The retinoblastoma protein RB and the transcription factor p53 are central tumor suppressors. They are often found inactivated in various tumor types. Both proteins play central roles in regulating the cell division cycle. RB forms complexes with the E2F family of transcription factors and downregulates numerous genes. Among the RB-E2F target genes, a large number code for key cell cycle regulators. Their transcriptional repression by the RB-E2F complex is released through phosphorylation of RB, leading to expression of the cell cycle regulators. The release from repression can be prevented by the cyclin-dependent kinase inhibitor p21/CDKN1A. The CDKN1A gene is transcriptionally activated by p53. Taken together, these elements constitute the p53-p21-RB signaling pathway. Following activation of p53, for example by viral infection or induction of DNA damage, p21 expression is upregulated. High levels of p21 then result in RB-E2F complex formation and downregulation of a large number of cell cycle genes. Thus, p53-dependent transcriptional repression is indirect. The reduced expression of the many regulators leads to cell cycle arrest. Examination of the p53-p21-RB targets and genes controlled by the related p53-p21-DREAM signaling pathway reveals that there is a large overlap of the two groups. Mechanistically this can be explained by replacing RB-E2F complexes with the DREAM transcriptional repressor complex at E2F sites in target promoters. In contrast to RB-E2F, DREAM can downregulate genes also through CHR transcription factor binding sites. This results in a distinct gene set controlled by p53-p21-DREAM signaling independent of RB-E2F. Furthermore, RB has non-canonical functions without binding to E2F and DNA. Such a role of RB supporting DREAM formation may be exerted by the RB-SKP2-p27-cyclin A/E-CDK2-p130-DREAM link. In the current synopsis, the mechanism of regulation by p53-p21-RB signaling is assessed and the overlap with p53-p21-DREAM signaling is examined.

Activation of the p53 tumor suppressor can lead to cell cycle arrest. The key mechanism of p53-mediated arrest is transcriptional downregulation of many cell cycle genes. In recent years it has become evident that p53-dependent repression is controlled by the p53–p21–DREAM–E2F/CHR pathway (p53–DREAM pathway). DREAM is a transcriptional repressor that binds to E2F or CHR promoter sites. Gene regulation and deregulation by DREAM shares many mechanistic characteristics with the retinoblastoma pRB tumor suppressor that acts through E2F elements. However, because of its binding to E2F and CHR elements, DREAM regulates a larger set of target genes leading to regulatory functions distinct from pRB/E2F. The p53–DREAM pathway controls more than 250 mostly cell cycle-associated genes. The functional spectrum of these pathway targets spans from the G₁ phase to the end of mitosis. Consequently, through downregulating the expression of gene products which are essential for progression through the cell cycle, the p53–DREAM pathway participates in the control of all checkpoints from DNA synthesis to cytokinesis including G₁/S, G₂/M and spindle assembly checkpoints. Therefore, defects in the p53–DREAM pathway contribute to a general loss of checkpoint control. Furthermore, deregulation of DREAM target genes promotes chromosomal instability and aneuploidy of cancer cells. Also, DREAM regulation is abrogated by the human papilloma virus HPV E7 protein linking the p53–DREAM pathway to carcinogenesis by HPV. Another feature of the pathway is that it downregulates many genes involved in DNA repair and telomere maintenance as well as Fanconi anemia. Importantly, when DREAM function is lost, CDK inhibitor drugs employed in cancer treatment such as Palbociclib, Abemaciclib and Ribociclib can compensate for defects in early steps in the pathway upstream from cyclin/CDK complexes. In summary, the p53–p21–DREAM–E2F/CHR pathway controls a plethora of cell cycle genes, can contribute to cell cycle arrest and is a target for cancer therapy.

Ki-67 serves as a prominent cancer marker. We describe how expression of the MKI67 gene coding for Ki-67 is controlled during the cell cycle. MKI67 mRNA and Ki-67 protein are maximally expressed in G₂ phase and mitosis. Expression is dependent on two CHR elements and one CDE site in the MKI67 promoter. DREAM transcriptional repressor complexes bind to both CHR sites and downregulate the expression in G₀/G₁ cells. Upregulation of MKI67 transcription coincides with binding of B-MYB-MuvB and FOXM1-MuvB complexes from S phase into G₂/M. Importantly, binding of B-MYB to the two CHR elements correlates with loss of CHR-dependent MKI67 promoter activation in B-MYB-knockdown experiments. In knockout cell models, we find that DREAM/MuvB-dependent transcriptional control cooperates with the RB Retinoblastoma tumor suppressor. Furthermore, the p53 tumor suppressor indirectly downregulates transcription of the MKI67 gene. This repression by p53 requires p21/CDKN1A. These results are consistent with a model in which DREAM, B-MYB-MuvB, and FOXM1-MuvB together with RB cooperate in cell cycle-dependent transcription and in transcriptional repression following p53 activation. In conclusion, we present mechanisms how MKI67 gene expression followed by Ki-67 protein synthesis is controlled during the cell cycle and upon induction of DNA damage, as well as upon p53 activation.

