The CHR site: definition and genome-wide identification of a cell cycle transcriptional element

Abstract

The cell cycle genes homology region (CHR) has been identified as a DNA element with an important role in transcriptional regulation of late cell cycle genes. It has been shown that such genes are controlled by DREAM, MMB and FOXM1-MuvB and that these protein complexes can contact DNA via CHR sites. However, it has not been elucidated which sequence variations of the canonical CHR are functional and how frequent CHR-based regulation is utilized in mammalian genomes. Here, we define the spectrum of functional CHR elements. As the basis for a computational meta-analysis, we identify new CHR sequences and compile phylogenetic motif conservation as well as genome-wide protein-DNA binding and gene expression data. We identify CHR elements in most late cell cycle genes binding DREAM, MMB, or FOXM1-MuvB. In contrast, Myb- and forkhead-binding sites are underrepresented in both early and late cell cycle genes. Our findings support a general mechanism: sequential binding of DREAM, MMB and FOXM1-MuvB complexes to late cell cycle genes requires CHR elements. Taken together, we define the group of CHR-regulated genes in mammalian genomes and provide evidence that the CHR is the central promoter element in transcriptional regulation of late cell cycle genes by DREAM, MMB and FOXM1-MuvB.

Figure 1.

DREAM can bind to non-canonical CHR elements in DNA affinity purification assays. (A) Analysis of DREAM complex binding to the isolated cyclin B2 CDE/CHR element flanked by irrelevant DNA and to a 220-bp cyclin B2 promoter probe. Biotinylated DNA probes based on the mouse Ccnb2 promoter (Ccnb2) and on the CDE/CHR element from the same promoter (CDE/CHR) were cloned in the pGL4.10 vector backbone and subjected to DNA affinity purifications with density-arrested NIH3T3 cells. Binding of DREAM components (p130, E2f4, Lin37, Lin54) to both probes was analyzed with polyclonal antibodies. As a non-DREAM binding negative control, a DNA probe based on the empty pGL4.10 vector was utilized (CTRL). All samples are from the same blot. (B) DREAM complex binding to CHR elements differing from the canonical sequence TTTGAA in one nucleotide analyzed in an in vitro DNA affinity purification assay. Biotinylated probes based on the CDE/CHR probe with all possible 1 bp permutations were prepared and subjected to DNA affinity purifications with nuclear extracts from density-arrested NIH3T3 cells. A DNA probe based on the empty pGL4.10 vector was used as a non-DREAM binding negative control (CTRL). Eluates were probed in western blots for the DREAM components p130, E2f4 and Lin9. All samples were processed in parallel.

Figure 2.

Identification of potential CHR elements in cell cycle genes. (A) Numbers of genes annotated as cell cycle-regulated by one or several of the data sets reported by Whitfield et al. (WF), Bar-Joseph et al. (BJ), Sadasivam et al. (SV) and Grant et al. (GT). The numbers of genes with identically annotated cell cycle phases of peak expression for each intersection are shown in parentheses. (B) Distribution of CHR-like elements in promoter regions of All Genes with (phylCons> = 0.9) or without (no phylCons) phylogenetical conservation. (C) Fold change of phylogenetically conserved CHR-like elements in the sets of DREAM-binding genes (D), late cell cycle-expressed genes (LCC), or late cell cycle genes binding DREAM (D+LCC). For all elements the fold change was calculated using their relative frequencies in the corresponding set divided by their relative frequencies in All Genes. A fold change above one indicates an enrichment of the element in the corresponding set.

Figure 3.

Genes with non-canonical CHR elements bind DREAM and are expressed in late cell cycle phases. (A) Alignments of putative CHR elements in Bub1, Chek2, Melk and Pold1 promoters from different mammals. CHRs are highlighted in gray. (B) Chromatin immunoprecipitations in density-arrested NIH3T3 cells to promoters with putative CHR elements. Antibodies targeting the representative DREAM components E2f4, p130 and Lin9 as well as a non-targeting rabbit antibody (IgG) were applied. Binding of DREAM components to the Gapdhs promoter was measured as a negative control. Precipitated DNA fragments were quantified by qPCR and normalized to the input. (C) mRNA expression of Bub1, Chek2, Melk and Pold1 in different cell cycle phases was measured in NIH3T3 cells synchronized by serum deprivation followed by serum re-stimulation. FACS analyses for the different time points are shown in Supplementary Figure S1.

Figure 4.

Non-canonical CHR elements are essential for cell cycle-dependent gene transcription and DREAM binding. (A) Promoter activity of Bub1, Chek2, Melk and Pold1 was analyzed with luciferase reporter assays in NIH3T3 cells synchronized by serum starvation followed by serum re-stimulation. Activities of wild-type promoters were measured in different cell cycle phases and compared to the activity of the corresponding CHR mutants (ΔCHR). pGL4.10 empty vector served as a negative control. FACS analyses for the different time points are shown in Supplementary Figure S1. (B) DREAM binding to promoters with non-canonical CHR elements was analyzed by DNA affinity purification followed by western blot. Proteins from nuclear extracts of density-arrested NIH3T3 cells binding to wild-type promoters (WT) and CHR mutants (ΔCHR) were probed with antibodies targeting p130, E2f4 and Lin37 as representative components of DREAM. A DNA probe of the Gapdhs promoter served as a negative control (CTRL).

