II. Circadian regulation of the transcriptional network

1. Genome-wide regulation by the clock

The core clock machinery drives circadian expression of an important fraction of the genome varying from 15% to 30% of all transcripts, depending on the tissue. This genome-wide regulation depends on the coordinate and global regulation of transcription and chromatin state. The study of liver circadian transcription provides a description of these molecular mechanisms. The activators of the circadian core loop (CLOCK:BMAL1 and NPAS2, which can act as a substitute for CLOCK) bind Eboxes on their targets genes (∼3000 unique genes) in a cyclic manner between the beginning of the subjective day (CT0, beginning of the active phase of diurnal animals) and the beginning of the subjective night (CT12, beginning of the rest phase of diurnal animals). The repressors of the loop (PER1, PER2, and CRY2) bind to the same sites with an opposite phase. CRY1 peaks at CT0. Interestingly, RNA transcription in the liver peaks at CT15, which is preceded by a peak of RNA polymerase II (RNAPII), occurring at CT14.5. In addition, the peaks of CLOCK:BMAL1 and CRY1 at CT0 coincide with a peak of the genome-wide occupancy of RNAPII in its initiation state (ie, phosphorylated on serine 5). This suggests that the activators CLOCK:BMAL1 can recruit and initiate RNAPII, whereas CRY represses the complex, resulting in a balanced state (Figure 3).
The timing of RNAPII recruitment also coincides with modifications of the chromatin state. Histone 3 lysine 4 trimethylation (H3K4me3), H3 lysine 9 acetylation (H3K9ac) and H3 lysine 27 acetylation (H3K27ac) are chromatin modifications associated with transcriptional activation. These specific modifications occur in a circadian rhythm in the majority of liver cycling genes. Genome-wide regulation by the clock machinery thus implies coordinate modification of chromatin state along with the recruitment and the initiation of RNAPII (Figure 3, Joseph Takahashi Lecture). In this way, the transcription of the genome is prepared daily to act in concert with the metabolic demands of the organism. This timing organization is favored by a specific spatial shaping of chromatin in functional nuclear territories. Circadian coregulated genes seem to cluster in nuclear locations allowing the sharing of common transcription factors (Paolo Sassone-Corsi Lecture).

2. Chromatin plasticity: an additional level of regulation of the circadian core machinery

Increasing evidence demonstrates a bidirectional regulation between circadian rhythms and metabolism. While clock proteins control the expression of several genes involved in cellular metabolism, the whole clock machinery seems to sense the energy state of the cell and modulate its transcriptional activity accordingly. Histone modifications are specific codes printed on a genomic locus to control
transcription. This regulation depends on the amino acids and on the modifications (acetylation, phosphorylation, and methylation). Components of the core clock machinery control chromatin modifications. CLOCK induces H3 lysine 9 and lysine 14 acetylations, leading to a permissive state for transcription, whereas PER and CRY recruit the histone deacetylases (HDACs) SIN3A-HDAC1 and SIN3B-HDAC1, respectively.
Rev-erb α is also able to recruit the NCOR-HDAC3 complex in a rhythmic manner. In addition to their transactivation capacity, circadian transcription factors can thus also modulate chromatin state (Figure 4).
Interestingly, chromatin modifications may link cellular metabolism and the circadian clock. Substrates for histone modifications are indeed provided by cellular metabolism. Glucose metabolism might impact circadian regulation via the production of metabolic substrates for histone modifications or via posttranslational modifications of clock proteins. Acetyl-CoA production from glucose depends on the adenosine triphosphate (ATP)-citrate lyase (ACLY), and the supply of methyl groups involves S-adenosyl methionine (SAM). Both proteins have been linked to circadian rhythms. In addition, the activities of CLOCK, BMAL1, and PER2 can be altered by O-linked N-acetylglucosamine (GlcNAc), which is produced by glucose metabolism. NAD+ is a pivotal metabolite for the epigenetic regulation of the circadian genome. The NAD+ rate-limiting enzyme nicotinamide phosphoribosyltransferase (NAMPT [also known as visfatin]) is a target of the activating complex CLOCK:BMAL1 (Figure 1). The circadian levels of NAD+ may thus impose circadian rhythmicity on the activity of NAD+-dependent enzymes, such as the sirtuins (SIRTs). SIRT3 is a mitochondrial enzyme involved in the regulation of fatty acid oxidation and may be the link between clock machinery and mitochondrial processes. SIRT1 targets histones and nonhistone proteins (such as BMAL1 and PER2), but is also able to control the activity of the methyltransferase MLL1 and acetyl-CoA synthetase 1 (AceCS1). The cyclic oscillation of NAD+ levels are then linked to acetylation and methylation of histone tails (Figure 4). SIRT6 coordinates CLOCK:BMAL1 recruitment to specific chromatin sites and has a key role in the control of fatty acid metabolism by the clock. SIRT1 functions as an HDAC targeting histone H3 and nonhistone circadian proteins, whereas SIRT6 organizes circadian chromatin recruitment of transcriptional machinery. SIRT1 and SIRT6 thus operate through distinct mechanisms and thereby
have diverging functions in controlling circadian gene expression and metabolism (Paolo Sassone-Corsi Lecture).

“The Islet and Metabolism Keep Time”
I. The circadian system
II. Circadian regulation of the transcriptional network
III. Circadian rhythms and metabolism
IV. Clock and the islet
V. Conclusion