The tumor suppressor p53 functions predominantly as a transcription factor by activating and downregulating gene expression, leading to cell cycle arrest or apoptosis. p53 was shown to indirectly repress transcription of the CCNB2, KIF23 and PLK4 cell cycle genes through the recently discovered p53-p21-DREAM-CDE/CHR pathway. However, it remained unclear whether this pathway is commonly used. Here, we identify genes regulated by p53 through this pathway in a genome-wide computational approach. The bioinformatic analysis is based on genome-wide DREAM complex binding data, p53-depedent mRNA expression data and a genome-wide definition of phylogenetically conserved CHR promoter elements. We find 210 target genes that are expected to be regulated by the p53-p21-DREAM-CDE/CHR pathway. The target gene list was verified by detailed analysis of p53-dependent repression of the cell cycle genes B-MYB (MYBL2), BUB1, CCNA2, CCNB1, CHEK2, MELK, POLD1, RAD18 and RAD54L. Most of the 210 target genes are essential regulators of G2 phase and mitosis. Thus, downregulation of these genes through the p53-p21-DREAM-CDE/CHR pathway appears to be a principal mechanism for G2/M cell cycle arrest by p53.

Regulatory T cells (Treg) expand during pregnancy and are present at the fetal-maternal interface at very early stages in pregnancy. The migration mechanisms of Treg to the pregnant uterus are still unclear. Human chorionic gonadotropin (hCG) is secreted by the blastocyst immediately after fertilization and has chemoattractant properties. Therefore, we sought to analyze whether hCG secreted by early trophoblasts attracts Treg to the uterus and hence contributes to maternal tolerance toward the fetus. Decidua and placenta tissue samples from patients having spontaneous abortions or ectopic pregnancies were employed to evaluate Treg and hCG levels. Age-matched samples from normal pregnant women served as controls. We further performed in vitro studies with primary first trimester trophoblast cells and a choriocarcinoma cell line (JEG-3) aiming to evaluate the ability of secreted hCG to attract Treg. Patients having miscarriages or ectopic pregnancy presented significantly decreased hCG mRNA and protein levels associated with decreased Foxp3, neuropilin-1, IL-10, and TGF-β mRNA levels as compared with normal pregnant women. Using migration assays we demonstrated that Treg were attracted by hCG-producing trophoblasts or choriocarcinoma cells. Treg migration toward cells transfected with hCG expression vectors confirmed the chemoattractant ability of hCG. Our data clearly show that hCG produced by trophoblasts attracts Treg to the fetal-maternal interface. High hCG levels at very early pregnancy stages ensure Treg to migrate to the site of contact between paternal Ags and maternal immune cells and to orchestrate immune tolerance toward the fetus.

There are nearly 50 forkhead (FOX) transcription factors encoded in the human genome and, due to sharing a common DNA binding domain, they are all thought to bind to similar DNA sequences. It is therefore unclear how these transcription factors are targeted to specific chromatin regions to elicit specific biological effects. Here, we used chromatin immunoprecipitation followed by sequencing (ChIP-seq) to investigate the genome-wide chromatin binding mechanisms used by the forkhead transcription factor FOXM1. In keeping with its previous association with cell cycle control, we demonstrate that FOXM1 binds and regulates a group of genes which are mainly involved in controlling late cell cycle events in the G₂ and M phases. However, rather than being recruited through canonical RYAAAYA forkhead binding motifs, FOXM1 binding is directed via CHR (cell cycle genes homology region) elements. FOXM1 binds these elements through protein-protein interactions with the MMB transcriptional activator complex. Thus, we have uncovered a novel and unexpected mode of chromatin binding of a FOX transcription factor that allows it to specifically control cell cycle-dependent gene expression.