The data presented here reveal miRNAs as key regulators of the circadian timing process. miR-219-1 is a clock-controlled gene that plays a role in regulating the length of the circadian day, whereas miR-132 is light-inducible and modulates the phase shifting capacity of light. The effects of miR-132 culminate at the level of mPer1 transcription and PER2 protein expression, thus identifying a potential route by which these miRNAs influence the transcriptional rhythm which lies at the core of the timing process. Our data also reveal that both miR-219 and miR-132 affect cellular excitability, which, in turn, might regulate both clock periodicity and clock entrainment. Collectively, our observations reveal a new and previously unexplored layer of modulation to the circadian clock: inducible translation control via microRNAs.
Biological oscillators such as the circadian clock (and the cell cycle clock) are modeled as interacting positive and negative feedback loops that drive the rhythmic expression of a few molecular determinants. In the case of circadian clocks, the molecules in question are CLOCK and BMAL1, which together activate transcription of Per and Cry genes. PER and CRY proteins subsequently work together to inhibit their own transcription. The established view is that rhythmic gene transcription is the underlying molecular basis for circadian rhythm generation. This model has been enriched by a series of findings (described below) indicating that a complex, integrated, set of physiological processes act in coordination with the transcription-based oscillator to drive highly precise SCN pacemaker activity.
In this context, microRNAs represent a route by which the circadian clock may be regulated. These small noncoding RNAs, which are impressively numerous and phylogenetically extensive (there are currently 364 mouse miRNA genes listed in the MicroRNA Registry [http://www.microrna.sanger.ac.uk/sequences/index.shtml
]), have been implicated in mRNA turnover of miRNA targets (Lim et al., 2005
), suppression of their protein translation (Lee et al., 1993; Wightman et al., 1993
), and, possibly, methylation of target genes (Bao et al., 2004
). A role of microRNAs in circadian timing processes has not been demonstrated until now (this report).
The use of SACO analysis, which permitted an unbiased and systematic analysis of transcription factor binding across an entire mammalian genome, revealed that a substantial number of CREB binding targets were noncoding (S.I., unpublished data). We analyzed one of these CREB targets, miR-132, for its potential involvement in the circadian clock, based on the rationale that the CREB/CRE transcriptional pathway plays a pivotal role in coupling light to changes in gene expression in the SCN that underlie entrainment (Dziema et al., 2003
). Similar to other CREB-regulated light-responsive immediate early genes (eg., Fos, Jun, and the core clock gene mPer1), miR-132 gene expression in the SCN is tightly regulated by light, but its induction is phase-restricted to the subjective night. It should be noted that the observed differences in pre-miR-132 levels are also reflected in the abundance of mature miRNA, ruling out the possibility that light has no functional consequence on miR-132 effects. As with other CREB-regulated genes, infusion of the MEK1/2 inhibitor, U0126, blocked light-inducible miR-132 expression, indicating that it is mediated by a MAPK-dependent signaling pathway. Multiple lines of evidence indicate MAPK signaling as a principal effector of light-induced clock entrainment (Obrietan et al., 1998
; Butcher et al., 2002
). The characteristics determined in this study (ie., regulation by CREB and MAPK, rapid light inducibility, phase restriction) raise the possibility that miR-132 contributes to the clock entrainment mechanism.
Conceivably, miRNAs within the SCN may be regulated not only by light but also by the molecular clock itself. As a starting point, we sought to identify potential miRNAs, within the CREB SACO subset of miRNAs, that are direct targets of CLOCK/BMAL1-mediated transcription. Using chromatin immunoprecipitation, the miR-219-1 locus was enriched in the CLOCK-binding DNA fraction and identified by a PCR-based approach. The murine miR-219-1 locus is architecturally analogous to the mPer1 and mPer2 loci, in that its 5′ region consists of an E-box motif as well as CRE consensus sites. The presence of an E-box motif and the CLOCK ChIP data strongly suggested to us that miR-219-1 is a clock-regulated gene. Similar to other E-box regulated core clock (ie., per1, per2, cry1, cry2) and clock output (eg., arginine vasopressin [AVP]) genes, we observed robust induction of miR-219 expression in CLOCK/BMAL1-overexpressing cells in vitro. In vivo
, miR-219 exhibits a circadian rhythm of expression in the SCN. Our observation that miR-219 levels peak in the early- to mid-subjective day lends further support for a CLOCK/BMAL1-dependent miR-219 transcriptional rhythm: this time window coincides with maximal mPer1
expression (mRNA levels peak by mid-day and decline substantially by CT 12) and precedes the mPer2 peak (mid- to late-subjective day) by several hours. Moreover, the trough in miR-219 expression occurs in the subjective night, a time when PER/CRY protein complexes have reached sufficient levels to inhibit CLOCK/BMAL1-mediated transcription. Interestingly, although miR-219-1 contains CRE consensus sequences in its promoter and was identified in the CREB SACO, it is not rapidly upregulated by light. In this respect, miR-219-1 is analogous to mPer2, which contains a CRE (in addition to an E-box), but its transcription is not elicited by CREB-activating stimuli, such as cAMP (Travnickova-Bendova et al., 2002
To define a functional role for miR-132 and miR-219 in the clock, we employed an intraventricular infusion technique to deliver antagomirs to the SCN. With this approach we detected a striking suppression of both clock-regulated (mir-219) and light-induced (miR-132) expression. miR-219 knockdown resulted in an increase in the length (tau) of the circadian clock period. Thus, period length increased by ~ 9 min. As noted above, this mean value underestimates the effects of the knockdown. Along these lines, tau lengthening
