Histone serotonylation regulates ependymoma tumorigenesis

Chen, Hsiao-Chi; He, Peihao; McDonald, Malcolm; Williamson, Michael R.; Varadharajan, Srinidhi; Lozzi, Brittney; Woo, Junsung; Choi, Dong-Joo; Sardar, Debosmita; Huang-Hobbs, Emmet; Sun, Hua; Ippagunta, Siri M.; Jain, Antrix; Rao, Ganesh; Merchant, Thomas E.; Ellison, David W.; Noebels, Jeffrey L.; Bertrand, Kelsey C.; Mack, Stephen C.; Deneen, Benjamin

doi:10.1038/s41586-024-07751-z

Article
Published: 31 July 2024

Histone serotonylation regulates ependymoma tumorigenesis
组蛋白色氨酸酰化调控室管膜瘤肿瘤发生

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Abstract 摘要

Bidirectional communication between tumours and neurons has emerged as a key facet of the tumour microenvironment that drives malignancy^1,2. Another hallmark feature of cancer is epigenomic dysregulation, in which alterations in gene expression influence cell states and interactions with the tumour microenvironment³. Ependymoma (EPN) is a paediatric brain tumour that relies on epigenomic remodelling to engender malignancy^4,5; however, how these epigenetic mechanisms intersect with extrinsic neuronal signalling during EPN tumour progression is unknown. Here we show that the activity of serotonergic neurons regulates EPN tumorigenesis, and that serotonin itself also serves as an activating modification on histones. We found that inhibiting histone serotonylation blocks EPN tumorigenesis and regulates the expression of a core set of developmental transcription factors. High-throughput, in vivo screening of these transcription factors revealed that ETV5 promotes EPN tumorigenesis and functions by enhancing repressive chromatin states. Neuropeptide Y (NPY) is one of the genes repressed by ETV5, and its overexpression suppresses EPN tumour progression and tumour-associated network hyperactivity through synaptic remodelling. Collectively, this study identifies histone serotonylation as a key driver of EPN tumorigenesis, and also reveals how neuronal signalling, neuro-epigenomics and developmental programs are intertwined to drive malignancy in brain cancer.
肿瘤与神经元之间的双向交流已成为推动恶性肿瘤发展的肿瘤微环境关键方面 ^1,2 。癌症的另一个标志性特征是表观基因组失调，其中基因表达的改变影响细胞状态及其与肿瘤微环境的相互作用 ³ 。室管膜瘤（EPN）是一种依赖表观遗传重塑来引发恶性变化的儿童脑肿瘤 ^4,5 ；然而，这些表观遗传机制如何在 EPN 肿瘤进展过程中与外部神经信号相互作用尚不清楚。在此，我们表明血清素能神经元的活性调控 EPN 肿瘤发生，而血清素本身也作为一种激活性组蛋白修饰发挥作用。我们发现抑制组蛋白的血清素化可阻断 EPN 肿瘤发生并调控一组核心发育转录因子的表达。通过高通量体内筛选这些转录因子，揭示 ETV5 促进 EPN 肿瘤发生，其功能是通过增强抑制性染色质状态实现的。神经肽 Y（NPY）是 ETV5 抑制的基因之一，其过表达通过突触重塑抑制了 EPN 肿瘤进展及肿瘤相关网络过度活跃。总体而言，本研究揭示了组蛋白血清素化作为 EPN 肿瘤发生的关键驱动因素，并阐明了神经信号传导、神经表观基因组学及发育程序如何交织在一起，推动脑癌的恶性发展。

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Main 主

Ependymomas (EPNs) are aggressive paediatric brain tumours that are resistant to chemotherapy and lack targeted therapies, leading to poor survival and neurocognitive outcomes⁶. EPNs can be divided into at least nine molecular subtypes associated with distinct genetic and epigenetic alterations. Approximately 70% of supratentorial EPNs (ST-EPNs) are characterized by a gene fusion between ZFTA and RELA (ZFTA–RELA fusion; hereafter, ZR^FUS)^7,8. Among ST-EPNs, those bearing ZR^FUS are particularly aggressive, and this is reflected in the decreased progression-free survival compared with other forms of ST-EPN that are driven by YAP1 fusions⁷. Paediatric brain tumours exhibit an exquisite dependency on epigenomic mechanisms, such as those controlled by core transcriptional regulatory proteins^4,5,9,10. Studies indicate that ZR^FUS functions as an aberrant transcription factor that recruits chromatin modifiers to activate oncogenic transcriptional programs that are linked to neural development^11,12,13,14. Although these global alterations have been detected, the precise mechanisms that drive aberrant epigenomic states in ZR^FUS EPN are obscure, and are likely to be multifactorial, encompassing both cell-intrinsic and cell-extrinsic factors. Separate investigations have shown that the neuromodulator serotonin can be integrated into histones and serve as an epigenetic mark that regulates gene expression in both neurons and astrocytes, ultimately controlling circuit-level activities in the brain^15,16. Together, these observations raise the possibility that neurons can remodel the tumour epigenome through the release of neuroactive compounds that are deposited on histones. Furthermore, previous studies have highlighted bidirectional signalling between brain tumours and neurons, and have identified paracrine factors, direct synaptic integration and circuit-specific infiltration as key drivers of tumorigenesis^{17,18,19,20,21,22,23,24,25}. Nevertheless, how neuronal activity influences tumour growth in EPN is incompletely understood, and whether neuromodulators act as epigenetic regulators of brain-tumour progression is unknown.
室管膜瘤（EPNs）是一类侵袭性强的儿童脑肿瘤，对化疗耐药且缺乏靶向治疗手段，导致生存率低及神经认知结果不佳 ⁶ 。EPNs 至少可分为九种分子亚型，各具独特的遗传和表观遗传改变。约 70%的幕上室管膜瘤（ST-EPNs）特征为 ZFTA 与 RELA 基因融合（ZFTA–RELA 融合；以下简称 ZR ^FUS ） ^7,8 。在 ST-EPNs 中，携带 ZR ^FUS 的肿瘤尤为侵袭性强，其无进展生存期较由 YAP1 融合驱动的其他 ST-EPNs 缩短 ⁷ 。儿童脑肿瘤对表观基因组机制表现出高度依赖性，如受核心转录调控蛋白控制的机制 ^4,5,9,10 。研究表明，ZR ^FUS 作为异常转录因子，招募染色质修饰因子激活与神经发育相关联的致癌转录程序 ^11,12,13,14 。尽管已检测到这些全球性的改变，但驱动 ZR ^FUS EPN 中异常表观基因组状态的确切机制仍不明确，且很可能是多因素的，涉及细胞内在和外在因素。独立研究表明，神经调节物质血清素能够整合入组蛋白并作为一种表观遗传标记，调控神经元和星形胶质细胞中的基因表达，最终控制大脑中的环路级活动 ^15,16 。这些观察结果共同提出了一个可能性：神经元通过释放沉积在组蛋白上的神经活性化合物，能够重塑肿瘤表观基因组。此外，先前研究强调了脑肿瘤与神经元之间的双向信号传导，并指出了旁分泌因子、直接突触整合及特定环路渗透作为肿瘤发生的关键驱动因素 ^{17,18,19,20,21,22,23,24,25} 。然而，神经活动如何影响 EPN 中的肿瘤生长尚不完全清楚，神经调节物质是否作为脑肿瘤进展的表观遗传调控因子也仍属未知。

Serotonergic neurons suppress EPN growth
血清素能神经元抑制嗜睡症结节增长

To understand how the ZR^FUS fusion promotes EPN tumorigenesis, we compared the transcriptomic profiles of patients with ZR^FUS-driven tumours with those lacking the fusion¹⁰. This comparison revealed a selective enrichment of gene ontology (GO) sets involved in neuronal function, synaptic organization and neurotransmission in EPN tumours that contain ZR^FUS (Fig. 1a and Supplementary Table 1). These pathways are enriched at the level of RNA sequencing (RNA-seq), and are supported by the accumulation of active H3K27ac chromatin marks, which we previously showed to be enriched in ion channels and neurotransmitter pathways¹⁰.
为了理解 ZR ^FUS 融合如何促进 EPN 肿瘤发生，我们将 ZR ^FUS 驱动肿瘤患者的转录组特征与缺乏该融合的患者进行了比较 ¹⁰ 。这一比较揭示了在含有 ZR ^FUS 的 EPN 肿瘤中，与神经功能、突触组织及神经传递相关的基因本体（GO）集有选择性富集（图 1a 及补充表 1）。这些通路在 RNA 测序（RNA-seq）层面得到富集，并得到活性 H3K27ac 染色质标记积累的支持，我们此前已证明这些标记在离子通道和神经递质通路中富集 ¹⁰ 。

Fig. 1: Stimulation of serotonergic neurons suppresses EPN tumorigenesis.
图 1：血清素能神经元的刺激抑制了 EPN 肿瘤的发生。

These findings implicate aberrant neuronal activity in the brain microenvironment in EPN tumorigenesis—a phenotype that has not been functionally evaluated in this disease. To test this hypothesis, we used our in utero electroporation (IUE) model of ZR^FUS EPN to perform DREADD-based activation of cortical neurons in the hemisphere ipsilateral to the tumour^11,19,26. Accordingly, we generated ZR^FUS tumours and then injected adeno-associated virus (AAV)-2/9 syn1-hM3Dq-mCherry at postnatal day (P) 5, stimulating neuronal activity by treating mice with either saline or clozapine N-oxide (CNO) twice a day for ten days, starting at P20 (Extended Data Fig. 1a). To confirm the activation of neurons, we used FOS staining in neurons as a proxy for increased neuronal activity, and confirmed increases in neurons at the peritumoral margin in CNO-treated mice (Extended Data Fig. 1h,i). Next, using the incorporation of bromodeoxyuridine (BrdU) as an index of cellular proliferation, we found an increase in BrdU staining in tumours treated with CNO (Extended Data Fig. 1b,c,g), coupled with a gross expansion in tumour size (Extended Data Fig. 1b). These findings indicate that the activation of cortical neurons promotes the proliferation of EPN tumours. To further analyse the neuronal subtypes that contribute to activity-dependent tumorigenesis, we used similar experimental paradigms, but instead selectively targeted excitatory (AAV-CaMKIIa promoter) or inhibitory (AAV-Dlx5/6) neurons (Fig. 1c and Extended Data Fig. 1d). The activation of excitatory neurons promoted tumour proliferation and gross expansion (Fig. 1d,e), whereas the activation of inhibitory neurons had no effect on tumour growth (Extended Data Fig. 1e,f). Together, these data indicate that the activation of cortical excitatory neurons promotes ZR^FUS EPN tumorigenesis.
这些发现暗示了脑微环境中异常的神经活动在 EPN 肿瘤发生中的作用——这一表型在此疾病中尚未得到功能性评估。为验证此假设，我们利用子宫内电穿孔（IUE）模型 ZR ^FUS EPN，在肿瘤同侧的大脑半球进行了基于 DREADD 的皮质神经元激活 ^11,19,26 。据此，我们生成了 ZR ^FUS 肿瘤，并在出生后第（P）5 天注射腺相关病毒（AAV）-2/9 syn1-hM3Dq-mCherry，从 P20 开始，每天两次用生理盐水或氯氮平 N-氧化物（CNO）处理小鼠，持续十天（扩展数据图 1a）。为确认神经元激活，我们采用 FOS 染色作为神经活动增强的标志，并证实 CNO 处理的小鼠肿瘤周围区域神经元活动增加（扩展数据图 1h,i）。接着，以溴脱氧尿苷（BrdU）掺入作为细胞增殖指标，我们发现 CNO 处理的肿瘤中 BrdU 染色增加（扩展数据图 1b,c,g），同时肿瘤体积显著扩大（扩展数据图 1b）。这些发现表明，皮质神经元的激活促进了 EPN 肿瘤的增殖。为了进一步分析参与活动依赖性肿瘤发生的神经元亚型，我们采用了类似的实验范式，但改为选择性靶向兴奋性（AAV-CaMKIIa 启动子）或抑制性（AAV-Dlx5/6）神经元（图 1c 及扩展数据图 1d）。兴奋性神经元的激活促进了肿瘤的增殖和整体扩张（图 1d,e），而抑制性神经元的激活对肿瘤生长无影响（扩展数据图 1e,f）。综上所述，这些数据表明皮质兴奋性神经元的激活促进了 ZR ^FUS EPN 的肿瘤发生。

