Genome-wide profiling of ?H2AX in Arabidopsis

The R-loop, composed of a DNA-RNA hybrid and the displaced single-stranded DNA, regulates diverse cellular processes. However, how cellular R-loops are sensed remain poorly understood. Here we report the discovery of the evolutionally conserved ALBA proteins (AtALBA1&2) functioning as the R-loops sensor of genic regions in higher plant Arabidopsis thaliana. AtALBA1 and 2 form a heterodimer protein complex and localize in the nucleus. While AtALBA1 binds to the DNA-RNA hybrid strand, AtALBA2 associates with single-stranded DNA in the R-loops. AtALBA1&2 preferentially binds to the genic R-loop regions that are associated with active epigenetic marks. Depletion of AtALBA1 or AtALBA2 results in hypersensitivity of plants to DNA damaging agents. Our results demonstrate the unprotected R-loops by loss of AtALBA1&2 are main source of DNA damage through ?H2AX ChIP-seq. Overall design: We conducted ?H2AX ChIP-seq with Arabidopsis wild-type (Col-0) and double mutant (alba1-1/alba2-1) seedlings to proflie the DNA damage .

由DNA-RNA杂交双链与置换出的单链DNA组成的R环（R-loop），可调控诸多细胞生命活动。然而，目前学界对细胞内R环的感知机制尚未得到清晰阐释。本研究报道了进化保守的ALBA蛋白（ALBA proteins）的功能：高等植物拟南芥中的AtALBA1与AtALBA2可作为基因区域R环的感知因子。AtALBA1与AtALBA2可形成异源二聚体蛋白复合物，并定位于细胞核中；其中AtALBA1结合DNA-RNA杂交双链，而AtALBA2则与R环中的单链DNA相结合。AtALBA1与AtALBA2优先结合带有活性表观遗传标记的基因区域R环。敲除或敲低AtALBA1或AtALBA2会使植物对DNA损伤试剂呈现超敏反应。通过γH2AX染色质免疫共沉淀测序（ChIP-seq）分析，本研究证实：AtALBA1与AtALBA2缺失后未受保护的R环是引发DNA损伤的主要来源。实验整体设计：我们以拟南芥野生型（Col-0）与双突变体（alba1-1/alba2-1）幼苗为材料，开展γH2AX染色质免疫共沉淀测序（ChIP-seq），以解析DNA损伤的分布特征。