Cyclin F drives proliferation through SCF-dependent degradation of the retinoblastoma-like tumor suppressor p130/RBL2

Cell cycle gene expression programs fuel proliferation and are universally dysregulated in cancer. The retinoblastoma (RB)-family of proteins, RB1, RBL1/p107 and RBL2/p130, coordinately repress cell cycle gene expression, inhibiting proliferation and suppressing tumorigenesis. Phosphorylation of RB-family proteins by cyclin dependent kinases is firmly established. Like phosphorylation, ubiquitination is essential to cell cycle control, and numerous proliferative regulators, tumor suppressors, and oncoproteins are ubiquitinated. However, little is known about the role of ubiquitin signaling in controlling RB-family proteins. A systems genetics analysis of CRISPR/Cas9 screens suggested the potential regulation of the RB-network by cyclin F, a substrate recognition receptor for the SCF family of E3 ligases. We demonstrate that RBL2/p130 is a direct substrate of SCFcyclin F. We map a cyclin F regulatory site to a flexible linker in the p130 pocket domain, and show that this site mediates binding, stability, and ubiquitination. Expression of a mutant version of p130, which cannot be ubiquitinated, severely impaired proliferative capacity and cell cycle progression. Consistently, we observed reduced expression of cell cycle gene transcripts, as well a reduced abundance of cell cycle proteins, analyzed by quantitative, iterative immunofluorescent imaging. These data suggest a key role for SCFcyclin F in the CDK-RB network and raise the possibility that aberrant p130 degradation could dysregulate the cell cycle in human cancers.

A DNA Repair and Cell Cycle Gene Expression Signature in Pediatric High-Grade Gliomas: Prognostic and Therapeutic Value

Background: Pediatric high-grade gliomas (pHGGs) are the leading cause of mortality in pediatric neuro-oncology, displaying frequent resistance to standard therapies. Profiling DNA repair and cell cycle gene expression has recently been proposed as a strategy to classify adult glioblastomas. To improve our understanding of the DNA damage response pathways that operate in pHGGs and the vulnerabilities that these pathways might expose, we sought to identify and characterize a specific DNA repair and cell-cycle gene expression signature of pHGGs. Methods: Transcriptomic analyses were performed to identify a DNA repair and cell-cycle gene expression signature able to discriminate pHGGs (n = 6) from low-grade gliomas (n = 10). This signature was compared to related signatures already established. We used the pHGG signature to explore already transcriptomic datasets of DIPGs and sus-tentorial pHGGs. Finally, we examined the expression of key proteins of the pHGG signature in 21 pHGG diagnostic samples and nine paired relapses. Functional inhibition of one DNA repair factor was carried out in four patients who derived H3.3 K27M mutant cell lines. Results: We identified a 28-gene expression signature of DNA repair and cell cycle that clustered pHGGs cohorts, in particular sus-tentorial locations, in two groups. Differential protein expression levels of PARP1 and XRCC1 were associated to TP53 mutations and TOP2A amplification and linked significantly to the more radioresistant pHGGs displaying the worst outcome. Using patient-derived cell lines, we showed that the PARP-1/XRCC1 expression balance might be correlated with resistance to PARP1 inhibition. Conclusion: We provide evidence that PARP1 overexpression, associated to XRCC1 expression, TP53 mutations, and TOP2A amplification, is a new theranostic and potential therapeutic target.

Cyclin F drives proliferation through SCF-dependent degradation of the retinoblastoma-like tumor suppressor p130/RBL2

Cell cycle gene expression programs fuel proliferation and are dysregulated in many cancers. The retinoblastoma-family proteins, RB, p130/RBL2 and p107/RBL1, coordinately repress cell cycle gene expression, inhibiting proliferation and suppressing tumorigenesis. Ubiquitin-dependent protein degradation is essential to cell cycle control, and numerous proliferative regulators, tumor suppressors, and oncoproteins are ubiquitinated. However, little is known about the role of ubiquitin signaling in controlling RB-family proteins. A systems genetics analysis of several hundred CRISPR/Cas9 loss-of-function screens suggested the potential regulation of the RB-network by cyclin F, a substrate recognition receptor for the SCF family of E3 ligases. We demonstrate that RBL2/p130 is a direct substrate of SCFcyclin F. We map a cyclin F regulatory site to a flexible linker in the p130 pocket domain, and show that this site mediates binding, stability, and ubiquitination. Expression of a non-degradable p130 represses cell cycle gene expression and strongly reduces proliferation. These data suggest that SCFcyclin Fplays a key role in the CDK-RB network and raises the possibility that aberrant p130 degradation could dysregulate the cell cycle in human cancers.