Further analysis of the neurotransmitter profiles enriched in ZR^FUS EPN highlighted the expression of monoamine, dopamine and catecholamine transporters (Extended Data Fig. 2a–e and Supplementary Table 1). To validate these findings, we stained human and mouse ZR^FUS EPN tumours and identified an increase in the expression of the serotonin transporter SLC6A4, but not in that of the dopamine transporter SLC6A3 (Fig. 1b and Extended Data Fig. 2f,g). These observations suggest that the levels of serotonin in the brain microenvironment influence EPN tumorigenesis. Therefore, to manipulate the levels of serotonin in the brain, we used DREADD-based approaches to activate serotonergic neurons in the dorsal raphe nucleus (dRN), which are the predominant source of serotoninergic input to the cortex and are in a brain region remote to the primary tumour^27,28. Similar to the above studies, we used IUE to generate tumours, injected AAV-2/9 syn1-hM3Dq-mCherry in the dRN and stimulated neuronal activity by treating mice with CNO (or saline control) twice a day for ten days, followed by BrdU injection before collection (Fig. 1f). Owing to the anatomical constraints of the perinatal mouse brain, our dRN injections occurred at P28, followed by CNO-based stimulation from P35 to P46 (Fig. 1f). Using FOS expression as a proxy for neuronal activity, we confirmed CNO-induced increases in FOS expression in serotonergic neurons residing in the dRN (Extended Data Fig. 1j–l). Analysis of BrdU staining revealed a marked decrease in cell proliferation in CNO-treated tumours (Fig. 1g,h), accompanied by a decrease in gross tumour volume (Fig. 1g). Next, we performed the complimentary experiment, in which we injected the dRN with AAV-2/9 syn1-hM4Di-mCherry, which enabled us to inhibit neuronal activity in the dRN (Fig. 1i). Analysis of ZR^FUS EPN tumours revealed an increase in proliferation, coupled with a gross expansion of the tumour after the inhibition of dRN neurons (Fig. 1j,k). To understand how these manipulations of the remote dRN influence local neurons in the cortex, we stained the peritumoral neurons with synaptic markers, and did not observe any gross changes in the synaptic constituency of the peritumoral margin after manipulation of dRN neurons (Extended Data Fig. 3a–h). Together, these findings show that the activity of serotonergic neurons in the dRN suppresses the progression of EPN tumours. Collectively, these observations reveal circuit-specific effects on EPN tumorigenesis, in which broad stimulation of cortical neurons promotes growth, whereas stimulation of serotonergic neurons in the dRN suppresses growth.
进一步分析富集于 ZR ^FUS EPN 的神经递质特征，突出了单胺、多巴胺和儿茶酚胺转运体的表达（扩展数据图 2a-e 及补充表 1）。为验证这些发现，我们对人及小鼠 ZR ^FUS EPN 肿瘤进行了染色，并发现 5-羟色胺转运体 SLC6A4 表达增加，而多巴胺转运体 SLC6A3 表达未见变化（图 1b 及扩展数据图 2f,g）。这些观察结果提示，脑微环境中 5-羟色胺水平影响 EPN 肿瘤发生。因此，为调控脑内 5-羟色胺水平，我们采用基于 DREADD 的方法激活背侧缝核（dRN）内的 5-羟色胺能神经元，该区域是向皮质提供 5-羟色胺能输入的主要来源，且位于远离原发肿瘤 ^27,28 的脑区。与上述研究类似，我们通过 IUE 生成肿瘤，向 dRN 注射 AAV-2/9 syn1-hM3Dq-mCherry，并通过每日两次给予 CNO（或生理盐水对照）处理十天来刺激神经活动，随后在收集前注射 BrdU（图 1f）。由于新生小鼠大脑的解剖学限制，我们的 dRN 注射在 P28 进行，随后从 P35 到 P46 进行基于 CNO 的刺激（图 1f）。利用 FOS 表达作为神经元活性的代理指标，我们确认了 CNO 诱导的位于 dRN 中的血清素能神经元 FOS 表达增加（扩展数据图 1j-l）。BrdU 染色分析显示，CNO 处理过的肿瘤细胞增殖显著减少（图 1g,h），伴随肿瘤总体积的减小（图 1g）。接下来，我们进行了补充实验，向 dRN 注射 AAV-2/9 syn1-hM4Di-mCherry，从而抑制 dRN 中的神经元活动（图 1i）。对 ZR ^FUS EPN 肿瘤的分析显示，在抑制 dRN 神经元后，增殖增加，肿瘤总体积扩大（图 1j,k）。为了理解对远端 dRN 的这些操作如何影响皮质中的局部神经元，我们对肿瘤周围神经元进行了突触标记染色，并未观察到在操纵 dRN 神经元后肿瘤边缘突触组成的显著变化（扩展数据图 3a-h）。这些发现共同表明，背侧中缝核（dRN）中血清素能神经元的活动抑制了 EPN 肿瘤的进展。综合来看，这些观察结果揭示了 EPN 肿瘤发生中的特定神经回路效应：广泛刺激皮层神经元促进肿瘤生长，而刺激 dRN 中的血清素能神经元则抑制肿瘤生长。

Histone serotonylation governs EPN
组蛋白色氨酸羟化调控内吞蛋白

The above observations implicate serotonergic signalling in EPN tumorigenesis and highlight the tumour-suppressive effects that are driven by serotonergic neurons. Therefore, we next asked how EPN cells respond to and process serotonin released from the brain microenvironment. Previous studies have shown that serotonin can be added directly to histones and serves as an epigenomic regulator of gene expression in neurons and astrocytes^15,16. Given that epigenomic dysregulation has a central role in EPN tumorigenesis, we next examined whether histone serotonylation occurs in our ZR^FUS EPN model by staining these tumours with antibodies specific for the serotonylated histone mark on histone H3 (H3K4me3Q5ser, hereafter referred to as H3-5HT). Immunostaining showed abundant expression of H3-5HT in the nucleus of human ZR^FUS EPN tumours and posterior fossa A (PFA, non-ZR^FUS) EPN tumours, indicating that this histone mark is present in both forms of EPN (Fig. 2a). Analysis of mouse ZR^FUS EPN tumours also demonstrated the expression of H3-5HT (Fig. 2b), and immunoblotting revealed a significant increase in H3-5HT tumours compared with normal, non-malignant cortical tissue (Fig. 2c). Moreover, manipulation of dRN neurons led to alterations in H3-5HT in ZR^FUS EPN tumours (Extended Data Fig. 3i–l). These observations led us to investigate whether histone serotonylation contributes to EPN tumorigenesis. To test the role of histone serotonylation, we overexpressed a dominant-negative form of H3.3 (H3.3-Q5A) that blocks H3Q5Ser in our ZR^FUS EPN model¹⁵ (Fig. 2d). Notably, overexpression of H3.3-Q5A suppressed tumour formation, with only 4 out of 17 mice forming tumours, compared with 9 out of 10 wild-type tumours expressing control H3.3 (Fig. 2e). In the few H3.3-Q5A-expression EPN tumours that were generated, we observed a reduction in the levels of H3-5HT, coupled with decreased proliferation (Extended Data Fig. 4a–d). These data show that serotonin has potentially disparate effects on EPN tumorigenesis, in contrast to its effects in the microenvironment, in which it suppresses growth (Fig. 1f–k), and that blockade of histone serotonylation in neoplastic cells considerably abrogates EPN tumorigenesis.
上述观察结果暗示了血清素信号在 EPN 肿瘤发生中的作用，并突出了血清素能神经元驱动的肿瘤抑制效应。因此，我们接下来探究 EPN 细胞如何响应并处理来自脑微环境的释放的血清素。先前研究表明，血清素可直接添加到组蛋白上，并在神经元和星形胶质细胞中作为表观基因组的基因表达调控因子 ^15,16 。鉴于表观基因组失调在 EPN 肿瘤发生中起核心作用，我们随后检测了在我们的 ZR ^FUS EPN 模型中是否存在组蛋白的血清素化现象，通过使用针对组蛋白 H3 上血清素化标记（H3K4me3Q5ser，以下简称 H3-5HT）的特异性抗体对这些肿瘤进行染色。免疫染色显示，在人 ZR ^FUS EPN 肿瘤及后颅窝 A 型（PFA，非 ZR ^FUS ）EPN 肿瘤的细胞核中，H3-5HT 表达丰富，表明这两种 EPN 形式中均存在此组蛋白标记（图 2a）。对小鼠 ZR ^FUS EPN 肿瘤的分析同样显示了 H3-5HT 的表达（图 2b），免疫印迹显示与正常非恶性皮质组织相比，H3-5HT 在肿瘤中的表达显著增加（图 2c）。此外，对 dRN 神经元的操控导致 ZR ^FUS EPN 肿瘤中 H3-5HT 发生改变（扩展数据图 3i-l）。这些观察结果促使我们探究组蛋白血清素化是否参与了 EPN 肿瘤的发生。为检验组蛋白血清素化的作用，我们在 ZR ^FUS EPN 模型 ¹⁵ 中过表达了一种阻断 H3Q5Ser 的 H3.3 显性负性形式（H3.3-Q5A）（图 2d）。值得注意的是，H3.3-Q5A 的过表达抑制了肿瘤形成，仅有 4/17 的小鼠形成了肿瘤，相比之下，表达对照 H3.3 的野生型肿瘤中有 9/10 形成了肿瘤（图 2e）。在少数表达 H3.3-Q5A 的 EPN 肿瘤中，我们观察到 H3-5HT 水平降低，同时增殖减少（扩展数据图 4a-d）。这些数据表明，与在微环境中抑制生长的作用相反（图 1f-k），血清素对 EPN 肿瘤发生可能具有不同的影响，并且阻断肿瘤细胞中的组蛋白血清素化显著抑制了 EPN 肿瘤的发生。

Fig. 2: Histone serotonylation is required for EPN tumorigenesis.
图 2：组蛋白色氨酸酰化对 EPN 肿瘤发生是必需的。

The robust effect of H3.3-Q5A on EPN tumorigenesis led us next to examine whether attenuating serotonin transport also affected tumorigenesis and H3-5HT status. Focusing on SLC6A4, because this serotonin transporter is expressed in EPN tumours (Fig. 1b), we used a CRISPR–Cas9 strategy to knock it out in our ZR^FUS EPN model. These studies revealed a modest improvement in overall survival, coupled with a decrease in cell proliferation and reduced levels of H3-5HT in tumours (Extended Data Fig. 5a–e). Next, we acutely treated mice bearing ZR^FUS EPN tumours with the selective serotonin reuptake inhibitor (SSRI) sertraline, and again found a decrease in the levels of H3-5HT in tumours (Extended Data Fig. 5h–j). These studies suggest that H3-5HT in EPN is mediated by transport from external sources, which is corroborated by our observations that key enzymes involved in serotonin synthesis are not expressed in EPN (Extended Data Fig. 5f,g). Together, these data indicate that manipulating serotonin transport into EPN tumours directly affects the levels of H3-5HT.
H3.3-Q5A 对 EPN 肿瘤发生的强效作用促使我们进一步探究，减弱血清素转运是否同样影响肿瘤发生及 H3-5HT 水平。鉴于 SLC6A4 编码的血清素转运蛋白在 EPN 肿瘤中表达（图 1b），我们采用 CRISPR-Cas9 技术在 ZR ^FUS EPN 模型中敲除该基因。研究发现，整体生存期略有改善，伴随细胞增殖减少及肿瘤内 H3-5HT 水平降低（扩展数据图 5a-e）。随后，我们对携带 ZR ^FUS EPN 肿瘤的小鼠急性施用选择性血清素再摄取抑制剂（SSRI）舍曲林，再次观察到肿瘤内 H3-5HT 水平下降（扩展数据图 5h-j）。这些研究提示，EPN 中的 H3-5HT 源自外部来源的转运，我们的观察结果也支持这一点，即参与血清素合成的主要酶在 EPN 中未表达（扩展数据图 5f,g）。综合数据表明，调控血清素向 EPN 肿瘤的转运直接影响 H3-5HT 水平。

