Abstract
In the molecular oscillatory mechanism governing circadian rhythms, positive regulators, including CLOCK and BMAL1, transactivate Per and Cry genes through E-box elements, and translated PER and CRY proteins negatively regulate their own transactivation. Like BMAL1, its paralog BMAL2 dimerizes with CLOCK to activate the E-box-dependent transcription, but the role of BMAL2 in the circadian clockwork is still elusive. Here we characterized BMAL2 function in NIH3T3 cells and found that the cellular rhythms monitored by Bmal1 promoter-driven bioluminescence signals were blunted by RNA interference-mediated suppression of Bmal2 as well as that of Bmal1. Transcription assays with a 2.1-kb mPer1 promoter revealed that CRY2 inhibited the transactivation mediated by BMAL1-CLOCK more strongly than that by BMAL2-CLOCK. In contrast, PER2 showed a stronger inhibitory effect on BMAL2-CLOCK than on BMAL1-CLOCK. The molecular link between BMAL2 and PER2 was further strengthened by the fact that PER2 exhibited a greater affinity for BMAL2 than for BMAL1 in co-immunoprecipitation experiments. These results indicate a functional partnership between BMAL2 and PER2 and reemphasize the negative role of PER2 in the circadian transcription. As a broad spectrum function, BMAL2-CLOCK activated transcription from a variety of SV40-driven reporters harboring various E/E'-box-containing sequences present in the upstream regions of clock and clock-controlled genes. Importantly, the efficiencies of BMAL2-CLOCK-mediated transactivation relative to that achieved by BMAL1-CLOCK were dependent heavily on the E-box-containing sequences, supporting distinguishable roles of the two BMALs. Collectively, it is strongly suggested that BMAL2 plays an active role in the circadian transcription.
Highlights
Odicity [1, 2]
Physiological importance of BMAL2 was investigated in the cellular clock system by monitoring the circadian rhythms of bioluminescence signals driven by 2.8-kb Bmal1 promoter
Transactivation of Period Promoters by BMAL1-CLOCK and BMAL2-CLOCK—We first asked whether BMAL2 contributes to transcriptional activation of mPer1 and mPer2 genes by examining regulation of luciferase reporters driven by 2.1-kb mPer1 promoter (Fig. 1A) and 1.6-kb mPer2 promoter (Fig. 1B) in HEK293 cells
Summary
Odicity [1, 2]. In mammals, the central clock is located in the hypothalamic suprachiasmatic nucleus, whereas peripheral clocks with self-sustainable oscillation machinery are located in many peripheral tissues [3]. Translated PER and CRY proteins interact with each other to enter into the nucleus (11, 20 –23), where these negative regulators form a multimeric complex, interact with the BMAL1-CLOCK complex, and suppress their own transactivation through the E-boxes [24, 25] In this way, transcription levels of these negative regulators exhibit robust circadian rhythms, closing the core circadian molecular loop The circadian rhythm of the locomotor activity is completely abolished in Bmal1-deficient mice [38], the two Bmal genes are both expressed in the mouse suprachiasmatic nucleus [31, 39] These observations indicate that Bmal does not compensate for Bmal, but it has been left undetermined whether Bmal plays an active role in the mechanism of rhythm generation. Molecular characterization by transcription assays together with physical interaction profiles among the relevant proteins revealed an intimate functional linkage between BMAL2 and PER2 and reemphasizes their roles as a positive and a negative regulator, respectively, in the E-box-dependent feedback loop of the molecular clock
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