Virtually all diffuse large cell lymphomas and a significant fraction of follicular lymphomas contain translocations and/or point mutations in the 5' non-coding region of the putative oncogene BCL-6, that are presumed to deregulate its expression. BCL-6 encodes a Cys2-His2 zinc finger transcriptional repressor with a POZ domain at its amino-terminus. The POZ (or BTB) domain, a 120-amino-acid motif, mediates homomeric and, in some proteins, heteromeric POZ-POZ interactions. In addition, the POZ domain is required for transcriptional repression of several proteins, including BCL-6. Using a yeast two-hybrid screen, we identified N-CoR and SMRT as BCL-6 interacting proteins. Both N-CoR and SMRT, which were originally identified as co-repressors for the unliganded nuclear thyroid hormone and retinoic acid receptors, are components of large complexes containing histone deacetylases. We show that the interaction between BCL-6 and these co-repressors is also detected in the more physiologically relevant mammalian two-hybrid assay. The POZ domain is necessary and sufficient for interaction with these co-repressors. BCL-6 and N-CoR co-localize to punctate regions of the nucleus. Furthermore, when BCL-6 is bound to its consensus recognition sequence in vivo, it can interact with N-CoR and SMRT. We find, in vitro, that POZ domains from a variety of other POZ domain-containing proteins, including the transcriptional repressor PLZF, as well as ZID, GAGA and a vaccinia virus protein, SalF17R, also interact with varying affinities with N-CoR and SMRT. We find that BCL-6 POZ domain mutations that disrupt the interaction with NCoR and SMRT no longer repress transcription. In addition, these mutations no longer self associate suggesting that self interaction is required for interaction with the co-repressors and for repression. More recently N-CoR has also been implicated in transcriptional repression by the Mad/Mxi proteins. Our demonstration that N-CoR and SMRT interact with the POZ domain containing proteins indicates that these co-repressors are likely involved in the mediation of repression by multiple classes of repressors and may explain, in part, how POZ domain containing repressors mediate transcriptional repression.
Bibliographical noteFunding Information:
We thank I Goldsmith for oligonucleotide synthesis; Christy Miller for construction of WT and MT1 reporters, and G Aviles for assistance with yeast assays. We thank T Miki for his generous gift of the BCL-6 cDNA; R Evans for pCMX – SMRT; MG Rosenfeld for pCMX-N-CoR; R Marais for EFplink2 and EFlacZ expression vectors; H Towle for mammalian GAL4 DNA binding domain expression construct and pCMVlacZ; R Baer for G5E1bLuc reporter plasmid; D Bernhlor for pAH205; G Smith for SalF17R DNA; D Zarkower for TRA-1 bait plasmid; and A Zelent for PLZF cDNA. VB would like to thank Richard Treisman in whose lab at the ICRF the BCL-6 site selection was performed. We thank G Evan for 9E10 antibody; S Elledge for yeast plasmids and advice; and K Gustafson for noticing the STAT binding site similarity. We thank D Zarkower, H Towle, B VanNess, G Li, P Siliciano, J Little and S Snyder for critical reading of the manuscript. VB was supported by the Medical Research Council of Canada while in R Treisman’s laboratory. This work was supported by the University of Minnesota Graduate School and NIH grant 5R29CA71540 from the National Cancer Institute.
- POZ domain
- Transcriptional repression