Time-resolved single-cell sequencing identifies multiple waves of mRNA decay during mitotic exit

Accurate control of the cell cycle is critical for development and tissue homeostasis and requires precisely-timed expression of many genes. Cell cycle gene expression is regulated through transcriptional and translational control, as well as through regulated protein degradation. Here, we show that widespread and temporally-controlled mRNA decay acts as an additional mechanism for gene expression regulation during the cell cycle. We find that two waves of mRNA decay occur sequentially during the mitosis-to-G1 phase transition, and identify the deadenylase CNOT1 as a factor that contributes to mRNA decay during this cell cycle transition. Collectively, our data show that, akin to protein degradation, scheduled mRNA decay helps to reshape cell cycle gene expression as cells move from mitosis into G1 phase.

Localized phosphorylation of RNA Polymerase II by G1 cyclin-Cdk promotes cell cycle entry

The cell cycle is thought to be initiated by cyclin-dependent kinases (Cdk) inactivating transcriptional inhibitors of cell cycle gene-expression. In budding yeast, the G1 cyclin Cln3-Cdk1 complex is thought to directly phosphorylate Whi5, thereby releasing the transcription factor SBF and committing cells to division. Here, we report that Cln3-Cdk1 does not phosphorylate Whi5, but instead phosphorylates the RNA Polymerase II subunit Rpb1 C-terminal domain (CTD) on S5 of its heptapeptide repeats. Cln3-Cdk1 binds SBF-regulated promoters and Cln3 function can be performed by the canonical S5 kinase Ccl1-Kin28 when synthetically recruited to SBF. Thus, Cln3-Cdk1 triggers cell division by phosphorylating Rpb1 at SBF-regulated promoters to activate transcription. Our findings blur the distinction between cell cycle and transcriptional Cdks to highlight the ancient relationship between these processes.

Oncogenic B-Myb Is Associated With Deregulation of the DREAM-Mediated Cell Cycle Gene Expression Program in High Grade Serous Ovarian Carcinoma Clinical Tumor Samples

Cell cycle control drives cancer progression and treatment response in high grade serous ovarian carcinoma (HGSOC). MYBL2 (encoding B-Myb), an oncogene with prognostic significance in several cancers, is highly expressed in most HGSOC cases; however, the clinical significance of B-Myb in this disease has not been well-characterized. B-Myb is associated with cell proliferation through formation of the MMB (Myb and MuvB core) protein complex required for transcription of mitotic genes. High B-Myb expression disrupts the formation of another transcriptional cell cycle regulatory complex involving the MuvB core, DREAM (DP, RB-like, E2F, and MuvB), in human cell lines. DREAM coordinates cell cycle dependent gene expression by repressing over 800 cell cycle genes in G0/G1. Here, we take a bioinformatics approach to further evaluate the effect of B-Myb expression on DREAM target genes in HGSOC and validate our cellular model with clinical specimens. We show that MYBL2 is highly expressed in HGSOC and correlates with expression of DREAM and MMB target genes in both The Cancer Genome Atlas (TCGA) as well as independent analyses of HGSOC primary tumors (N = 52). High B-Myb expression was also associated with poor overall survival in the TCGA cohort and analysis by a DREAM target gene expression signature yielded a negative impact on survival. Together, our data support the conclusion that high expression of MYBL2 is associated with deregulation of DREAM/MMB-mediated cell cycle gene expression programs in HGSOC and may serve as a prognostic factor independent of its cell cycle role. This provides rationale for further, larger scale studies aimed to determine the clinical predictive value of the B-Myb gene expression signature for treatment response as well as patient outcomes.

Short-term perinatal oxygen exposure may impair lung development in adult mice

Background Hyperoxia at resuscitation increases oxidative stress, and even brief exposure to high oxygen concentrations during stabilization may trigger organ injury with adverse long-term outcomes in premature infants. We studied the long-term effects of short-term perinatal oxygen exposure on cell cycle gene expression and lung growth in adult mice. Methods We randomized mice litters at birth to 21, 40, or 100%O2 for 30 min and recovered in room air for 4 or 12 weeks. Cell cycle gene expression, protein analysis, and lung morphometry were assessed at 4 and 12 weeks. Results The principal component analysis demonstrated a high degree of correlation for cell cycle gene expression among the three oxygen groups. Lung elastin was significantly lower in the 100%O2 groups at 4 weeks. On lung morphometry, radial alveolar count, alveolar number, and septal count were similar. However, the mean linear intercept (MLI) and septal length significantly correlated among the oxygen groups. The MLI was markedly higher in the 100%O2 groups at 4 and 12 weeks of age, and the septal length was significantly lower in the 100%O2 groups at 12 weeks. Conclusion Short-term exposure to high oxygen concentrations lead to subtle changes in lung development that may affect alveolarization. The changes are related explicitly to secondary crest formation that may result in alteration in lung elastin. Resuscitation with high oxygen concentrations may have a significant impact on lung development and long-term outcomes such as BPD in premature infants.

