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

Abstract

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 (ATRXADD) 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 ATRXADD and the L12 loop of macroH2A. A swapping mutation in the L12 loop of macroH2A disrupts ATRX binding, whereas the reverse mutation in H2A confers binding capacity with ATRX. Notably, ATRXADD 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 ATRXADD-associating proteins, with CDH4 mimicking H3 in its direct interaction with ATRXADD. These findings elucidate the molecular basis of ATRX's interaction with macroH2A and NuRD, and also demonstrate the versatility of ATRXADD in recognizing diverse chromatin regulators, providing insights into ATRX's multifaceted roles in epigenetic regulation and pathogenesis.

1. Introduction

ATRX (Alpha Thalassemia/Mental Retardation Syndrome X-linked) is a critical chromatin remodeling protein initially identified in connection with alpha-thalassemia and intellectual disability (ATR-X syndrome) [1]. ATRX plays central roles in transcription regulation, telomere maintenance, heterochromatin organization, and the DNA damage response [2]. In mouse models, complete ATRX knockout results in early embryonic lethality [3], while its conditional deletion in the central nervous system causes postnatal death [4,5]. Mutations in ATRX are the primary cause of ATR-X syndrome [6,7] and are frequently observed in various cancers, particularly pediatric gliomas and pancreatic neuroendocrine tumors [8].

Disease-causing mutations in ATRX are frequently clustered in two structural domains with distinct functions: the ATPase domain and the ADD (ATRX-DNMT3-DNMT3L) domain [4,9]. The ATPase domain, a SNF2 (Sucrose Non-Fermenting)-type chromatin remodeling domain, exhibits an ATP hydrolysis-dependent DNA translocase activity [10,11]. By leveraging this translocase activity, the ATRX complex can mobilize nucleosomes, facilitating chromatin remodeling and the transcription of specific genes, especially the α-globin gene [11,12]. Additionally, the remodeling activity of ATRX resolves G-quadruplex structures, preventing replication fork stalling and maintaining genome stability [13‒15].

The ADD domain of ATRX (ATRXADD) is a specialized zinc finger domain that recognizes histone modifications. ATRXADD specifically binds a dual marker consisting of unmodified lysine 4 (H3K4me0) and trimethylated lysine 9 (H3K9me3) on histone H3 [16‒18]. This selective interaction enables ATRX to localize to heterochromatin regions [18]. To date, histone H3 remains the only well-characterized binding partner of ATRXADD. The limited interaction spectrum of ATRXADD stands in sharp contrast to its vital role in heterochromatin organization and its frequent mutations in ATR-X syndrome. Whether ATRXADD engages with other proteins beyond histone H3 to fulfil additional regulatory roles remains an open question.