title: Dual modification of BMAL1 by SUMO2/3 and ubiquitin promotes circadian activation of the CLOCK/BMAL1 complex.
abstract: Heterodimers of BMAL1 and CLOCK drive rhythmic expression of clock-controlled genes, thereby generating circadian physiology and behavior. Posttranslational modifications of BMAL1 play a key role in modulating the transcriptional activity of the CLOCK/BMAL1 complex during the circadian cycle. Recently, we demonstrated that circadian activation of the heterodimeric transcription factor is accompanied by ubiquitin-dependent proteolysis of BMAL1. Here we show that modification by SUMO localizes BMAL1 exclusively to the promyelocytic leukemia nuclear body (NB) and simultaneously promotes its transactivation and ubiquitin-dependent degradation. Under physiological conditions, BMAL1 was predominantly conjugated to poly-SUMO2/3 rather than SUMO1, and the level of these conjugates underwent rhythmic variation, peaking at times of maximum E-box-mediated circadian transcription. Interestingly, mutation of the sumoylation site (Lys(259)) of BMAL1 markedly inhibited both its ubiquitination and its proteasome-mediated proteolysis, and these effects were reversed by covalent attachment of SUMO3 to the C terminus of the mutant BMAL1. Consistent with this, SUSP1, a SUMO protease highly specific for SUMO2/3, abolished ubiquitination, as well as sumoylation of BMAL1, while the ubiquitin protease UBP41 blocked BMAL1 ubiquitination but induced accumulation of polysumoylated BMAL1 and its localization to the NB. Furthermore, inhibition of proteasome with MG132 elicited robust nuclear accumulation of SUMO2/3- and ubiquitin-modified BMAL1 that was restricted to the transcriptionally active stage of the circadian cycle. These results indicate that dual modification of BMAL1 by SUMO2/3 and ubiquitin is essential for circadian activation and degradation of the CLOCK/BMAL1 complex.
authors.affiliation: School of Biological Sciences, Seoul National University, Seoul 151-742, Republic of Korea.
authors.forename: Jiwon
authors.initials: J
authors.lastname: Lee
authors.forename: Yool
authors.initials: Y
authors.lastname: Lee
authors.forename: Min Joo
authors.initials: MJ
authors.lastname: Lee
authors.forename: Eonyoung
authors.initials: E
authors.lastname: Park
authors.forename: Sung Hwan
authors.initials: SH
authors.lastname: Kang
authors.forename: Chin Ha
authors.initials: CH
authors.lastname: Chung
authors.forename: Kun Ho
authors.initials: KH
authors.lastname: Lee
authors.forename: Kyungjin
authors.initials: K
authors.lastname: Kim
mesh_terms: D056930:ARNTL Transcription Factors; D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000378:metabolism*; D056926:CLOCK Proteins; D019556:COS Cells; D002522:Chlorocebus aethiops; D002940:Circadian Rhythm; D019281:Dimerization; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D041681:NIH 3T3 Cells; D011499:Protein Processing, Post-Translational; D025841:Small Ubiquitin-Related Modifier Proteins / Q000378:metabolism*; D015534:Trans-Activators / Q000378:metabolism*; D014452:Ubiquitins / Q000378:metabolism*
chemical_list: D056930:ARNTL Transcription Factors; D051792:Basic Helix-Loop-Helix Proteins; D056926:CLOCK Proteins; D025841:Small Ubiquitin-Related Modifier Proteins; D015534:Trans-Activators; D014452:Ubiquitins; C538960:BMAL1 protein, human; C538961:Bmal1 protein, mouse; C538943:CLOCK protein, human; C538944:Clock protein, mouse