Centro Andaluz de Biología Molecular y Medicina Regenerativa CABIMER, Universidad de Sevilla, 41092 Seville, Spain.
Genes Dev. 2014 Apr 1;28(7):735-48. doi: 10.1101/gad.234070.113. Epub 2014 Mar 17.
FACT (facilitates chromatin transcription) is a chromatin-reorganizing complex that swaps nucleosomes around the RNA polymerase during transcription elongation and has a role in replication that is not fully understood yet. Here we show that recombination factors are required for the survival of yeast FACT mutants, consistent with an accumulation of DNA breaks that we detected by Rad52 foci and transcription-dependent hyperrecombination. Breaks also accumulate in FACT-depleted human cells, as shown by γH2AX foci and single-cell electrophoresis. Furthermore, FACT-deficient yeast and human cells show replication impairment, which in yeast we demonstrate by ChIP-chip (chromatin immunoprecipitation [ChIP] coupled with microarray analysis) of Rrm3 to occur genome-wide but preferentially at highly transcribed regions. Strikingly, in yeast FACT mutants, high levels of Rad52 foci are suppressed by RNH1 overexpression; R loops accumulate at high levels, and replication becomes normal when global RNA synthesis is inhibited in FACT-depleted human cells. The results demonstrate a key function of FACT in the resolution of R-loop-mediated transcription-replication conflicts, likely associated with a specific chromatin organization.
FACT（促进染色质转录）是一种染色质重排复合物，在转录延伸过程中围绕 RNA 聚合酶交换核小体，在复制过程中具有尚未完全理解的作用。在这里，我们表明重组因子是酵母 FACT 突变体存活所必需的，这与我们通过 Rad52 焦点和转录依赖性超重组检测到的 DNA 断裂积累一致。在 FACT 耗尽的人类细胞中也会积累断裂，如 γH2AX 焦点和单细胞电泳所示。此外，FACT 缺陷型酵母和人类细胞显示出复制损伤，我们在酵母中通过 Rrm3 的 ChIP-chip（染色质免疫沉淀 [ChIP] 与微阵列分析）证明这种损伤是全基因组的，但在转录活跃区域更为明显。引人注目的是，在酵母 FACT 突变体中，高水平的 Rad52 焦点被 RNH1 过表达抑制；R 环积累水平升高，当抑制 FACT 耗尽的人类细胞中的全局 RNA 合成时，复制恢复正常。结果表明 FACT 在解决 R 环介导的转录-复制冲突方面具有关键作用，可能与特定的染色质组织有关。