A toxin-antitoxin system associated transcription factor of Caulobacter crescentus can influence cell cycle-regulated gene expression during the SOS response

Toxin-antitoxin (TA) systems are widespread in bacterial chromosomes but their functions remain enigmatic. Although many are transcriptionally upregulated by stress conditions, it is unclear what role they play in cellular responses to stress and to what extent the role of a given TA system homologue varies between different bacterial species. In this work we investigate the role of the DNA damage-inducible TA system HigBA of Caulobacter crescentus in the SOS response and discover that in addition to the toxin HigB affecting cell cycle gene expression through inhibition of the master regulator CtrA, HigBA possesses a transcription factor third component, HigC, which both auto-regulates the TA system and acts independently of it. Through HigC, the system exerts downstream effects on antibiotic (ciprofloxacin) resistance and cell cycle gene expression. HigB and HigC had inverse effects on cell cycle gene regulation, with HigB reducing and HigC increasing the expression of CtrA-dependent promoters. Neither HigBA nor HigC had any effect on formation of persister cells in response to ciprofloxacin. Rather, their role in the SOS response appears to be as transcriptional and post-transcriptional regulators of cell cycle-dependent gene expression, transmitting the status of the SOS response as a regulatory input into the cell cycle control network via CtrA.ImportanceAlmost all bacteria respond to DNA damage by upregulating a set of genes that helps them to repair and recover from the damage, known as the SOS response. The set of genes induced during the SOS response varies between species, but frequently includes toxin-antitoxin systems. However, it is unknown what the consequence of inducing these systems is, and whether they provide any benefit to the cells. We show here that the DNA damage-induced TA system HigBA of the asymmetrically dividing bacterium Caulobacter crescentus affects the cell cycle regulation of this bacterium. HigBA also has a transcription factor encoded immediately downstream of it, named here HigC, which controls expression of the TA system and potentially other genes as well. Therefore, this work identifies a new role for TA systems in the DNA damage response, distinct from non-specific stress tolerance mechanisms which had been proposed previously.

Cell Cycle Genes Are Potential Diagnostic and Prognostic Biomarkers in Hepatocellular Carcinoma

Background. The cell cycle pathway genes are comprised of 113 members which are critical to the maintenance of cell cycle and survival of tumor cells. This study was performed to investigate the diagnostic and prognostic values of cell cycle gene expression in hepatocellular carcinoma (HCC) patients. Methods. Clinical features and cell cycle pathway gene expression data were obtained from the Gene Expression Omnibus and The Cancer Genome Atlas databases. Differentially expressed genes (DEGs) were determined by the student t-test between HCC and noncancerous samples. Kaplan-Meier survival, univariate, and multivariate survival analyses and validation analysis were performed to characterize the associations between cell cycle gene expression and patients’ overall survival and recurrence-free survival. Results. 47 and 5 genes were significantly upregulated and downregulated genes in HCC samples, respectively. The high expression of BUB3, CDK1, and CHEK1 was associated with increased mortality (adjusted P value = 0.04, odds ratio (OR): 1.89 (95% confidence interval (CI): 1.04-3.46); adjusted P value = 0.02, OR: 2.06 (95% CI:1.15-3.75); and adjusted P value = 0.04, OR: 1.84 (%95 CI: 1.03-3.32), respectively). The expression of PTTG2 and RAD21 was significantly associated with cancer recurrence (adjusted P value = 0.01, OR: 2.17 (95% CI: 1.24-3.86); adjusted P value = 0.03, OR: 1.88[95% CI:1.08-3.28], respectively), while the low expression of MAD1L1 was associated with cancer recurrence (adjusted P value = 0.03, OR: 0.53 (%95 CI: 0.3-0.93)). Conclusions. The present study demonstrated that BUB3, CDK1, and CHEK1 may serve as a prognostic biomarker for HCC patients. PTTG2, RAD21, and MAD1L1 expression is a major factor affecting the recurrence of HCC patients.