The LAZ3(BCL-6) oncoprotein recruits a SMRT/mSIN3A/histone deacetylase containing complex to mediate transcriptional repression

doi:10.1093/nar/26.20.4645

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Nucleic Acids Research Pages 4645-4651

The LAZ3(BCL-6) oncoprotein recruits a SMRT/mSIN3A/histone deacetylase containing complex to mediate transcriptional repression
Introduction
Materials And Methods
   Immunoprecipitations and western blot analysis
   Yeast two-hybrid and GST pulldown assays
   Transfection experiments
   HDAC assays
Results
Discussion
Acknowledgements
References

The LAZ3(BCL-6) oncoprotein recruits a SMRT/mSIN3A/histone deacetylase containing complex to mediate transcriptional repression

Philippe Dhordain*, Richard J. Lin¹, Sabine Quief, Danièle Lantoine, Jean-Pierre Kerckaert, Ronald M. Evans¹ and Olivier Albagli

U124 INSERM/IRCL, Place de Verdun, F-59045 Lille cedex, France and ¹Howard Hughes Medical Institute and The Salk Institute for Biological Studies, La Jolla, CA 92037, USA

Received June 26, 1998; Revised and Accepted September 7, 1998

ABSTRACT

Recent works demonstrated that some transcriptional repressors recruit histone deacetylases (HDACs) either through direct interaction, or as a member of a multisubunit repressing complex containing other components referred to as corepressors. For instance, the bHLH-Zip transcriptional repressors MAD/MXI recruit HDACs together with the mSIN3 corepressors, whereas unliganded nuclear receptors contact another corepressor, SMRT (or its relative N-CoR), which, in turn, associates with both mSIN3 and HDACs to form the repressor complex. Recently, we reported that SMRT also directly associates with LAZ3(BCL-6), a POZ/Zn finger transcriptional repressor involvedin the pathogenesis of non-Hodgkin lymphomas. However, whether LAZ3 recruits the HDACs-containing repression complex is currently unknown. We report here that LAZ3 associates with corepressor mSIN3A both in vivo and in vitro, and found that a central region, which harbours autonomous repression activity, is mainly responsible for this interaction. Conversely, the N-terminal half of mSIN3A is both necessary and sufficient to bind LAZ3. Moreover, we show that LAZ3 also interacts with an HDAC (HDAC-1) through its POZ domain in vitro while the immunoprecipitation of LAZ3 results in the coretention of an endogenous HDAC activity in vivo. Finally, inhibitors of HDACs significantly reduce the LAZ3-mediated repression. Taken together, we conclude that LAZ3 recruits a repressing complex containing SMRT, mSIN3A and a HDAC, and that its full repressing potential on transcription requires HDACs activity. Our results identify HDACs as molecular targets of LAZ3 oncogene and further strengthen the connection between aberrant chromatin acetylation and human cancers.

INTRODUCTION

Transcriptional repression plays a central role in many biological processes. Recently, a large body of evidence highlighted the importance of histone deacetylases (HDACs) in transcriptional repression (1-3). Indeed, HDACs are recruited by several transcriptional repressors such as yeast UME6, or, in vertebrates, by the bHLH-ZIP MAD protein, the zinc finger protein YY1, the tumor-suppressor pRB and by certain unliganded nuclear receptors (1,4-6). Moreover, deletion of the yeast HDAC-encoding gene rpd3 impedes the UME6-mediated transcriptional repression (7). Similarly, specific inhibitors of HDACs decrease the repressing activity of MAD, pRb and unliganded nuclear receptors (5,6,8,9). Collectively, these biochemical and genetic data indicate that the targeting of HDACs to specific regulatory regions is an important feature of various transcriptional repressors.

What appears to differ, however, between these transcriptional repressors is the way they recruit HDACs. In some cases, the recruitment presumably occurs through a direct binding of the transcriptional repressor to HDACs, as illustrated by the cases of both pRB and YY-1 (4-6,10-12). In other cases, HDACs are recruited as an integral component of a larger repressing complex containing other proteins collectively referred to as corepressors. This is exemplified by MAD proteins whose transcriptional repressing activity has been correlated to the recruitment of HDACs together with the mammalian orthologs of the yeast SIN3 corepressor, mSIN3A and mSIN3B (collectively referred to as mSIN3) (9,13-16). A further level of complexity is reached by certain unliganded nuclear receptors which directly bind to another corepressor called SMRT (or its relative N-CoR), which, in turn, associates with both mSIN3 and HDACs (8,17). Thus, SMRT (or N-CoR) behaves as a molecular bridge allowing unliganded nuclear receptors to recruit HDAC-containing repressing complex.