Because H3-5HT serves as an active histone mark that drives gene expression, we next used chromatin immunoprecipitation followed by sequencing (ChIP–seq) to evaluate its distribution throughout the genome in ZR^FUS EPN tumours. We found that H3-5HT enrichment was closely aligned with H3K27 acetylation (Fig. 2f,g), an active transcriptional mark, and bound at sites colocalized with ZR^FUS protein (Fig. 2f,g). To identify intersectional regulatory networks between H3-5HT and ZR^FUS, we focused on genes with adjacent peaks and performed transcription factor (TF) motif analysis, which identified a host of developmental TFs regulating their expression (Fig. 2h and Supplementary Table 2). Comparing TFs associated with H3-5HT peaks and core developmental TFs previously identified in patients with ZR^FUS EPN, we prioritized 12 TFs that are conserved between datasets¹⁰ (Fig. 2i,j, Extended Data Fig. 6a and Supplementary Table 3). Collectively, these results indicate that H3-5HT has an essential role in EPN tumorigenesis and is tightly associated with gene-regulatory programs established by the ZR^FUS protein.
由于 H3-5HT 作为一种驱动基因表达的活性组蛋白标记，我们随后采用染色质免疫沉淀后测序（ChIP-seq）技术，评估其在 ZR ^FUS EPN 肿瘤基因组中的分布情况。我们发现，H3-5HT 的富集与 H3K27 乙酰化（图 2f,g）这一活跃的转录标记紧密相关，并定位于与 ZR ^FUS 蛋白共定位的位点（图 2f,g）。为了揭示 H3-5HT 与 ZR ^FUS 之间的交叉调控网络，我们聚焦于邻近峰位的基因，并进行了转录因子（TF）基序分析，识别出一系列调控这些基因表达的发育相关转录因子（图 2h 及补充表 2）。通过比较与 H3-5HT 峰位相关的转录因子与先前在 ZR ^FUS EPN 患者中鉴定出的核心发育转录因子，我们筛选出 12 个在数据集 ¹⁰ 间保守的转录因子（图 2i,j，扩展数据图 6a 及补充表 3）。综合来看，这些结果表明 H3-5HT 在 EPN 肿瘤发生中扮演着关键角色，并与由 ZR ^FUS 蛋白建立的基因调控程序紧密关联。

ETV5 regulates EPN tumour progression
ETV5 调控 EPN 肿瘤进展

The key role of H3-5HT in EPN tumorigenesis and its regulation of developmental TFs led us to further investigate their contribution to tumorigenesis. To examine the functional effects of these core developmental TFs on EPN tumorigenesis, we performed a barcoded overexpression screen by generating a PiggyBac-based, barcoded library containing 38 of these developmental TFs^10,23 (Fig. 3a and Supplementary Table 4). After introducing the TF library into our ZR^FUS EPN model, we collected tumour-bearing mice at P70 and performed barcode sequencing, using barcode enrichment as a proxy for relative abundance in the tumour. Our screen identified LIM homeobox 2 (LHX2), LIM homeobox 4 (LHX4), ETS variant transcription factor 5 (ETV5) and KLF transcription factor 12 (KLF12) as enriched in EPN tumours. To validate the role of these nominated candidates in EPN tumorigenesis, we individually overexpressed them in our ZR^FUS EPN model and used overall survival as a surrogate for tumorigenesis. We found that overexpression of ETV5 decreased overall survival and enhanced proliferation, as measured by BrdU incorporation (Fig. 3b and Extended Data Fig. 7a,d,e,g,h), whereas overexpression of LHX2, LHX4 or KLF12 did not affect overall survival (Extended Data Figs. 7a–c and 8). Next, we used a CRISPR–Cas9 strategy to knock out Etv5 from ZR^FUS EPN tumours, and found a complementary increase in overall survival and decrease in proliferation (Fig. 3b and Extended Data Fig. 7d–h). These complementary gain-of-function (ETV5-GOF) and loss-of-function (ETV5-LOF) experiments implicate ETV5 as an important transcriptional regulator of EPN tumorigenesis that we prioritized for further downstream analysis. Moreover, the Etv5 locus exhibits local H3-5HT- and ZR^FUS-binding sites (Fig. 2j) and its expression is reduced in H3.3-Q5A-expressing tumours (Extended Data Fig. 4e,f), reinforcing the point that it is regulated by histone serotonylation and ZR^FUS.
H3-5HT 在 EPN 肿瘤发生中的关键作用及其对发育转录因子的调控促使我们进一步探究它们对肿瘤发生的贡献。为考察这些核心发育转录因子对 EPN 肿瘤发生的功能影响，我们通过构建基于 PiggyBac 的条形码文库，包含 38 种发育转录因子，进行了条形码过表达筛选（图 3a 及补充表 4）。将转录因子文库引入我们的 ZR EPN 模型后，在 P70 时收集携带肿瘤的小鼠并进行条形码测序，以条形码富集程度作为肿瘤中相对丰度的代理指标。筛选结果显示，LIM 同源盒 2（LHX2）、LIM 同源盒 4（LHX4）、ETS 变异转录因子 5（ETV5）及 KLF 转录因子 12（KLF12）在 EPN 肿瘤中富集。为验证这些提名候选因子在 EPN 肿瘤发生中的作用，我们在 ZR EPN 模型中分别过表达它们，并采用总体生存期作为肿瘤发生的替代指标。研究发现，ETV5 的过表达降低了总体生存期并增强了增殖能力，通过 BrdU 掺入量衡量（图 3b 及扩展数据图）。相比之下，LHX2、LHX4 或 KLF12 的过度表达并未影响总体生存率（扩展数据图 7a-c 和 8）。随后，我们采用 CRISPR-Cas9 技术从 ZR ^FUS EPN 肿瘤中敲除 Etv5，并观察到总体生存率有所提高及增殖减少（图 3b 及扩展数据图 7d-h）。这些增益功能（ETV5-GOF）和失能功能（ETV5-LOF）实验共同表明，ETV5 是 EPN 肿瘤发生的重要转录调控因子，我们将其列为下游分析的优先研究对象。此外，Etv5 位点显示出局部 H3-5HT 和 ZR ^FUS 结合位点（图 2j），并且在表达 H3.3-Q5A 的肿瘤中其表达降低（扩展数据图 4e,f），这进一步强化了其受组蛋白血清素化和 ZR ^FUS 调控的观点。

Fig. 3: ETV5 regulates EPN progression and repressive chromatin states.
图 3：ETV5 调控 EPN 进展及抑制性染色质状态。

The role of ETV5 in EPN tumorigenesis has not previously been investigated. We therefore next examined the mechanisms by which it regulates gene expression and chromatin states. Because epigenetic dysregulation has a central role in the progression of EPN, we assessed active (H3K27ac) and repressive (H3K27me3) chromatin marks by ChIP–seq in ETV5-GOF tumours. These studies revealed a reduction in H3K27ac peaks (52,658 peaks in control and 27,197 peaks in ETV5-GOF tumours), coupled with an increase in H3K27me3 peaks (227 peaks in control and 1343 peaks in ETV5-GOF tumours), in ETV5-GOF EPN tumours (Fig. 3c, Extended Data Fig. 7j and Supplementary Tables 5–8), whereas immunoblotting showed that there were no differences in the overall levels of these marks (Extended Data Fig. 7i). To understand how ETV5 regulates these epigenomic states, we performed immunoprecipitation followed by mass spectrometry (IP–MS) on ZR^FUS EPN and on healthy brain cortex, and identified 297 proteins that specifically interact with ETV5 in EPN tumours (Fig. 3d–f, Extended Data Fig. 7k and Supplementary Tables 9 and 10). A subset of these proteins have established roles in regulating the polycomb repressive complex (PRC), and using co-immunoprecipitation, we validated interactions between ETV5 and chromobox 3 (CBX3) and histone deacetylase 1 (HDAC1) specifically in ZR^FUS EPN (Fig. 3g). Collectively, these data indicate that ETV5 promotes a repressive H3K27 trimethylation signature in ZR^FUS EPN tumours, through the recruitment of PRC-associated proteins.
ETV5 在 EPN 肿瘤发生中的作用此前未有研究。因此，我们接下来探讨了其调控基因表达和染色质状态的机制。鉴于表观遗传失调在 EPN 进展中起核心作用，我们通过 ChIP-seq 技术评估了 ETV5-GOF 肿瘤中活性（H3K27ac）和抑制性（H3K27me3）染色质标记。这些研究显示，ETV5-GOF EPN 肿瘤中 H3K27ac 峰减少（对照组 52,658 个峰，ETV5-GOF 肿瘤 27,197 个峰），同时 H3K27me3 峰增多（对照组 227 个峰，ETV5-GOF 肿瘤 1343 个峰）（图 3c，扩展数据图 7j 及补充表 5-8），而免疫印迹显示这些标记的整体水平无差异（扩展数据图 7i）。为理解 ETV5 如何调控这些表观遗传状态，我们对 ZR ^FUS EPN 及健康脑皮质进行了免疫沉淀后质谱分析（IP-MS），鉴定出 297 种特异性地与 ETV5 在 EPN 肿瘤中相互作用的蛋白质（图 3d-f，扩展数据图 7k 及补充表 9 和 10）。这些蛋白质中的一部分已明确在调控多梳抑制复合体（PRC）中发挥作用，通过共免疫沉淀技术，我们验证了 ETV5 与染色盒蛋白 3（CBX3）及组蛋白去乙酰化酶 1（HDAC1）在 ZR ^FUS EPN 中特异性的相互作用（图 3g）。综合这些数据表明，ETV5 通过招募 PRC 相关蛋白，在 ZR ^FUS EPN 肿瘤中促进 H3K27 三甲基化抑制标记的形成。

ETV5 suppresses NPY gene expression
ETV5 抑制 NPY 基因表达

Having established that ETV5 can remodel the epigenome in ZR^FUS EPN, we next sought to identify downstream effector genes that mediate its role in tumorigenesis. We performed RNA-seq on ETV5-GOF tumours (and controls), and found that the vast majority of the differentially expressed genes (DEGs) in the ETV5-GOF tumours were downregulated (85.7%; 258 total) (Fig. 3h and Supplementary Table 11), corroborating our observations that ETV5 increases epigenomic repressive states. Next, we integrated these RNA-seq data with the H3K27me3 ChIP–seq data and found a subset of genes that are both transcriptionally repressed and acquire H3K27me3 modification when ETV5 is overexpressed (Fig. 3i and Supplementary Table 12). GO analysis of this subset revealed an association with chemical synaptic transmission and neuropeptide signalling pathways (Fig. 3j and Supplementary Tables 12 and 13). Furthermore, cross-comparison of these gene lists with ChIP–seq data from ETV5-GOF tumours further resolved this list of genes (Extended Data Fig. 6b and Supplementary Tables 11 and 14). Focusing on the neuropeptide gene sets, we validated the downregulation of neuropeptide Y (NPY) in human ZR^FUS EPN compared with normal brain, and used immunostaining to show that it is downregulated in mouse ETV5-GOF tumours (Fig. 3k–m and Extended Data Fig. 7l). This downregulation of NPY in ETV5-GOF tumours is complemented by increased H3K27me3 repressive marks at the Npy locus (Extended Data Fig. 7m), implicating the PRC complex as an important regulator of NPY expression.
在确认 ETV5 能够重塑 ZR ^FUS EPN 的表观基因组后，我们接下来致力于鉴定介导其在肿瘤发生中作用的下游效应基因。我们对 ETV5-GOF 肿瘤（及对照组）进行了 RNA-seq 分析，发现 ETV5-GOF 肿瘤中绝大多数差异表达基因（DEGs）呈现下调趋势（85.7%；总计 258 个）（图 3h 及补充表 11），这印证了我们关于 ETV5 增加表观基因组抑制状态的观察。随后，我们将这些 RNA-seq 数据与 H3K27me3 ChIP-seq 数据整合，发现当 ETV5 过表达时，有一部分基因同时表现出转录抑制并获得 H3K27me3 修饰（图 3i 及补充表 12）。对这一基因子集进行 GO 分析显示，它们与化学突触传递及神经肽信号通路相关联（图 3j 及补充表 12 和 13）。此外，将这些基因列表与 ETV5-GOF 肿瘤的 ChIP-seq 数据交叉比对，进一步细化了该基因列表（扩展数据图 6b 及补充表 11 和 14）。聚焦于神经肽基因集，我们验证了与正常脑组织相比，人类 ZR ^FUS EPN 中神经肽 Y（NPY）的下调，并通过免疫染色显示其在小鼠 ETV5-GOF 肿瘤中同样下调（图 3k-m 及扩展数据图 7l）。ETV5-GOF 肿瘤中 NPY 的这种下调现象，伴随着 Npy 位点上 H3K27me3 抑制标记的增加（扩展数据图 7m），暗示 PRC 复合体作为 NPY 表达的重要调控因子。

