay of lysates of control CB33 cells and

Ramos cells left untreated or treated for 6 h with 20 mM etoposide (Etop) in the presence of MG132, then

immunoprecipitated (IP) with anti-Bcl-6 (C19) or control IgG antibody and analyzed by immunoblot with MPM-2

and anti-Bcl-6 (D8). Data are representative of at least three independent experiments.

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As Pin1 is known to recognize phosphorylated S/T-P peptide bondsspecifically, we tested whether the interaction of Pin1 with Bcl-6 wasdependent on Bcl-6 phosphorylation. SDS-PAGE analysis showed thatthe Pin1-bound fraction of Bcl-6 underwent a substantial shift inmigration after treatment with protein phosphatases (Fig. 3f), sug-gesting that Pin1 interacts with phosphorylated Bcl-6. Furthermore,treatment of the cell lysates with phosphatase impaired the ability ofBcl-6 to interact with glutathione S-transferase–tagged Pin1 (GST-Pin1) in vitro (Fig. 3g). To identify the Bcl-6 phosphorylation sitesresponsible for its interaction with Pin1, we generated Bcl-6 deriva-tives containing substitutions of alanine for the relevant residues of theS/T-P motif (T190, S250 and S260) in the Pin1-interacting domain ofBcl-6 (amino-terminal amino acids 120–300) and assessed their abilityto bind GST-Pin1 in vitro after expression in etoposide-treated cells. Inthese conditions, single or double substitution of T190 and S250 didnot affect the interaction of Bcl-6 with Pin1, but Bcl-6 mutants withsubstitution of S260 bound much less Pin1 (Fig. 3h). However, thefull-length Bcl-6 S260A mutant was only partially resistant toetoposide-mediated degradation when introduced into humanembryonic kidney 293T cells (data not shown), suggesting thatadditional S-T/P sites may be involved in the interaction between

Bcl-6 and Pin1. These results collectively indicate that Pin1 interactswith Bcl-6 and that this interaction is dependent on Bcl-6 phos-phorylation, most likely at multiple S-T/P sites, including S260.

DNA damage induces Bcl-6–Pin1 interactions

The observations that DNA damage induced Bcl-6 phosphorylation atS/T-P motifs and that Pin1 recognized and bound phosphorylatedBcl-6 led us to investigate whether this interaction is regulated byDNA damage. To address this issue, we examined the interaction ofBcl-6 derived from nuclear extracts of Ramos cells with GST-Pin1 inan in vitro precipitation assay. As expected, DNA damage reduced thesteady-state abundance of endogenous Bcl-6 (Fig. 4a, top). However,the proportion of Bcl-6 that bound GST-Pin1 was much greater inetoposide-treated cells than in untreated cells (Fig. 4a, bottom). Weobtained similar results with extracts from many other B cell linesexposed to various DNA-damaging agents (data not shown). Theseresults indicate that the interaction of Bcl-6 with Pin1 is enhancedconsiderably by DNA damage.

To confirm the physiological relevance of this interaction, wedetermined whether DNA damage also increased the association ofendogenous Bcl-6 and Pin1 polypeptides in Ramos cells. We did

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Figure 3 Pin1 interacts with Bcl-6. (a) Immunoprecipitates from Ramos or control (Bcl-6-negative) CB33 cells, obtained with anti-Bcl-6 or control antibody

(Control IgG), analyzed by immunoblot with anti-Pin1. (b) Immunoprecipitates from normal germinal center B cells, obtained with anti-Bcl-6 or control

antibody, analyzed by immunoblot with anti-Pin1. (c) Constructs of hemagglutinin-tagged (HA–) Bcl-6 and its derivatives lacking the poxvirus zinc finger

domain (DPOZ), zinc finger domain (DZF) or PEST domain (DPEST) or consisting of the zinc finger domain only (ZF), as well as Flag-tagged Pin1 and mutant

Flag-tagged Pin1 lacking the WW domain (DWW). (d) Immunoassay of lysates from 293T cells transiently transfected with vector expressing hemagglutinin-

tagged Bcl-6, its truncated mutants or Flag-tagged Pin1, immunoprecipitated with M2 beads and then analyzed by immunoblot (IB) with anti-hemagglutinin

