ABSTRACT
The cohesin complex regulates higher order chromosome architecture through maintaining sister chromatid cohesion and folding chromatin by DNA loop extrusion. Impaired cohesin function underlies a heterogeneous group of genetic syndromes and is associated with cancer. Here, we mapped the genetic dependencies of human cell lines defective of cohesion regulators DDX11 and ESCO2. The obtained synthetic lethality networks are strongly enriched for genes involved in DNA replication and mitosis and support the existence of parallel sister chromatid cohesion establishment pathways. Among the hits, we identify the chromatin binding, BRCT-domain containing protein PAXIP1 as a novel cohesin regulator. Depletion of PAXIP1 severely aggravates cohesion defects in ESCO2 mutant cells, leading to mitotic cell death. PAXIP1 promotes global chromatin association of cohesin, independent of DNA replication, a function that cannot be explained by indirect effects of PAXIP1 on transcription or DNA repair. Cohesin regulation by PAXIP1 requires its binding partner PAGR1 and a conserved FDF motif in PAGR1. PAXIP1 co-localizes with cohesin on multiple genomic loci, including active gene promoters and enhancers. Possibly, this newly identified role of PAXIP1-PAGR1 in regulating cohesin occupancy on chromatin is also relevant for previously described functions of PAXIP1 in transcription, immune cell maturation and DNA repair.
ABSTRACT
The cohesin complex facilitates faithful chromosome segregation by pairing the sister chromatids after DNA replication until mitosis. In addition, cohesin contributes to proficient and error-free DNA replication. Replisome progression and establishment of sister chromatid cohesion are intimately intertwined processes. Here, we review how the key factors in DNA replication and cohesion establishment cooperate in unperturbed conditions and during DNA replication stress. We discuss the detailed molecular mechanisms of cohesin recruitment and the entrapment of replicated sister chromatids at the replisome, the subsequent stabilization of sister chromatid cohesion via SMC3 acetylation, as well as the role and regulation of cohesin in the response to DNA replication stress.
Subject(s)
Cell Cycle Proteins/genetics , Chromatids/genetics , Chromosomal Proteins, Non-Histone/genetics , DNA Replication/genetics , Saccharomyces cerevisiae Proteins/genetics , Acetylation , Cell Cycle Proteins/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Chromosome Segregation/genetics , Mitosis/genetics , Saccharomyces cerevisiae/genetics , Sister Chromatid Exchange/genetics , CohesinsABSTRACT
Cells employ transcription-coupled repair (TCR) to eliminate transcription-blocking DNA lesions. DNA damage-induced binding of the TCR-specific repair factor CSB to RNA polymerase II (RNAPII) triggers RNAPII ubiquitylation of a single lysine (K1268) by the CRL4CSA ubiquitin ligase. How CRL4CSA is specifically directed towards K1268 is unknown. Here, we identify ELOF1 as the missing link that facilitates RNAPII ubiquitylation, a key signal for the assembly of downstream repair factors. This function requires its constitutive interaction with RNAPII close to K1268, revealing ELOF1 as a specificity factor that binds and positions CRL4CSA for optimal RNAPII ubiquitylation. Drug-genetic interaction screening also revealed a CSB-independent pathway in which ELOF1 prevents R-loops in active genes and protects cells against DNA replication stress. Our study offers key insights into the molecular mechanisms of TCR and provides a genetic framework of the interplay between transcriptional stress responses and DNA replication.