NPY suppresses brain hyperactivity
NPY 抑制大脑过度活跃

NPY is a potent modulator of neuronal activity in the brain and also has separate functions in many other tissues outside the central nervous system^{29,30,31,32,33,34}. NPY has also been implicated in a host of malignancies; however, its role in EPN remains undefined^35,36. Because ETV5 promotes tumour progression and NPY is downregulated in ETV5-GOF tumours, we hypothesized that its overexpression would suppress EPN tumour growth. Indeed, NPY-GOF in our ZR^FUS EPN model extended overall survival and suppressed cell proliferation, as assayed by BrdU incorporation (Fig. 4a–c and Extended Data Figs. 8 and 9a). To understand how NPY suppresses tumorigenesis, we performed RNA-seq on NPY-GOF tumours (and control), and—similar to ETV5—we found that the vast majority of the DEGs (89%; 995 total) were downregulated (Fig. 4d). GO analysis identified synaptic development and synapse signalling as the top gene sets downregulated in NPY-overexpressing tumours (Extended Data Fig. 9e and Supplementary Tables 15 and 16). These results suggest that the release of NPY from EPN tumours influences the synaptic constituency of the microenvironment. Consistent with this notion, immunostaining of the peritumoral margin revealed nominal expression of NPY receptor 2 (NPY2R) in mouse EPN tumours and high expression in neurons surrounding the tumour (Extended Data Fig. 9b–d).
NPY 是一种强效的大脑神经活动调节剂，同时在中枢神经系统外的许多组织中具有独立功能 ^{29,30,31,32,33,34} 。NPY 还与多种恶性肿瘤有关；然而，其在 EPN 中的作用尚不明确 ^35,36 。由于 ETV5 促进肿瘤进展且 NPY 在 ETV5-GOF 肿瘤中表达下调，我们假设其过表达将抑制 EPN 肿瘤生长。确实，在我们的 ZR ^FUS EPN 模型中，NPY-GOF 延长了总体生存期并抑制了细胞增殖，通过 BrdU 掺入检测证实（图 4a-c 及扩展数据图 8 和 9a）。为探究 NPY 如何抑制肿瘤发生，我们对 NPY-GOF 肿瘤（及对照组）进行了 RNA 测序，与 ETV5 相似，我们发现绝大多数差异表达基因（DEGs，89%；共 995 个）被下调（图 4d）。GO 分析显示，突触发育和突触信号传导是 NPY 过表达肿瘤中下调最多的基因集（扩展数据图 9e 及补充表 15 和 16）。这些结果表明，EPN 肿瘤中 NPY 的释放影响了微环境的突触组成。与这一观点一致，对肿瘤周围边缘进行免疫染色显示，小鼠 EPN 肿瘤中 NPY 受体 2（NPY2R）表达量极低，而肿瘤周围的神经元则呈现高表达（扩展数据图 9b-d）。

Fig. 4: NPY suppresses EPN progression.
图 4：NPY 抑制 EPN 进展。

These observations suggest that NPY exerts its effect on EPN tumour growth through paracrine interactions with neurons in the microenvironment, which prompted us to assess excitatory and inhibitory synapses at the peritumoral margin in NPY-GOF EPN tumours. These studies showed a decrease in excitatory synapses (VGLUT1 and PSD95) and an increase in inhibitory synapses (VGAT) at the peritumoral margins in NPY-GOF tumours (Fig. 4e–h). The synaptic constituency at the peritumoral margin can influence brain hyperactivity and tumour progression. Our observations of synaptic remodelling towards decreased excitatory synapses, coupled with previous studies showing that NPY suppresses brain hyperactivity³¹, led us to examine whether overexpression of NPY in EPN suppresses progressive brain hyperactivity. We performed serial electroencephalography (EEG) starting at P30 and recording for 48 h, every 10 days (Fig. 5b). Mice bearing ZR^FUS EPN control tumours exhibited increased brain network hyperactivity as early as P32, which led to 50% of mice exhibiting observable seizures by P42 (Fig. 5i–k and Extended Data Fig. 9f). By contrast, across the P30–P52 recording interval, mice bearing NPY-GOF EPN tumours did not exhibit seizures and had a significant delay in the onset of network hyperactivity (Fig. 5i–k). To confirm that overexpression of NPY leads to increased inhibitory synaptic activity, we generated NPY-GOF tumours, injected AAV-2/9 syn1-mCherry to label peritumoral neurons and performed whole-cell recordings on labelled cortical neurons from tumour-bearing brain slices (Fig. 5a). These studies revealed an increase in the frequency of inhibitory post-synaptic currents (IPSCs), coupled with a decrease in the frequency of excitatory post-synaptic current (EPSCs) (Fig. 5c–h), indicating an overall increase in inhibitory activity in the brains of mice bearing NPY-GOF tumours. Together, these data show that NPY suppresses EPN tumour progression by remodelling the brain microenvironment towards synaptic inhibition, which, in turn, blunts brain hyperactivity and impedes tumour progression.
这些观察结果表明，NPY 通过与微环境中的神经元进行旁分泌相互作用来影响 EPN 肿瘤生长，这促使我们评估了 NPY-GOF EPN 肿瘤周边缘的兴奋性和抑制性突触。这些研究表明，在 NPY-GOF 肿瘤的周边缘，兴奋性突触（VGLUT1 和 PSD95）减少，而抑制性突触（VGAT）增加（图 4e-h）。肿瘤周边缘的突触组成可以影响大脑过度活跃和肿瘤进展。我们观察到突触重塑趋向于减少兴奋性突触，结合先前研究显示 NPY 抑制大脑过度活跃的结果，促使我们探究在 EPN 中过度表达 NPY 是否能抑制渐进性大脑过度活跃。我们从 P30 开始进行连续脑电图（EEG）监测，每 10 天记录 48 小时（图 5b）。携带 ZR EPN 对照肿瘤的小鼠早在 P32 就表现出脑网络过度活跃，到 P42 时，50%的小鼠出现可观察到的癫痫发作（图 5i-k 及扩展数据图 9f）。相比之下，在 P30 至 P52 的记录期间，携带 NPY-GOF EPN 肿瘤的小鼠未出现癫痫发作，并且网络过度活跃的开始时间显著延迟（图 5i-k）。为证实 NPY 的过度表达导致抑制性突触活动增强，我们生成了 NPY-GOF 肿瘤，注射 AAV-2/9 syn1-mCherry 以标记肿瘤周围神经元，并对来自肿瘤携带脑片的标记皮质神经元进行全细胞记录（图 5a）。这些研究揭示了抑制性突触后电流（IPSCs）频率的增加，同时伴随兴奋性突触后电流（EPSCs）频率的降低（图 5c-h），表明携带 NPY-GOF 肿瘤的小鼠大脑中抑制性活动整体增强。综上所述，这些数据表明 NPY 通过重塑大脑微环境向突触抑制方向发展，从而抑制 EPN 肿瘤的进展，进而减弱大脑过度活跃并阻碍肿瘤进展。

Fig. 5: NPY suppresses EPN-induced brain hyperactivity.
图 5：NPY 抑制 EPN 诱导的大脑过度活跃。

Discussion 讨论

Investigating neuron–tumour interactions in autochthonous models of ZR^FUS-driven EPN, we found that the activation of specific neuronal subpopulations differentially affects tumour progression: local cortical neurons drive progression, whereas serotonergic neurons in the dRN suppress progression (Fig. 5l). Previous studies identified cortical projection neurons, glutamatergic signalling, sensory input and remote areas with high activity as driving glioma progression, supporting the notion that heightened neuronal activity is pro-tumorigenic^{17,18,19,20,21,22,23,24,25}. By contrast, our findings indicate that the activation of discrete subtypes of neurons suppresses EPN tumorigenesis, highlighting the need to decipher how neuronal-subtype- and circuit-specific interactions affect brain-tumour progression. Although our results suggest that serotonin from dRN neurons is directly transported into EPN cells, it remains possible that these effects are mediated through subsets of cortical interneurons that express the serotonin receptor HTR3A (refs. ^37,38,39,40) (Fig. 5l). Mechanistically, we found that serotonin serves as an essential histone modification in EPN, associating with oncogenic transcriptional networks (Fig. 5l). Given that histone serotonylation occurs in astrocytes and neurons^15,16, it is possible that this phenomenon operates in other types of brain tumours.
在研究 ZR ^FUS 驱动的 EPN 自发性模型中神经元与肿瘤相互作用时，我们发现特定神经元亚群的激活对肿瘤进展产生差异化影响：局部皮质神经元促进进展，而背侧中缝核（dRN）中的血清素能神经元则抑制进展（图 5l）。先前研究已确认皮质投射神经元、谷氨酸能信号传导、感觉输入及高活性远程区域均推动胶质瘤进展，这支持了神经元活动增强具有促瘤性的观点 ^{17,18,19,20,21,22,23,24,25} 。然而，我们的发现表明，特定神经元亚型的激活反而抑制 EPN 肿瘤发生，凸显了解析神经元亚型及环路特异性相互作用如何影响脑肿瘤进展的重要性。尽管结果提示 dRN 神经元释放的血清素可能直接转运至 EPN 细胞内，但也不能排除这些效应是通过表达血清素受体 HTR3A 的皮质中间神经元亚群介导的可能性（参考文献 ^37,38,39,40 ）（图 5l）。从机制上讲，我们发现血清素在 EPN 中作为一种重要的组蛋白修饰发挥作用，与致癌转录网络相关联（图 5l）。鉴于组蛋白血清素化现象在星形胶质细胞和神经元中均有发生，这一现象有可能在其他类型的脑肿瘤中也存在。

Examining how neuronal signalling influences epigenomic states in EPN, we found that the developmental TF ETV5 is a target of histone serotonylation and a key regulator of EPN tumorigenesis (Fig. 5l). ETV5 has a central role in astrocyte development⁴¹, whereas interactions between progenitor populations and neurons have a key role in brain development^42,43,44,45. Consequently, these relationships might influence other epigenomic and developmental states that are manifested in EPN. Moreover, we found that ETV5 promotes repressive chromatin states, implicating gene repression as a driver of EPN progression. Among the repressed ETV5 targets is NPY, which functions to suppress EPN progression and brain hyperactivity through paracrine signalling with neurons (Fig. 5l). However, we cannot rule out a possible autocrine mechanism, because NPY functions in this manner in other systems^46,47,48. Brain tumours release factors that remodel synapses towards hyperactivity²³, whereas we found that EPN tumours can release factors that suppress excitatory synaptic remodelling and that repressing this mechanism is essential for progression. Collectively, these studies highlight the intersection between neurodevelopment, chromatin regulation and neuronal signalling, reinforcing the importance of defining specific interactions between neuroactive compounds, circuits and brain tumours.
探究神经信号如何影响 EPN 中的表观基因组状态，我们发现发育转录因子 ETV5 是组蛋白血清素化的靶点，也是 EPN 肿瘤发生的关键调控因子（图 5l）。ETV5 在星形胶质细胞发育中起核心作用 ⁴¹ ，而前体细胞群与神经元间的相互作用对脑发育至关重要 ^42,43,44,45 。因此，这些关系可能影响 EPN 中其他表观基因组和发育状态。此外，我们发现 ETV5 促进抑制性染色质状态，暗示基因抑制是 EPN 进展的驱动因素。在受抑制的 ETV5 靶点中，NPY 通过与神经元的旁分泌信号作用抑制 EPN 进展和脑过度活跃（图 5l）。然而，我们不能排除自分泌机制的可能性，因为 NPY 在其他系统中以这种方式发挥功能 ^46,47,48 。脑肿瘤释放重塑突触向过度活跃的因子 ²³ ，而我们发现 EPN 肿瘤能释放抑制兴奋性突触重塑的因子，抑制这一机制对 EPN 进展至关重要。这些研究共同强调了神经发育、染色质调控与神经信号传导之间的交集，突显了界定神经活性化合物、神经回路与脑肿瘤之间特定相互作用的重要性。

Methods 方法

Analysis of human RNA-seq data
人类 RNA 测序数据分析

The DEGs from human EPN samples were obtained from our previous study¹⁰. To find genes enriched in ZR^FUS versus non-ZR^FUS EPN, GO term enrichment was performed using the R package Clusterprofiler⁴⁹. For further detailed analysis of monoamine-transport-related genes, the gene set corresponding to the GO term was obtained from GO project datasets from MGI.
从我们之前的研究中获得了人类 EPN 样本的 DEGs ¹⁰ 。为了找出在 ZR ^FUS 与非 ZR ^FUS EPN 中富集的基因，使用 R 包 Clusterprofiler ⁴⁹ 进行了 GO term 富集分析。对于进一步详细分析单胺转运相关基因，从 MGI 的 GO 项目数据集中获取了与 GO term 对应的基因集。

Acquisition of samples from patients with EPN
从 EPN 患者中获取样本

EPN samples were obtained from patients undergoing surgical resection at Le Bonheur Children’s Hospital and St. Jude Children’s Research Hospital. Informed consent was obtained from all patients with approval for human tissue use from reviews from Institutional Review Boards. Diagnoses were confirmed by a neuropathologist. For histology, samples were paraffin embedded.
从勒邦赫尔儿童医院和圣裘德儿童研究医院接受手术切除的患者中获取了 EPN 样本。所有患者均获得知情同意，并经机构审查委员会批准使用人体组织。诊断结果由神经病理学家确认。组织学检查样本采用石蜡包埋。

