E2f2 Attenuates Apoptosis of Activated T Lymphocytes and Protects from Immune-Mediated Injury through Repression of Fas and FasL

Targeted disruption of E2f2 in mice causes T-cell hyperactivation and a disproportionate cell cycle entry upon stimulation. However, E2f2−/− mice do not develop a lymphoproliferative condition. We report that E2f2 plays a Fas-dependent anti-apoptotic function in vitro and in vivo. TCR-stimulated murine E2f2−/− T cells overexpress the proapoptotic genes Fas and FasL and exhibit enhanced apoptosis, which is prevented by treatment with neutralizing anti-FasL antibodies. p53 pathway is activated in TCR-stimulated E2f2−/− lymphocytes, but targeted disruption of p53 in E2f2−/− mice does not abrogate Fas/FasL expression or apoptosis, implying a p53-independent apoptotic mechanism. We show that E2f2 is recruited to Fas and FasL gene promoters to repress their expression. in vivo, E2f2−/− mice are prone to develop immune-mediated liver injury owing to an aberrant lymphoid Fas/FasL activation. Taken together, our results suggest that E2f2-dependent inhibition of Fas/FasL pathway may play a direct role in limiting the development of immune-mediated pathologiesThis work was supported by grants from the MCIU/AEI/FEDER, UE (RTI2018-097497-B-100 and RED2018-102723-T) and Basque Government, Department of Education (IT1257-19) to A.M.Z. N.M. is recipient of a University of the Basque Country (UPV/EHU) Postdoctoral Fellowship. J.M. is recipient of an Ikerbasque Research Foundation Fellowship

Targeting E2F Sensitizes Prostate Cancer Cells to Drug-Induced Replication Stress by Promoting Unscheduled CDK1 Activity

E2F1/E2F2 expression correlates with malignancy in prostate cancer (PCa), but its functional significance remains unresolved. To define the mechanisms governed by E2F in PCa, we analyzed the contribution of E2F target genes to the control of genome integrity, and the impact of modulating E2F activity on PCa progression. We show that silencing or inhibiting E2F1/E2F2 induces DNA damage during S phase and potentiates 5-FU-induced replication stress and cellular toxicity. Inhibition of E2F downregulates the expression of E2F targets involved in nucleotide biosynthesis (TK1, DCK, TYMS), whose expression is upregulated by 5-FU. However, their enzymatic products failed to rescue DNA damage of E2F1/E2F2 knockdown cells, suggesting additional mechanisms for E2F function. Interestingly, targeting E2F1/E2F2 in PCa cells reduced WEE1 expression and resulted in premature CDK1 activation during S phase. Inhibition of CDK1/CDK2 prevented DNA damage induced by E2F loss, suggesting that E2F1/E2F2 safeguard genome integrity by restraining CDK1/CDK2 activity. Importantly, combined inhibition of E2F and ATR boosted replication stress and dramatically reduced tumorigenic capacity of PCa cells in xenografts. Collectively, inhibition of E2F in combination with drugs targeting nucleotide biosynthesis or DNA repair is a promising strategy to provoke catastrophic levels of replication stress that could be applied to PCa treatment.This work was supported by grants from the MCIU/AEI/FEDER, UE (RTI2018-097497-B-100, PID2021-122922OB-100 and RED2018-102723-T) to A.M.Z., MCIU/AEI/FEDER, UE (PID2021-124425OB-I00) to P.A., Basque Government, Department of Education (IT1257-19 and IT1547-22) to A.M.Z and Basque Government, Department of Education (IT1476-2) to P.A. M.H. is recipient of the Asociación Española Contra el Cancer (AECC) predoctoral fellowship. J.M. is recipient of an Ikerbasque Research Foundation Fellowship

Efficient pre-mRNA cleavage prevents replication-stress-associated genome instability

Cellular mechanisms that safeguard genome integrity are often subverted in cancer. To identify cancer-related genome caretakers, we employed a convergent multi-screening strategy coupled to quantitative image-based cytometry and ranked candidate genes according to multivariate readouts reflecting viability, proliferative capacity, replisome integrity, and DNA damage signaling. This unveiled regulators of replication stress resilience, including components of the pre-mRNA cleavage and polyadenylation complex. We show that deregulation of pre-mRNA cleavage impairs replication fork speed and leads to excessive origin activity, rendering cells highly dependent on ATR function. While excessive formation of RNA:DNA hybrids under these conditions was tightly associated with replication-stress-induced DNA damage, inhibition of transcription rescued fork speed, origin activation, and alleviated replication catastrophe. Uncoupling of pre-mRNA cleavage from co-transcriptional processing and export also protected cells from replication-stress-associated DNA damage, suggesting that pre-mRNA cleavage provides a mechanism to efficiently release nascent transcripts and thereby prevent gene gating-associated genomic instability

