MRG15-mediated tethering of PALB2 to unperturbed chromatin protects active genes from genotoxic stress

Abstract

The partner and localiser of BRCA2 (PALB2) plays important roles in the maintenance of genome integrity and protection against cancer. Although PALB2 is commonly described as a repair factor recruited to sites of DNA breaks, recent studies provide evidence that PALB2 also associates with unperturbed chromatin. Here, we investigated the previously poorly described role of chromatin-associated PALB2 in undamaged cells. We found that PALB2 associates with active genes through its major binding partner, MRG15, which recognizes histone H3 trimethylated at lysine 36 (H3K36me3) by the SETD2 methyltransferase. Missense mutations that ablate PALB2 binding to MRG15 confer elevated sensitivity to the topoisomerase inhibitor camptothecin (CPT) and increased levels of aberrant metaphase chromosomes and DNA stress in gene bodies, which were suppressed by preventing DNA replication. Remarkably, the level of PALB2 at genic regions was frequently decreased, rather than increased, upon CPT treatment. We propose that the steady-state presence of PALB2 at active genes, mediated through the SETD2/H3K36me3/MRG15 axis, ensures an immediate response to DNA stress and therefore effective protection of these regions during DNA replication. This study provides a conceptual advance in demonstrating that the constitutive chromatin association of repair factors plays a key role in the maintenance of genome stability and furthers our understanding of why PALB2 defects lead to human genome instability syndromes.nherited mutations in the partner and localiser of BRCA2 (PALB2) gene predispose to breast and pancreatic cancer (1-3) and also congenital malformations, growth retardation, and early childhood cancer in a rare subgroup of Fanconi anemia (FA-N) patients (4, 5). The best characterized function of PALB2 is to physically link the two main breast cancer susceptibility gene products, BRCA1 and BRCA2, at sites of DNA damage (6-8), thus playing a pivotal role in the repair of DNA double-strand breaks (DSBs) by homologous recombination (HR). BRCA1 facilitates DNA-end resection to produce single-stranded (ss) DNA (9) and concomitantly attracts PALB2, together with BRCA2 and RAD51, to DSB sites (6-8). In turn, BRCA2 promotes the loading of the RAD51 recombinase onto ssDNA (10-12), which is critical for the strand invasion and exchange phase of HR (13). This mechanism of repair factor recruitment is important for the timely activation of HR at sites of DNA damage, and its perturbation in individuals with impaired PALB2 function is presumed to cause human pathologies. Although PALB2 is commonly described as a repair factor recruited to sites of DNA breaks, we and others have provided evidence that PALB2 also associates with chromatin in the absence of DNA damage (14-17). A recent genome-wide analysis of PALB2 chromatin occupancy revealed enrichment at highly active genes, with PALB2 occupying the entire body of these genes (18). PALB2 was shown to support the transcription of a subset of NF-κB-and retinoic acid-responsive genes. However, PALB2 does...

KAT2-mediated acetylation switches the mode of PALB2 chromatin association to safeguard genome integrity

“…So far, our analyses showed that ChAM acetylation promotes PALB2 association with nucleosomes/chromatin ( Fig. 6A), whereas PALB2 dissociates from active genes upon CPT treatment (Bleuyard et al, 2017b). These observations prompted us to further investigate whether ChAM acetylation is dynamically controlled during the DDR.…”

“…Moreover, PALB2 intrinsic chromatin association is reinforced by the chromatin-association motif (ChAM), an evolutionarily conserved domain uniquely found in PALB2 orthologues, which directly binds to nucleosomes (Bleuyard et al, 2012). Notably, our locus-specific chromatin immunoprecipitation (ChIP) analyses revealed a decrease in PALB2 association with active genes following an exposure to a topoisomerase inhibitor camptothecin (CPT), suggesting that the mode of PALB2 chromatin association is actively regulated (Bleuyard et al, 2017b). Despite these observations, the regulatory mechanism by which PALB2 is switched between different modes of chromatin association (i.e.…”

“…HEK293T cells were grown in Dulbecco's Modified Eagle's Medium (DMEM, Sigma) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 0.1 mg/mL streptomycin. U2OS Flp-In T-REx P2shRNA cell line (Bleuyard et al, 2017b), carrying a doxycycline-inducible shRNA targeting the endogenous PALB2 3' UTR, referred to as U2OS-shPALB2, were used to generate stable isogenic cell lines with constitutive expression of N3xFLAG-PALB2 variants.…”

“…U2OS-shPALB2 were co-transfected with pOG44 and pcDNA5/FRT GW/N3×FLAG-PALB2 (7Q or 7R), and resultant stable cell lines were selected in DMEM supplemented with 10% FBS, 100 U/mL penicillin, 0.1 mg/mL streptomycin, 10 µg/mL blasticidin, 200 μg/mL hygromycin B (100 µg/mL to maintain the cell lines), and 1 µg/mL puromycin. Stable U2OS-shPALB2 lines carrying the empty pcDNA5/FRT GW/N3×FLAG vector or the pcDNA5/FRT GW/N3×FLAG-PALB2 WT vector have been previously described (Bleuyard et al, 2017b).…”

“…We have recently revealed a repair-independent role of PALB2 in protecting transcriptionally active chromatin from DNA damage arising from replicative stress (Bleuyard et al, 2017b). This role of PALB2 is mediated through its high-affinity binding partner the MORF-related gene 15 protein (MRG15), which recognises an epigenetic marker of active genes, histone H3 trimethylated at lysine 36 (H3K36me 3 ), via its N-terminal chromodomain (Bleuyard et al, 2017b;Hayakawa et al, 2010;Sy et al, 2009a). Moreover, PALB2 intrinsic chromatin association is reinforced by the chromatin-association motif (ChAM), an evolutionarily conserved domain uniquely found in PALB2 orthologues, which directly binds to nucleosomes (Bleuyard et al, 2012).…”