IUE mouse ZR^FUS EPN model
IUE 小鼠 ZR ^FUS EPN 模型

To generate mouse ZR^FUS EPN tumours, we performed IUE in the CD-1 IGS mouse background at E16.5 as previously described^11,50. In brief, the uterine horns of the pregnant mice were exposed for injecting plasmid mixture into the embryonic lateral ventricles with Fast Green dye as the indicator, followed by electroporation using BTW Tweezertrodes connected to the pulse generator (BTX 8300; parameters set at 33 V and 55 ms per pulse six times at 100-ms intervals). The plasmid mixture included helper plasmid pGLAST-PBase (2.0 μg μl⁻¹), PBCAG-GFP (1.0 μg μl⁻¹), PBCAG-ZR^FUS-HA (1.0 μg μl⁻¹) and a CRISPR–Cas9 construct with gRNA against Trp53 (1.5 μg μl⁻¹). Co-electroporation of PBCAG-GFP allowed fluorescent visualization of tumours. GOF studies were performed by co-electroporating the human variant gene of interest in PBCAG constructs (1.0 μg μl⁻¹). LOF studies used a CRISPR–Cas9 construct with Trp53 gRNA and gRNA against the gene of interest (1.5 μg μl⁻¹). The knockout efficiency was validated by the mismatch-cleavage SURVEYOR assay (IDT, 706020) on genomic DNA acquired from mouse tumours. Primer sequences for Surveyor assays and functional studies validation are listed in Supplementary Table 17. All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at Baylor College of Medicine (BCM) and conform to the US Public Health Service Policy on Human Care and Use of Laboratory Animals.
为生成小鼠 ZR ^FUS EPN 肿瘤，我们在 CD-1 IGS 小鼠背景中于 E16.5 进行宫内电穿孔（IUE），操作如前所述 ^11,50 。简言之，通过暴露孕鼠的子宫角，将质粒混合物注射入胚胎侧脑室，以快绿染料作为指示剂，随后使用连接到脉冲发生器（BTX 8300；参数设定为 33 V 和 55 ms 每脉冲，间隔 100 ms 重复六次）的 BTW Tweezertrodes 进行电穿孔。质粒混合物包括辅助质粒 pGLAST-PBase（2.0 μg/μl ⁻¹ ）、PBCAG-GFP（1.0 μg/μl ⁻¹ ）、PBCAG-ZR ^FUS -HA（1.0 μg/μl ⁻¹ ）以及针对 Trp53 的 gRNA 的 CRISPR-Cas9 构建体（1.5 μg/μl ⁻¹ ）。共电穿孔 PBCAG-GFP 便于肿瘤的荧光可视化。功能获得（GOF）研究通过共电穿孔感兴趣的人类变异基因的 PBCAG 构建体（1.0 μg/μl ⁻¹ ）进行。功能丧失（LOF）研究则采用含 Trp53 gRNA 及针对目标基因 gRNA 的 CRISPR-Cas9 构建体（1.5 μg/μl ⁻¹ ）。敲除效率通过从小鼠肿瘤获取的基因组 DNA 进行的错配切割 SURVEYOR 分析（IDT，706020）验证。 Surveyor 分析和功能研究验证的引物序列列于补充表 17。所有程序均已获得贝勒医学院（Baylor College of Medicine, BCM）机构动物护理和使用委员会（IACUC）的批准，并符合美国公共卫生署关于实验室动物护理和使用的政策。

AAV generation, AAV-DREADD delivery and CNO treatment
AAV 制备、AAV-DREADD 递送及 CNO 处理

All AAVs were generated at the Neuroconnectivity Core at BCM and detailed information is included in Supplementary Table 17. AAV was diluted with loading dye (10x Fast Green, 2 mg ml⁻¹) and loaded into a microdispenser (Drummond Scientific, 13-681-460) before injection. For ipsilateral cortical neural stimulation, AAV was injected at a rate of 7 nl per s, to give a total volume of 1.5 μl, into the ventricle of P5 mice. For the dRN neural stimulation experiment, the dRN was injected with 750 nl AAV at a rate of 200 nl per minute (injection coordinates: −4.5 mm A/P, 0 mm M/L and −2.2 mm ventral⁵¹) and the micropipette was left in place for 5 min after injection before being slowly removed. Mice were weighed daily before injecting with CNO (Tocris, 4936) at 0.5 mg per kg or 5 mg per kg intraperitoneally, or an equivalent volume of saline for controls. Mice received CNO or saline injection twice per day before collecting tissues for further analyses.
所有 AAV 均在贝勒医学院的神经连接核心实验室制备，详细信息见补充表 17。AAV 用加载染料（10x Fast Green，2 mg ml ⁻¹ ）稀释后，装入微量分配器（Drummond Scientific, 13-681-460）再进行注射。对于同侧皮层神经刺激，将 AAV 以每秒 7 nl 的速度注入 P5 小鼠的脑室，总注射量为 1.5 μl。在 dRN 神经刺激实验中，dRN 以每分钟 200 nl 的速度注射 750 nl AAV（注射坐标：-4.5 mm A/P, 0 mm M/L 和 -2.2 mm 腹侧 ⁵¹ ），注射后微管针保留 5 分钟，然后缓慢拔出。小鼠每日称重后，按 0.5 mg/kg 或 5 mg/kg 腹腔注射 CNO（Tocris, 4936），或等量生理盐水作为对照。小鼠每日两次接受 CNO 或生理盐水注射，随后收集组织进行进一步分析。

Mouse survival analyses, bromodeoxyuridine labelling and tumour collection
小鼠生存分析、溴脱氧尿苷标记及肿瘤收集

Mice were monitored for suggestive symptoms of tumours, including lethargy, hunched posture, decreased grooming, trembling, partial limb paralysis, abnormal gait and hydrocephalus, representing the IACUC permitted end-point. Male and female mice were both included in this study. Mice were humanely euthanized once the disease symptoms showed. Survival dates were recorded for Kaplan–Meier survival curve analyses. For BrdU labelling, mouse brains at P70 were collected 4 h after BrdU pulsing (100 mg per kg body weight in phosphate-buffered saline (PBS)) by intraperitoneal injection. For biochemical and molecular studies, tumour tissues were dissected using GFP before further processing in other experiments described below.
对小鼠进行了肿瘤相关症状的监测，包括嗜睡、弓背姿势、梳理减少、颤抖、部分肢体瘫痪、步态异常及脑积水，这些症状代表了经机构动物护理和使用委员会（IACUC）批准的终止点。本研究涵盖了雄性和雌性小鼠。一旦出现疾病症状，即对小鼠实施安乐死。记录生存日期以进行 Kaplan-Meier 生存曲线分析。对于 BrdU 标记，在 P70 时收集小鼠脑组织，采集时间为 BrdU 脉冲处理（100 mg/kg 体重，溶于磷酸盐缓冲液（PBS））后 4 小时，通过腹腔注射给药。进行生化和分子研究时，利用 GFP 标记先分离肿瘤组织，随后进行下述实验处理。

Immunofluorescence staining
免疫荧光染色

For paraffin sectioning, mouse brains were fixed through intracardial perfusion and overnight incubation with 4% paraformaldehyde. After dehydration using 70% ethanol, samples were sent to the Pathology Core of the Breast Center at Baylor College of Medicine for paraffin embedding. Ten-micrometre sections were deparaffinized as follows before further treatments: 3 × 3 min in xylene, 3 × 3 min in 100% ethanol, 3 × 3 min in 95% ethanol, 3 min in 80% ethanol, 3 min in 70% ethanol, 3 min in 50% ethanol, 5 min in ddH₂O and 5 min in PBS.
对于石蜡切片，小鼠大脑通过心内灌注固定，并经 4%多聚甲醛过夜孵育。脱水处理采用 70%乙醇后，样本送至贝勒医学院乳腺中心病理核心进行石蜡包埋。随后，10 微米厚的切片按以下步骤进行脱蜡处理以备进一步处理：在二甲苯中浸泡 3 次，每次 3 分钟；在 100%乙醇中浸泡 3 次，每次 3 分钟；在 95%乙醇中浸泡 3 次，每次 3 分钟；在 80%乙醇中浸泡 3 分钟；在 70%乙醇中浸泡 3 分钟；在 50%乙醇中浸泡 3 分钟；在双蒸水中浸泡 5 分钟，以及在磷酸盐缓冲液（PBS）中浸泡 5 分钟。

For cryosectioning, mouse brains were fixed through intracardial perfusion and incubated overnight with 4% paraformaldehyde. Subsequent incubation with 20% sucrose was done overnight before embedding tissues in Tissue Plus OCT compound (Thermo Fisher Scientific, 4585). Thirty-micrometre sections were mounted onto slides before further processing.
为了进行冰冻切片，小鼠大脑通过心内灌注固定，并在 4%多聚甲醛中孵育过夜。随后在 20%蔗糖中孵育过夜，然后使用 Tissue Plus OCT 复合物（赛默飞世尔科技，4585）包埋组织。将 30 微米厚的切片贴附于载玻片上，以进行后续处理。

The antigen exposure step was done by incubating the sections in 10 mM sodium citrate (pH 6.5) at 95 °C for 20 min. After 1 h of serum blocking, slides were incubated with primary antibodies overnight at 4 °C. The next day, slides were rinsed with PBS, incubated with secondary antibodies conjugated with fluorophore for 1 h and rinsed with PBS again. After counterstaining with Hoechst 33342 nucleic acid stain (Thermo Fisher Scientific, H3570), coverslips were mounted with VECTASHIELD PLUS Antifade Mounting Medium (Vector Laboratories, H-1900). Antibody information is provided in Supplementary Table 17.
抗原修复步骤通过将切片在 10 mM 柠檬酸钠缓冲液（pH 6.5）中于 95°C 孵育 20 分钟完成。血清阻断 1 小时后，载玻片在 4°C 下与一抗共孵育过夜。次日，载玻片用 PBS 冲洗，与偶联荧光素的二抗孵育 1 小时，再次用 PBS 冲洗。随后用 Hoechst 33342 核酸染料（赛默飞世尔科技，H3570）进行反染，并用 VECTASHIELD PLUS 防褪色封片剂（Vector Laboratories，H-1900）封片。抗体信息见补充表 17。

Confocal imaging 共聚焦成像

Immunofluorescent images were acquired using a Zeiss Axio Zeiss LSM980 confocal microscope with a 20× or 63× oil objective. A total of three images per sample were acquired in each experiment at 0.5-μm intervals over a 5-μm depth. The immunostaining experiment was performed on the same day for both control and experimental groups and images were acquired using an identical laser power setting for comparison. BrdU⁺ and Ki67⁺ nuclei, and the total number of nuclei, were recorded using the Analyze Particles function for each field of view in ImageJ/Fiji. Synapse counts and colocalization were calculated using the Synapse Counter plug-in (SynPuCo) in ImageJ/Fiji (https://github.com/SynPuCo/SynapseCounter). NPY intensity was measured within each field of view, whereas H3-5HT, ETV5, TPH2 and NPY2R intensity were measured from at least ten cells from each field of view in each sample followed by normalizing to DAPI.
使用 Zeiss Axio Zeiss LSM980 共聚焦显微镜配备 20×或 63×油镜进行免疫荧光图像采集。每次实验中，每个样本共获取三张图像，间隔 0.5 微米，覆盖 5 微米深度。对照组与实验组的免疫染色实验在同一天进行，并采用相同的激光功率设置进行图像采集以供比较。利用 ImageJ/Fiji 软件中的 Analyze Particles 功能记录每个视野内的 BrdU ⁺ 和 Ki67 ⁺ 核以及总核数。通过 ImageJ/Fiji 中的 Synapse Counter 插件（SynPuCo）计算突触计数及共定位情况（插件地址：https://github.com/SynPuCo/SynapseCounter）。NPY 强度在每个视野内测量，而 H3-5HT、ETV5、TPH2 和 NPY2R 强度则从每个样本的每个视野中至少十个细胞中测得，随后归一化至 DAPI。

SSRI treatment SSRI 治疗

Mice started to receive an SSRI intraperitoneally from P28 for three weeks. Mice received sertraline (2.5 mg per kg, in 7% dimethyl sulfoxide in PBS, MedChem Express, HY-B0176A) or vehicle injection three days per week before collecting tissues for further analyses.
从 P28 开始，小鼠连续三周通过腹腔注射接受 SSRI。在收集组织进行进一步分析前，小鼠每周三次接受舍曲林（2.5 毫克/千克，溶于 7%二甲基亚砜 PBS 溶液，MedChem Express，HY-B0176A）或对照注射。

Preparation of protein lysates and histone extracts, and western blotting
蛋白质裂解液与组蛋白提取物的制备，以及蛋白质印迹法

