SummaryInhibitor of growth 1 (ING1) is implicated in oncogenesis, DNA damage repair and apoptosis. Mutations within the ING1 gene and altered expression levels of ING1 are found in multiple human cancers. Here, we show that both DNA repair and apoptotic activities of ING1 require the interaction of the C-terminal plant homeodomain (PHD) finger with trimethylated at Lys 4 histone H3 (H3K4me3). The ING1 PHD finger recognizes methylated H3K4 but not other histone modifications as revealed by the peptide microarrays. The molecular mechanism of the histone recognition is elucidated based on a 2.1 Å resolution crystal structure of the PHD-H3K4me3 complex. The K4me3 occupies a deep hydrophobic pocket formed by the conserved Y212 and W235 residues that make cation-π contacts with the trimethylammonium group. Both aromatic residues are essential in the H3K4me3 recognition, as substitution of these residues with Ala disrupts the interaction. Unlike the wild type ING1, the W235A mutant, overexpressed in the stable clones of melanoma cells or in HT1080 cells, was unable to stimulate DNA repair after UV irradiation or promote DNA-damage induced apoptosis, indicating that H3K4me3 binding is necessary for these biological functions of ING1. Furthermore, N216S, V218I and G221V mutations, found in human malignances, impair the ability of ING1 to associate with H3K4me3 or to induce nucleotide repair and cell death, linking the tumorigenic activity of ING1 with epigenetic regulation. Together, our findings reveal the critical role of the H3K4me3 interaction in mediating cellular responses to genotoxic stresses and offer new insight into the molecular mechanism underlying the tumor suppressive activity of ING1.

Polymerase‐blocking DNA lesions are thought to elicit a checkpoint response via accumulation of single‐stranded DNA at stalled replication forks. However, as an alternative to persistent fork stalling, re‐priming downstream of lesions can give rise to daughter‐strand gaps behind replication forks. We show here that the processing of such structures by an exonuclease, Exo1, is required for timely checkpoint activation, which in turn prevents further gap erosion in S phase. This Rad9‐dependent mechanism of damage signaling is distinct from the Mrc1‐dependent, fork‐associated response to replication stress induced by conditions such as nucleotide depletion or replisome‐inherent problems, but reminiscent of replication‐independent checkpoint activation by single‐stranded DNA. Our results indicate that while replisome stalling triggers a checkpoint response directly at the stalled replication fork, the response to replication stress elicited by polymerase‐blocking lesions mainly emanates from Exo1‐processed, postreplicative daughter‐strand gaps, thus offering a mechanistic explanation for the dichotomy between replisome‐ versus template‐induced checkpoint signaling.

Highlights d RPA foci induced by MMS or UV irradiation arise in postreplicative repair territories d Translesion synthesis and homologous recombination mediate resolution of RPA foci d Replication stress induced by damaged templates is resolved behind replication forks d Spontaneous replication problems are solved largely behind replication forks as well