Antibodies.

The FA/BRCA pathway safeguards DNA replication by repairing interstrand crosslinks (ICL) and maintaining replication fork stability. Chromatin structure, which is in part regulated by histones posttranslational modifications (PTMs), has a role in maintaining genomic integrity through stabilization of the DNA replication fork and promotion of DNA repair. An appropriate balance of PTMs, especially acetylation of histones H4 in nascent chromatin, is required to preserve a stable DNA replication fork. To evaluate the acetylation status of histone H4 at the replication fork of FANCA deficient cells, we compared histone acetylation status at the DNA replication fork of isogenic FANCA deficient and FANCA proficient cell lines by using accelerated native immunoprecipitation of nascent DNA (aniPOND) and in situ protein interactions in the replication fork (SIRF) assays. We found basal hypoacetylation of multiple residues of histone H4 in FA replication forks, together with increased levels of Histone Deacetylase 1 (HDAC1). Interestingly, high-dose short-term treatment with mitomycin C (MMC) had no effect over H4 acetylation abundance at the replication fork. However, chemical inhibition of histone deacetylases (HDAC) with Suberoylanilide hydroxamic acid (SAHA) induced acetylation of the FANCA deficient DNA replication forks to levels comparable to their isogenic control counterparts. This forced permanence of acetylation impacted FA cells homeostasis by inducing DNA damage and promoting G2 cell cycle arrest. Altogether, this caused reduced RAD51 foci formation and increased markers of replication stress, including phospho-RPA-S33. Hypoacetylation of the FANCA deficient replication fork, is part of the cellular phenotype, the perturbation of this feature by agents that prevent deacetylation, such as SAHA, have a deleterious effect over the delicate equilibrium they have reached to perdure despite a defective FA/BRCA pathway.

范可尼贫血/乳腺癌易感基因（FA/BRCA）通路通过修复链间交联（interstrand crosslinks, ICL）并维持DNA复制叉（DNA replication fork）稳定性，保障DNA复制进程。染色质结构在一定程度上受组蛋白翻译后修饰（histones posttranslational modifications, PTMs）调控，其可通过稳定DNA复制叉、促进DNA修复以维持基因组完整性。维持恰当的组蛋白翻译后修饰平衡，尤其是新生染色质（nascent chromatin）中组蛋白H4的乙酰化水平，是稳定DNA复制叉的必要条件。为评估FANCA缺陷细胞复制叉处组蛋白H4的乙酰化状态，本研究采用新生DNA加速原生免疫沉淀（accelerated native immunoprecipitation of nascent DNA, aniPOND）与复制位点原位蛋白质相互作用（in situ protein interactions in the replication fork, SIRF）实验技术，对比了同基因背景的FANCA缺陷型与FANCA功能正常型细胞系在DNA复制叉处的组蛋白乙酰化状态。研究发现，FA通路缺陷的复制叉中存在组蛋白H4多个位点的基础低乙酰化现象，同时伴随组蛋白去乙酰化酶1（Histone Deacetylase 1, HDAC1）水平升高。值得注意的是，高剂量短期丝裂霉素C（mitomycin C, MMC）处理并未对复制叉处的H4乙酰化丰度产生显著影响。然而，采用辛二酰苯胺异羟肟酸（Suberoylanilide hydroxamic acid, SAHA）对组蛋白去乙酰化酶（histone deacetylases, HDAC）进行化学抑制，可将FANCA缺陷细胞的DNA复制叉乙酰化水平恢复至与其同基因对照相当的程度。这种强制维持的乙酰化状态会干扰FA细胞的稳态：一方面诱导DNA损伤，另一方面促进G2期细胞周期阻滞（G2 cell cycle arrest）。综合来看，这会导致RAD51焦点（RAD51 foci）形成减少，并加剧复制应激相关标志物（包括磷酸化RPA-S33（phospho-RPA-S33））的表达水平升高。综上，FANCA缺陷型复制叉的低乙酰化是细胞表型的一部分；通过辛二酰苯胺异羟肟酸等阻断去乙酰化的试剂扰动这一特征，会对FA/BRCA通路缺陷细胞为维持存活所建立的微妙平衡产生有害影响。