For preparing whole-cell lysates, tissues were washed with cold PBS three times, followed by dissociation in radio immunoprecipitation assay (RIPA) lysis buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% deoxycholate, 0.1% SDS and 1% NP-40; with protease inhibitors) using a pellet homogenizer on ice.
为制备全细胞裂解物，组织用冷 PBS 洗涤三次，随后在冰上使用研磨器将其分散于放射免疫沉淀测定（RIPA）裂解缓冲液中（50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5%脱氧胆酸, 0.1% SDS 和 1% NP-40；含蛋白酶抑制剂）。

For extracting histone lysates, tissues were washed with cold PBS three times, followed by dissociation using the Histone Extraction Kit (Abcam, ab113476) according to the manufacturer’s instructions.
为了提取组蛋白裂解物，组织先用冷 PBS 洗涤三次，随后按照制造商的说明使用组蛋白提取试剂盒（Abcam，ab113476）进行解离。

Before performing western blot analysis, protein quantification was performed using the Bradford protein assay (Bio-Rad, 5000006), and 40 μg of the protein lysates was applied to SDS gel electrophoresis, and wet transferred to a nitrocellulose membrane at 350 mA for 65 min. The membranes were blocked in 5% non-fat milk in Tris-buffered-saline with Tween20 (TBST) and incubated with primary antibodies overnight at 4 °C. The next day, membranes were washed with TBST, incubated with horseradish-peroxidase–conjugated secondary antibodies for 1 h at room temperature, and washed again with TBST before subsequent development using luminol reagent (Santa Cruz Biotechnology, sc2048). Antibody information is provided in Supplementary Table 17.
在进行 Western blot 分析之前，采用 Bradford 蛋白测定法（Bio-Rad，5000006）进行蛋白定量，并将 40 μg 的蛋白裂解物应用于 SDS 凝胶电泳，随后以 350 mA 电流湿转至硝酸纤维素膜上，持续 65 分钟。膜在含 5%脱脂牛奶的 Tris 缓冲盐溶液（含 Tween20，TBST）中封闭，并在 4°C 下与一抗孵育过夜。次日，膜经 TBST 洗涤后，与辣根过氧化物酶标记的二抗在室温下孵育 1 小时，再次用 TBST 洗涤，然后使用 luminol 试剂（Santa Cruz Biotechnology，sc2048）进行后续显影。抗体信息见补充表 17。

ChIP 染色质免疫沉淀

Mouse tumours were dissociated in cold PBS using a pellet homogenizer on ice. Chromatin cross-linking was performed by incubating samples with freshly prepared 1.1% formaldehyde solution at room temperature for 10 min, followed by stopping the reaction with the addition of 0.1 M glycine. After collecting cell pellets by centrifugation, samples were washed with PBS and stored at −80 °C until further processing. Pellets were resuspended in PBS/PMSF with 0.5% Igepal to release nuclei before washing with cold ChIP buffer (0.25% Triton X, 10 mM EDTA, 0.5 mM EGTA and 10 mM HEPES (pH 6.5)). Nuclei were lysed with ChIP lysis buffer (0.5% SDS, 5 mM EDTA and 25 mM Tris-HCl (pH 8.0)) for 15–20 min at room temperature and sonicated into 250–350 bp using the Diagenode Bioruptor. The fragmented chromatin concentration was quantified using the Quant-iT double-stranded DNA (dsDNA) assay kit (Thermo Fisher Scientific, Q33120), diluted five-fold with ChIP dilution buffer (2 mM EDTA, 150 mM NaCl, 1% Triton X-100 and 20 mM Tris-HCl (pH 8.0); with protease inhibitors). Immunoprecipitation was then performed by incubating fragmented chromatin with antibodies overnight at 4 °C with rotation. The next day, protein A/G magnetic beads (Thermo Fisher Scientific, 88802) were added for a 6-h incubation at 4 °C with rotation. Beads were washed with Tris-SDS-EDTA-I buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 150 mM NaCl and 20 mM Tris-HCl (pH 8.0)), Tris-SDS-EDTA-II buffer (TSEI buffer with 500 mM NaCl), LiCl buffer (250 mM LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA and 10 mM Tris-HCl (pH 8.0)) and Tris-EDTA buffer (10 mM Tris-HCl (pH 8.0) and 1 mM EDTA). Beads were incubated in ChIP elution buffer (1% SDS and 0.1 M NaHCO₃) for 20 min at 65 °C twice for eluting pull-down samples. Eluates from beads were treated with proteinase K (0.4 mg ml⁻¹; Thermo Fisher Scientific, AM2546) and NaCl (0.125 M) overnight at 65 °C for reverse cross-linking. ChIP-DNA was purified using a PCR purification kit (Qiagen, 28104) and quantified using the Quant-iT dsDNA assay kit. A 10–12-ng quantity of ChIP-DNA was used for ChIP–seq library preparation as described below. Antibody information and antibody:chromatin ratios used for immunoprecipitation are provided in Supplementary Table 17.
小鼠肿瘤组织在冰上使用研磨器于冷磷酸盐缓冲液（PBS）中解离。通过将样品与新鲜配制的 1.1%甲醛溶液在室温下孵育 10 分钟进行染色质交联，随后加入 0.1 M 甘氨酸终止反应。收集细胞沉淀后，样品用 PBS 洗涤并储存于-80°C 待进一步处理。沉淀物在含 0.5% Igepal 的 PBS/PMSF 中重新悬浮以释放核，之后用冷 ChIP 缓冲液（0.25% Triton X，10 mM EDTA，0.5 mM EGTA 和 10 mM HEPES，pH 6.5）洗涤。核用 ChIP 裂解缓冲液（0.5% SDS，5 mM EDTA 和 25 mM Tris-HCl，pH 8.0）在室温下裂解 15-20 分钟，并使用 Diagenode Bioruptor 超声处理成 250-350 bp 片段。使用 Quant-iT 双链 DNA（dsDNA）定量试剂盒（Thermo Fisher Scientific，Q33120）测定片段化染色质浓度，并用 ChIP 稀释缓冲液（2 mM EDTA，150 mM NaCl，1% Triton X-100 和 20 mM Tris-HCl，pH 8.0；含蛋白酶抑制剂）稀释五倍。随后，通过将片段化染色质与抗体在 4°C 下旋转孵育过夜进行免疫沉淀。次日，向样品中加入蛋白 A/G 磁珠（Thermo Fisher Scientific，88802），在 4°C 下旋转孵育 6 小时。磁珠依次用 Tris-SDS-EDTA-I 缓冲液（0.1% SDS，1% Triton X-100，2 mM EDTA，150 mM NaCl 和 20 mM Tris-HCl（pH 8.0））、Tris-SDS-EDTA-II 缓冲液（含 500 mM NaCl 的 TSEI 缓冲液）、LiCl 缓冲液（250 mM LiCl，1% NP-40，1%脱氧胆酸，1 mM EDTA 和 10 mM Tris-HCl（pH 8.0））以及 Tris-EDTA 缓冲液（10 mM Tris-HCl（pH 8.0）和 1 mM EDTA）洗涤。磁珠在 ChIP 洗脱缓冲液（1% SDS 和 0.1 M NaHCO3）中于 65°C 下洗脱两次，每次 20 分钟，以释放捕获的样本。洗脱液经蛋白酶 K（0.4 mg/ml；Thermo Fisher Scientific，AM2546）和 NaCl（0.125 M）在 65°C 下过夜处理，进行反向交联。ChIP-DNA 通过 PCR 纯化试剂盒（Qiagen，28104）纯化，并使用 Quant-iT dsDNA 定量试剂盒进行定量。取 10–12 ng 的 ChIP-DNA 用于后续的 ChIP-seq 文库制备，具体步骤如下。免疫沉淀所用的抗体信息及抗体与染色质的比率列于补充表 17 中。

ChIP–seq library preparation, sequencing and bioinformatic analysis
ChIP-seq 文库制备、测序及生物信息学分析

The TruSeq ChIP Library Preparation Kit (Illumina, IP-202-1012) was used to prepare ChIP–seq libraries. Around 250–350 bp of libraries were purified from agarose gel using the QIAquick Gel Extraction Kit (Qiagen, 28706), amplified with PCR and extracted using AMPure XP beads (Beckman Coulter Life Science, A63882). The quality of the libraries was analysed using the Standard Sensitivity NGS Fragment Analysis Kit (Agilent, formerly AATI, DNF-473-0500) on a 12-Capillary Fragment Analyzer. After using the Quant-iT dsDNA assay kit for quantification, an equal concentration (2 nM) of each library was pooled for single end (1 × 150) sequencing using the High Output v2 kit (Illumina, FC-404-2002) on an Illumina NextSeq550 System (around 60 million–80 million reads per sample). All ChIP–seq experiments were performed in at least two independent biological replicates.
使用 TruSeq ChIP 文库制备试剂盒（Illumina, IP-202-1012）制备 ChIP-seq 文库。约 250-350 bp 的文库通过 QIAquick 凝胶提取试剂盒（Qiagen, 28706）从琼脂糖凝胶中纯化，经 PCR 扩增后使用 AMPure XP 磁珠（Beckman Coulter Life Science, A63882）提取。文库质量采用标准灵敏度 NGS 片段分析试剂盒（Agilent，原 AATI, DNF-473-0500）在 12 通道片段分析仪上进行分析。使用 Quant-iT dsDNA 定量试剂盒进行定量后，各文库等浓度（2 nM）混合，采用高输出 v2 试剂盒（Illumina, FC-404-2002）在 Illumina NextSeq550 系统上进行单端（1×150）测序（每样本约 6000 万至 8000 万条读取）。所有 ChIP-seq 实验均至少在两个独立生物学重复中进行。

For H3-5HT and ETV5 ChIP–seq analysis, the raw paired-end reads were adapter and quality trimmed using TrimGalore (v.0.6.7). The resulting reads were aligned to mouse genome mm10 using bowtie2 (v.2.3.5.1, parameters: –local -D 20 -R 3 -N 0 -L 20 -i S,1,0.50 –no-unal –no-mixed –no-discordant –phred33 -I 10 -X 700)⁵². Duplicated reads were then marked and removed using picard MarkDuplicates (v.2.26.6) and SAMtools (v.1.14)⁵³, respectively. DeepTools (v.3.5.1) was used to convert all the resulting BAM files to Bigwig format for visualization⁵⁴. MACS2 (v.2.2.7.1) was used to call peaks, on the resulting BAM files, with a q-value threshold of 0.05 (ref. ⁵⁵). The ZR^FUS-binding sites obtained from a previous study were used to determine overlap with H3-5HT sites using computeMatrix¹¹. De novo motif analysis was performed using the HOMER (v.4.4) software suite.
对于 H3-5HT 和 ETV5 的 ChIP-seq 分析，原始双端读取序列通过 TrimGalore（v.0.6.7）进行适配器和质量修剪。修剪后的读取序列使用 bowtie2（v.2.3.5.1，参数：–local -D 20 -R 3 -N 0 -L 20 -i S,1,0.50 –no-unal –no-mixed –no-discordant –phred33 -I 10 -X 700） ⁵² 对小鼠基因组 mm10 进行比对。随后，利用 picard MarkDuplicates（v.2.26.6）和 SAMtools（v.1.14） ⁵³ 分别标记并移除重复读取序列。DeepTools（v.3.5.1）用于将所有生成的 BAM 文件转换为 Bigwig 格式以便可视化 ⁵⁴ 。MACS2（v.2.2.7.1）以 0.05 的 q 值阈值对生成的 BAM 文件进行峰识别（参考 ⁵⁵ ）。从前研究获得的 ZR ^FUS 结合位点用于通过 computeMatrix ¹¹ 确定与 H3-5HT 位点的重叠。利用 HOMER（v.4.4）软件套件进行从头基序分析。

For identifying core TFs, H3K27ac data were obtained from our previous study¹¹, and super-enhancers were first annotated using ROSE⁵⁶ with a stitching distance of 12.5 kb and with the exclusion of peaks within 2.5 kb of a promoter. To infer core TFs, the super-enhancers overlapping open chromatin regions marked by ATAC peaks were used. The resulting TFs were compared against the TFs identified in a ZR^FUS-specific context in our previous work¹⁰.
为了鉴定核心转录因子，我们从先前研究中获取了 H3K27ac 数据 ¹¹ ，并首先使用 ROSE ⁵⁶ 对超级增强子进行注释，拼接距离设定为 12.5 kb，并排除距离启动子 2.5 kb 内的峰。通过分析与 ATAC 峰标记的开放染色质区域重叠的超级增强子，推断出核心转录因子。随后，将这些转录因子与我们在先前工作中针对 ZR ^FUS 特异性环境所识别的转录因子进行对比 ¹⁰ 。

