Cell cycle regulation: p53-p21-RB signaling

Abstract

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.

Fig. 1. p53-p21-RB signaling.

Following p53 activation, transcription of p21/CDKN1A is strongly induced as a direct target of p53. The cyclin-dependent kinase inhibitor p21 then blocks activity of several cyclin-CDK complexes. This results in hypophosphorylation of RB, which fosters RB-E2F complex formation and their binding to E2F sites in target promoters. Many target genes are downregulated as a consequence of this mechanism of indirect p53-dependent transcriptional repression. As most repressed genes are involved in cell cycle progression, their downregulation causes cell cycle arrest.

Fig. 2. Gene ontology terms biological processes.

RB-E2F target genes (488 genes, Table 1) were subjected to GO analysis using DAVID with the criteria: fold-enrichment ≥5; p value ≤10⁻¹⁰; false discovery rate ≤10⁻¹⁰; Fisher’s exact test ≤10⁻¹⁰ [83, 84].

Fig. 3. Many genes activated by p53 also bind p53 in their promoters. A large fraction of genes repressed upon p53 induction binds RB-E2F, but essentially all of these genes do not bind p53 in their promoters.

Venn diagram depicting p53 and RB binding to promoters of genes regulated by p53. Genes activated (“activated genes”) following p53 induction had a p53 expression score of ≥ 5, genes downregulated (“repressed genes”) required a p53 expression score of ≤ −5 to be included. Chromatin immunoprecipitation (ChIP) binding of p53 to the promoters of target genes (“p53 ChIP”) had to score ≥5 (Supplementary Table S2 in [56]). The group of RB-E2F targets (“RB ChIP”) are the same 488 genes binding RB and E2F1 as the in “RB” column of Table 1.

Fig. 4. Genes regulated by p53-p21-RB signaling mostly bind DREAM.

“RB”: A dataset of 488 genes (100%) selected for RB and E2F1 ChIP binding was used as a basis for the analysis (Table 1). “DREAM”: Genes included if 4 out of 9 ChIP datasets for DREAM complex components scored positive [56]. Out of the 488 RB-E2F targets, 410 out of 488 genes scored positive for DREAM. “p53”: Target genes were selected for downregulation of their mRNA levels following p53 overexpression, nutlin-3a, or doxorubicin treatment (Table 1; 465 out of 488 genes). “p21”: p21 requirement for mRNA downregulation following nutlin-3a or doxorubicin treatment (Table 1; 437 out of 488 genes). “p53 ChIP”: Genes with binding of p53 by ChIP in the promoter region 2.5 kb up- or downstream from the transcriptional start site (Table 1; 32 out of 488 genes). “p53-p21-RB”: Overlap of “RB”, “p53”, and “p21” groups (Table 1; 415 out of 488 genes). Represents p53-p21-RB targets.

Fig. 5. Regulation by RB-E2F complexes is exclusively through E2F promoter sites, but DREAM can contact DNA and repress transcription through four types of promoter sites. B-MYB-FOXM1-MuvB complexes activate genes through CHR sites.

A Even in the presence of CHR or CLE promoter elements, the RB-E2F complex can only repress transcription through E2F binding elements. B Also transcriptional activation, after dissociation of hyperphosphorylated RB from E2F transcription factors, is mediated exclusively through E2F sites. C The DREAM repressor complex employs two kinds of subunits for DNA binding. E2F4/5-DP heterodimers bind to E2F and CDE sites. The MuvB core complex component LIN54 contacts CHR and CLE sites. Thus, DREAM can bind DNA through four single or combinations of promoter elements: E2F sites, E2F/CLE, CDE/CHR, or CHR sites. CDE and CLE sites only support DREAM binding. Affinity of DREAM or LIN54-MuvB to CDE and CLE sites alone is not sufficient for productive binding. D Following the switch from DREAM to binding of B-MYB and FOXM1 to MuvB, only LIN54 is left as a DNA-binding component. Therefore, gene activation by B-MYB-FOXM1-MuvB complexes is controlled solely through CHR sites.

Fig. 6. Non-canonical function of RB. Indirect regulation of DREAM formation by RB via the RB-SKP2-p27-cyclin A/E-CDK2-p130-DREAM link.

RB can form complexes with SKP2 preventing SKP2 from supporting ubiquitination and degradation of p27 (KIP1, CDKN1B). The CDK inhibitor p27 is then available to block cyclin A/E-CDK2 activity. These cyclin-CDK complexes have a preference for p130 as substrate. Inhibiting p130 phosphorylation yields hypophosphorylated p130, which then enables formation of the DREAM transcriptional repressor.