(HA) and anti-Pin1 (top two blots); below, immunoblot analysis of whole-cell extracts. (e) Immunoassay of lysates of 293T cells transiently transfected with

vector expressing hemagglutinin-tagged Bcl-6, Flag-tagged Pin1 or Flag-tagged Pin1 mutant lacking the WW domain, immunoprecipitated with M2 beads

and then analyzed by immunoblot with anti-Bcl-6 and anti-Flag. (f) Immunoblot of Pin1-immunoprecipitates (obtained with M2 beads) from 293T cells

transfected with vectors expressing hemagglutinin-tagged Bcl-6 and Flag-tagged Pin1 and treated with phosphatase (l-PPase), analyzed with anti-Bcl-6 and

anti-Pin1. (g) Immunoassay of lysates of 293T cells expressing hemagglutinin-tagged Bcl-6; cells were treated with calf intestinal phosphatase (CIP),

incubated with GST-Pin1, immunoprecipitated with anti-Bcl-6 (CD19), then analyzed by immunoblot with anti-Bcl-6 (D8) and anti-GST. (h) Immunoassay of

lysates of 293T cells treated for 6 h with 20 mM etoposide, transiently transfected with vectors expressing hemagglutinin-tagged Bcl-6 ‘N300’ (WT) and its

Ser-to-Ala and Thr-to-Ala point substitution mutants, then precipitated with GST-Pin1 and analyzed by immunoblot with anti-Bcl-6 and anti-Pin1. Input, total

cell lysate. Data are representative of at least three independent experiments.

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coimmunoprecipitation analysis of etoposide-treated Ramos cellsexposed to the MG132 proteasome inhibitor, which blocks Bcl-6degradation and thus allows comparison of Bcl-6–Pin1 interactionsin the presence of similar amounts of Bcl-6 (Fig. 4b, left). We detectedPin1 only in Bcl-6 immunoprecipitates from etoposide-treated cells(Fig. 4b, right). Thus, DNA damage induces the interaction betweenBcl-6 and Pin1 in physiological conditions in B cells.

Interaction with Pin1 is required for Bcl-6 degradation

Having established that DNA damage enhanced the interaction ofBcl-6 with Pin1, we determined whether Pin1 is involved in DNAdamage–induced degradation of Bcl-6 by testing the effect of Pin1overexpression on Bcl-6 stability in H1299cells (lung carcinoma cells lacking endogen-ous p53) transfected with Bcl-6 and Pin1

expression vectors. In these cells, etoposide treatment did not inducesubstantial degradation of overexpressed Bcl-6 polypeptides (Fig. 4c,lane 3 versus lane 2), probably because of the stoichiometric excess ofexogenous Bcl-6 relative to the endogenous molecules necessary forthe process. Nonetheless, overexpression of Pin1, but not of a mutantunable to bind Bcl-6 (Pin1 lacking the WW domain), restored Bcl-6degradation in a dose-dependent way (Fig. 4c, lanes 4–7). To furtherconfirm these results, we evaluated DNA damage–induced degrada-tion of Bcl-6 in Ramos cells in which Pin1 expression was ablated bytransduction of a Pin1-specific small interfering RNA (siRNA). Wefound that whereas inhibition of Pin1 expression had almost no effecton Bcl-6 expression in basal conditions, it substantially reduced the

Etoposide

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Figure 4 The interaction of Bcl-6 with Pin1 is enhanced after genotoxic insults

and is required for DNA damage–induced degradation. (a) Immunoassay of lysates

of Ramos cells left untreated or treated for 6 h with 20 mM etoposide and then

incubated with GST-Pin1, followed by immunoprecipitation with anti-Bcl-6 (C19)and immunoblot analysis with anti-Bcl-6 (D8) and anti-GST. (b) Immunoassay of

nuclear extracts of Ramos cells left untreated or treated for 6 h with increasing

doses of etoposide (0 mM (–), 1 mM (+), 5 mM (++) and 20 mM (+++)), in the

presence (+) or absence (–) of 50 mM MG132, then immunoprecipitated with anti-

Bcl-6 or control IgG antibody and analyzed by immunoblot with anti-Bcl-6 and

anti-Pin1. L, light chain. (c) Immunoblot of H1299 cells transiently transfected

with various vectors (above lanes; more plusses indicate increasing amounts) and

left untreated or treated for 6 h with 20 mM etoposide, analyzed with anti-Bcl-6 (D8), anti-Flag, anti-GFP and anti-b-actin. (d) Immunoblot of Ramos cells

transfected with control or Pin1-specific siRNA and then left untreated or treated for 6 h with 20 mM etoposide, analyzed with anti-Bcl-6, anti-Pin1 and anti-

GFP (left). Right, quantification of Bcl-6 and GFP signal intensities by densitometry. Bcl-6 expression is normalized to that of GFP and is relative to that of

cells transfected with control siRNA and left untreated (set as 100%). Data are representative of at least three independent experiments.

