Joseph, F. M.; Holt, M. V.; Jerome, J. M.; Zhang, L.; Boice, A. G.; Castro, P. D.; Aramburu, S. I.; Dere, R. L.; Rosenberg, S. M.; Rowley, D. R.; Young, N. L.

Phosphorylation of histone H2AX at serine 139 ({gamma}H2AX) by ATM/ATR kinases is a central marker of the DNA damage response (DDR), widely used to detect DNA double-strand breaks. However, the molecular basis for tissue- and context-specific variation in {gamma}H2AX signaling remains poorly defined. Here we discover a post-translational truncation of H2AX, catalyzed by lysine demethylase 4A (KDM4A), which removes two C-terminal amino acids critical for ATM/ATR-dependent phosphorylation. This truncation renders H2AX refractory to {gamma}H2AX formation, effectively bypassing canonical DDR signaling. Truncated H2AX accumulates in select cell lines, primary cells, solid tumors, and normal tissues. Genetic knockdown or pharmacologic inhibition of KDM4A reduces H2AX truncation, restores {gamma}H2AX induction, and enhances DNA repair capacity. Conversely, KDM4A overexpression promotes H2AX truncation, impairs {gamma}H2AX signaling, and exacerbates DNA damage accumulation. This previously unrecognized regulatory axis implicates KDM4A catalyzed H2AX truncation as a superseding mechanism that represses the canonical DDR and disrupts the correlation between {gamma}H2AX and DNA damage. This dioxygenase-based protease mechanism represents a new class of proteases and is the first example of c-terminal dipeptide protein truncation. This discovery has broad implications in the basic science of genome maintenance, wound healing, cancer, combinatorial therapy, precision medicine, and technologies such as gene editing.