We also extend 30 fragmentary gene predictions from WB to longer pseudogene sequences

enes ATAD2, Blm and BRCA1 could not be verified to be functional in the reporter assays. The CHR- Nucleic Acids Research, 2014, Vol. 42, No. 16 10339 like element in NEK2 is not conserved between human and mouse genes. We cloned the promoters of both ortholog genes and showed that their cell cycle-dependent transcription was mediated by these elements. While the wild-type promoters showed a pattern of activity that is typical for late-expressed cell cycle genes, mutation of the CHR-like elements resulted PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19819297 in deregulation. However, although binding of DREAM components to the promoters in ChIP assays was detected, we did not observe binding of the proteins in DNA affinity purifications. Similar observations were made for the CHR-like element ATCGAA in the mouse Exo1 promoter. Taken together, the CHR-like sequences exhibit features of functional CHRs and were shown to be central elements for cell cycle-dependent regulation in the DREAM-bound promoters of NEK2 and Exo1 genes. Yet, we do not designate them as CHRs since direct DREAM binding to the CHR-like elements could not be shown in vitro. Nevertheless, we could identify two novel functional noncanonical CHR elements with two-nucleotide variations in the promoters of Rad18 and Rad54l. Both CHRs are highly conserved and located close to the transcription start. Human RAD18 and RAD54L mRNA were already shown to be differentially expressed during the cell cycle. We confirmed the cell cycle-dependent expression with a peak in G2 /M phases for Rad18 in the mouse NIH3T3 cell system and observed a similar pattern for Rad54l. More importantly, we detected binding of DREAM components to the region close to the TSS by ChIP. Subsequently, we analyzed the regions 500 bp upstream from the translational starts of the both genes and found that these promoters were repressed in G0 and early G1 and became activated when the cells progressed to S phase. When the CHR-like elements were mutated, cell cycle-dependent regulation was completely abolished and DREAM binding was lost. Thus, the cell cycle-dependent expression of Rad18 and Rad54l genes is regulated through noncanonical CHRs that deviate by two nucleotides from the canonical element. CHR elements are OPC 8212 chemical information enriched in promoters binding DREAM, MMB or FOXM1 The MuvB core complex participates in DNA binding of DREAM, MMB and FOXM1-MuvB by differentially associating with E2F4-DP1-p130, B-MYB or FOXM1. With E2F elements, CHRs, MBS elements and FBS elements, four different elements have been suggested as binding sites for MuvBcontaining complexes. However, it appears that not all interactions of those transcription factors with their canonical DNA binding sites are necessary for the MuvBcontaining complexes to function. For instance, although a significant overlap of genes bound by MMB and FOXM1 is observed, no enrichment of MYB-binding sites or forkhead-binding sites was detected in the data set representing FOXM1-binding genes. Here, the presence of all 10 identified CHR sequences in combination with either E2F, MBS or FBS elements was analyzed and compared in sets of promoters bound by DREAM, MMB and FOXM1. We observed a substantial enrichment of promoters containing single CHR or E2F elements or combinations of both in the data set representing genes bound by DREAM compared to the All Genes data set. CHR elements are even more enriched in promoters bound by MMB or FOXM1 and accumulate to 82% in the set of genes bound by DREAM, MMB and FOXM1. In contrast, th