ABSTRACT
The AML-1/ETO fusion protein, created by the (8;21) translocation in M2-type acute myelogenous leukemia (AML), is a dominant repressive form of AML-1. This effect is due to the ability of the ETO portion of the protein to recruit co-repressors to promoters of AML-1 target genes. The t(11;17)(q21;q23)-associated acute promyelocytic leukemia creates the promyelocytic leukemia zinc finger PLZFt/RAR alpha fusion protein and, in a similar manner, inhibits RAR alpha target gene expression and myeloid differentiation. PLZF is expressed in hematopoietic progenitors and functions as a growth suppressor by repressing cyclin A2 and other targets. ETO is a corepressor for PLZF and potentiates transcriptional repression by linking PLZF to a histone deacetylase-containing complex. In transiently transfected cells and in a cell line derived from a patient with t(8;21) leukemia, PLZF and AML-1/ETO formed a tight complex. In transient assays, AML-1/ETO blocked transcriptional repression by PLZF, even at substoichiometric levels relative to PLZF. This effect was dependent on the presence of the ETO zinc finger domain, which recruits corepressors, and could not be rescued by overexpression of co-repressors that normally enhance PLZF repression. AML-1/ETO also excluded PLZF from the nuclear matrix and reduced its ability to bind to its cognate DNA-binding site. Finally, ETO interacted with PLZF/RAR alpha and enhanced its ability to repress through the RARE. These data show a link in the transcriptional pathways of M2 and M3 leukemia. (Blood. 2000;96:3939-3947)
Subject(s)
DNA-Binding Proteins/genetics , Leukemia, Promyelocytic, Acute/genetics , Oncogene Proteins, Fusion/pharmacology , Proto-Oncogene Proteins , Transcription Factors/genetics , Transcription Factors/pharmacology , Transcription, Genetic/drug effects , Binding Sites , Cell Line , Core Binding Factor Alpha 2 Subunit , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/pharmacology , Gene Expression Regulation/drug effects , Humans , Kruppel-Like Transcription Factors , Leukemia, Myeloid, Acute/etiology , Leukemia, Myeloid, Acute/genetics , Leukemia, Promyelocytic, Acute/etiology , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Nuclear Matrix/drug effects , Oncogene Proteins, Fusion/genetics , Oncogene Proteins, Fusion/metabolism , Promyelocytic Leukemia Zinc Finger Protein , Protein Binding , RUNX1 Translocation Partner 1 Protein , Repressor Proteins/metabolism , Repressor Proteins/pharmacology , Transcription Factors/antagonists & inhibitors , Transcription Factors/metabolismABSTRACT
The promyelocytic leukemia zinc finger (PLZF) protein is a transcription factor disrupted in patients with t(11;17)(q23;q21)-associated acute promyelocytic leukemia. PLZF contains an N-terminal BTB/POZ domain which is required for dimerization, transcriptional repression, formation of high-molecular-weight DNA-protein complexes, nuclear sublocalization, and growth suppression. X-ray crystallographic data show that the PLZF BTB/POZ domain forms an obligate homodimer via an extensive interface. In addition, the dimer possesses several highly conserved features, including a charged pocket, a hydrophobic monomer core, an exposed hydrophobic surface on the floor of the dimer, 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 integrity of the charged-pocket region was crucial for proper folding of the BTB/POZ domain. In addition, the pocket was critical for the ability of the BTB/POZ domain to repress transcription. Alteration of charged-pocket residue arginine 49 to a glutamine (mutant R49Q) yields a domain that can still dimerize but activates rather than represses transcription. In the context of full-length PLZF, a properly folded BTB/POZ domain was required for all PLZF functions. However, PLZF with the single pocket mutation R49Q repressed transcription, while the double mutant D35N/R49Q could not, despite its ability to dimerize. These results indicate that PLZF requires the BTB/POZ domain for dimerization and the charged pocket for transcriptional repression.
Subject(s)
DNA-Binding Proteins/chemistry , Repressor Proteins/chemistry , Transcription Factors/chemistry , Transcription, Genetic , Amino Acid Motifs , Amino Acid Sequence , Arginine/chemistry , Cell Line , Circular Dichroism , DNA, Complementary/metabolism , Dimerization , Escherichia coli/metabolism , Fluorescent Antibody Technique , Genes, Reporter , Glutamine/chemistry , Humans , Kruppel-Like Transcription Factors , Molecular Sequence Data , Mutagenesis, Site-Directed , Plasmids/metabolism , Point Mutation , Promyelocytic Leukemia Zinc Finger Protein , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , Sequence Homology, Amino Acid , Structure-Activity Relationship , Temperature , Trypsin/pharmacology , Two-Hybrid System TechniquesABSTRACT
The ETO protein was originally identified by its fusion to the AML-1 transcription factor in translocation (8;21) associated with the M2 form of acute myeloid leukemia (AML). The resulting AML-1-ETO fusion is an aberrant transcriptional regulator due to the ability of ETO, which does not bind DNA itself, to recruit the transcriptional corepressors N-CoR, SMRT, and Sin3A and histone deacetylases. The promyelocytic leukemia zinc finger (PLZF) protein is a sequence-specific DNA-binding transcriptional factor fused to retinoic acid receptor alpha in acute promyelocytic leukemia associated with the (11;17)(q23;q21) translocation. PLZF also mediates transcriptional repression through the actions of corepressors and histone deacetylases. We found that ETO is one of the corepressors recruited by PLZF. The PLZF and ETO proteins associate in vivo and in vitro, and ETO can potentiate transcriptional repression by PLZF. The N-terminal portion of ETO forms complexes with PLZF, while the C-terminal region, which was shown to bind to N-CoR and SMRT, is required for the ability of ETO to augment transcriptional repression by PLZF. The second repression domain (RD2) of PLZF, not the POZ/BTB domain, is necessary to bind to ETO. Corepression by ETO was completely abrogated by histone deacetylase inhibitors. This identifies ETO as a cofactor for a sequence-specific transcription factor and indicates that, like other corepressors, it functions through the action of histone deactylase.
