Preferential Inhibition of BMAL2-CLOCK Activity by PER2 Reemphasizes Its Negative Role and a Positive Role of BMAL2 in the Circadian Transcription

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 promoterdriven 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.A variety of organisms from bacteria to humans show circadian rhythms in physiology and behavior under the regulation of endogenous circadian clocks oscillating with an ϳ24-h periodicity (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). Even cultured fibroblasts were shown to retain the cellular clocks (4), and hence they have been used for studies on the oscillatory mechanism of peripheral clocks. The molecular clockwork in mammals centers on transcription/translation-based autoregulatory feedback loops of clock genes, to which bHLH 3 -PAS proteins, BMAL1 and CLOCK, contribute as positive regulators of the transcription (2, 5). BMAL1-CLOCK complex activates transcription through CACGTG-type E-box and its related sequences found in promoter regions of clock genes and clock-controlled genes such as Per1 (6), Per2 (CACGTT E-box-like or EЈ-box sequence; see Ref. 7), plasminogen activator inhibitor-1 (PAI-1) (8), and Rev-Erb␣/␤ (9, 10). The E-box-dependent transactivation mediated by the BMAL1-CLOCK complex is suppressed by an expanding number of negative regulators, including PER1 (11, 12), PER2 (13-15), PER3 (16,1...

Unwinding the differences of the mammalian PERIOD clock proteins from crystal structure to cellular function

“…The PAS domains mediate homo-and heterodimeric interactions between the mPER homologues (5)(6)(7)(8) as well as interactions of the mPERs with mBMAL1/2, mCLOCK, and NPAS2 (9)(10)(11)(12). These interactions regulate the stability and cellular localization of the mPERs and modulate the activity of the mBMAL1/mCLOCK transcription factor complex.…”

Circadian clock and vascular disease

“…7 However, little is known about the redundancy or the dynamic function of BMALs in the vasculature, although CLIF/BMAL2 was shown to have a higher affinity to PER2 than BMAL1. 8 In addition to this core feedback loop, the nuclear receptor REV-ERBa is also transactivated by the CLOCK/BMAL1 heterodimer.…”

Association between ARNTL (BMAL1) rs2278749 polymorphism T >C and susceptibility to Alzheimer disease in a Chinese population

“…Neuronal PAS domain protein 2 (NPAS2) can substitute for CLOCK and aryl hydrocarbon receptor nuclear translocator-like 2 (ARNTL2 or BMAL2) for ARNTL. Paired CLOCK/NPAS2-ARNTL/ARNTL2 heterodimers then activate the transcription of their target genes (Reick et al, 2001;Oishi et al, 2003;Sasaki et al, 2009). Genetic variations in circadian rhythms genes have been associated with sleep, mood, and metabolic disorders.…”