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. 1997 Sep 30;94(20):10762–10767. doi: 10.1073/pnas.94.20.10762

Figure 3.

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 (13–18). (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).

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