Preferential inhibition of BMAL2-CLOCK activity by PER2 reemphasizes its negative role and a positive role of BMAL2 in the circadian transcription

Momoko Sasaki, Hikari Yoshitane, Ngoc Hien Du, Toshiyuki Okano, Yoshitaka Fukada

    Research output: Contribution to journalArticle

    32 Citations (Scopus)

    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.

    Original languageEnglish
    Pages (from-to)25149-25159
    Number of pages11
    JournalJournal of Biological Chemistry
    Volume284
    Issue number37
    DOIs
    Publication statusPublished - 2009 Sep 11

    Fingerprint

    E-Box Elements
    Transcription
    Transcriptional Activation
    Clocks
    Circadian Rhythm
    RNA Interference
    Immunoprecipitation
    Genes
    Bioluminescence
    Interference suppression
    Assays
    RNA
    Proteins
    Experiments

    ASJC Scopus subject areas

    • Biochemistry
    • Cell Biology
    • Molecular Biology

    Cite this

    Preferential inhibition of BMAL2-CLOCK activity by PER2 reemphasizes its negative role and a positive role of BMAL2 in the circadian transcription. / Sasaki, Momoko; Yoshitane, Hikari; Du, Ngoc Hien; Okano, Toshiyuki; Fukada, Yoshitaka.

    In: Journal of Biological Chemistry, Vol. 284, No. 37, 11.09.2009, p. 25149-25159.

    Research output: Contribution to journalArticle

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    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.",
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