CRISPR screens in sister chromatid cohesion defective cells reveal PAXIP1-PAGR1 as regulator of chromatin association of cohesin.

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.

Fanconi anemia is associated with a defect in the BRCA2 partner PALB2.

The Fanconi anemia and BRCA networks are considered interconnected, as BRCA2 gene defects have been discovered in individuals with Fanconi anemia subtype D1. Here we show that a defect in the BRCA2-interacting protein PALB2 is associated with Fanconi anemia in an individual with a new subtype. PALB2-deficient cells showed hypersensitivity to cross-linking agents and lacked chromatin-bound BRCA2; these defects were corrected upon ectopic expression of PALB2 or by spontaneous reversion.

Loss-of-Function FANCL Mutations Associate with Severe Fanconi Anemia Overlapping the VACTERL Association.

The diagnosis of VACTERL syndrome can be elusive, especially in the prenatal life, due to the presence of malformations that overlap those present in other genetic conditions, including the Fanconi anemia (FA). We report on three VACTERL cases within two families, where the two who arrived to be born died shortly after birth due to severe organs' malformations. The suspicion of VACTERL association was based on prenatal ultrasound assessment and postnatal features. Subsequent chromosome breakage analysis suggested the diagnosis of FA. Finally, by next-generation sequencing based on the analysis of the exome in one family and of a panel of Fanconi genes in the second one, we identified novel FANCL truncating mutations in both families. We used ectopic expression of wild-type FANCL to functionally correct the cellular FA phenotype for both mutations. Our study emphasizes that the diagnosis of FA should be considered when VACTERL association is suspected. Furthermore, we show that loss-of-function mutations in FANCL result in a severe clinical phenotype characterized by early postnatal death.

The DNA helicase BRIP1 is defective in Fanconi anemia complementation group J.

The protein predicted to be defective in individuals with Fanconi anemia complementation group J (FA-J), FANCJ, is a missing component in the Fanconi anemia pathway of genome maintenance. Here we identify pathogenic mutations in eight individuals with FA-J in the gene encoding the DEAH-box DNA helicase BRIP1, also called FANCJ. This finding is compelling evidence that the Fanconi anemia pathway functions through a direct physical interaction with DNA.

Warsaw breakage syndrome, a cohesinopathy associated with mutations in the XPD helicase family member DDX11/ChlR1.

The iron-sulfur-containing DNA helicases XPD, FANCJ, DDX11, and RTEL represent a small subclass of superfamily 2 helicases. XPD and FANCJ have been connected to the genetic instability syndromes xeroderma pigmentosum and Fanconi anemia. Here, we report a human individual with biallelic mutations in DDX11. Defective DDX11 is associated with a unique cellular phenotype in which features of Fanconi anemia (drug-induced chromosomal breakage) and Roberts syndrome (sister chromatid cohesion defects) coexist. The DDX11-deficient patient represents another cohesinopathy, besides Cornelia de Lange syndrome and Roberts syndrome, and shows that DDX11 functions at the interface between DNA repair and sister chromatid cohesion.

X-linked inheritance of Fanconi anemia complementation group B.

Fanconi anemia is an autosomal recessive syndrome characterized by diverse clinical symptoms, hypersensitivity to DNA crosslinking agents, chromosomal instability and susceptibility to cancer. Fanconi anemia has at least 11 complementation groups (A, B, C, D1, D2, E, F, G, I, J, L); the genes mutated in 8 of these have been identified. The gene BRCA2 was suggested to underlie complementation group B, but the evidence is inconclusive. Here we show that the protein defective in individuals with Fanconi anemia belonging to complementation group B is an essential component of the nuclear protein 'core complex' responsible for monoubiquitination of FANCD2, a key event in the DNA-damage response pathway associated with Fanconi anemia and BRCA. Unexpectedly, the gene encoding this protein, FANCB, is localized at Xp22.31 and subject to X-chromosome inactivation. X-linked inheritance has important consequences for genetic counseling of families with Fanconi anemia belonging to complementation group B. Its presence as a single active copy and essentiality for a functional Fanconi anemia-BRCA pathway make FANCB a potentially vulnerable component of the cellular machinery that maintains genomic integrity.

MMS22L-TONSL functions in sister chromatid cohesion in a pathway parallel to DSCC1-RFC.