Subject(s)
DNA Damage , DNA Repair , Peptide Elongation Factor 1/metabolism , RNA Polymerase II/metabolism , Ubiquitin-Protein Ligases/metabolism , Ubiquitination , CRISPR-Cas Systems , Cell Line, Tumor , DNA Helicases/genetics , DNA Helicases/metabolism , DNA Repair Enzymes/genetics , DNA Repair Enzymes/metabolism , Humans , Peptide Elongation Factor 1/genetics , Poly-ADP-Ribose Binding Proteins/genetics , Poly-ADP-Ribose Binding Proteins/metabolism , Protein Binding , Protein Interaction Domains and Motifs , RNA Polymerase II/genetics , Transcription Elongation, Genetic , Transcription Factors/genetics , Transcription Factors/metabolism , Ubiquitin-Protein Ligases/geneticsABSTRACT
Warsaw Breakage Syndrome (WABS) is a rare disorder related to cohesinopathies and Fanconi anemia, caused by bi-allelic mutations in DDX11. Here, we report multiple compound heterozygous WABS cases, each displaying destabilized DDX11 protein and residual DDX11 function at the cellular level. Patient-derived cell lines exhibit sensitivity to topoisomerase and PARP inhibitors, defective sister chromatid cohesion and reduced DNA replication fork speed. Deleting DDX11 in RPE1-TERT cells inhibits proliferation and survival in a TP53-dependent manner and causes chromosome breaks and cohesion defects, independent of the expressed pseudogene DDX12p. Importantly, G-quadruplex (G4) stabilizing compounds induce chromosome breaks and cohesion defects which are strongly aggravated by inactivation of DDX11 but not FANCJ. The DNA helicase domain of DDX11 is essential for sister chromatid cohesion and resistance to G4 stabilizers. We propose that DDX11 is a DNA helicase protecting against G4 induced double-stranded breaks and concomitant loss of cohesion, possibly at DNA replication forks.
Subject(s)
Abnormalities, Multiple/etiology , DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/metabolism , DNA Helicases/genetics , DNA Helicases/metabolism , G-Quadruplexes , Sister Chromatid Exchange , Abnormalities, Multiple/genetics , Abnormalities, Multiple/pathology , Cell Proliferation , DEAD-box RNA Helicases/chemistry , DNA Helicases/chemistry , Fanconi Anemia Complementation Group Proteins/genetics , Fanconi Anemia Complementation Group Proteins/metabolism , Humans , Male , Middle Aged , Mutation, Missense , Protein Stability , Pseudogenes , RNA Helicases/genetics , RNA Helicases/metabolism , Rad51 Recombinase/genetics , Rad51 Recombinase/metabolism , Syndrome , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolismABSTRACT
Premature loss of sister chromatid cohesion at metaphase is a diagnostic marker for different cohesinopathies. Here, we report that metaphase spreads of many cancer cell lines also show premature loss of sister chromatid cohesion. Cohesion loss occurs independently of mutations in cohesion factors including SA2, a cohesin subunit frequently inactivated in cancer. In untransformed cells, induction of DNA replication stress by activation of oncogenes or inhibition of DNA replication is sufficient to trigger sister chromatid cohesion loss. Importantly, cell growth under conditions of replication stress requires the cohesin remover WAPL. WAPL promotes rapid RAD51-dependent repair and restart of broken replication forks. We propose that active removal of cohesin allows cancer cells to overcome DNA replication stress. This leads to oncogene-induced cohesion loss from newly synthesized sister chromatids that may contribute to genomic instability and likely represents a targetable cancer cell vulnerability.
Subject(s)
Carrier Proteins/metabolism , Chromatids/genetics , DNA Repair , Nuclear Proteins/metabolism , Proto-Oncogene Proteins/metabolism , ras Proteins/metabolism , Animals , Carrier Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Cells, Cultured , Chromosomal Proteins, Non-Histone/metabolism , DNA Replication , HEK293 Cells , Humans , Mice , Nuclear Proteins/genetics , Proto-Oncogene Proteins/genetics , CohesinsABSTRACT
In a process linked to DNA replication, duplicated chromosomes are entrapped in large, circular cohesin complexes and functional sister chromatid cohesion (SCC) is established by acetylation of the SMC3 cohesin subunit. Roberts Syndrome (RBS) and Warsaw Breakage Syndrome (WABS) are rare human developmental syndromes that are characterized by defective SCC. RBS is caused by mutations in the SMC3 acetyltransferase ESCO2, whereas mutations in the DNA helicase DDX11 lead to WABS. We found that WABS-derived cells predominantly rely on ESCO2, not ESCO1, for residual SCC, growth and survival. Reciprocally, RBS-derived cells depend on DDX11 to maintain low levels of SCC. Synthetic lethality between DDX11 and ESCO2 correlated with a prolonged delay in mitosis, and was rescued by knockdown of the cohesin remover WAPL. Rescue experiments using human or mouse cDNAs revealed that DDX11, ESCO1 and ESCO2 act on different but related aspects of SCC establishment. Furthermore, a DNA binding DDX11 mutant failed to correct SCC in WABS cells and DDX11 deficiency reduced replication fork speed. We propose that DDX11, ESCO1 and ESCO2 control different fractions of cohesin that are spatially and mechanistically separated.
