ATRX ADD domain is a versatile module for recognizing macroH2A, H3, and beyond.

Alpha Thalassemia/Mental developmental retardation, X-linked (ATRX) is an important heterochromatin regulator, frequent mutated in ATR-X syndrome and various cancers. ATRX binds a histone variant macroH2A, forming a functional axis crucial for transcription regulation and genome stability. However, the molecular mechanism underlying the ATRX-macroH2A interaction remains obscure. Here we demonstrate that the ADD domain of ATRX (ATRX _ADD) specifically binds the histone-fold domain of macroH2A, but not the canonical H2A. The binding specificity is mediated by a D/E-rich loop of ATRX _ADD and the L ₁₂ loop of macroH2A. A swapping mutation in the L ₁₂ loop of macroH2A disrupts ATRX binding, whereas the reverse mutation in H2A confers binding capacity with ATRX. Notably, ATRX _ADD employs a conserved interface to recognize both macroH2A and H3, leading to competition between macroH2A and H3 for ATRX binding. Furthermore, affinity purification and mass spectrometry identify NuRD components as the potential ATRX _ADD-associating proteins, with CDH4 mimicking H3 in its direct interaction with ATRX _ADD. These findings elucidate the molecular basis of ATRX's interaction with macroH2A and NuRD, and also demonstrate the versatility of ATRX _ADD in recognizing diverse chromatin regulators, providing insights into ATRX's multifaceted roles in epigenetic regulation and pathogenesis.