B220+

CD

95

(14)104

104

103

103

102

102

101

101100

100

104

104

103

103

102

102

101

101100

100

104

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103

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102

102

101

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100

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104

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103

103

102

102

101

101100

100

104

104

103

103

102

102

101

101100

100

(31) (26)

6.5%

Bcl-6

7

GC

cel

ls (

% C

D95

+P

NA

+)

6

5

4

3

2

1

0

4.6%3.4%

3.1% 4.0% 6.0%

PNA

(28) (35) (33)

Pin1+/+ Pin1+/– Pin1 –/–

P = 0.03

P = 0.009

Pin1+/

+

Pin1+/

–

Pin1–/

–

Pin1+/+ Pin1 –/–Pin1 +/–

a

b

Figure 5 Increased germinal center formation in

Pin1�/� mice immunized with sheep red blood

cells. (a) Flow cytometry of splenic B cells from

Pin1+/+, Pin1+/� and Pin1�/� mice immunized

with sheep red blood cells (n ¼ 2 mice for

each genotype, with identification numbers

in parentheses); cells were labeled with

phycoerythrin-conjugated anti-CD95 (Fas) and

fluorescein isothiocyanate–conjugated anti-PNA.

Numbers above outlined areas indicate percent

CD95hiPNAhi germinal center B cells in the

B220+ population. Right, cumulative analysis

(n ¼ 3 mice per genotype; average + s.d.).Data are representative of two independent

experiments. (b) Immunohistochemical analysis

of spleen sections from Pin1+/+, Pin1+/� and

Pin1�/� mice immunized with sheep red blood

cells; sections are stained with anti-Bcl-6.

Original magnification, �40. Data are

representative of three experiments.

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degradation of Bcl-6 induced by etoposide treatment (up to 85%;Fig. 4d). We also obtained analogous results when we exposed cells tothree different siRNA constructs targeting Pin1 and to other DNA-damaging agents (data not shown). These results collectively providedirect evidence that Pin1 is required for the regulation of Bcl-6 proteinstability in response to genotoxic stress.

The Bcl-6–Pin1 interaction suggested that Pin1 be involved inregulating the development of germinal centers through Bcl-6 degra-dation. Thus, we evaluated germinal center responses and Bcl-6expression in Pin1–/– and wild-type littermate mice before and afterimmunization with sheep red blood cells. Other than the phenotypesreported before41,42, unimmunized Pin1–/– mice did not have anyobvious abnormality in the architecture of their lymphoid organs orin their distribution of B cell and T cell subpopulations (data notshown). However, flow cytometry of germinal center B cells, identifiedby double immunostaining for the specific germinal center markersPNA and CD95, showed a substantial increase in this cell populationin immunized Pin1–/– mice (Fig. 5a). In fact, immunized Pin1–/– micehad more and larger germinal centers (Fig. 5b), a phenotype similar tothat of mice with constitutive Bcl-6 expression27. These data collec-tively indicate that Pin1-deficient mice show enhanced germinalcenter formation, consistent with involvement of Pin1 in controllingthe expression of Bcl-6 in vivo.

DISCUSSION

Studies have identified several distinct mechanisms for negativeregulation of Bcl-6 gene expression. CD40 signaling regulates BCL6transcription; Bcl-6 function is regulated by acetylation, which inacti-vates its trans-repressive function; and B cell receptor signalingregulates Bcl-6 protein stability14,17,18. Here we have shown thatDNA damage–induced degradation of Bcl-6 represents an additionaland distinct mode of negative regulation.

