HIRA protects telomeres against R-loop-induced instability in ALT cancer cells [ATAC-seq]. HIRA protects telomeres against R-loop-induced instability in ALT cancer cells [ATAC-seq]

Alterations to chromatin modifiers, such as the inactivating mutations in the ATRX-DAXX chromatin remodeling/histone H3.3 deposition complex, are implicated as drivers of the cancer-specific Alternative Lengthening of Telomeres (ALT) pathway. Prior studies revealed that HIRA adapts to compensate for ATRX-DAXX loss to sustain ALT cancer cell survival. However, the specific mechanisms underlying HIRA’s ability to rescue telomeres from the consequences of ATRX-DAXX loss remain unclear. Here, using ATAC-seq and CUT&RUN, we demonstrate that HIRA-mediated deposition of new H3.3 is essential to maintain chromatin accessibility and to prevent the detrimental accumulation of nucleosome-free single-stranded DNA (ssDNA) at telomeres in ATRX-deficient ALT cancer cells. We provide evidence that the timely deposition of new H3.3 by HIRA and interacting partners UBN1 and UBN2 is crucial to prevent unwarranted TERRA R-loop formation and transcription-replication conflicts (TRCs) at telomeres. Furthermore, we determined that the delivery of H3.3 to telomeric chromatin by HIRA may link the phosphorylation of an H3.3-specific amino acid, serine 31, by Chk1 with mechanisms that promote productive ALT. Therefore, these studies identify a role for HIRA-mediated histone H3.3 deposition in TERRA R-loop homeostasis that we propose is essential for ensuring the survival of ALT cancer cells where the ATRX-DAXX complex is activated. Overall design: ATAC-seq samples from human IMR90 WT (CTRL) or ATRX KO (ALT) cells treated with HIRA siRNA or control siRNA treatment. Sets of samples in biological triplicate (n=3).

染色质修饰因子的异常改变，例如ATRX-DAXX染色质重塑/组蛋白H3.3沉积复合物的失活突变，被证实为癌症特异性端粒替代延长（Alternative Lengthening of Telomeres，ALT）通路的驱动因素。既往研究表明，HIRA可通过代偿机制弥补ATRX-DAXX的缺失，以维持ALT癌细胞的存活。然而，HIRA能够挽救端粒免受ATRX-DAXX缺失所带来的不良影响的具体机制仍不明确。
本研究通过转座酶可及性测序测定（ATAC-seq）与靶标切割与释放测序（CUT&RUN），证实：在ATRX缺陷型ALT癌细胞中，HIRA介导的新生H3.3沉积对于维持染色质开放性、防止端粒处无核小体单链DNA（single-stranded DNA，ssDNA）的有害积累至关重要。
我们的研究证据显示，HIRA及其相互作用蛋白UBN1与UBN2及时沉积新生H3.3，对于阻止端粒处异常的TERRA R-loop形成以及转录-复制冲突（transcription-replication conflicts，TRCs）至关重要。
此外，本研究明确，HIRA将H3.3递送至端粒染色质的过程，可能将Chk1对H3.3特异性氨基酸丝氨酸31的磷酸化，与促进功能性ALT发生的机制相关联。
因此，本研究揭示了HIRA介导的组蛋白H3.3沉积在TERRA R-loop稳态调控中的作用，我们认为该过程对于ATRX-DAXX复合物被激活的ALT癌细胞的存活至关重要。
实验整体设计：取自人类IMR90野生型（WT，CTRL）或ATRX敲除（KO，ALT）细胞的ATAC-seq样本，分别经HIRA小干扰RNA（siRNA）或对照siRNA处理。所有样本均设置生物学重复三次（n=3）。