Corepressor SMRT binds the BTB/POZ repressing domain of the LAZ3/BCL6 oncoprotein

Abstract

The LAZ3/BCL6 (lymphoma-associated zinc finger 3/B cell lymphomas 6) gene frequently is altered in non-Hodgkin lymphomas. It encodes a sequence-specific DNA binding transcriptional repressor that contains a conserved N-terminal domain, termed BTB/POZ (bric-à-brac tramtrack broad complex/pox viruses and zinc fingers). Using a yeast two-hybrid screen, we show here that the LAZ3/BCL6 BTB/POZ domain interacts with the SMRT (silencing mediator of retinoid and thyroid receptor) protein. SMRT originally was identified as a corepressor of unliganded retinoic acid and thyroid receptors and forms a repressive complex with a mammalian homolog of the yeast transcriptional repressor SIN3 and the HDAC-1 histone deacetylase. Protein binding assays demonstrate that the LAZ3/BCL6 BTB/POZ domain directly interacts with SMRT in vitro. Furthermore, DNA-bound LAZ3/BCL6 recruits SMRT in vivo, and both overexpressed proteins completely colocalize in nuclear dots. Finally, overexpression of SMRT enhances the LAZ3/BCL6-mediated repression. These results define SMRT as a corepressor of LAZ3/BCL6 and suggest that LAZ3/BCL6 and nuclear hormone receptors repress transcription through shared mechanisms involving SMRT recruitment and histone deacetylation.

Figure 1

Interaction between the LAZ3/BCL6 BTB/POZ domain and SMRT in the yeast two-hybrid assay. (A) Schematic drawing of the LAZ3/BCL6 and SMRT chimeras used in the two-hybrid experiments in B (Upper) and C (Lower). The SMRT protein is shown, with its nuclear receptors interaction domains (NRID) (black boxes) and its two SMRT repressive domains (SRD-1 and SRD-2) (gray boxes) (–26) to point out the SMRT(cl2) cloned in the two-hybrid screen. In the LAZ3/BCL6 protein, the N-terminal BTB/POZ domain and the six C-terminal zinc fingers are shown. GAL4dbd, black ovals; GAL4act, open ovals. (B) Both full-length LAZ3/BCL6 and LAZ(1–181) proteins interact with SMRT(cl2). The proteins expressed (+) or not expressed (−) in the corresponding yeast two-hybrid test are mentioned. β-Gal activity was determined by liquid assay and expressed as a unit defined in ref. . The average of three experiments in triplicate are plotted. SD are shown. (C) The BTB/POZ domain mediates the interaction between LAZ3/BCL6 and SMRT(cl2) in the yeast two-hybrid system. The proteins expressed (+) or not expressed (−) in the corresponding test are mentioned. The average of three experiments in triplicate are plotted. Results are expressed as in B. All GAL4act chimeras are expressed in yeast at similar level as indicated by Western blot analyses using an anti-GAL4act antibody (data not shown).

Figure 2

The BTB/POZ domain of LAZ3/BCL6 directly interacts with SMRT in GST pulldown assays. In vitro translated and ³⁵S-methionine-labeled SMRT (lane 1, I represents 40% of the SMRT amount used in the pulldown experiments) was incubated with either GST alone (lane 2), or GST-LAZ(BTB/POZ) (lane 4). Interaction between SMRT and GST-RARα is shown as a positive control (lane 3). Molecular masses are indicated on the left.