The observation that DNA damage–induced degradation of Bcl-6was blocked by caffeine as well as by a specific ATM inhibitor suggeststhat the first step in the signaling pathway is the sensing of DNAdamage by ATM. However, we were unable to exclude the possibilityof involvement of ATR, as we could not specifically inhibit its activity,possibly because ATR can also be activated ‘downstream’ of ATM43.Although ATM is a damage-inducible kinase capable of direct sub-strate phosphorylation, its effect on Bcl-6 is probably indirect, throughphosphorylation of an intermediate kinase, as DNA damage–inducedphosphorylation of Bcl-6 occurs at S/T-P motifs, whereas the ‘pre-ferred’ motifs for ATM and ATR are S/Q and T/Q sequences36. Ourresults exclude the possibility of Chk2 as the intermediate kinase, as wefound that downregulation of Bcl-6 was still inhibited in the presenceof phosphorylated Chk2, consistent with the observation that thiskinase can be activated by etoposide-induced DNA breaks in a ATM-ATR–independent way44. Further studies are therefore needed toidentify the putative downstream kinase. Candidates for this includeChk1, whose activation is prevented in ATM-inhibited Ramos cellslacking Bcl-6 downregulation, and MAPKAP2, a DNA damage–checkpoint kinase that phosphorylates S/T-P motifs and whose abla-tion leads to enlarged germinal centers in mice45, consistent with anegative regulatory effect on Bcl-6.

After Bcl-6 phosphorylation, the second critical event leading toDNA damage–induced degradation of Bcl-6 is the interaction betweenphosphorylated Bcl-6 and Pin1. These bonds have the unique poten-tial to exist in two distinct isomers (cis and trans) in folded proteins,and the interconversion of these isomers is especially important inprocesses such as phosphorylation or dephoshorylation and proteo-lysis, as many key enzymes can specifically recognize cis or trans

conformations of their substrates46. The cis-trans interconversionreaction is intrinsically slow but can be catalyzed by Pin1, leading todistinct functional modifications of many important polypeptides,including p53 and c-Myc47–49. Our data have shown that prolyl-isomerization by Pin1 was required for Bcl-6 degradation in responseto DNA damage, although, as with other molecules interacting withPin1, the steps between isomerization and degradation have not beenidentified. Although Pin1 has been proposed as a potential dominantoncogene46, the established function of Pin1 in stabilizing p53(refs. 48,49) and in degrading Bcl-6 indicate an inhibitory effect oncell proliferation in the germinal center. This possibility was in factconfirmed by the observation that Pin1–/– mice had more germinalcenter formation. Although the possibility of an effect of Pin1 onother molecules regulating the development of germinal centerscannot be excluded, these results are entirely consistent with theidea that Pin1 may affect the development of germinal centers throughregulation of Bcl-6 stability, as shown in Ramos cells.

Our results have implications for the mechanisms regulating thegerminal center reaction, as the development of this structure is tightlycontrolled by the abundance of Bcl-6. Germinal centers do not form inthe absence of Bcl-6 (refs. 5,6), whereas constitutive expression of Bcl-6leads to increased germinal center formation27. Thus, the observationthat DNA damage directly and specifically regulated Bcl-6 expressionindicates that the extent of genotoxic stress may be a principal signalcontrolling the development of germinal centers. Although our resultshere were based on the induction of DNA breaks by etoposide, weobtained analogous effects on Bcl-6 expression with a range ofgenotoxic agents, suggesting a general mechanism linked to the sensingof DNA damage. In physiological conditions, this damage may resultfrom the extraordinarily high proliferation rate of germinal centercentroblasts, leading to replicative as well as oxidative damage, andfrom the accumulation of DNA breaks associated with SHM and CSR.

Published studies have indicated that Bcl-6 provides some toleranceto DNA damage by suppressing both p53-dependent and p53-independent apoptotic and cell cycle–arrest responses28,29. Thatobservation has been extended to Bcl-6-mediated modulation ofDNA-damage sensing through transcriptional suppression of ATR30.This pathway would allow germinal center B cells to sustain thegenotoxic stress associated with their proliferative and genome-remodeling functions without eliciting p53-related responses. Ourresults here suggest that the Bcl-6-mediated tolerance to genotoxicstress is not limitless, as larger amounts of DNA damage that lead toBcl-6 degradation would alleviate Bcl-6 repression and restore normalp53-dependent and p53-independent apoptotic and cell cycle–arrestresponses in the germinal center. Given the known difficulty inculturing normal germinal center B cells30,33,34, we could not quanti-tatively define the exact physiological threshold of DNA damagenecessary to induce Bcl-6 degradation. Nonetheless, the number ofDNA breaks in normal germinal center B cells was within the range inwhich Bcl-6 stability is experimentally regulated by etoposide inRamos cells, suggesting that the pathway identified here has physio-logical relevance. Therefore, we suggest a model in which Bcl-6 wouldremain stable and suppress apoptotic and cell cycle responses in theface of moderate genotoxic stress engendered by SHM, CSR and rapidcell proliferation. However, larger amounts of DNA damage sufficientto activate the ATM-ATR–mediated pathway in germinal center B cellswould promote Bcl-6 degradation and thereby allow cell cycle arrestand/or death responses to occur through derepression of TP53 andCDKN1A. This model suggests that the amount of genotoxic stressmay represent a signal for terminating the germinal center reaction bydownregulating Bcl-6 expression.

