HIRA protects telomeres against R-loop-induced instability in ALT cancer cells [CUT&RUN]. HIRA protects telomeres against R-loop-induced instability in ALT cancer cells [CUT&RUN]

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: CUT&RUN 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，被认定为癌症特异性端粒替代延长（Alternative Lengthening of Telomeres, ALT）通路的驱动因素。既往研究显示，HIRA会通过代偿机制弥补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分子机制仍未阐明。本研究借助ATAC-seq与CUT&RUN技术，证实了在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、防止端粒区域无核小体单链DNA（single-stranded DNA, ssDNA）的病理性积累至关重要。我们的研究证据表明，HIRA及其相互作用蛋白UBN1与UBN2及时沉积新生H3.3，对于抑制端粒区域异常的TERRA R环形成以及转录-复制冲突（transcription-replication conflicts, TRCs）具有关键作用。此外，我们发现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的磷酸化修饰，与促进有效ALT发生的分子机制联系起来。因此，本研究明确了HIRA介导的组蛋白H3.3沉积在TERRA R环稳态调控中的功能，我们提出该调控过程对于确保ATRX-DAXX复合物功能缺陷的ALT癌细胞的存活至关重要。总体实验设计：本研究的CUT&RUN样本来自经HIRA小干扰RNA（small interfering RNA, siRNA）或对照siRNA处理的人源IMR90野生型（WT，即对照组CTRL）或ATRX敲除（KO）细胞，其中ATRX KO细胞即为ALT模型细胞，所有样本均设置3次生物学重复（n=3）。