Embryonic stem cell factors DPPA2/4 amplify active H3K4me3-H2AK119ub chromatin domains in non-small cell lung cancer (ATAC-seq)

Embryonic regulators are often re-expressed in cancers, however the functional and molecular significance of this is not always understood. The epigenetic priming factors Developmental Pluripotency Associated 2 and 4 (DPPA2/4) have crucial roles in early development and are implicated in cancer pathogenesis. We reveal in non-small cell lung cancer (NSCLC), DPPA2/4 co-expression is associated with poorly differentiated tumours and impaired patient outcomes. Biochemically, human DPPA2/4 multimerise for their protein stability and enhanced nucleosome binding activity. In NSCLC cells, DPPA2/4 bind CG-rich sequences including promoters of developmental, Wnt signaling and catabolic genes. Chromatin state modelling revealed DPPA2/4 preferentially bind active H3K4me3 and H3K27ac domains that were intriguingly also enriched for PRC1 and its product H2AK119ub, validated by H3K4me3-H2AK119ub sequential ChIP. Knockdown experiments revealed DPPA2/4 were required to maintain RING1B and H2AK119ub at these domains. Surprisingly, despite the presence of PRC2.1, these regions lacked any detectable H3K27me3, suggesting an uncoupling between the recruitment of PRC2 to chromatin and its catalytic product. When exogenously over-expressed in NSCLC cells where they are not normally present, DPPA2/4 bind to and promote active chromatin states, resulting in an increase in vivo xenograft tumour growth. Our results demonstrate how in NSCLC cells, DPPA2/4 act as molecular amplifiers of active and poised chromatin. Together, this highlights how aberrant re-activation of embryonic factors in cancers may take on new functions, promoting tumourigenesis. Overall design: The NCI-H661 cell line is derived from lung large cell carcinoma (subtype of non-small cell lung cancer) metastases and expresses both DPPA2 and DPPA4 highly at the mRNA and protein level. In order to investigate the functional consequence of peturbing DPPA2/4 we performed shRNA-mediated knockdown of DPPA2 or/and DPPA4 in NCI-H661 cells followed by ATAC-seq analyses. Analyses were conducted for shRNA models following 7 days of induction with either H20 vehicle control or 2ug/mL doxycyline, for control shRNA (shREN), shDPPA2, shDPPA4 or both shDPPA2+4. All conditions were done in biological duplicates.

胚胎发育调控因子常于癌症中异常重表达，但其对应的功能与分子意义尚未完全明确。表观遗传预激活因子（epigenetic priming factors）发育多能性相关蛋白2与4（Developmental Pluripotency Associated 2 and 4，DPPA2/4）在早期胚胎发育中发挥核心作用，且被证实参与癌症发生发展。本研究在非小细胞肺癌（non-small cell lung cancer, NSCLC）中发现，DPPA2/4共表达与低分化肿瘤及患者不良预后显著相关。生化实验显示，人源DPPA2/4通过形成多聚体维持蛋白稳定性，并增强其与核小体的结合活性。在非小细胞肺癌细胞中，DPPA2/4可结合富含CG的序列区域，包括发育相关基因、Wnt信号通路基因及分解代谢基因的启动子区域。染色质状态建模分析显示，DPPA2/4优先结合活性组蛋白修饰区域组蛋白H3赖氨酸4三甲基化（H3K4me3）与组蛋白H3赖氨酸27乙酰化（H3K27ac）结构域；有趣的是，这些区域同时富集多梳抑制复合体1（Polycomb Repressive Complex 1，PRC1）及其催化产物组蛋白H2A赖氨酸119单泛素化（H2AK119ub），该结果经H3K4me3-H2AK119ub串联染色质免疫沉淀（sequential Chromatin Immunoprecipitation, sequential ChIP）验证。敲低实验结果表明，DPPA2/4是维持上述染色质区域中RING指蛋白1B（RING finger protein 1B，RING1B）与H2AK119ub水平的必需因子。令人意外的是，尽管这些区域存在多梳抑制复合体2.1（Polycomb Repressive Complex 2.1，PRC2.1），却未检测到任何组蛋白H3赖氨酸27三甲基化（H3K27me3）修饰，提示PRC2招募至染色质与其催化产物的形成之间存在解偶联现象。当DPPA2/4在正常情况下不表达的非小细胞肺癌细胞中外源过表达时，它们可结合染色质并促进活性染色质状态的建立，最终导致体内异种移植瘤的生长速度加快。本研究结果证实，在非小细胞肺癌细胞中，DPPA2/4可作为活性染色质与预备性染色质（poised chromatin）的分子放大器。综上，本研究揭示了癌症中胚胎因子的异常重表达如何获得全新功能，进而促进肿瘤发生发展。

实验设计：NCI-H661细胞系源自肺大细胞癌（非小细胞肺癌的亚型）转移灶，其在mRNA与蛋白水平均高表达DPPA2与DPPA4。为探究敲低DPPA2/4的功能效应，本研究通过短发夹RNA（short hairpin RNA, shRNA）介导在NCI-H661细胞中分别或共同敲低DPPA2、DPPA4，随后进行转座酶可及性测序（Assay for Transposase-Accessible Chromatin using sequencing, ATAC-seq）分析。所有shRNA模型均经7天诱导处理，诱导剂为2μg/mL多西环素（doxycycline），对照组为H2O溶剂对照，涵盖的实验组包括对照shRNA（shREN）、shDPPA2、shDPPA4以及shDPPA2+4双敲组，所有实验条件均设置生物学重复两次。