The breast cancer susceptibility proteins BRCA1 and BRCA2 have emerged as key stabilizing factors for the maintenance of replication fork integrity following replication stress. In their absence, stalled replication forks are extensively degraded by the MRE11 nuclease, leading to chemotherapeutic sensitivity. Here we report that BRCA proteins prevent nucleolytic degradation by protecting replication forks that have undergone fork reversal upon drug treatment. The unprotected regressed arms of reversed forks are the entry point for MRE11 in BRCA-deficient cells. The CtIP protein initiates MRE11-dependent degradation, which is extended by the EXO1 nuclease. Next, we show that the initial limited resection of the regressed arms establishes the substrate for MUS81 in BRCA2-deficient cells. In turn, MUS81 cleavage of regressed forks with a ssDNA tail promotes POLD3-dependent fork rescue. We propose that targeting this pathway may represent a new strategy to modulate BRCA2-deficient cancer cell response to chemotherapeutics that cause fork degradation.

Summary Acute treatment with replication-stalling chemotherapeutics causes reversal of replication forks. BRCA proteins protect reversed forks from nucleolytic degradation, and their loss leads to chemosensitivity. Here, we show that fork degradation is no longer detectable in BRCA1-deficient cancer cells exposed to multiple cisplatin doses, mimicking a clinical treatment regimen. This effect depends on increased expression and chromatin loading of PRIMPOL and is regulated by ATR activity. Electron microscopy and single-molecule DNA fiber analyses reveal that PRIMPOL rescues fork degradation by reinitiating DNA synthesis past DNA lesions. PRIMPOL repriming leads to accumulation of ssDNA gaps while suppressing fork reversal. We propose that cells adapt to repeated cisplatin doses by activating PRIMPOL repriming under conditions that would otherwise promote pathological reversed fork degradation. This effect is generalizable to other conditions of impaired fork reversal (e.g., SMARCAL1 loss or PARP inhibition) and suggests a new strategy to modulate cisplatin chemosensitivity by targeting the PRIMPOL pathway.

Methylation of histone H3 lysine 4 (H3K4) in Saccharomyces cerevisiae is implemented by Set1/COMPASS, which was originally purified based on the similarity of yeast Set1 to human MLL1 and Drosophila melanogaster Trithorax (Trx). While humans have six COMPASS family members, Drosophila possesses a representative of the three subclasses within COMPASS-like complexes: dSet1 (human SET1A/SET1B), Trx (human MLL1/2), and Trr (human MLL3/4). Here, we report the biochemical purification and molecular characterization of the Drosophila COMPASS family. We observed a one-to-one similarity in subunit composition with their mammalian counterparts, with the exception of LPT (lost plant homeodomains [PHDs] of Trr), which copurifies with the Trr complex. LPT is a previously uncharacterized protein that is homologous to the multiple PHD fingers found in the N-terminal regions of mammalian MLL3/4 but not Drosophila Trr, indicating that Trr and LPT constitute a split gene of an MLL3/4 ancestor. Our study demonstrates that all three complexes in Drosophila are H3K4 methyltransferases; however, dSet1/COMPASS is the major monoubiquitination-dependent H3K4 di-and trimethylase in Drosophila. Taken together, this study provides a springboard for the functional dissection of the COMPASS family members and their role in the regulation of histone H3K4 methylation throughout development in Drosophila.Histone H3 lysine 4 methylation (H3K4me) is associated with the transcriptionally active regions of the genome in yeast, flies, and mammals (3,23,35). Set1 was identified as a component of a macromolecular protein complex named COMPASS (complex of proteins associated with Set 1), as the first H3K4 methylase, and it is responsible for all mono-, di-, and trimethylation of H3K4 in yeast (22,31,40,52). In Drosophila melanogaster, four SET domain-containing proteins, namely, Trithorax (Trx), Trithorax-related (Trr), dSet1, and Ash1, have been reported to implement H3K4 methylation (10). All but Ash1, which has subsequently been demonstrated to be an H3K36 methyltransferase (49,59), are related to subunits of the six COMPASS and COMPASS-like complexes in mammals. trx was originally characterized as a gene that when mutated caused homeotic transformations (6, 18). Detailed genetic and molecular analyses showed that Trx is required to maintain activation states of its target genes throughout development and counteracts the repressive effects of the Polycomb group proteins (PcG) (39, 41). Trr was identified based on sequence similarity to Trx but was shown to function in the regulation of hormone-responsive gene expression (42). dSet1 was identified based on sequence homology to the Saccharomyces cerevisiae and mammalian Set1 proteins (53, 58).In mammals, there are at least six SET1-related proteins that form COMPASS-like complexes, namely, SET1A, SET1B, and MLL1 to MLL4. SET1A and SET1B are orthologous to dSet1; MLL1 and MLL2 are orthologous to Drosophila Trx; MLL3 and MLL4 (also known as ALR) are orthologous to Drosophila Trr (33,43,45). All of the m...

To define the molecular regulators required for differential pattern of H3K79 methylation by Dot1, we performed a GPS screen and discovered that the components of the cell cycle-regulated SBF complex were required for normal levels of H3K79 di- but not trimethylation. Genome-wide mapping revealed that H3K79 di- and trimethylation to present a mutually exclusive pattern on chromatin with M/G1 cell-cycle-regulated genes significantly enriched for H3K79 dimethylation. Since H3K79 trimethylation requires prior monoubiquitination of H2B, we performed genome-wide profiling of H2BK123 monoubiquitination and showed that H2BK123 monoubiquitination is excluded from cell cycle regulated genes and sites containing H3K79me2 but not from H3K79me3 containing regions. A genome-wide screen for factors responsible for the establishment/removal of H3K79 dimethylation resulted in the identification of several genes including NRM1 and WHI3, which both impact the transcription by the SBF, and MBF complexes, further linking the regulation of H3K79’s methylation status to the cell cycle.

Histone H3 lysine27-to-methionine (H3K27M) gain-of-function mutations occur in highly aggressive pediatric gliomas. Here, we establish a Drosophila animal model for the pathogenic histone H3K27M mutation and show that its overexpression resembles Polycomb repressive complex 2 (PRC2) loss-of-function phenotypes, causing de-repression of PRC2 target genes and developmental perturbations. Similarly, a H3K9M mutant depletes H3K9 methylation levels and suppresses position-effect variegation in various Drosophila tissues. The histone H3K9 demethylase KDM3B/JHDM2 associates with H3K9M nucleosomes and its overexpression in Drosophila results in loss of H3K9 methylation levels and heterochromatic silencing defects. Here we establish histone lysine-to-methionine mutants as robust in vivo tools for inhibiting methylation pathways that also function as biochemical reagents for capturing site-specific histone-modifying enzymes, thus providing molecular insight into chromatin-signaling pathways.