Slumber of the Cell
Quiescence vs. Senescence
Entry before exit
In addition to cyclins and CDKs, vertebrates possess 8 E2Fs (E2F1-8), three dimerization partners (DP1-3), and three pocket proteins (pRB, p107, p130) that conduct an orchestra of transcriptional programs which regulate cell cycle entry and exit. E2F1-3 were first characterized as activator transcription factors, and were subsequently classified as 'activator' E2Fs. This classification would prove inaccurate as repressive roles were later observed for the same transcription factors. Thus far, atypical E2Fs are characterized as repressors. Atypical E2Fs lack a transactivation domain, and are not regulated by pocket proteins.
General Senescence Intro
Senescent cells exit the cell cycle irreversibly, acquire a large and flat morphology, accumulate senescence-associated β-galactosidase (SA-β-gal), and undergo changes in gene expression linked to cell cycle inhibition and inflammation.
Exclusive pRB-dependent repression of E2F Targets during Senescence
Pocket proteins suppress E2F targets to maintain cytostasis during quiescence or senescence. Specific targets of pRB can vary between different growth states. pRB is dispensible for proper cell cycle exit during quiescence because other RB family members act redundantly to control key E2F targets, Genome-wide analysis reveals pRB has a non-redundant role in binding and repressing E2F target genes directly involved in DNA replication, particularly during senescence. During senescence pRB, demonstrates exclusive responsibility to suppress specific E2F targets involved in DNA replication, including components of the preRC, and cyclin E1. During oncogenic ras-induced senescence, absence of pRB (via shRNA knockdown) is not fully compensated by p130 or p107; cyclin E1 recruits MCMs to the chromatin-bound preRC (in a CDK-independent manner), followed by unscheduled DNA synthesis, with some degree of endoreplication which increases 4N and 8N DNA content. None of the aforementioned defects are observed in the absence (shRNA knockdown) of p130 or p107. Senescence-induced cells lacking pRB eventually arrest via a second proliferation barrier mediated by p53-activated p21.
The pRB-specific repression of E2F targets may be explained by requirement of recruitment pRB-specific chromatin remodelling factors (CRF), as an pRB mutant defective for CRF binding (delta L) fails to repress DNA replication during oncogene-induced senescence, but not quiescence or terminal differentiation. Activator E2Fs (E2F1-3) upregulate genes involved in the G1-S-phase transition. Atypical E2Fs (E2F7 and E2F8) downregulate these targets during late S, G2, and M-phase upon which functional execution by these targets is complete. Temporal inactivation of atypical E2Fs is then mediated by the anaphase-promoting complex/cyclosome (APC/C) in a feedback loop in which atypical E2Fs repress the APC/C inhibitors cyclin A, cyclin E, and Emi1 (Boekhout et. al. 2016 EMBO). APC/C then targets E2F7/8 for ubiquitin-mediated proteosomal degredation.
cell cycle/mitotic E2F targets: cdc2, cyclin A, cyclin B
p130 is the most abundanct RB family member bound to E2F targets in quiescent cells
In quiescent cells (human diploid fibroblasts: IMR90 serum starved), shRNA knockdown of all 3 pocket proteins is required to de-repress E2F targets down-regulated during quiescence
ChIP-Seq reveals in the absence of pRB (via shRNA knockdown), p130 replaces pRB at repressed E2F targets during quiescence, but not during senescence, indicating pRB-p130 binding equilibrium in quiescent cells not found in senescent cells.
Entry before exit
In addition to cyclins and CDKs, vertebrates possess 8 E2Fs (E2F1-8), three dimerization partners (DP1-3), and three pocket proteins (pRB, p107, p130) that conduct an orchestra of transcriptional programs which regulate cell cycle entry and exit. E2F1-3 were first characterized as activator transcription factors, and were subsequently classified as 'activator' E2Fs. This classification would prove inaccurate as repressive roles were later observed for the same transcription factors. Thus far, atypical E2Fs are characterized as repressors. Atypical E2Fs lack a transactivation domain, and are not regulated by pocket proteins.
General Senescence Intro
Senescent cells exit the cell cycle irreversibly, acquire a large and flat morphology, accumulate senescence-associated β-galactosidase (SA-β-gal), and undergo changes in gene expression linked to cell cycle inhibition and inflammation.
Exclusive pRB-dependent repression of E2F Targets during Senescence
Pocket proteins suppress E2F targets to maintain cytostasis during quiescence or senescence. Specific targets of pRB can vary between different growth states. pRB is dispensible for proper cell cycle exit during quiescence because other RB family members act redundantly to control key E2F targets, Genome-wide analysis reveals pRB has a non-redundant role in binding and repressing E2F target genes directly involved in DNA replication, particularly during senescence. During senescence pRB, demonstrates exclusive responsibility to suppress specific E2F targets involved in DNA replication, including components of the preRC, and cyclin E1. During oncogenic ras-induced senescence, absence of pRB (via shRNA knockdown) is not fully compensated by p130 or p107; cyclin E1 recruits MCMs to the chromatin-bound preRC (in a CDK-independent manner), followed by unscheduled DNA synthesis, with some degree of endoreplication which increases 4N and 8N DNA content. None of the aforementioned defects are observed in the absence (shRNA knockdown) of p130 or p107. Senescence-induced cells lacking pRB eventually arrest via a second proliferation barrier mediated by p53-activated p21.
The pRB-specific repression of E2F targets may be explained by requirement of recruitment pRB-specific chromatin remodelling factors (CRF), as an pRB mutant defective for CRF binding (delta L) fails to repress DNA replication during oncogene-induced senescence, but not quiescence or terminal differentiation. Activator E2Fs (E2F1-3) upregulate genes involved in the G1-S-phase transition. Atypical E2Fs (E2F7 and E2F8) downregulate these targets during late S, G2, and M-phase upon which functional execution by these targets is complete. Temporal inactivation of atypical E2Fs is then mediated by the anaphase-promoting complex/cyclosome (APC/C) in a feedback loop in which atypical E2Fs repress the APC/C inhibitors cyclin A, cyclin E, and Emi1 (Boekhout et. al. 2016 EMBO). APC/C then targets E2F7/8 for ubiquitin-mediated proteosomal degredation.
cell cycle/mitotic E2F targets: cdc2, cyclin A, cyclin B
p130 is the most abundanct RB family member bound to E2F targets in quiescent cells
In quiescent cells (human diploid fibroblasts: IMR90 serum starved), shRNA knockdown of all 3 pocket proteins is required to de-repress E2F targets down-regulated during quiescence
ChIP-Seq reveals in the absence of pRB (via shRNA knockdown), p130 replaces pRB at repressed E2F targets during quiescence, but not during senescence, indicating pRB-p130 binding equilibrium in quiescent cells not found in senescent cells.