Replicated chromatin curtails 53BP1 recruitment in BRCA1-proficient and BRCA1-deficient cells

DNA double-strand breaks can be repaired by non-homologous end-joining or homologous recombination. Which pathway is used depends on the balance between the tumor suppressors 53BP1 and BRCA1 and on the availability of an undamaged template DNA for homology-directed repair. How cells switch from a 53BP1-dominated to a BRCA1-governed homologous recombination response as they progress through the cell cycle is incompletely understood. Here we reveal, using high-throughput microscopy and applying single cell normalization to control for increased genome size as cells replicate their DNA, that 53BP1 recruitment to damaged replicated chromatin is inefficient in both BRCA1-proficient and BRCA1-deficient cells. Our results substantiate a dual switch model from a 53BP1-dominated response in unreplicated chromatin to a BRCA1–BARD1–dominated response in replicated chromatin, in which replication-coupled dilution of 53BP1’s binding mark H4K20me2 functionally cooperates with BRCA1–BARD1–mediated suppression of 53BP1 binding. More generally, we suggest that appropriate normalization of single cell data, for example, to DNA content, provides additional layers of information, which can be critical for quantifying and interpreting cellular phenotypes

Cell cycle resolved measurements of poly(ADP-ribose) formation and DNA damage signaling by quantitative image-based cytometry

Formation of poly(ADP-ribose) (PAR) marks intracellular stress signaling and is notably induced upon DNA damage. PAR polymerases (PARPs) catalyze PAR synthesis upon genotoxic stress and thereby recruit multiple proteins to damaged chromatin. PAR induction is transient and antagonized by the action of PAR glycohydrolase (PARG). Given that poly(ADP-ribosyl)ation (PARylation) is involved in genome integrity maintenance and other vital cellular functions, but also in light of the recent approval of PARP inhibitors for cancer treatments, reliable measurements of intracellular PAR formation have gained importance. Here we provide a detailed protocol for PAR measurements by quantitative image-based cytometry. This technique combines the high spatial resolution of single-cell microscopy with the advantages of cell population measurements through automated high-content imaging. Such upscaling of immunofluorescence-based PAR detection not only increases the robustness of the measurements through averaging across large cell populations but also allows for the discrimination of subpopulations and thus enables multivariate measurements of PAR levels and DNA damage signaling. We illustrate how this technique can be used to assess the dynamics of the cellular response to oxidative damage as well as to PARP inhibitor-induced genotoxicity in a cell cycle resolved manner. Due to the possibility to use any automated microscope for quantitative image-based cytometry, the presented method has widespread applicability in the area of PARP biology and beyond

Replication-coupled dilution of H4K20me2 guides 53BP1 to pre-replicative chromatin

The bivalent histone modification reader 53BP1 accumulates around DNA double-strand breaks (DSBs), where it dictates repair pathway choice decisions by limiting DNA end resection. How this function is regulated locally and across the cell cycle to channel repair reactions toward non-homologous end joining (NHEJ) in G1 and promote homology-directed repair (HDR) in S/G2 is insufficiently understood. Here, we show that the ability of 53BP1 to accumulate around DSBs declines as cells progress through S phase and reveal that the inverse relationship between 53BP1 recruitment and replicated chromatin is linked to the replication-coupled dilution of 53BP1's target mark H4K20me2. Consistently, premature maturation of post-replicative chromatin restores H4K20me2 and rescues 53BP1 accumulation on replicated chromatin. The H4K20me2-mediated chromatin association of 53BP1 thus represents an inbuilt mechanism to distinguish DSBs in pre- versus post-replicative chromatin, allowing for localized repair pathway choice decisions based on the availability of replication-generated template strands for HDR

Analysis of PARP inhibitor toxicity by multidimensional fluorescence microscopy reveals mechanisms of sensitivity and resistance