For H3K27ac and H3K27me3 ChIP–seq analysis in Fig. 3, sequencing files were downloaded and merged before performing quality control using fastQC (v.0.11.7) and MultiQC (v.1.6). Reads were then mapped to the mouse genome (mm10 assembly) using bowtie2 (v.2.2.6)⁵² before generating bedgraph files and tag directories using the HOMER (v.4.4) software suite⁵⁷. To filter ChIP peaks enriched over input control, the findPeaks command in histone mode was conducted using parameters set as follows: H3K27ac ChIP–seq: default; H3K27me3 ChIP–seq: -L 0 -C 0 -size 2000 -minDist 4000. Enriched peaks were annotated using annotatePeaks with mm10 assembly. To visualize peak distribution along the mouse genome, Integrated Genome Browser-compatible files were made using SAMtools (v.0.1.19), sort and index, deepTools (v.3.2.0) and bamCompare (v.3.2.0)^53,54. ETS-specific motif analysis was done using seq2profile.pl at 100 bp from the peak centre. Overlapping and differentially bound peaks between biological replicates were obtained using getDifferentialPeaks, and peaks were visualized using computeMatrix and plotHeatmap.
对于图 3 中的 H3K27ac 和 H3K27me3 ChIP-seq 分析，首先下载并合并测序文件，然后使用 fastQC（v.0.11.7）和 MultiQC（v.1.6）进行质量控制。随后，利用 bowtie2（v.2.2.6）将读段映射到小鼠基因组（mm10 组装版本）上，之后通过 HOMER（v.4.4）软件套件生成 bedgraph 文件和标签目录。为了筛选出相对于输入对照富集的 ChIP 峰，采用 histone 模式的 findPeaks 命令，参数设置如下：H3K27ac ChIP-seq：默认；H3K27me3 ChIP-seq：-L 0 -C 0 -size 2000 -minDist 4000。使用 annotatePeaks 结合 mm10 组装对富集峰进行注释。为在小鼠基因组上可视化峰的分布，利用 SAMtools（v.0.1.19）、sort 和 index，以及 deepTools（v.3.2.0）和 bamCompare（v.3.2.0）制作了 Integrated Genome Browser 兼容文件。ETS 特异性基序分析在峰中心 100 bp 处通过 seq2profile.pl 完成。通过 getDifferentialPeaks 获取生物学重复间的重叠及差异结合峰，并使用 computeMatrix 和 plotHeatmap 进行可视化。

In vivo barcoded screen 体内条形码筛选

A piggyBac transposable vector (piggyBac-CAG-GFP-T2A-attR1-attR4) was constructed from the piggyBac-eGFP plasmid⁵⁸. The eGFP stop codon was removed for inserting an in-frame T2A sequence followed by attR1 and attR4 Gateway cloning sites, which flanked with chloramphenicol and ccdB selection cassettes. A V5 tag sequence was inserted downstream of the attR4 site. Human variant open reading frames (ORFs) of the developmental TFs were cloned with a unique barcode sequence using the HiTMMoB method⁵⁹. A total of 38 barcoded TF ORFs and 12 barcoded mCherry controls were pooled at equal moles, ethanol precipitated and reconcentrated in ddH₂O to make the pooled plasmid library for IUE. This plasmid library was diluted to 1 μg μl⁻¹ for final injection cocktail.
构建了由 piggyBac-eGFP 质粒衍生的 piggyBac 转座载体（piggyBac-CAG-GFP-T2A-attR1-attR4） ⁵⁸ 。移除了 eGFP 终止密码子，以便插入与 attR1 和 attR4 Gateway 克隆位点相连的 T2A 序列，两侧为氯霉素和 ccdB 筛选盒。在 attR4 位点下游插入了 V5 标签序列。利用 HiTMMoB 方法，将发育转录因子（TFs）的人源变异开放阅读框（ORFs）与独特条形码序列克隆 ⁵⁹ 。总共 38 个条形码标记的 TF ORFs 和 12 个条形码标记的 mCherry 对照品按等摩尔混合，经乙醇沉淀并在 ddH ₂ O 中重新浓缩，制备成用于 IUE 的混合质粒文库。该质粒文库最终稀释至 1 μg/μl ⁻¹ ，用于注射混合液。

Mouse tumours were dissected and rinsed with PBS for the subsequent preparation of genomic DNA (gDNA) using the EZNA Tissue DNA Kit (Omega BioTek, D3396), according to the manufacturer’s instructions. The purified gDNA was used for constructing sequencing libraries as previously described²³. In brief, 50 ng of gDNA from mouse tumours and 2 ng of pooled IUE injection cocktail (input control) were used for amplifying barcoded sequences using PCR. Platinum Super Mix (Thermo Fisher Scientific, 12532016) with primers targeting upstream and downstream of the barcodes was used in this PCR reaction with the cycling parameters stated as follows: (94 °C, 4 min) × 1; (94 °C, 1 min; 54 °C, 1 min; 68 °C, 1 min) × 35; (68 °C, 10 min) × 1; hold at 4 °C. The PureLink PCR Purification Kit (Thermo Fisher Scientific, K310001) was used to purify the PCR products for further processing using the Ion Plus Library Kit (Thermo Fisher Scientific, 4471252). Samples were purified and ligated to unique Ion Xpress Barcode Adaptors (Thermo Fisher Scientific, 4474517) to generate the barcoded libraries. After amplification and purification, the resulting libraries of each sample were pooled for PGM sequencing (318 V2 Chip) according to the manufacturer’s recommendations. Raw data were concatenated into a single reference file and indexed using the Burrows–Wheeler alignment tool for aligning amplicon barcode sequences. Barcode enrichment level was calculated by quantifying the number of detected reads for each barcode relative to the total number of sequencing reads. Data were presented by normalizing values to input control and the standard error of the mean (s.e.m.) was calculated across replicates and plotted as error bars on the graphs.
小鼠肿瘤组织被解剖并用 PBS 冲洗，随后按照制造商的说明使用 EZNA Tissue DNA Kit（Omega BioTek，D3396）制备基因组 DNA（gDNA）。纯化的 gDNA 用于构建测序文库，如前所述 ²³ 。简言之，来自小鼠肿瘤的 50 ng gDNA 和 2 ng 混合 IUE 注射液（输入对照）用于通过 PCR 扩增带条形码的序列。使用含有针对条形码上下游引物的 Platinum Super Mix（Thermo Fisher Scientific，12532016）进行 PCR 反应，循环参数如下：（94 °C，4 min）× 1；（94 °C，1 min；54 °C，1 min；68 °C，1 min）× 35；（68 °C，10 min）× 1；保持在 4 °C。使用 PureLink PCR Purification Kit（Thermo Fisher Scientific，K310001）纯化 PCR 产物，进一步使用 Ion Plus Library Kit（Thermo Fisher Scientific，4471252）进行处理。样本被纯化并连接到独特的 Ion Xpress 条形码适配器（Thermo Fisher Scientific，4474517），以生成带条形码的文库。经过扩增和纯化后，各样本的文库按照制造商的建议合并，用于 PGM 测序（318 V2 芯片）。原始数据被合并成一个参考文件，并使用 Burrows-Wheeler 对齐工具进行索引，以对扩增子条形码序列进行比对。条形码富集水平通过计算每个条形码检测到的读数相对于总测序读数的数量来量化。数据通过归一化至输入对照值来呈现，并计算了各重复间的平均标准误差（s.e.m.），在图表上以误差条形式绘制。

IP–MS and co-immunoprecipitation
IP-MS 与共免疫沉淀

Tissues were washed with cold PBS three times and dissociated on ice using a pellet homogenizer. Nuclear lysates were extracted using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, 78833) according to the manufacturer’s instructions.
组织用冷 PBS 洗涤三次，并在冰上使用研杵均浆器进行解离。根据制造商的说明，使用 NE-PER 核与细胞质提取试剂（赛默飞世尔科技，78833）提取核裂解物。

For IP–MS, the immunoprecipitation and mass spectrometry were performed as described earlier⁵⁰. In brief, 3 mg of nuclear extracts were immunoprecipitated with anti-ETV5 antibody for 1 h at 4 °C. The protein complex was run on SDS–PAGE and in-gel-digested using trypsin protease. The liquid chromatography–mass spectrometry analysis was performed on a nanoLC1000 system coupled to an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific). The MS raw data were searched against the Mus musculus protein database from NCBI RefSeq (updated 24 March 2020). The ZR^FUS protein and human ETV5 protein sequence were added to the database as well. The raw data processing, label-free based quantification and differential analysis were performed as described before⁵⁰.
对于 IP-MS 分析，免疫沉淀与质谱检测按照先前描述的方法进行 ⁵⁰ 。简言之，取 3 毫克核提取物在 4°C 下与抗 ETV5 抗体共同孵育 1 小时进行免疫沉淀。随后，蛋白质复合物通过 SDS-PAGE 电泳分离，并采用胰蛋白酶进行胶内消化。液相色谱-质谱分析在 nanoLC1000 系统上进行，该系统与 Orbitrap Fusion 质谱仪（赛默飞世尔科技）联用。MS 原始数据比对至 NCBI RefSeq 的 Mus musculus 蛋白数据库（更新于 2020 年 3 月 24 日）。同时，ZR ^FUS 蛋白及人类 ETV5 蛋白序列也被添加至该数据库中。原始数据处理、基于无标记定量及差异分析均依照先前所述方法进行 ⁵⁰ 。

For co-immunoprecipitation, nuclear lysates were prepared from tumour tissues as described above. Immunoprecipitation was performed by incubating nuclear lysates with antibodies or anti-IgG overnight at 4 °C with rotation. The next day, recombinant protein G agarose (Thermo Fisher Scientific, 15920010) was added for subsequent pull-down at 4 °C for 4 h. After collecting and washing steps, beads were boiled in 2× SDS gel loading dye for 10 min at 95 °C to elute immunoprecipitated proteins. Eluates were subjected to further analyses by western blot as described above. Antibody information is provided in Supplementary Table 17.
为了进行共免疫沉淀，按照上述方法从肿瘤组织中制备核裂解物。通过将核裂解物与抗体或抗 IgG 在 4°C 下旋转孵育过夜来执行免疫沉淀。次日，加入重组蛋白 G 琼脂糖（Thermo Fisher Scientific，15920010），在 4°C 下进行 4 小时的下拉操作。收集并洗涤后，珠子在 95°C 下用 2×SDS 凝胶加载染料煮沸 10 分钟以释放免疫沉淀的蛋白质。洗脱物随后通过如上所述的 Western blot 进行进一步分析。抗体信息见补充表 17。

RNA purification and RT–qPCR
RNA 纯化与 RT-qPCR

Mouse tumours were dissected, rinsed with PBS and dissociated in TRIzol (Thermo Fisher Scientific, 15596018). Total RNA was purified using the RNeasy Mini Kit (Qiagen, 74106) following the manufacturer’s instructions. Five hundred nanograms of RNA was converted to complementary DNA (cDNA) using iScript Reverse Transcriptase Supermix (Bio-Rad, 1708841). qPCR reaction mix included 2 ng cDNA, 250 nM primers and 1× PerfeCTa SYBR Fast Mix (Quantabio, 95072-012), and the reaction was performed on a Bio-Rad CFX96 Touch Real-Time PCR Detection System (95 °C for 30 s, 40 cycles of 95 °C for 5 s and 60 °C for 30 s, with subsequent melting curve analysis). The transcript expression of target genes was normalized to Actb. Primer sequences are provided in Supplementary Table 17.
小鼠肿瘤被解剖，用 PBS 冲洗并在 TRIzol（赛默飞世尔科技，15596018）中分离。总 RNA 采用 RNeasy Mini 试剂盒（Qiagen，74106）按照制造商说明书纯化。500 纳克 RNA 使用 iScript 逆转录酶 Supermix（Bio-Rad，1708841）转化为互补 DNA（cDNA）。qPCR 反应混合物包含 2 纳克 cDNA、250 nM 引物及 1× PerfeCTa SYBR Fast Mix（Quantabio，95072-012），反应在 Bio-Rad CFX96 Touch 实时 PCR 检测系统上进行（95 °C 30 秒，40 个循环的 95 °C 5 秒和 60 °C 30 秒，随后进行熔解曲线分析）。目标基因的转录表达量以 Actb 为基准进行归一化。引物序列见补充表 17。

RNA-seq library preparation, sequencing and bioinformatic analysis
RNA-seq 文库制备、测序及生物信息学分析

RNA extraction was performed as described above. Three hundred nanograms of total RNA was used for constructing Illumina sequencing libraries with 6-bp single indices by the TruSeq Stranded mRNA kit (Illumina, RS-122-2101). An equal concentration (2 nM) of each library was pooled together for paired-end sequencing using the Mid Output v2 kit (Illumina, 20024904) on an Illumina NextSeq550 System (around 20 million–30 million reads per sample).
如上所述进行 RNA 提取。使用 300 纳克总 RNA 通过 TruSeq Stranded mRNA 试剂盒（Illumina, RS-122-2101）构建含 6 碱基单标签的 Illumina 测序文库。各文库以等浓度（2 nM）混合，采用 Mid Output v2 试剂盒（Illumina, 20024904）在 Illumina NextSeq550 系统上进行双端测序（每样本约 2000 万至 3000 万条读取）。