The leading strand-oriented alternative PCNA clamp loader DSCC1-RFC functions in DNA replication, repair, and sister chromatid cohesion (SCC), but how it facilitates these processes is incompletely understood. Here, we confirm that loss of human DSCC1 results in reduced fork speed, increased DNA damage, and defective SCC. Genome-wide CRISPR screens in DSCC1-KO cells reveal multiple synthetically lethal interactions, enriched for DNA replication and cell cycle regulation. We show that DSCC1-KO cells require POLE3 for survival. Co-depletion of DSCC1 and POLE3, which both interact with the catalytic polymerase ε subunit, additively impair DNA replication, suggesting that these factors contribute to leading-strand DNA replication in parallel ways. An additional hit is MMS22L, which in humans forms a heterodimer with TONSL. Synthetic lethality of DSCC1 and MMS22L-TONSL likely results from detrimental SCC loss. We show that MMS22L-TONSL, like DDX11, functions in a SCC establishment pathway parallel to DSCC1-RFC. Because both DSCC1-RFC and MMS22L facilitate ESCO2 recruitment to replication forks, we suggest that distinct ESCO2 recruitment pathways promote SCC establishment following either cohesin conversion or de novo cohesin loading.

BIRC2-BIRC3 amplification: a potentially druggable feature of a subset of head and neck cancers in patients with Fanconi anemia.

Head-and-neck squamous cell carcinomas (HNSCCs) are relatively common in patients with Fanconi anemia (FA), a hereditary chromosomal instability disorder. Standard chemo-radiation therapy is not tolerated in FA due to an overall somatic hypersensitivity to such treatment. The question is how to find a suitable alternative treatment. We used whole-exome and whole genome mRNA sequencing to identify major genomic and transcriptomic events associated with FA-HNSCC. CRISPR-engineered FA-knockout models were used to validate a number of top hits that were likely to be druggable. We identified deletion of 18q21.2 and amplification of 11q22.2 as prevailing copy-number alterations in FA HNSCCs, the latter of which was associated with strong overexpression of the cancer-related genes YAP1, BIRC2, BIRC3 (at 11q22.1-2). We then found the drug AZD5582, a known small molecule inhibitor of BIRC2-3, to selectively kill FA tumor cells that overexpressed BIRC2-3. This occurred at drug concentrations that did not affect the viability of untransformed FA cells. Our data indicate that 11q22.2 amplifications are relatively common oncogenic events in FA-HNSCCs, as holds for non FA-HNSCC. Therefore, chemotherapeutic inhibition of overexpressed BIRC2-3 may provide the basis for an approach to develop a clinically realistic treatment of FA-HNSCCs that carry 11q22.2 amplifications.

Biallelic BUB1 mutations cause microcephaly, developmental delay, and variable effects on cohesion and chromosome segregation.

Budding uninhibited by benzimidazoles (BUB1) contributes to multiple mitotic processes. Here, we describe the first two patients with biallelic BUB1 germline mutations, who both display microcephaly, intellectual disability, and several patient-specific features. The identified mutations cause variable degrees of reduced total protein level and kinase activity, leading to distinct mitotic defects. Both patients' cells show prolonged mitosis duration, chromosome segregation errors, and an overall functional spindle assembly checkpoint. However, while BUB1 levels mostly affect BUBR1 kinetochore recruitment, impaired kinase activity prohibits centromeric recruitment of Aurora B, SGO1, and TOP2A, correlating with anaphase bridges, aneuploidy, and defective sister chromatid cohesion. We do not observe accelerated cohesion fatigue. We hypothesize that unresolved DNA catenanes increase cohesion strength, with concomitant increase in anaphase bridges. In conclusion, BUB1 mutations cause a neurodevelopmental disorder, with clinical and cellular phenotypes that partially resemble previously described syndromes, including autosomal recessive primary microcephaly, mosaic variegated aneuploidy, and cohesinopathies.

Fancm-deficient mice reveal unique features of Fanconi anemia complementation group M.

The Fanconi anemia (FA) core complex member FANCM remodels synthetic replication forks and recombination intermediates. Thus far, only one FA patient with FANCM mutations has been described, but the relevance of these mutations for the FA phenotype is uncertain. To provide further experimental access to the FA-M complementation group we have generated Fancm-deficient mice by deleting exon 2. FANCM deficiency caused hypogonadism in mice and hypersensitivity to cross-linking agents in mouse embryonic fibroblasts (MEFs), thus phenocopying other FA mouse models. However, Fancm(Delta2/Delta2) mice also showed unique features atypical for FA mice, including underrepresentation of female Fancm(Delta2/Delta2) mice and decreased overall and tumor-free survival. This increased cancer incidence may be correlated to the role of FANCM in the suppression of spontaneous sister chromatid exchanges as observed in MEFs. In addition, FANCM appeared to have a stimulatory rather than essential role in FANCD2 monoubiquitination. The FA-M mouse model presented here suggests that FANCM functions both inside and outside the FA core complex to maintain genome stability and to prevent tumorigenesis.