Phosphorylation of Histone H2A.X by DNA-dependent Protein Kinase Is Not Affected by Core Histone Acetylation, but It Alters Nucleosome Stability and Histone H1 Binding

Abstract

Phosphorylation of the C-terminal end of histone H2A.X is the most characterized histone post-translational modification in DNA double-stranded breaks (DSB). DNA-dependent protein kinase (DNA-PK) is one of the three phosphatidylinositol 3 kinase-like family of kinase members that is known to phosphorylate histone H2A.X during DNA DSB repair. There is a growing body of evidence supporting a role for histone acetylation in DNA DSB repair, but the mechanism or the causative relation remains largely unknown. Using bacterially expressed recombinant mutants and stably and transiently transfected cell lines, we find that DNA-PK can phosphorylate Thr-136 in addition to Ser-139 both in vitro and in vivo. Furthermore, the phosphorylation reaction is not inhibited by the presence of H1, which in itself is a substrate of the reaction. We also show that, in contrast to previous reports, the ability of the enzyme to phosphorylate these residues is not affected by the extent of acetylation of the core histones. In vitro assembled nucleosomes and HeLa S3 native oligonucleosomes consisting of non-acetylated and acetylated histones are equally phosphorylated by DNA-PK. We demonstrate that the apparent differences in the extent of phosphorylation previously observed can be accounted for by the differential chromatin solubility under the MgCl 2 concentrations required for the phosphorylation reaction in vitro. Finally, we show that although H2A.X does not affect nucleosome conformation, it has a de-stabilizing effect that is enhanced by the DNA-PK-mediated phosphorylation and results in an impaired histone H1 binding. DNA double-stranded break (DSB)3 repair is an important cellular process that is critical for the maintenance of genome integrity. If improperly repaired, DSBs can have detrimental consequences leading to cancer via chromosomal aberrations such as chromosomal breaks, translocation, and aneuploidy (1).In the eukaryotic cell the repair mechanism requires the participation of chromatin components. One of the chromatin hallmarks involved is the phosphorylation of histone H2A.X at its highly conserved C-terminal SQE motif (Fig. 1A) (2-4). This phosphorylated form of H2A.X is commonly denoted as ␥-H2A.X (2). It is well known that histone H2A.X is phosphorylated ubiquitously at DNA DSB regardless of the source of the damage and of the pathway used in its repair: homologous recombination or non-homologous-end joining (NHEJ). The enzymes responsible for the DSB-induced H2A.X phosphorylation are all members of the PIKK family, such as the DNA-dependent protein kinase (DNA-PK), ataxia telangiectasia mutated (ATM) and ATM and Rad3-related (ATR) (4, 5). The DNA-PK catalytic subunit has been recently shown to play a dominant role in regulating the DNA damage-mediated phosphorylation of H2A.X (6).In the yeast H2A.X ortholog, Thr-126 and Ser-129 can be both phosphorylated during DSB in a way that appears to depend on the repair pathway. It has been shown that Thr-126 is important for homologous recombination but dispensable for NHEJ ...

High mobility group protein-mediated transcription requires DNA damage marker γ-H2AX

“…Accordingly, it has been reported that promoters of active (or poised) genes are either nucleosome-free or marked with unstable nucleosomes that contain specific histone variants such as H3.3 and H2A.Z [28]. Although H2AX does not affect nucleosome conformation, it has a destabilizing effect that is enhanced by its phosphorylation and results in an impaired linker histone H1 binding [29]. HMGA proteins were shown to dynamically compete with H1 for binding to the linker DNA, thereby loosening the chromatin [30,31].…”

Recognition, signaling, and repair of DNA double-strand breaks produced by ionizing radiation in mammalian cells: The molecular choreography

“…The presence of H2AX in nucleosomes has a modest de-stabilizing influence that is enhanced in vitro by DNA-PKmediated phosphorylations at Thr136 and Ser139, resulting in reduced histone H1 binding and reduced compaction [352]. This phosphorylation occurs independently of core histone acetylation.…”

“…A recently described site of H2AX C-terminal phosphorylation is Thr136 [352]. The presence of H2AX in nucleosomes has a modest de-stabilizing influence that is enhanced in vitro by DNA-PKmediated phosphorylations at Thr136 and Ser139, resulting in reduced histone H1 binding and reduced compaction [352].…”

DNA damage responsive miR-33b-3p promoted lung cancer cells survival and cisplatin resistance by targeting p21WAF1/CIP1

“…The phosphorylation of histone H2A.X at Ser139 (gH2A.X) marked the sites of DNA lesions caused by irradiation, UV and alkylation agents, and provided a nucleation site for recruitment of DNA damage checkpoint and DNA repair proteins. [20][21][22] Thus, we examined the gH2A.X levels in A549 cells transfected with miR-33b-3p mimics or NC. As the result revealed, A549 cells transfected with miR-33b-3p mimics displayed lower phosphorylated levels of gH2A.X compared with that in NC transfected cells after cisplatin treatment (Fig.…”