Figure 3

The BTB/POZ domain mediates the interaction between LAZ3/BCL6 and SMRT(cl2) in mammalian cells. (A) Schematic representation of the mutants used in B (Upper) and C (Lower). VP16 activating domain (open boxes) and GAL4dbd (black ovals) are shown (see Fig. 1 A for the other legend conventions). (B) DNA-tethered BTB/POZ recruits SMRT(cl2) in mammalian cells. GAL4dbd, (GAL4dbd)LAZ(BTB/POZ), or (GAL4dbd)LAZ(ΔBTB/POZ) were coexpressed with either the isolated VP16 activating domain (VP16) (empty bars) or the VP16 activating domain fused to SMRT(cl2) [(VP16)SMRT(cl2)] (filled bars). The G5-TATA-Luc reporter construct (33) is shown above the panel. Only the coexpression of (GAL4dbd)LAZ(BTB/POZ) with the (VP16)SMRT(cl2) construct elicits a dramatic increase in the activity of the G5-TATA-Luc reporter (33). Either pSG424, pSG424-LAZ(BTB/POZ), or pSG424-LAZ(ΔBTB/POZ) (34) (0.2 μg) was transfected together with either VP16 or (VP16)SMRT(cl2) expressing pSG-FNV vectors (0.2 μg) (36) and with the G5-TATA-Luc (1.6 μg) reporter (33). Normalized luciferase activity are plotted (arbitrary units). The results obtained with the coexpression of the isolated GAL4dbd with the VP16 activating domain are arbitrarily taken as 1. Note that we observed a repressive effect as previously described when the VP16 alone was coexpressed with the GAL4dbd LAZ3/BCL6 chimeras (–18). (C) DNA-bound LAZ3/BCL6 recruits SMRT(cl2) in mammalian cells. Full-length LAZ3/BCL6 was coexpressed with either the isolated VP16 activating domain (empty bar) or with the (VP16)SMRT(cl2) chimera (filled bar). The B6BS-tk-LUC reporter (Upper) contains one LAZ3/BCL6 binding site upstream of the minimal tk promoter (18). The pTL1-LAZ3/BCL6 expression vector (0.2 μg) was cotransfected along with the B6BStkLuc reporter vector (1.6 μg) (18) and either pCMX-VP16 or pCMX-VP16-SMRT(cl2) expression vector (0.2 μg). Results are expressed as normalized luciferase activity. The activity obtained for the coexpression of LAZ3/BCL6 and VP16 was arbitrarily taken as 1. (D) LAZ3/BCL6 and SMRT colocalize in nuclear dots. A SMRT and an epitope-tagged LAZ3/BCL6 (LAZ3/BCL6-flag) encoding vectors were cotransfected in C2 cells. The nucleus of a cotransfected C2 cell is shown for the LAZ3/BCL6-flag pattern in green (Left) and for the SMRT pattern in red (Middle). Both patterns are completely identical as shown by the resulting yellow image after superimposition of both staining (Right). Note that the same results were obtained with two different anti-SMRT polyclonal antibodies, or when SMRT was cotransfected with the “self-detectable” LAZ3/BCL6-GFP (green fluorescent protein) chimera ruling out that the observed colocalization results from antibody crossreactions (data not shown).

Figure 4

Overexpression of SMRT potentiates the LAZ3/BCL6-mediated repression. The LAZ3/BCL6 encoding vector (pTL1-LAZ3/BCL6) was transfected without (−) or with (+) the SMRT encoding vector (pCMX-SMRT) (hatched bars). The coexpression of LAZ3/BCL6 and SMRT elicits a ≈ 2-fold increase in the LAZ3/BCL6-mediated repression of the B6BS reporter activity (18). Cotransfection of pCMX-SMRT with the empty pTL1 vector results in a slight decrease of the B6BS reporter activity, as compared with the mock transfected cells (empty bars). This is presumably due to the weak expression of the endogenous LAZ3/BCL6 protein in C2 cells (ref. , data not shown). Either empty pTL or pTL-LAZ3/BCL6 (17) expression vector was transfected together with B6BS-tkLuc (1.5 μg) (18), and with either empty pCMX or pCMX-SMRT expression vector (0.2 μg). pSG5-β-gal (0.1 μg) was cotransfected in each assay to correct for variation in transfection efficiency. Results are expressed as normalized luciferase activity.