Systematic Identification of Functional Residues in Mammalian Histone H2AX

Abstract

dThe histone variant H2AX is a principal component of chromatin involved in the detection, signaling, and repair of DNA double-strand breaks (DSBs). H2AX is thought to operate primarily through its C-terminal S139 phosphorylation, which mediates the recruitment of DNA damage response (DDR) factors to chromatin at DSB sites. Here, we describe a comprehensive screen of 67 residues in H2AX to determine their contributions to H2AX functions. Our analysis revealed that H2AX is both sumoylated and ubiquitylated. Individual residues defective for sumoylation, ubiquitylation, and S139 phosphorylation in untreated and damaged cells were identified. Specifically, we identified an acidic triad region in both H2A and H2AX that is required in cis for their ubiquitylation. We also report the characterization of a human H2AX knockout cell line, which exhibits DDR defects, including p53 activation, following DNA damage. Collectively, this work constitutes the first genetic complementation system for a histone in human cells. Finally, our data reveal new roles for several residues in H2AX and define distinct functions for H2AX in human cells. N uclear DNA is bound by histones within nucleosomes to form chromatin (1). Core nucleosomes consist of two copies each of four canonical histones (H2A, H2B, H3, and H4) in an octamer that contains ϳ146 bp of DNA wrapped around the histone protein core. In mammalian genomes, several histone variants resembling core histones exist, such as the histone variant H2AX, which is nearly identical to H2A except for a divergent and extended C terminus. Histones can be modified on specific amino acid residues by various posttranslational chemical modifications (PTMs), including methylation, acetylation, and phosphorylation (2-4). In addition, lysine residues can be modified by the covalent attachment of small polypeptides such as ubiquitin (Ub) and SUMO (small ubiquitin-like modifier) (5). These various PTMs are catalyzed by "writer" enzymes and are removed by additional enzymes that act to "erase" these marks (3). Together, these enzymes and chromatin binding proteins dynamically regulate the structure and functions of chromatin, which in turn regulates fundamental nuclear processes, such as chromosome replication and segregation, transcription, and DNA repair.The protection of our genetic material is paramount for averting various human diseases, and chromatin plays an important role in coordinating the repair of nuclear DNA (6, 7). Cells have evolved a complex network of diverse cellular pathways, termed the DNA damage response (DDR), which detects damaged DNA, signals its presence, and promotes DNA repair (6, 7). DNA double-strand breaks (DSBs) represent a particularly challenging and cytotoxic form of DNA damage. DSBs create discontinuities in chromosomal DNA that, if not repaired or repaired incorrectly, result in mutations, chromosome loss, and/or ongoing genome instability. DSBs are predominantly repaired by either homologous recombination (HR) or nonhomologous end joining (NHEJ) (8). DSB rep...

Endogenous APOBEC3A DNA Cytosine Deaminase Is Cytoplasmic and Nongenotoxic

“…A3A expression also caused increased levels of the DNA damage marker ␥-H2AX. Interestingly, the increase was most obvious for the monoubiquitinated form of ␥-H2AX, which is an important mediator of the DNA damage response (41)(42)(43). In contrast, endogenous A3A was not toxic, despite the high levels of A3A induction by type I IFN seen in primary CD14…”

Screen identifies bromodomain protein ZMYND8 in chromatin recognition of transcription-associated DNA damage that promotes homologous recombination

“…For example, gH2AX (i.e., phosphorylated H2AX on Ser139), is induced at DSBs and recruits the DDR protein MDC1 to damaged chromatin. This event promotes accumulation of many DDR factors at DSBs that orchestrate several DDR functions (Polo and Jackson 2011;Chen et al 2013). Transcription-associated H3K36 methylation promotes DNA repair Carvalho et al 2014;Jha and Strahl 2014;Pai et al 2014;Pfister et al 2014).…”

Sumoylated Human Histone H4 Prevents Chromatin Compaction by Inhibiting Long-range Internucleosomal Interactions

“…Histone sumoylation (modification by the SUMO-1 or 2/3 family of proteins) occurs in parasites (9), plants (10), yeast (11,12), and humans (13,14). Similar to the range of histone targets of ubiquitin (Ub), SUMO is conjugated to all four core histones (11), the linker histone H1 (15), and histone variants H2A.Z (12) and H2A.X (14).…”

“…Similar to the range of histone targets of ubiquitin (Ub), SUMO is conjugated to all four core histones (11), the linker histone H1 (15), and histone variants H2A.Z (12) and H2A.X (14). Among the core histones, H4 is the primary target for modification by SUMO-1, and SUMO-3 in human 293T cells and B-lymphocytes (13).…”