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 ubiquitindependent 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 ubiquitindependent 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.Circadian rhythms are fundamental mechanisms that optimize the physiology and behavior of most organisms. Extensive genetic and molecular studies have shown that these biological rhythms are driven by molecular clockwork composed of autoregulatory transcription-translation feedback loops involving several clock gene products (27,36). In mammals, the heterodimeric transcription factor CLOCK/ BMAL1 activates transcription of its own negative regulators, including cryptochromes (CRYs) and periods (PERs), and the products of the newly synthesized transcripts form complexes that block their own transcription by binding to the heterodimeric transcription activator. Of the negative regulators, the CRYs play a central role in this feedback inhibition by interacting directly with the C terminus of BMAL1 (15,26,29).There is, however, some evidence that this canonical feedback mechanism is insufficient to account for the robust oscillation of the circadian clock. During the circadian cycle, the mRNA profiles of the key negative regulators, the CRYs, exhibit weak oscillations compared to those of other clock genes controlled by the CLOCK/BMAL1 complex (14,31,35). Moreover, the abundance of BMAL1 and CLOCK proteins in the nucleus reaches a minimum at the time that expression of...

Significant lethality following liver resection in A20 heterozygous knockout mice uncovers a key role for A20 in liver regeneration

“…40,41 We traced back higher DBP mRNA levels to higher protein (not mRNA) levels of its transcriptional regulator ARNTL in HET livers. This latter result suggests that hepatic ARNTL is directly or indirectly regulated at the posttranslational level by ubiquitin-editing A20, which would agree with ARNTL incurring posttranslational sumoylation and ubiquitination, 42 and its protein levels being modulated by proteasomal activity. 43 Increased ARNTL levels in HET livers correlated with a significant increase in mRNA levels of its other target gene Wee1 (Supplementary Figure 5), a cyclic kinase that inactivates cdc2 to preclude mitosis.…”

Sequential and Compartment-specific Phosphorylation Controls the Life Cycle of the Circadian CLOCK Protein

“…These modifications may involve phosphorylation or alternatively sumoylation. BMAL1 was shown to be sumoylated (43,44), and this modification is known to control transcription factor activities by targeting to specific subnuclear compartments (45,46). Storage of CLK in nuclear foci provides an explanation for the puzzling observation that overexpression, as well as ectopic expression of Clk from a per-promoter in anti-phase to its endogenous rhythm is able to sustain normal clock function (15).…”

The intricate dance of post-translational modifications in the rhythm of life

“…Regulation of CLOCK-BMAL1 activity through modulation of protein turnover is not limited to phosphorylation. SUMOylation at Lys259 induces ubiquitination of BMAL1 and increases transactivation and degradation 91,92 . In a similar manner, SUMOylation at Lys67 and Lys851 of human CLOCK increases the activity of CLOCK 93 , although it is unclear whether this modification destabilizes CLOCK.…”