The LAZ3(BCL-6) gene has been cloned because of its frequent structural alterations in a large subset of non-Hodgkin lymphomas (NHLs) (18-21). Consistent with its implication in lymphoproliferative disorders, LAZ3 is expressed and functionally required in lymphoid tissues, since its disruption through knock-out experiments prevents germinal center formation and alters the Th2 mediated immune response (22-24). It encodes a sequence-specific transcriptional repressor belonging to the POZ/zinc finger protein family (25-29). The POZ domain is required for the full LAZ3 transcriptional repressing activity and acts as a potent autonomous repressing domain when fused to a heterologous DNA binding domain (25-29). In addition, another autonomous repression domain within the central part of LAZ3 has also been identified (25-27,29,30). Recently, we reported that the SMRT corepressor directly binds the POZ domain of LAZ3 (31). Moreover, SMRT (or N-CoR) has been shown to also associate with the POZ domain of the PLZF transcriptional repressor, a relative of LAZ3 that is fused to the RAR[alpha] nuclear receptor in a subset of acute promyelocytic leukemias (32-36). That SMRT is a corepressor shared by unliganded nuclear receptors and POZ/zinc finger proteins raises the possibility that these two families of transcription factors silence transcription through the recruitment of a similar repressing complex. To directly address this hypothesis, we examined whether LAZ3 can also recruit the mSIN3 and HDACs.

Here we report that LAZ3 and mSIN3A are associated in vivo. In addition, using yeast two hybrids and glutathione S-transferase (GST) pull-down assays, we demonstrate that LAZ3 binds both mSIN3A and a HDAC (HDAC-1). We further show that the central repressing domain of LAZ3, and, albeit to a lesser extent, the POZ domain, autonomously interact with mSIN3A. Moreover, we observe that LAZ3 is able to contact HDAC-1 in vitro through its POZ domain. Finally, we show that LAZ3 is associated with a HDAC activity in vivo while inhibitors of HDACs significantly reduce the LAZ3-mediated transcriptional repression. These findings indicate that LAZ3 recruits a SMRT, mSIN3A and HDAC-containing complex to mediate transcriptional repression.

MATERIALS AND METHODS

Immunoprecipitations and western blot analysis

The H7 cell line is derived from the UTA6 clone which expressed the TET-VP16 chimeric activating protein (37). For western blot analysis, nuclear proteins of ~1.2 × 10⁶ H7 cells were prepared according to Andrews and Faller (38) and separated by SDS-PAGE and electrotransferred onto a nitrocellulose membrane (Schleicher & Schuell). Western blot was carried out according to current protocols. Primary affinity-purified rabbit anti-LAZ3 or anti-SIN3A polyclonal antibodies (N3 or K20, respectively, Santa-Cruz) were used at 1/100 dilution. After washes, an affinity-purified, horseradish peroxidase conjugated, goat anti-rabbit secondary antibody (Amersham) was used at 1/1000 dilution in both cases. Protein detection was performed using chimioluminescent assays (NEN) followed by autoradiography. Co-immunoprecipitation and western blot analysis were carried out as follows: nuclear extracts of H7 cells (corresponding to ~4.5 × 10⁶ cells per immunoprecipitation) were immunoprecipitated on ice for 2 h in the immunoprecipitation buffer (20 mM HEPES-KOH pH 7.9, 420 mM NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 0.5 mM DTT, 25% glycerol, protease inhibitors) using the anti-GAL4 antibody (39) that serves as a negative control or the K20 anti-SIN3A antibody. Then, protein A + protein G agarose (1/1, Boehringer Mannheim) was added and the tubes were gently agitated for 40 min at 4°C. After three washes in NET-N buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 1.5 mM MgCl₂, 1 mM EDTA, 0.5% NP-40, 10% glycerol) containing protease inhibitors (8), immunoprecipitated proteins were separated by SDS-PAGE and subjected to western blot analysis using the anti-LAZ3 N3 antibody.

Yeast two-hybrid and GST pulldown assays

Yeast cells (Y190 strain from Clontech) were transformed as described previously (31). After 3 days of growing at 30°C on selective medium, colonies were transferred on filters (Whatman), freeze-thawed in liquid nitrogen and incubated in LacZ buffer (Clontech) containing 0.34 mg/ml X-gal (5-bromo-4-chloro-3-indolyl-[beta]-d-galactopyranoside) (Sigma) for 4-5 h at 30°C, then coloration analyses were performed.

GST pulldown experiments were performed as described previously (31).

