In-depth mutational analysis of the promyelocytic leukemia zinc finger BTB/POZ domain reveals motifs and residues required for biological and transcriptional functions

The promyelocytic leukemia zinc finger (PLZF) protein is a transcription fa ctor disrupted in patients with t(11;17) (q23;q21)-associated acute promyel ocytic leukemia, PLZF contains an N-terminal BTB/POZ domain which is requir ed for dimerization, transcriptional repression, formation of high-molecula r-weight DNA-protein complexes, nuclear sublocalization, and growth suppres sion. X-ray crystallographic data show that the PLZF BTB/POZ domain farms a n obligate homodimer via an extensive interface. In addition, the dimer pos sesses several highly conserved features, including a charged pocket, a hyd rophobic monomer core, an exposed hydrophobic surface on the floor of the d imer, and two negatively charged surface patches. To determine the role of these structures, mutational analysis of the BTB/POZ domain was performed. We found that point mutations in conserved residues that disrupt the dimer interface or the monomer core result in a misfolded nonfunctional protein. Mutation of key residues from the exposed hydrophobic surface suggests that these are also important for the stability of PLZF complexes. The integrit y of the charged-pocket region was crucial for proper folding of the BTB/PO Z domain. In addition, the pocket was critical for the ability of the BTB/P OZ domain to repress transcription. Alteration of charged-pocket residue ar ginine 49 to a glutamine (mutant R49Q) yields a domain that can still dimer ize but activates rather than represses transcription. In the context of fu ll-length PLZF, a properly folded BTB/POZ domain was required for all PLZF functions, However, PLZF with the single pocket mutation R49Q repressed tra nscription, while the double mutant D35N/R49Q could not, despite its abilit y to dimerize. These results indicate that PLZF requires the BTB/POZ domain for dimerization and the charged pocket for transcriptional repression.