Exploiting the full potential of anti-cancer drugs necessitates a detailed understanding of their cytotoxic effects. While standard omics approaches are limited to cell population averages, emerging single cell techniques currently lack throughput and are not applicable for compound screens. Here, we employed a versatile and sensitive high-content microscopy-based approach to overcome these limitations and quantify multiple parameters of cytotoxicity at the single cell level and in a cell cycle resolved manner. Applied to PARP inhibitors (PARPi) this approach revealed an S-phase-specific DNA damage response after only 15 min, quantitatively differentiated responses to several clinically important PARPi, allowed for cell cycle resolved analyses of PARP trapping, and predicted conditions of PARPi hypersensitivity and resistance. The approach illuminates cellular mechanisms of drug synergism and, through a targeted multivariate screen, could identify a functional interaction between PARPi olaparib and NEDD8/SCF inhibition, which we show is dependent on PARP1 and linked to PARP1 trapping

Basal CHK1 activity safeguards its stability to maintain intrinsic S-phase checkpoint functions

The DNA replication machinery frequently encounters impediments that slow replication fork progression and threaten timely and error-free replication. The CHK1 protein kinase is essential to deal with replication stress (RS) and ensure genome integrity and cell survival, yet how basal levels and activity of CHK1 are maintained under physiological, unstressed conditions is not well understood. Here, we reveal that CHK1 stability is controlled by its steady-state activity during unchallenged cell proliferation. This autoactivatory mechanism, which depends on ATR and its coactivator ETAA1 and is tightly associated with CHK1 autophosphorylation at S296, counters CHK1 ubiquitylation and proteasomal degradation, thereby preventing attenuation of S-phase checkpoint functions and a compromised capacity to respond to RS. Based on these findings, we propose that steady-state CHK1 activity safeguards its stability to maintain intrinsic checkpoint functions and ensure genome integrity and cell survival

Phase separation of 53BP1 determines liquid-like behavior of DNA repair compartments

The DNA damage response (DDR) generates transient repair compartments to concentrate repair proteins and activate signaling factors. The physicochemical properties of these spatially confined compartments and their function remain poorly understood. Here, we establish, based on live cell microscopy and CRISPR/Cas9‐mediated endogenous protein tagging, that 53BP1‐marked repair compartments are dynamic, show droplet‐like behavior, and undergo frequent fusion and fission events. 53BP1 assembly, but not the upstream accumulation of γH2AX and MDC1, is highly sensitive to changes in osmotic pressure, temperature, salt concentration and to disruption of hydrophobic interactions. Phase separation of 53BP1 is substantiated by optoDroplet experiments, which further allowed dissection of the 53BP1 sequence elements that cooperate for light‐induced clustering. Moreover, we found the tumor suppressor protein p53 to be enriched within 53BP1 optoDroplets, and conditions that disrupt 53BP1 phase separation impair 53BP1‐dependent induction of p53 and diminish p53 target gene expression. We thus suggest that 53BP1 phase separation integrates localized DNA damage recognition and repair factor assembly with global p53‐dependent gene activation and cell fate decisions.ISSN:0261-4189ISSN:1460-207

Phase separation of 53 BP 1 determines liquid‐like behavior of DNA repair compartments

The DNA damage response (DDR) generates transient repair compartments to concentrate repair proteins and activate signaling factors. The physicochemical properties of these spatially confined compartments and their function remain poorly understood. Here, we establish, based on live cell microscopy and CRISPR/Cas9-mediated endogenous protein tagging, that 53BP1-marked repair compartments are dynamic, show droplet-like behavior, and undergo frequent fusion and fission events. 53BP1 assembly, but not the upstream accumulation of γH2AX and MDC1, is highly sensitive to changes in osmotic pressure, temperature, salt concentration and to disruption of hydrophobic interactions. Phase separation of 53BP1 is substantiated by optoDroplet experiments, which further allowed dissection of the 53BP1 sequence elements that cooperate for light-induced clustering. Moreover, we found the tumor suppressor protein p53 to be enriched within 53BP1 optoDroplets, and conditions that disrupt 53BP1 phase separation impair 53BP1-dependent induction of p53 and diminish p53 target gene expression. We thus suggest that 53BP1 phase separation integrates localized DNA damage recognition and repair factor assembly with global p53-dependent gene activation and cell fate decisions