DAXX-ATRX-H3.3 Regulation of p53 Chromatin Binding and DNA Damage Response [ATAC-Seq]

DAXX and ATRX are tumor suppressor proteins that form a complex with histone H3.3 chaperone and are frequently mutated in cancers with the alternative lengthening of telomeres (ALT), such as pediatric glioblastoma. Rapid loss of function of either DAXX or ATRX are not by themselves sufficient to induce the ALT phenotype. However, cells lacking DAXX or ATRX can be readily selected for ALT-like features. Here, we show that a key feature of ALT selected DAXX and ATRX null glioblastoma cells is the attenuation of p53 function. RNA-seq analysis of DAXX or ATRX null U87 glioblastoma cells with ALT-like features revealed that p53 pathway is among perturbed. ALT-selected DAXX and ATRX-null cells had aberrant response to DNA damaging agent etoposide. Both DAXX and ATRX-null ALT cells showed a loss of p53 binding at a subset of response elements. Complementation of DAXX null cells with wt DAXX rescued p53 binding and transcription, while the tumor associated mutation L130R, that disrupts ATRX binding, was incapable of rescuing p53 chromatin binding. We show that histone H3.3 binding is reduced in DAXX-null cells especially at subtelomeric p53 binding sites and telomere repeats. These findings indicate that DAXX and ATRX function to enable p53 chromatin binding through modulation of histone H3.3 binding, especially at sub-telomeric sites. ATAC-seq in DAXX/ATRX WT and DAXX KO or ATRX KO conditions in control or etoposide treated cells

DAXX与ATRX均为肿瘤抑制蛋白（tumor suppressor protein），可与组蛋白H3.3伴侣蛋白（histone H3.3 chaperone）形成复合物，在携带端粒替代延长（alternative lengthening of telomeres, ALT）表型的癌症（如儿童胶质母细胞瘤）中常发生突变。仅快速丧失DAXX或ATRX其中任一的功能，不足以诱导ALT表型的产生。但缺失DAXX或ATRX的细胞可被筛选出类ALT特征。本研究发现，经ALT筛选的DAXX和ATRX双敲除（knockout, KO）胶质母细胞瘤细胞的核心特征为p53功能衰减。对携带类ALT特征的DAXX或ATRX敲除U87胶质母细胞瘤细胞进行RNA测序（RNA-seq）分析后发现，p53通路处于紊乱状态。经ALT筛选的DAXX和ATRX双敲除细胞对DNA损伤剂依托泊苷（etoposide）的应答存在异常。DAXX与ATRX双敲除的ALT细胞在部分应答元件上出现p53结合能力丧失的现象。用野生型（wild-type, wt）DAXX对DAXX敲除细胞进行互补实验，可恢复p53结合与转录功能；而与肿瘤相关的L130R突变（该突变可破坏ATRX结合）则无法恢复p53的染色质结合能力。本研究证实，DAXX敲除细胞中组蛋白H3.3的结合能力降低，尤其在亚端粒p53结合位点与端粒重复序列区域。上述结果表明，DAXX与ATRX可通过调控组蛋白H3.3的结合，尤其是在亚端粒区域，实现p53的染色质结合功能。本研究对对照组及依托泊苷处理条件下的DAXX/ATRX野生型、DAXX敲除或ATRX敲除细胞进行了转座酶可及性测序（ATAC-seq）