Figure 5.

The Rad18 and Rad54l CHRs deviate in two nucleotides from the consensus TTTGAA element but are still functional. (A) Comparison of nucleotide sequences in Rad18 and Rad54l promoters from several mammals. (B)Rad18 and Rad54l mRNA expression measured in synchronized NIH3T3 cells at indicated time points after serum starvation and serum re-stimulation. (C)In vivo binding of DREAM components (E2f4, p130, Lin9) in density-arrested NIH3T3 cells to the Rad18 and Rad54l promoters measured by ChIP. A non-targeting rabbit antibody (IgG) served as a negative control. Binding of DREAM proteins to Rad18 and Rad54l was quantified by qPCR and compared to binding to the Gapdhs promoter, which is not targeted by DREAM. (D) Promoter activity of Rad18 and Rad54l measured in synchronized NIH3T3 cells with luciferase reporter assays. Activity of the wild-type promoters is compared to CHR mutants (ΔCHR). (E) DNA affinity purification from nuclear extracts of density-arrested NIH3T3 cells tested for representative DREAM components E2f4, p130 and Lin37 by western blot. A DNA probe of the Gapdhs promoter served as a negative control (CTRL).

Figure 6.

CHR elements are enriched in promoters of genes bound by DREAM, MMB and FOXM1. (A) Percentage of promoters with or without CHR elements (+CHR or –CHR) and with or without E2F sites (+E2F or –E2F) in different promoter subsets: all promoters (All Genes), DREAM-bound promoters (D), promoters bound by the MMB components B-MYB and LIN9 (MMB), promoters bound by FOXM1 (FOXM1) and promoters bound by two or all three of the complexes. (B) Fraction of promoters with or without Myb-binding sites (+MBS or –MBS) and with or without CHRs (+CHR, –CHR) in different promoter subsets. (C) Percentage of promoters with or without forkhead-binding sites (+FBS or –FBS) and with or without CHRs (+CHR, –CHR) in different promoter subsets. (D) Binding of MMB to the isolated cyclin B2 CDE/CHR site flanked by irrelevant DNA in comparison to a 220 bp cyclin B2 promoter probe. Biotinylated DNA probes derived from the mouse Ccnb2 promoter (Ccnb2) and from the cyclin B2 CDE/CHR element cloned into the pGL4.10 vector backbone (CDE/CHR) were subjected to DNA affinity purifications with nuclear extracts of proliferating F9 cells which do not form DREAM. MMB components B-Myb, Lin54, Lin37 and Lin9 were detected by western blot. To control the absence of DREAM binding to the probes, E2f4 as part of the DREAM complex was analyzed. As a non-MMB-binding negative control, a DNA probe based on the empty pGL4.10 vector was employed (CTRL).

Figure 7.

CHR genes bound by DREAM, MMB and FOXM1 are cell cycle regulated with peak expression in late cell cycle phases. (A) Correlation of the time point of maximal gene expression with the occurrence of CHR elements (+CHR or –CHR) and E2F sites (+E2F or –E2F) in promoters. Enriched functional clusters obtained using each of the four subsets along with their P-values are given. (B) Peak expression of CHR genes bound by DREAM, MMB and FOXM1 in early and late cell cycle phases. Numbers of genes in the different subsets are given. (C) Rate of CHR sites in genes bound by DREAM, MMB and FOXM1. Percentages of genes with (CHR) and without (no CHR) CHR element are given.

Figure 8.

CHR sites predominantly have the sequence TTTGAA and are found close to the transcription start site. (A) Alignment of functional CHR elements. (B) Relative occurrence of the 10 validated CHR sequences in DREAM-bound genes expressed during the late cell cycle (D+LCC). (C) Positions of the identified CHR elements relative to the transcription start site (TSS) in DREAM-bound genes expressed during the late cell cycle (D+LCC). (D) Sequence logo derived from verified CHR sequences identified in promoters of the D+LCC data set with flanking nucleotides.

Figure 9.

Regulation of late cell cycle genes through sequential binding of DREAM, MMB and FOXM1-MuvB to the CHR. In the derived model, all complexes are recruited to CHR elements via MuvB. Expression of late cell cycle genes is repressed in G₀ and G₁ by DREAM. CDE elements can support binding of DREAM to the CHR. In early S phase, MMB binds to the CHR. Later, MMB recruits FOXM1, which results in initiation of transcription. In G₂ and M phases, B-MYB is degraded and expression of late cell cycle genes reaches its maximum through activation by FOXM1-MuvB.