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The idea of a ‘rheostatic switch’ for the transition from a Bcl-6-regulated (or damage-resistant) pathway to a p53-regulated (or DNAdamage–sensitive) pathway in germinal center B cells is supported bythe existence of multiple mechanisms for opposing regulation of thesetwo molecules, which include the transcriptional suppression of TP53by Bcl-6 (ref. 28), their antagonistic regulation (activation of p53 andinactivation of Bcl-6) by the same two deacetylation pathways (HDACand Sir2)19,50,51, and the ATM-ATR–dependent phosphorylation andPin1-mediated isomerization (both of which activate p53)48,49 shownhere. Our results suggest that an important signal regulating the switchis the amount of DNA damage.

Our results also have implications for Bcl-6-driven lymphomagen-esis. Although a subset of DLBCLs and follicular lymphomas haveBCL6 alleles that are deregulated at the transcriptional level, most havevariable expression of Bcl-6, suggesting that the Bcl-6 degradationpathway may have been inactivated in some cases. In fact, inactivatingmutations of ATM have been reported in limited surveys52. Theidentification of the pathway components provided here will allowits structural and functional integrity to be conclusively assessed inlymphoma cells. In addition, pharmacological activation of the Bcl-6destruction pathway may represent an approach for inactivating Bcl-6and reactivating apoptotic responses in tumor cells.

METHODSExpression vectors, reagents and antibodies. The mammalian expression

vectors encoding hemagglutinin-tagged Bcl-6 and its derivatives have been

described19,28. Hemagglutinin-tagged Bcl-6 ‘N300’ (amino-terminal amino

acids 1–300) plasmids encoding point substitutions at T190, S250 and S260 were

generated with the QuickChange site-directed mutagenesis kit (Stratagene).

Plasmids encoding Flag-tagged Pin1 or the Flag-tagged Pin1 derivative lacking

the WW domain and those encoding GST-Pin1 were constructed by subcloning

of the corresponding human cDNA amplified by PCR into the pcDNA3 vector

(Invitrogen) and pGEX vector (Promega), respectively. For the generation of

Pin1-specific siRNA viral vectors, double-stranded oligonucleotides encoding

siRNA specifically targeting the Pin1 transcript (Pin1-siRNA1, 5¢-GCCGAGTG

TACTACTTCAA-3¢; Pin1-siRNA2, 5¢-GCTACATCCAGAAGATCAA-3¢) were

first cloned into the pSilencer vector (Ambion). The fragment containing

U6-Pin1-siRNA was then excised from pSilencer by digestion with BamHI, then

its ends were made blunt and it was cloned into the c-FUGW lentiviral vector53.

All final vectors were verified by enzymatic digestion and confirmed by DNA

sequencing. Etoposide, caffeine, MG132 and N-ethylmaleimide were from

Sigma. KU-55933 was from Calbiochem. Anti-Bcl-6 (N3, C19 and D8), anti-

Oct-2 (DO-1), anti-Chk1 (G-4), anti-Chk2 (H-300), anti-ATM (2C1) and anti-

Pin1 (H123) were from Santa Cruz Biotechnology. The following antibodies

were also used: anti-Pin1 (3722), antibody to phosphorylated Chk1 (2341S),

antibody to phosphorylated Chk2 (2661S), anti-Bcl6 (4242; all from Cell

Signaling Technology); antibody to phosphorylated ATM (S1981; Rockland);

anti-gH2AX (JBW301) and MPM-2 (05-368; both from Upstate Biotechnol-

ogy); anti-GST (30001; Pierce); anti-b-actin (AC-15), anti-Flag (F3165) and

anti-M2 beads (A2220; all from Sigma); anti-hemagglutinin (3F10; Roche); and

antibody to green fluorescent protein (GFP; Becton Dickinson).

Purification of human germinal center B cells, cell lines and cell cultures.

