Transcriptional architecture of the mammalian circadian clock

Abstract

Circadian clocks are endogenous oscillators that control 24-hour physiological and behavioural processes in organisms. These cell-autonomous clocks are composed of a transcription-translation-based autoregulatory feedback loop. With the development of next-generation sequencing approaches, biochemical and genomic insights into circadian function have recently come into focus. Genome-wide analyses of the clock transcriptional feedback loop have revealed a global circadian regulation of processes such as transcription factor occupancy, RNA polymerase II recruitment and initiation, nascent transcription, and chromatin remodelling. The genomic targets of circadian clocks are pervasive and are intimately linked to the regulation of metabolism, cell growth and physiology.

Figure 1

Circadian rhythms are adaptations of organismal physiology to resonate with the 24-hour solar energetic cycle on earth. a) The earth’s 24-hour rotation leads to a diurnal cycle of light and darkness that drive an energy harvesting and energy storage daily rhythm. Solar irradiation also imposes a cycle of DNA damage and recovery. Reproduced with permission from REF.. b) Animals have behavioral rhythms of sleep-wake and feeding and fasting occur on a 24-hour basis in synchrony with the solar day. Reproduced with permission from REF.. c) A conserved network motif of circadian clocks involves a transcription-translation negative feedback loop with delay. d) In mammals, circadian clocks are cell autonomous and are found in all major organ systems and tissues of the body. A hierarchical organization exists in which the hypothalamic suprachiasmatic nucleus (SCN) acts as a master pacemaker to synchronize behavioral and physiological rhythms throughout the body. Modified with permission from REF..

Figure 2

The circadian gene network in mammals. a) At the core, CLOCK and BMAL1 activate the Per1, Per2, Cry1 and Cry2 genes, whose protein products interact and repress their own transcription. The stability of the PER and CRY proteins are regulated by parallel E3 ubiquitin ligase pathways. CLOCK and BMAL1 also regulate the nuclear receptors, Rev-erbα/β, which rhythmically repress the transcription of Bmal1 and Nfil3 that is driven by the activators, RORa/b. NFIL3 in turn represses the PAR-bZip factor, DBP, to regulate a rhythm in the ROR nuclear receptors. These three interlocked transcriptional feedback loops represent the three major transcriptional regulators of the majority of cycling genes. Different combinations of these factors generate different phases of transcriptional rhythms as exemplified by the RNA profiles of Dbp, Per2, Cry1 and Bmal1 in the mouse liver (b). Additional rhythmic output genes (so call “Clock-controlled genes or Ccg’s) are transcriptionally regulated by the three loops acting on E-box, RRE and D-box elements in the regulatory regions of target genes.

Figure 3

The circadian cistrome in the mouse liver. a) UCSC genome browser view of ChIP-seq profiles of circadian transcription factors at the Per1 gene at 6 circadian times of day [0, 4, 8, 12, 16, 20 CT (hr)]. The colors of the wiggle plots of ChIP-seq occupancy indicate the following transcriptional regulators: BMAL1 (blue), CLOCK (green), PER1 (orange), PER2 (gold), CRY1 (red), CRY2 (pink). KO indicates a ChIP-seq sample from a knockout mouse for each transcriptional regulator. b) Heat map views of genome-wide DNA binding for BMAL1, CLOCK, PER1, PER2, CRY1 and CRY2 measured over 500 bp fragments encompassing the binding sites. Each peak in the genome is represented as a horizontal line, ordered vertically by signal strength. Six time points are shown beginning at CT0 and ending at CT20 from left to right. Knockout (K) mouse control is shown on the far right of each panel. The number of peaks in the genome is indicated at the bottom of each panel. The blue-red gradient indicates the coverage for all binding sites in the genome. The number of binding sites (N) in the genome are indicated at the bottom of each heat map. c) Chow-Ruskey diagram showing the 6-way overlap of BMAL1, CLOCK, PER1, PER2, CRY1 and CRY2 peaks in the genome. The red circle in the middle represents the overlap of all six factors. Lighter shades of red, orange and yellow represent fewer overlaps of subsets. The boundaries for each protein are color coded: BMAL1 (blue), CLOCK (green), PER1 (orange), PER2 (brown), CRY1 (red) and CRY2 (purple), and the areas of each domain are proportional to the number of binding sites. Modified/Reproduced with permission from REF..

Figure 4

Whole-transcriptome RNA-seq analysis of circadian gene expression in the mouse liver. a) Heat map view of intron (left) and exon (right) RNA cycling genes. Each gene is represented as a horizontal line, ordered vertically by phase in hours indicated at the top. Twelve time points are shown beginning at CT0 and ending at CT44 from left to right. “N” indicates the number of cycling genes in each category. b) Venn diagram of intron and exon cycling genes showing only 22% overlap. c) The phase distribution of cycling genes. The phase of each transcript rhythm is represented in a dot plot (top) and histogram plot (bottom). d) Heat map view of cycling RNAPII-8WG16 occupancy across the genome of the liver with a peak occurring at night. Modified/Reproduced with permission from REF..

Figure 5

Circadian chromatin states in the mouse liver. a) UCSC genome browser view of histone methylation and acetylation at the Per1 gene at 6 circadian times of day [0, 4, 8, 12, 16, 20 CT (hr)]. The colors of the wiggle plots of ChIP-seq signal indicate the following: BMAL1 (blue), H3K4me1 (red), H3K4me3 (pink), H3K9ac (aqua), H3K27ac (orange), H3K36me3 (light green), H3K79me2 (dark green). b) Binding profiles of RNAPII-8WG16 and H3K4me3 at the transcription start site (TSS) +/− 3kb in 12,680 expressed genes (top row), 8,945 unexpressed genes (second row), 1371 intron cycling genes (third row) and 5,839 non-cycling genes (bottom row). Modified/Reproduced with permission from REF..

Figure 6

Circadian transcriptional landscape in the mouse liver. Histograms show the phase distributions of each factor as a function of time of day. Reproduced with permission from REF.

Figure 7

Circadian transcriptional regulation at enhancer and promoter sites during the CLOCK:BMAL1 activation phase in the daytime. CLOCK:BMAL1 act as pioneer transcription factors at enhancer sites. Circadian recruitment and initiation (as seen by RNAPII-Ser5 marks) of RNA polymerase II occurs at both distal enhancers and proximal promoter sites. However, the circadian regulation of pausing factors (NELF, DSIF) and pause release factors (P-TEFb) remain to be determined. Modified with permission from REF..