title: Interactive Organization of the Circadian Core Regulators PER2, BMAL1, CLOCK and PML.
abstract: The BMAL1 and CLOCK heterodimer in the mammalian circadian transcriptional complex is thought to be repressed by PER2 and CRY1 via direct interactions. We recently reported that PER2 is largely cytosolic in Pml(-/-) cells and did not co-immunoprecipitate (co-IP) with BMAL1 or CLOCK. Here, using multi-color immunofluorescence (IF) staining and co-IP, we observed a nuclear distribution of BMAL1 and a predominately cytosolic distribution of CLOCK in Pml(-/-) MEF. In the presence of WT PML, PER2 co-localized with BMAL1 in the nucleus. In Pml(-/-) MEF transfected with mutant K487R PML, we observed that BMAL1 and PER2 co-localized with K487R PML in the cytosol. Furthermore, cytosolic CLOCK and PER2 displayed a significant non-overlapping IF staining pattern. In Bmal1(-/-) MEF, CLOCK was primarily cytosolic while PML and PER2 were nuclear. Together, our studies suggest that PML mediates the binding of PER2 to BMAL1 in the BMAL1/CLOCK heterodimer and is an important component in the organization of a functional clock complex in the nucleus. Our studies also support that BMAL1 is important for CLOCK nuclear localization.
authors.affiliation: Department of Biochemistry and Molecular Biology, Medical School, University of Texas Health Science Center-Houston, Houston, Texas 77030, USA.
authors.forename: Takao
authors.initials: T
authors.lastname: Miki
authors.affiliation: Department of Biochemistry and Molecular Biology, Medical School, University of Texas Health Science Center-Houston, Houston, Texas 77030, USA.
authors.forename: Zhaoyang
authors.initials: Z
authors.lastname: Zhao
authors.affiliation: Department of Biochemistry and Molecular Biology, Medical School, University of Texas Health Science Center-Houston, Houston, Texas 77030, USA.
authors.forename: Cheng Chi
authors.initials: CC
authors.lastname: Lee
mesh_terms: D056930:ARNTL Transcription Factors / Q000378:metabolism*; D000818:Animals; D056926:CLOCK Proteins / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D057906:Circadian Clocks; D005455:Fluorescent Antibody Technique; D006801:Humans; D047468:Immunoprecipitation; D051379:Mice; D008954:Models, Biological; D056950:Period Circadian Proteins / Q000378:metabolism*; D000071156:Promyelocytic Leukemia Protein / Q000378:metabolism*; D011485:Protein Binding
chemical_list: D056930:ARNTL Transcription Factors; C538961:Bmal1 protein, mouse; C513312:Per2 protein, mouse; D056950:Period Circadian Proteins; C513293:Pml protein, mouse; D000071156:Promyelocytic Leukemia Protein; D056926:CLOCK Proteins