is counter to the effects of the infusion of non-target antisense, or drug vehicle, where period shortening
was consistently observed. Likewise, some animals exhibited > 2X the mean tau lengthening. This animal-to-animal variability in the effects of the antagomir may result from variability in the efficacy of the infusion. In some respects, the relative effect of miR-219 antagomir on circadian period vs. miR-132 on light-induced phase-shifting is reminiscent of the relative difference between period and phase responses of the circadian system: light-induced phase shifts are on the order of up to several hours, whereas changes in period to a single light pulse are measured in minutes (Comas et al., 2006
When placed in a broad context, the tau
shift resulting from miR-219 knockdown suggests that rhythmic miR-219 plays a significant role in determining phase length. Along these lines, genetic deletion of a number of protein factors that have proven
roles in core clock timing processes or input pathways to the clock, nevertheless, has a subtle impact on the circadian system. The absence of core clock proteins mPer1, Cry1, Cry2, and Clock singly does not result in arrhythmicity under constant conditions. Another example is Rev-Erbα, a critical regulator of Bmal transcription, that, in its absence, produces only modest changes in period length and phase-shifting properties but does not alter circadian rhythm generation (Preitner et al., 2002
). In fact, there are only a handful of examples in which the absence of a single factor (eg., Bmal1 and mPer2) has a profound impact on the circadian timing system. Thus, these findings indicate that the clock is quite resistant to molecular perturbations; either the clock loses all capacity to keep time (arrhythmic), or the clock compensates with typically a modest phenotypic effect. Thus, placed in this context of these observations, we conclude that miR-219 has a role in regulating period length.
Knockdown of miR-132 by antagomir infusion enhanced the phase-resetting effects of light by nearly 2-fold. The difference in response may be attributed to 1) the strength of the inputs to the circadian pacemaker (eg., strength of the signaling pathways that are activated by light), 2) circadian pacemaker amplitude, which determines whether or not the same stimulus is more or less effective at phase-shifting the clock, or 3) a combination of both. While our study did not address the role of miR-132 in clock gene rhythms (point #2), our observation that light-induced mPer1 expression is affected by miR-132 (discussed later) lends support to a change in input strength (point #1). In addition, bearing in mind the fact that light induces expression of miR-132, one should further note that light suppresses clock responsiveness: this is observed as a low-amplitude phase response curve (PRC) under an LD cycle vs. a high-amplitude PRC in DD-adapted animals (Refinetti 2003). Even a single 1-hr light pulse in dark-adapted mice has been shown to reduce the magnitude of phase-shifting elicited by a second light pulse (Refinetti 2003). This raises the attractive possibility that the physiological significance of miR-132 induction by light is to act as a feedback inhibitor of photic responsiveness.
The target genes that mediate the effects of miR-219 on clock timing and miR-132 on clock entrainment are unknown. The challenge of identifying specific genes and determining how they relate to a complex physiological process, such as SCN timing, can be appreciated by examining the large number of predicted targets. To emphasize this point, the miRanda software predicts 114 and 265 targets for miR-219 and miR-132, respectively. Likewise, there is often little overlap between targets predicted by different algorithms (reviewed in Rajewsky, 2006
), and only a limited number of predicted targets have been confirmed. In this light, our in vitro
identification of SCOP and RFX4 as miR-219 and miR-132 targets, respectively, is a first step in this characterization. Thus, rather than exclusively focusing on direct miRNA targets, we turned our attention on three well-characterized (and experimentally conducive) physiological effectors of clock timing and entrainment: cellular excitability, clock gene expression and clock protein stability/abundance.
To assess the effects of microRNAs on cellular excitability, we measured neuronal Ca2+
responses to depolarizing agents. Our studies were conducted in cortical neurons in order to circumvent the issue of varied phasing of SCN cells in vitro (Honma et al., 1998
). Our studies indicate that miR-132 overexpression enhances neuronal excitability, whereas miR-219 has a modest, but significant, suppressive effect. The literature supports a role of membrane excitability in diverse aspects of clock physiology (McMahon and Block, 1987
; Meredith et al., 2006
). In this context, the effect of miR-219 and miR-132 on neuronal excitability may, in part, contribute to the behavioral phenotypes elicited by their in vivo
To address the effect of microRNAs on clock gene expression, we used complementary in vitro
gain-of-function and in vivo
loss-of-function approaches. Using a luciferase reporter system in vitro
, we demonstrated that CLOCK/BMAL1- and depolarization-dependent mPer1
transcription is augmented by miR-219 and miR-132. Conversely, in the case of miR-132, antagomir-mediated knockdown resulted in an attenuation of light-induced mPer1
expression. Whether or not the effects of miR-132 on mPer1 expression are causal to the potentiation in photic resetting in antagomir-infused animals, or is merely coincident with the behavioral phenotype, remains to be determined. However, it is interesting to note that mPer1−/−
mice exhibit exaggerated phase delays in response to prolonged light exposure (Masubuchi et al 2005
), and in general phenocopies the behavior of miR-132-antagomir-infused mice.