Regulation of clock-controlled genes in mammals

Abstract

The complexity of tissue- and day time-specific regulation of thousands of clock-controlled genes (CCGs) suggests that many regulatory mechanisms contribute to the transcriptional output of the circadian clock. We aim to predict these mechanisms using a large scale promoter analysis of CCGs.Our study is based on a meta-analysis of DNA-array data from rodent tissues. We searched in the promoter regions of 2065 CCGs for highly overrepresented transcription factor binding sites. In order to compensate the relatively high GC-content of CCG promoters, a novel background model to avoid a bias towards GC-rich motifs was employed. We found that many of the transcription factors with overrepresented binding sites in CCG promoters exhibit themselves circadian rhythms. Among the predicted factors are known regulators such as CLOCKratioBMAL1, DBP, HLF, E4BP4, CREB, RORalpha and the recently described regulators HSF1, STAT3, SP1 and HNF-4alpha. As additional promising candidates of circadian transcriptional regulators PAX-4, C/EBP, EVI-1, IRF, E2F, AP-1, HIF-1 and NF-Y were identified. Moreover, GC-rich motifs (SP1, EGR, ZF5, AP-2, WT1, NRF-1) and AT-rich motifs (MEF-2, HMGIY, HNF-1, OCT-1) are significantly overrepresented in promoter regions of CCGs. Putative tissue-specific binding sites such as HNF-3 for liver, NKX2.5 for heart or Myogenin for skeletal muscle were found. The regulation of the erythropoietin (Epo) gene was analysed, which exhibits many binding sites for circadian regulators. We provide experimental evidence for its circadian regulated expression in the adult murine kidney. Basing on a comprehensive literature search we integrate our predictions into a regulatory network of core clock and clock-controlled genes. Our large scale analysis of the CCG promoters reveals the complexity and extensiveness of the circadian regulation in mammals. Results of this study point to connections of the circadian clock to other functional systems including metabolism, endocrine regulation and pharmacokinetics.

Figure 1. Sequential procedure of our study.

The data collection consisted of a CCG study search, meta-analysis of the genes and promoter sequence collection. The meta-analysis allowed hierarchical separation of the gene list into subsets of genes expressed in 4 different tissues (heart, liver, SCN, skeletal muscle) and within each tissue into genes with the peak of circadian expression falling into 1 of 4 defined time intervals. Together with the full list of genes and a subset of genes robustly oscillating, those 22 lists were used in a TFBS overrepresentation search. The total number of predicted sites in a promoter set of interest was compared to the mean number of predictions in an iteratively sampled background promoter set (see Materials and Methods). Z-score has been used as a measure of a motif overrepresentation.

Figure 2. GC-content distribution of the selected subset of 167 CCG promoters versus all mouse gene promoters.

Mean values of the GC-ratio and standard deviations of both distributions are indicated in the inserted box.

Figure 3. Distribution of the number of predicted TFBS.

The histograms illustrate the distributions of number of predicted hits of several motifs in sampled background promoter sets with a GC-content matched to the selected subset of 167 CCGs. X-axis indicates numbers of predicted sites, the height of vertical bars corresponds to the percentage of sampled background sets containing the given number of predictions. The number of hits in the foreground set of 167 CCGs is marked with an arrow. All shown motifs are overrepresented in the analyzed CCG set (have z-scores over 2). The corresponding z-score values are given in Tables 1 and 2.

Figure 4. Sequence logos of binding sites showing strong similarity to a D-box.

Both D-boxes and E-boxes (supplementary Figure S2) are known to regulate clock genes and clock output pathways. Motifs highly similar to D-boxes and E-boxes are found to be overrepresented in the promoters of CCGs.

Figure 5. Analysis of circadian regulation of Epo.

(A) Quantitative PCR analysis of circadian Epo and Per2 mRNA expression in the adult murine kidney over a time period of 24 hours after release in constant darkness and normoxia. Epo mRNA (filled circles) and Per2 (open circles) mRNA transcript levels were normalized to Gapdh mRNA levels. Values are given as means±SD. (B) Analysis of the activity of a reporter gene construct harboring the 5′ promoter, first intron and 3′ enhancer of the human Epo gene (upper panel) or an E-Box reporter construct (lower panel) both co-transfected with Clock or Bmal1 alone or combined in human neuronal precursor (SH-SY5Y) cells. As positive control for the Epo reporter activation cells were also co-transfected with a HIF-1α expression plasmid (upper panel). A renilla luciferase vector was used for the normalization of transfection efficiencies. Values are shown as means±SD of three independent experiments, each performed in duplicate.

Figure 6. A putative network of circadian regulation.

The figure illustrates our computational predictions nested in other regulatory interactions reported in the literature. Solid line boxes contain transcription factors predicted by our study to regulate clock controlled genes. Dashed line boxes contain other factors involved in the regulation of clock controlled genes as provided by the literature. Transcription factors in bold letters are those reported to be clock controlled by at least one of the microarray studies mentioned in the main text or in other publications , . Black arrows indicate activation, red dead-end lines inhibition and blue simple lines represent interaction. References to the literature reporting on particular interactions are indicated next to each connecting line. The full reference list can be found in the supplementary material. Several functional groups are highlighted: core clock proteins in green, proteins related to metabolism and detoxification in red, immune system related proteins in grey and muscle-specific in blue.