hrough the CHR and that mutation of the element results not only in an increased promoter activity in G0 /G1, but also in a decreased activity in G2 /M. Furthermore, knockdown of B-MYB leads to a reduced activity of the wild-type promoter, but not of the CHR mutant. Thus, the CHR mediates repression as well as activation of the cyclin B2 gene, most likely through an interaction with Lin54. In contrast, the activity of promoters like Chek2, Melk and Pold1 was lower than the respective CHR mutants in all phases of the cell cycle. In addition, only for a subgroup of CHR promoters bound by DREAM an additional binding of MMB and FOXM1 and a reduced expression after knockdown of MMB components was reported. Thus, it remains open if activation by MMB and FOXM1-MuvB is a general feature of all CHR genes. However, in 95% of late cell cycle genes bound by DREAM and MMB as well as by FOXM1, we have identified evolutionary conserved CHR elements, which supports a general mechanism for this group of genes: If late cell cycle genes are regulated by sequential binding of DREAM, MMB and FOXM1-MuvB, then interaction of the complexes with the promoters is SB203580 price mediated through CHR elements. Binding of DREAM to non-cell cycle genes In ChIP-chip analyses, DREAM binds also to non-cell cycle-regulated genes. It is not clear whether these binding events contribute to transcription or if they represent just non-functional proteinDNA interactions. Moreover, bound transcription factors may become functional only in certain tissues or after triggering by specific stimuli. Interestingly, it was shown for the DREAM component E2F4 that protein binding to promoters is highly conserved between tissues of one species, while only 20% of bound genes overlap between human and mouse. Consistent with our observations, in the group of promoters bound in both species, genes with PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19818716 functions in cell cycle regulation and proliferation were highly overrepresented. Important functions of DREAM in cell cycle regulation are therefore conserved between species and tissues, while other interactions of DREAM with DNA may contribute to unknown species-specific regulation or, more likely, may be non-functional in most cases. This hypothesis is supported by the finding that only 25% of genes bound by MMB in their promoters vary in their expression after depletion of MMB by siRNA in Drosophila. In agreement with the concept that functional binding of transcription factors to their binding elements is 
often evolutionary conserved, we find CHR elements identified in human to be functional in mouse as well. Even in cases in which we observed differences in CHR nucleotide sequences of ortholog 10348 Nucleic Acids Research, 2014, Vol. 42, No. 16 human and mouse genes, these variations did not impair function as shown for the CHRs in the mouse and human Pold1 promoters,. In contrast to the group of genes only bound by DREAM, we found a massive enrichment of cell cycle genes in the group additionally bound by MMB or FOXM1. We identified only three genes holding an evolutionary conserved CHR and being bound by DREAM and MMB or FOXM1 that were not cell cycle-regulated in our analysis: HNRNPH2, LSM5 and RBMX. It remains to be clarified if binding of the complexes to these non-cell cycle genes has a physiological significance. In a recent publication, DREAM was implicated in the repression of genes during osteoblast differentiation. In undifferentiated proliferating MC3T3-E1 cells, DREAM was reported to bi
