Impaired Cell Fate by Gain-of-function Mutations in a Chromatin Reader

We previously identified the YEATS domain-containing protein ENL as a reader of histone acetylation. Recently, hotspot mutations in ENL were frequently found in Wilms' tumor, the most common type of pediatric kidney cancer. Here, we report that these cancer-associated mutations in the ENL YEATS domain confer gain of functions in transcriptional control and impair kidney differentiation by driving self-reinforced chromatin targeting. Ectopic expression of ENL mutants in kidney cell lines resulted in transcriptional changes of genes enriched in embryonic nephron progenitors and Wilms' tumor. When tested in a nephrogenesis assay, ENL mutant expression led to undifferentiated structures resembling those observed in human Wilms' tumor. Genome-wide analyses revealed that while mutant ENL bound to largely similar genomic loci as wild-type ENL, they exhibited increased occupancy at a subset of targets, including developmentally critical genes such as the HOXA cluster. The cancer-associated mutations enabled self-reinforced recruitment of ENL on chromatin by promoting self-association, resulting in the formation of discrete nuclear puncta that are characteristic of phase-separated biomolecular condensates. Enhanced occupancy of ENL mutants led to a marked increase in the recruitment and activity of transcription elongation machinery that enforces active transcription from target loci­­­­. Collectively, our studies represent, to our knowledge, the first discovery that cancer-associated mutations in a chromatin reader drive self-reinforced chromatin targeting, which in turns, perturbs developmental programs and derails normal cell fate control during mammalian development towards an oncogenic path. Overall design: Established stable cell lines to ectopic express either wild type or mutant human ENL in HEK293 and HK2 cell lines, and used these cell lines for Flag, CDK9 and Pol II S2P ChIP-seq and RNA-seq analysis.

我们此前已鉴定出含YEATS结构域（YEATS domain）的蛋白ENL为组蛋白乙酰化阅读器。近期研究发现，在儿童最常见的肾脏恶性肿瘤肾母细胞瘤（Wilms' tumor）中，ENL常存在热点突变。本研究表明，ENL的YEATS结构域上的这些癌症相关突变可获得转录调控相关的功能增益，并通过驱动自我强化的染色质靶向作用损害肾脏分化过程。在肾细胞系中异位表达ENL突变体，可使富集于胚胎肾祖细胞及肾母细胞瘤的基因出现转录表达变化。在肾发生实验中检测时，ENL突变体的表达会形成未分化结构，其形态与人类肾母细胞瘤中观察到的结构相似。全基因组分析结果显示，尽管突变型ENL与野生型ENL的结合基因组位点大体相似，但突变型ENL在部分靶位点的结合占据率有所升高，其中包括发育关键基因如HOXA基因簇（HOXA cluster）。这类癌症相关突变可通过促进ENL自身缔合，使其在染色质上实现自我强化的招募，进而形成离散的核斑点结构——这正是相分离生物分子凝聚体的典型特征。ENL突变体结合占据率的升高，会显著增强转录延伸复合物的招募及其活性，从而促使靶位点维持活跃转录状态。综上，据我们所知，本研究首次发现：染色质阅读器（chromatin reader）上的癌症相关突变可驱动自我强化的染色质靶向作用，进而干扰发育程序，在哺乳动物发育过程中破坏正常细胞命运调控，最终走向致癌通路。
实验设计：在HEK293与HK2细胞系中构建分别异位表达野生型或突变型人源ENL的稳定细胞系，随后利用这些细胞系开展Flag标签蛋白、CDK9以及磷酸化S2位点的RNA聚合酶II（Pol II S2P）的染色质免疫共沉淀测序（ChIP-seq）与转录组测序（RNA-seq）分析。