Most organisms from bacteria to humans exhibit circadian rhythms, which generate biochemical, physiological and behavioral oscillations to enable biological functions to function optimally at the most appropriate time of day. In eukaryotes, the circadian system is based on cell-autonomous molecular oscillators that rely on transcriptional feedback loops (Fig. 1). In mammals, the transcription factor BMAL1 acts as a dimer with either CLOCK or Neuronal PAS domain protein 2 (NPAS2) to activate the transcription of many genes, including the transcriptional repressors Period (Per1, Per2 and Per3) and Cryptochrome (Cry1 and Cry2). The PERs and CRYs are expressed, post-translationally modified, feedback to inhibit their own transcription and are then rhythmically degraded to lead to a new round of BMAL1:CLOCK or BMAL1:NPAS2 -mediated transcription (reviewed in Partch et al., 2013). Another feeback loop involves the transcription factors Rev-Erb α and ß as well as Ror α, ß and γ, and regulates the rhythmic transcription of Bmal1 and Npas2. This temporal regulation of clock gene transcription oscillates with a period of about 24-hr and underlies much of circadian biology.

         Molecular clocks in mammals are present in virtually every cell and contribute to the rhythmic expression of a large number of genes, i.e., more than 50% of the genome is rhythmically expressed in at least one tissue. (Fig. 1).

Figure 1: Molecular circadian clockwork in mammals

Rhythmic gene expression is highly tissue specific, reflecting the various tissue-specific biological functions they govern (Panda et al., 2002; Storch et al., 2002). Over the last 15 years, characterization of clock gene mutant mice highlighthed the role of the molecular clock in driving circadian rhythmicity at the molecular, physiological and behavioral levels. Surprisingly however, these mice also develop a wide spectra of pathological disorders, revealing a wider impact of biological clocks in the regulation of gene expression and biological functions. Research in the Menet lab aims at uncovering the mechnanisms by which the mammalian circadian clock regulates its target gene expression, as this will be instrumental to further delineate why clock dysfunction leads to the development of pathological disorders.

1. Contribution of transcription in genome-wide rhythmic gene expression
1.1. Nascent-Seq, a powerful technique to assay transcription at the genome-wide level

Rhythmic gene expression has essentially been assessed by analyzing temporal changes of steady-state mRNA levels, and it has been assumed that rhythms in mRNA expression directly result from temporal changes in transcription. Because of increasing evidence showing that post-transcriptional regulation contributes to rhythmic mRNA expression, we sought to address the genome-wide contribution of transcriptional and post-transcriptional regulation to mammalian mRNA rhythms. 

          To this end, we applied the Nascent-Seq technique (high-throughput sequencing of nascent RNA; Carrillo Oesterreich et al., 2010; Khodor et al., 2011) to assay global rhythmic transcription in mouse liver. Analysis of our Nascent-Seq dataset revealed that this technique is a powerful index of transcriptional activation (Fig. 2). Indeed, Nascent-Seq signal exhibits higher intron signal, a 5'-to-3' gradient and extends downstream the polyadenylation site. Moreover, difference in the relative signal between Nascent-Seq and RNA-Seq correlates with RNA half-life (Menet et al., 2012).

Figure 2: Comparison between mouse liver Nascent-Seq signal (brown) and RNA-Seq signal (red).
Adapted from Menet et al., 2012, eLife 1:e00011.
1.2. Post-transcriptional regulation significantly contributes to rhythmic mRNA expression

To address the genome-wide contribution of transcriptional and post-transcriptional regulation to mammalian mRNA rhythms, we performed a parallel analysis of rhythmic nascent RNA expression (Nascent-Seq) with mRNA expression (RNA-Seq) and compared the two sequencing datasets. Although many genes are rhythmically transcribed in the mouse liver (~15% of all detected genes), only 42% of these rhythmically transcribed genes show mRNA oscillations (Fig. 3). More importantly, about 70% of the genes that exhibit rhythmic mRNA expression do not show transcriptional rhythms, suggesting that post-transcriptional regulation plays a major role in defining the rhythmic mRNA landscape (Fig. 3). These results, that have been reported in other studies (Koike et al., 2012; Le Martelot et al., 2012), indicate that rhythmic transcription does not solely account for rhythmic mRNA expression, and underscore the role of post-transcriptional regulation in driving circadian gene expression. Research in our lab aims at investigating the underlying mechanisms, which remain for the most part unknown.