Normal germinal center B cells were purified from human tonsils (discarded

after tonsillectomy) as described54. Human tonsils were obtained with approval

from the Columbia University Institutional Review Board as discarded ‘left-

overs’ from tonsillectomies at New York Presbyterian Hospital. The human

embryonic kidney 293 cell line and its derivative 293T cells and H1299 cells

were maintained in DMEM supplemented with 10% (vol/vol) FBS and

antibiotics. All B cell lines were maintained in Iscove’s modified Dulbecco’s

medium supplemented with 10% (vol/vol) FBS and antibiotics.

Transfection and immunoprecipitation. The 293T and H1299 cells were

transiently transfected by the calcium-phosphate precipitation method as

described19,28. Protein-protein interactions were detected essentially as

described29. GST precipitation assays were done as reported48.

Treatment with DNA damage–inducing agents. For B cell lines, cells seeded at

a density of 0.5 � 106 cells per ml were treated with DNA damage–inducing

agents at various times and doses. MG132 (50 mM), caffeine (0–5 mM) and

KU-55933 (2 mM and 10 mM) were added to the medium 25–30 min before

treatment with DNA damage–inducing agents. For 293T cells, transfected cells

were treated with etoposide (5 mM) at 24 h after transfection and were collected

for analysis after 12–14 h.

Lentiviral infection. Lentiviral supernatants were produced by transfection of

the vectors c-FUGW, the HIV-1 packaging vector D8.9 and the VSVg envelope

glycoprotein together into 293T cells as described55. For infection, Ramos cells

(5 � 105 cells/ml) were resuspended in viral supernatants supplemented with

polybrene (0.5 mg/ml) and were centrifuged for 1 h at 450g. GFP expression in

Ramos cells was detected by flow cytometry with a FACSCalibur (Becton

Dickinson) and the transduced GFP+ cells were selected by cell sorting with a

FACStar (Becton Dickinson).

Immunofluorescence analysis. Immunofluorescence analysis was done essen-

tially as described12,27 with anti-Bcl-6 (N3), anti-g-H2AX and anti-Oct-2.

Mathlab software was used for quantitative analysis.

Analysis of lymphoid tissues. Lymphoid organs from Pin1–/–, Pin1+/– and

Pin1+/+ mice were examined before and after (day +10) immunization with

sheep red blood cells by histological analysis of formalin-fixed, paraffin-

embedded sections and flow cytometry of single-cell suspensions as

described12. The appropriate fluorochrome-conjugated antibody combinations

were used for characterization of B cell and T cell subpopulations in the bone

marrow, spleen and thymus5. Germinal center cells were also counted by

four-color staining with anti-B220 (553091; BD Pharmingen), anti-PNA (FL-

1071; Vector Laboratories), anti-Fas (CD95; 556641; BD Pharmingen), immu-

noglobulin G1 (IgG1; 553485; BD Pharmingen) and phycoerythrin-conjugated

IgD (11-26; Southern Biotechnology). Data were acquired on a FACSCalibur

(Becton Dickinson) and were analyzed with the FlowJo software.

Statistical analysis. Student’s t-test was used to measure whether the differ-

ences in cell subset numbers and distribution between mice of different

genotype were significant.

Note: Supplementary information is available on the Nature Immunology website.

ACKNOWLEDGMENTSWe thank P. Le, M. Uranishi, Q. Shen, P.M. Smith and T. Mo for technicalsupport; I. Schieren for help in cell sorting; G. Cattoretti for pathologyconsultation in the analysis of Pin1–/– mice; A. Melnick and S. Ranuncolo fordiscussions; and R. Baer, L. Pasqualucci and D. Dominguez-Sola for discussionsand critical reading of the manuscript. Supported by the National Institutes ofHealth (R.T.P. and R.D.-F.) and the Leukemia Lymphoma Society (SpecializedCenter of Research Grant).

AUTHOR CONTRIBUTIONSR.T.P. and M.S. did the experiments in Figures 1–5 (R.T.P.) and Figures 2d and3a,b (M.S.); Y.K. isolated centroblasts from tonsil biopsies; A.R.M. providedPin1–/– mice; and R.D.-F. provided project planning and supervision.

COMPETING INTERESTS STATEMENTThe authors declare no competing financial interests.

Published online at http://www.nature.com/natureimmunology

Reprints and permissions information is available online at http://npg.nature.com/

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