Promyelocytic leukemia zinc finger (PLZF) protein is a sequence-specific DNA-binding protein that represses the transcriptional activity of target genes such as those for cyclin A and the interleukin-3 receptor ␣ chain. The PLZF gene becomes fused to the retinoic acid receptor ␣ gene as a result of the t(11, 17)(q23;q21) chromosomal translocation that is associated with acute promyelocytic leukemia. We now show that endogenous PLZF in human promyelocytic leukemia HL-60 cells is modified by conjugation with SUMO-1 (small ubiquitin-related modifier-1) and that PLZF colocalizes with SUMO-1 in the nucleus of transfected human embryonic kidney 293T cells. Site-directed mutagenesis identified lysine 242 in the RD2 domain of human PLZF as the sumoylation site. A luciferase reporter gene assay suggested that SUMO-1 modification of this residue is required for transcriptional repression by PLZF, and an electrophoretic mobility shift assay showed that this modification increases the DNA binding activity of PLZF. PLZFmediated regulation of the cell cycle and transcriptional repression of the cyclin A2 gene were also dependent on sumoylation of PLZF on lysine 242. These results demonstrate that PLZF is modified by SUMO-1 conjugation and that this modification regulates the biological functions of PLZF.Promyelocytic leukemia zinc finger protein (PLZF) 1 is a sequence-specific DNA-binding transcriptional regulator. It comprises 673 amino acids and contains nine Kruppel-like C 2 H 2 zinc finger domains and an NH 2 -terminal POZ (Pox virus and zinc finger) or BTB (broad complex, tramtrack, bric-a-brac) domain (1, 2). PLZF is expressed in human CD34 ϩ myeloid progenitor cells and primitive multipotent cell lines, and its gene is fused to that of retinoic acid receptor ␣ (RAR␣) in a variant of acute promyelocytic leukemia (APL) associated with the t(11, 17)(q23;q21) chromosomal translocation (2). Whereas RAR␣ activates key genes required for normal myelopoiesis, the PLZF-RAR␣ fusion protein represses the expression of some of these genes in a dominant negative manner. PLZF colocalizes with the PML (promyelocytic leukemia) protein, suggesting a possible link with the pathogenesis of the more common t(15, 17)-associated form of APL (3-5).In its role as a transcriptional repressor, PLZF binds to the promoter of target genes, including those for cyclin A2 and the interleukin-3 receptor ␣ chain (IL-3R␣), and interacts with the corepressors N-CoR, SMRT, and Sin3A as well as histone deacetylases. The POZ/BTB domain overlaps with a transcriptional repression domain (RD1) and mediates PLZF self-association (2, 4) and interaction with histone deacetylases. A second transcriptional repression domain (RD2) is located downstream of the POZ domain and interacts with the ETO-AML1 protein in t(8, 21)-associated acute myelocytic leukemia; this protein may function as a PLZF corepressor (6, 7).During development, PLZF contributes to patterning of the limb and axial skeleton by acting as an upstream regulator of Hox gene expression (8 -10). PLZF is...