Figure 3: Post-transcriptional events contribute to rhythmic mRNA expression.
Adapted from Menet et al., 2012, eLife 1:e00011.
2. Disconnect between CLOCK:BMAL1 DNA binding and its transcriptional output

CLOCK:BMAL1 binding to DNA is rhythmic, with higher binding during the light phase for all target genes (Rey et al., 2011; Koike et al., 2012). By taking advantage of our Nascent-Seq dataset, we investigated how rhythmic CLOCK:BMAL1 DNA binding directly impacts on its target genes transcription (Menet et al., 2012). Although maximal binding occurs at an apparently uniform phase, the peak transcriptional phases of CLOCK:BMAL1 target genes are surprisingly heterogeneous, indicating that CLOCK:BMAL1 DNA binding is disconnected to its transcriptional output (Fig. 4). Importantly, this disconnect is observed for core clock genes (name written in red in Fig. 4), and suggests that other transcription factors and/or mechanisms collaborate with CLOCK:BMAL1 binding and are critical to determine the clock gene transcriptional output. We are currently extending this finding further, and are more particularly examining the newly described role of CLOCK:BMAL1 in regulating rhythmic chromatin remodeling (see below).

Figure 4: Rhythmic CLOCK:BMAL1 DNA binding profile does not determine the transcriptional phase of most target genes.
BMAL1 DNA binding profile (from Rey et al., 2011), transcription (Nascent-Seq) and mRNA expression (RNA-Seq) for CLOCK:BMAL1 target genes. Clock genes are written in red. Adapted from Menet et al., 2012, eLife 1:e00011.
3. CLOCK:BMAL1 promotes rhythmic chromatin opening

The disconnect between the phase of CLOCK:BMAL1 binding to DNA and the phase of its transcriptional output suggests that CLOCK:BMAL1 may regulate gene expression by additional mechanisms (i.e., in addition to directly promoting the recruitment of the transcription machinery to its DNA binding sites). We recently uncovered such a mechanism by addressing nucleosome occupancy at CLOCK:BMAL1 DNA binding sites (Menet et al., 2014). Indeed, by performing mouse liver MNase-Seq at 6 different time points in wild-type and Bmal1-/- mice, we found that nucleosome signal at CLOCK:BMAL1 DNA binding sites is rhythmic in wild-type mice, and low when CLOCK and BMAL1 are bound to DNA during the light phase (Fig. 5). Importantly, nucleosome signal is arrhythmic and at high levels in Bmal1-/- mice, suggesting that CLOCK:BMAL1 promotes nucleosome removal when it binds to DNA. Moreover, the amplitude of rhythmic nucleosome signal is higher at enhancers that at transcription start sites (Fig. 5A).

A
B

          We addressed the mechanisms by which CLOCK:BMAL1 can promote nucleosome removal and found that: 1) CLOCK:BMAL1 can bind to mononucleosomes, 2) CLOCK:BMAL1 rhythmic DNA binding is associated with rhythmic histone modification such as incorporation of the histone variant H2A.Z, and 3) CLOCK:BMAL1 rhythmic chromatin remodeling mediates the rhythmic binding of other transcription factors adjacent to CLOCK:BMAL1 (Fig 5B). Altogether, these data suggest that CLOCK:BMAL1 function relies more on controling the temporal regulation of its target genes’ chromatin rather than directly impacting their transcriptional activation (Menet et al., 2014).

          Current research in our lab aims at extanding these findings and addresses:

1) the mechanisms by which CLOCK:BMAL1 promotes rhythmic chromatin remodeling, and 2) whether CLOCK:BMAL1 regulation of transcription relies preferentially on the rhythmic regulation of chromatin accessibility.

Figure 5: Rhythmic CLOCK:BMAL1 DNA binding promotes rhythmic chromatin remodeling.
A: CLOCK:BMAL1 promotes the rhythmic removal of nucleosomes at its DNA-binding sites. Average nucleosome signal at the top 25% of CLOCK:BMAL1 DNA-binding sites (+/- 0.8 kb) located in intergenic regions in mouse livers during the light phase (ZT2, ZT6, and ZT10; green) and dark phase (ZT14, ZT18, and ZT22; red/orange) of wild-type mice and in Bmal1-/- mice (average signal for six time points; black). From Menet et al., Genes Dev., 2014.
B: Model describing CLOCK:BMAL1-mediated rhythmic chromatin remodeling.

Menet lab, BSBW 301, Texas A&M University, College Station, TX77843-3258

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