Transfection experiments

H7 cells were transfected in 35 mm dishes, using 7 µl of lipofectamine (Gibco BRL), with 1 µg of the B6Bs-tk-Luc reporter construct, for 5 h in 2.5 ml OPTIMEM (Gibco BRL) plus tetracycline (2 µg/ml). Then, the medium was replaced by DF10 plus teracycline. Sixteen hours after transfection, the cells were trypsinized and equally distributed under the different culture conditions in 24 well plates. Trichostatin A (TSA) (30 nM; BIOMOL) was added 24 h before the harvesting of the cells. Twenty-four hours after plating, cells were washed once in PBS and lysed in universal lysis buffer (Promega). Luciferase activity was measured as described (31), and normalized to the protein amount in each experiment. Similar results were obtained by adding TSA (300 nM, BIOMOL) 8 h before harvesting the cells (data not shown).

CV1 cells were transfected as previously described (36). Briefly, 10⁵ cells were transfected, using DOTAP (Boehringer Mannheim), with the indicated GAL4-LAZ chimera encoding vectors (in pSG424; 0.2 µg), together with 4xMH100-tk-Luc (0.2 µg) and pCMX-[beta]-Gal (0.5 µg) to correct for variations in transfection efficiency. After transfections, cells were washed twice with PBS and placed in fresh medium. Forty-eight hours after transfection, cells were lysed. Luciferase and [beta]-galactosidase activities were measured as described (36).

C2 cells were transfected in 6 well plates, using 12 µl of lipofectamine (Gibco BRL) for 6 h in 1 ml OPTIMEM (Gibco BRL). Cells were then placed in a fresh 1:1 (v/v) mixture of DMEM/MCDB202 containing 20% FCS. Either GAL4 (pSG424) or GAL4-LAZ3 (pSG424-LAZ3; 0.2 µg) encoding plasmids were cotransfected with 4xMH100-tk-Luc (1.7 µg), and pSG5-[beta]-gal (0.05 µg) to correct for variations in transfection efficiency. Cells were then treated (or not) with either 5 mM sodium butyrate (CALBIOCHEM) or trichostatin A (TSA, BIOMOL) 8 h before harvesting. Luciferase activity was measured and normalized to the [beta]-gal activity level as described previously (31).

HDAC assays

CV1 cells transfected with indicated plasmids (10 µg/10⁶ cells) were lysed by mild sonication in lysis buffer (50 mM Tris pH 8.0, 150 mM NaCl, 10 mM MgCl₂, 10% glycerol, 0.2% NP-40, 1 mM PMSF and protease inhibitors). The lysate (500-600 µl per immunoprecipitation, corresponding to 3 × 10⁷ cells) were immunoprecipitated by incubation with 30-40 µl of M2-agarose beads overnight. Then, the beads were washed three times in with the lysis buffer, resuspended in HDAC assay buffer and incubated for 2.5 h with shaking according to Taunton et al. (13). The substrate was ³H-labeled histones isolated from HeLa cells (40).

RESULTS

Unliganded nuclear receptors have been shown to recruit a SMRT/mSIN3 complex in vivo (17). As SMRT is also a corepressor for LAZ3 (30), we first investigated whether LAZ3 could also associate with mSIN3A in vivo. To this end we performed a co-immunoprecipitation assay in a human osteosarcoma (U2OS)-derived clone, H7, which is stably transfected with an inducible version of LAZ3 under the control of the bacterial Tet operator. In this clone, an epitope (flag)-tagged LAZ3 protein is expressed upon tetracycline withdrawal (Fig. 1A) (`tet-off' system; P.Dhordain, D.Lantoine, C.Englert and O.Albagli, manuscript in preparation). Nuclear extracts of induced (72 h after tetracycline withdrawal) or uninduced H7 cells were immunoprecipitated using either: (i) an affinity-purified rabbit polyclonal antibody recognizing mSIN3A, which is expressed in H7 cells irrespective of the presence of tetracycline (Fig. 1A and B, lower panels), or (ii) as a negative control, an anti GAL4 antibody (39). After washing and SDS-PAGE of the immunoprecipitated proteins, western blot analysis was carried out using an affinity purified polyclonal anti-LAZ3 antibody. As shown in Figure 1B, the immunoprecipitate obtained with anti-mSIN3A specific antibody contains LAZ3, whereas the immunoprecipitate obtained with the irrelevant GAL4 antibody does not. This experiment reveals the existence of nuclear complexes containing both LAZ3 and mSIN3A. Moreover, we found that in vitro translated full length mSIN3A is specifically retained on matrix-bound protein of GST fused to amino acids 1-517 of LAZ3 (Fig. 1C). This result argues in favor of a direct interaction between mSIN3A and LAZ3, although we cannot rule out that some bridging proteins may be present in reticulocyte lysates.

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