The complexity of tissue- and day time-specific regulation of thousands of
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. tissue-specific binding sites such as HNF-3 for liver, NKX2.5 for heart or Myogenin for skeletal muscle mass were found. The regulation of the erythropoietin (genes (and genes (and in a separate opinions loop through ROR regulatory elements. Light input to the SCN and intercellular coupling between SCN neurons is usually mediated by CREB binding motifs in the promoters of clock genes such as approach to the question of regulatory mechanisms of the clock output pathways. We based our study on a meta-analysis of DNA-array data from rodent tissues. As illustrated in Physique 1 we selected six microarray studies containing total gene annotation and full information on phases and levels of expression of genes with an oscillating circadian pattern , , , , , . We noticed that the promoter regions of the put together 2065 CCGs are relatively GC-rich (Physique 2). In order to avoid a bias towards GC-rich motifs we employed a novel background model. Previous promoter studies without compensation of the GC-content detected primarily GC-rich motifs , . Using a stringent control of the false discovery rate  we predicted transcription factor binding sites (TFBSs) in the annotated promoter regions for all available TRANSFAC matrices. The frequencies of predicted binding sites in promoters of CCGs were compared with promoters of randomly sampled units of mouse genes with the same GC-content 23313-21-5 supplier which allows the use of z-scores as a measure of overrepresentation. This procedure resulted in relatively large lists of overrepresented motifs. We focus our study on transcription factors that are themselves reported as circadian expressed and on factors whose known target genes belong to our list of 2065 CCGs. By applying the analysis on lists of CCGs separated according to their tissue-specific expression, we found candidate factors involved in tissue-specific gene regulation. Physique 1 Sequential process of our study. Physique 2 GC-content distribution of the selected subset of 167 CCG promoters versus all mouse gene promoters. Results Promoter regions of clock-controlled genes are GC-rich As explained in Materials and Methods we extracted 2065 CCGs from published microarray studies. Among them we selected a subset of 167 genes that appear in at least three published gene lists, as illustrated in Physique 1. Since oscillations of these genes have been detected by independent experiments, we expect their strong circadian expression. Previous promoter studies , , ,  detected clock-related gene promoter (for details see supplementary Text S1). Mammalian promoter regions are highly heterogeneous regarding their base composition. Thus, the detection of overrepresented TFBSs requires careful consideration of the appropriate background model. In Physique 23313-21-5 supplier 2 the GC-content of our set of selected CCG promoter regions is usually compared with the corresponding regions of all 25764 mouse genes available in EnsEmbl. The comparison discloses that CCGs have relatively GC-rich promoters. A naive comparison of predicted TFBSs with all mouse genes as a background would therefore lead to a bias in predictions towards GC-rich motifs such as E-boxes (consensus sequence: peak of expression. Thus, each gene could be assigned to its proper expression 23313-21-5 supplier peak bin. In order to limit the number of predictions based on z-scores, we Rabbit Polyclonal to CDH11 exploit the put together list of 2065 CCGs. We focused our study on vertebrate transcription factors that were present in our set of CCGs and on the TFs with clock-controlled target genes, as annotated by TRANSFAC. To our surprise, many of the transcription factors with high z-scores have been reported as clock-controlled. Target genes of numerous other overrepresented transcription factors are rhythmically expressed (e.g. of EVI-1, 23313-21-5 supplier HNF-4, MYCMAX, IPF1, LXR, NRF-1, GFI1, GATA-1 or NFAT). The complete results of our bioinformatic analysis are available in the supplementary Table S1 in the form of 22 lists of overrepresented and clock-related TRANSFAC matrices. The precise criteria for the matrix selection were the following: Z-score of the matrix higher than 2. This threshold allows us to focus on the significantly overrepresented motifs. The lowest z-score among the known circadian-related motifs is the score of the ROR motif – 2.07. We do not observe known circadian regulatory motifs below this threshold. Only the top 5% of the matrices are considered (at most 41 out of all 815 TRANSFAC.