Sequencing files were downloaded in fastq file format and quality control was performed using fastQC (v.0.11.7) and MultiQC (v.1.6) before mapping reads to the mouse genome (mm10 assembly) using STAR (v.2.5.0a)⁶⁰. Mapped reads were then used to build count matrices using the Bioconductor packages GenomicAlignments (v.1.26.0) and GenomicFeatures (v.1.42.3)⁶¹ in R (v.4.0.3). University of California Santa Cruz transcripts were downloaded from Illumina iGenomes in GTF file format. DESeq2 (v.1.30.1)⁶² was used to normalize and analyse differential gene expression. Enrichr was used to determine GO terms with P < 0.05 and ggplot2 (v.3.3.5) was used to visualize the results.
测序文件以 fastq 格式下载，并使用 fastQC（v0.11.7）和 MultiQC（v1.6）进行质量控制，随后通过 STAR（v2.5.0a）将读段映射到小鼠基因组（mm10 组装版本） ⁶⁰ 。映射后的读段接着用于构建计数矩阵，借助 R（v4.0.3）中的 Bioconductor 包 GenomicAlignments（v1.26.0）和 GenomicFeatures（v1.42.3） ⁶¹ 。从 Illumina iGenomes 下载的加州大学圣克鲁兹分校转录本以 GTF 文件格式提供。采用 DESeq2（v1.30.1） ⁶² 进行基因表达量的归一化与差异分析。Enrichr 用于确定 P 值小于 0.05 的 GO 条目，而 ggplot2（v3.3.5）则用于结果的可视化展示。

Slice recording for EPSCs and IPSCs
EPSCs 和 IPSCs 的切片记录

Mice were anaesthetized with isoflurane at P30 to isolate and submerge the brains in ice-cold artificial cerebrospinal fluid (ACSF) solution (130 mM NaCl, 24 mM NaHCO₃, 1.25 mM NaH₂PO₄, 3.5 mM KCl, 1.5 mM CaCl₂,1.5 mM MgCl₂ and 10 mM D(+)-glucose, pH 7.4). Three-hundred-millimetre brain slices were cut using a vibratome (DSK Linear Slicer) oxygenated in ACSF at room temperature for 1 h and then acclimated at room temperature with continuous perfusion with ASCF solution (2 ml per min). Slices were placed in the recording chamber, and target cells were identified with an upright Olympus microscope with a 60× water immersion objective with infrared differential interference contrast optics. Whole-cell recording was performed from cortical neurons at room temperature with pCLAMP 10 and a MultiClamp 700B amplifier (Axon Instruments, Molecular Devices). The holding potential was −60 mV and the pipette resistance was typically 5–8 MΩ. The pipette was filled with an internal solution: 135 mM CsMeSO₄, 8 mM NaCl, 10 mM HEPES, 0.25 mM EGTA, 1 mM Mg‐ATP, 0.25 mM Na₂‐GTP and 30 mM QX‐314, pH 7.2 with CsOH (278–285 mOsmol) for EPSC measurement; 135 mM CsCl, 4 mM NaCl, 0.5 mM CaCl₂, 10 mM HEPES, 5 mM EGTA, 2 mM Mg-ATP, 0.5 mM Na₂-GTP and 30 mM QX-314, pH 7.2 with CsOH (278–285 mOsmol) for IPSC measurement. IPSC measurement was performed with ionotropic glutamate receptor antagonists, (2R)-amino-5-phosphonovaleric acid (APV) (50 mM, Tocris) and cyanquixaline (CNQX) (20 mM, Tocris). Electrical signals were digitized and sampled at 50-ms intervals with Digidata 1550B and a MultiClamp 700B amplifier (Molecular Devices) on pCLAMP 10.7 software. Data were filtered at 2 kHz and the recorded current was analysed with ClampFit 10.7 software.
在 P30 时，小鼠通过异氟烷麻醉，以冰冷的人工脑脊液（ACSF）溶液（130 mM NaCl，24 mM NaHCO ₃ ，1.25 mM NaH ₂ PO ₄ ，3.5 mM KCl，1.5 mM CaCl ₂ ，1.5 mM MgCl ₂ 及 10 mM D(+)-葡萄糖，pH 7.4）分离并浸泡大脑。使用振动切片机（DSK Linear Slicer）切取 300 毫米厚的大脑切片，在室温下用 ACSF 充氧 1 小时，然后在室温下适应，并持续用 ASCF 溶液（每分钟 2 毫升）灌流。将切片置于记录室中，目标细胞通过带有 60×水浸物镜和红外差分干涉对比光学系统的奥林巴斯显微镜进行识别。在室温下，使用 pCLAMP 10 和 MultiClamp 700B 放大器（Axon Instruments，Molecular Devices）对皮质神经元进行全细胞记录。保持电位为−60 mV，电极阻抗通常为 5–8 MΩ。电极内充填内部溶液：135 mM CsMeSO ₄ ，8 mM NaCl，10 mM HEPES，0.25 mM EGTA，1 mM Mg‐ATP，0.25 mM Na ₂ ‐GTP 及 30 mM QX‐314，pH 7。用于 EPSC 测量的溶液：135 mM CsCl，4 mM NaCl，0.5 mM CaCl，10 mM HEPES，5 mM EGTA，2 mM Mg-ATP，0.5 mM Na-GTP，30 mM QX-314，pH 7.2，用 CsOH 调节至 278–285 mOsmol。用于 IPSC 测量的溶液：135 mM CsCl，4 mM NaCl，0.5 mM CaCl，10 mM HEPES，5 mM EGTA，2 mM Mg-ATP，0.5 mM Na-GTP，30 mM QX-314，pH 7.2，用 CsOH 调节至 278–285 mOsmol。IPSC 测量中使用了离子型谷氨酸受体拮抗剂，(2R)-氨基-5-膦基戊酸(APV)(50 mM，Tocris)和氰基喹啉(CNQX)(20 mM，Tocris)。电信号通过 Digidata 1550B 数字化并每隔 50 毫秒采样一次，使用 MultiClamp 700B 放大器(Molecular Devices)在 pCLAMP 10.7 软件上进行处理。数据以 2 kHz 的频率过滤，记录的电流通过 ClampFit 10.7 软件进行分析。

EEGs 脑电图

Mice at the age of P21-25 were implanted with intracranial EEG electrodes as per previously published protocols²³. Forty-eight hours of continuous monitoring sessions were performed every ten days from P30. In each recording session, mice were connected to a tethered wire and housed in a recording chamber in which food and water were provided ad libitum. The recording room was maintained at 20–22 °C with 40% humidity and featured 12-h light–dark cycles. EEG signals were acquired at 2 kHz using BioAmp (ADI), digitized and analysed using LabChart Pro software. Cortical spikes were quantified using a built-in function in LabChart Pro with optimized detection parameters for each event. Seizure event was calculated from the waveform data and corresponding video-recorded behaviours by visual inspection.
P21-25 龄的小鼠按照先前发表的协议植入颅内 EEG 电极 ²³ 。从 P30 开始，每十天进行一次连续 48 小时的监测。在每次记录期间，小鼠通过系绳连接，并安置在提供自由取食和饮水的记录室内。记录室保持在 20-22°C，湿度 40%，并设有 12 小时的光暗循环。使用 BioAmp（ADI）以 2 kHz 频率采集 EEG 信号，通过 LabChart Pro 软件进行数字化和分析。利用 LabChart Pro 内置功能，针对每次事件优化检测参数，对皮质尖波进行量化。通过视觉检查波形数据及相应视频记录的行为来计算癫痫发作事件。

Quantification and statistical analysis
定量与统计分析

Sample sizes and statistical details are listed in all figure legends. All analyses were performed blind to experimental conditions. Mice from each cohort were randomly distributed to experimental groups. Analyses were conducted using ImageJ/Fiji, GraphPad Prism 9 and RStudio. For immunofluorescent imaging, each experiment was performed in at least three independent pairs of mice using the same sets of staining and confocal imaging conditions. Representative images with similar results are shown in the figures. For Kaplan–Meier survival analysis, we used the log-rank test to compare the differences between groups. For the other datasets, data were tested for normal distribution using the Shapiro–Wilk test and for homogeneity of variance using the Levene test. Parametric tests were used for normally distributed datasets and for data with a small sample size (n = 3). For comparing two groups, unpaired or paired Student’s t-tests were used, and for comparing three groups, the one-way analysis of variance (ANOVA) followed by Tukey’s test were used. If data were not normally distributed, non-parametric Wilcoxon rank sum tests were performed. Data are presented as mean ± s.e.m. n refers to the number of mice. Levels of statistical significance are indicated as follows: *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.
样本量及统计细节均在各图例中列出。所有分析均在盲法条件下进行，即不知晓实验条件。每组小鼠随机分配至各实验组。分析采用 ImageJ/Fiji、GraphPad Prism 9 及 RStudio 软件进行。免疫荧光成像实验中，每项实验至少在三对独立的小鼠上重复进行，使用相同的染色和共聚焦成像条件。具有代表性的相似结果图像展示于图中。对于 Kaplan-Meier 生存分析，我们采用对数秩检验比较各组间的差异。对于其他数据集，首先使用 Shapiro-Wilk 检验测试数据的正态分布，并使用 Levene 检验检验方差齐性。对于正态分布的数据集及样本量较小的数据（n=3），采用参数检验。比较两组时，使用非配对或配对的学生 t 检验；比较三组时，先进行单因素方差分析（ANOVA），随后进行 Tukey 检验。若数据非正态分布，则执行非参数的 Wilcoxon 秩和检验。数据以均值±标准误（s.e.m.）形式呈现。 n 表示小鼠数量。统计显著性水平标记如下：*P < 0.05，**P < 0.01，***P < 0.001 及 ****P < 0.0001。

Reporting summary 报告摘要

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
有关研究设计的更多信息，请参阅与本文关联的《自然》系列期刊报告摘要。

Data availability 数据可用性

ChIP–seq and RNA-seq data have been deposited in the NCBI Gene Expression Omnibus under the accession number GSE246033. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD048170. All other data in this article are available from the corresponding authors on reasonable request. Uncropped immunoblots associated with Figs. 2, 3 and Extended Data Fig. 7 are provided in the supplementary information. Source data are provided with this paper.
ChIP-seq 和 RNA-seq 数据已存入 NCBI 基因表达综合数据库，存取号为 GSE246033。质谱蛋白质组学数据已通过 PRIDE 合作伙伴存储库提交至 ProteomeXchange 联盟，数据集标识符为 PXD048170。本文中其他所有数据可根据合理请求从通讯作者处获得。与图 2、图 3 及扩展数据图 7 相关的未裁剪免疫印迹图附于补充信息中。源数据随本文提供。

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Acknowledgements

This work was supported by US National Institutes of Health grants R35-NS132230 and R01-NS124093 to B.D., R01-CA284455 to B.D. and S.C.M., R01-CA223388 to B.D. and J.L.N., and R01NS128184, R01CA280203 and U01CA281823 to S.C.M.; the National Cancer Institute Cancer Target Discovery and Development grant U01-CA217842 to B.D.; DOD-IDEA (CA220510), and an DOD-IMPACT (CA220247) award to S.C.M.; National Cancer Institute Cancer Center Support Grant, P30 CA021765, St. Jude Children’s Research Hospital Research Collaborative on Transcription Regulation in Pediatric Cancer Grant, Alex’s Lemonade Stand Foundation ‘A’ Award to S.C.M; S.C.M is supported by funding from the National Brain Tumor Society and CERN Foundation; the ALSAC Foundation; and the Cancer Prevention Research Institute of Texas (CPRIT) (RP210027 to H.-C.C., F31-CA243382 to E.H.-H., T32-5T32HL092332-19 to B.L., 1K99-DC019668 to D.S. and AHA-23POST1019413 to M.R.W.). We are thankful for support from the David and Eula Wintermann Foundation and the Adrienne Helis Malvin Medical Research Foundation. We thank I. Maze for providing the H3.3-Q5A constructs, and we acknowledge the Optogenetics and Viral Vectors Core at the Jan and Dan Duncan Neurological Research Institute. BCM Mass Spectrometry Proteomics Core is supported by a Dan L. Duncan Comprehensive Cancer Center NIH award (P30 CA125123) and a CPRIT Core Facility Award (RP210227). Research reported in this publication was supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health under award number P50HD103555 for use of the Microscopy Core facilities and the Animal Phenotyping and Preclinical Endpoints Core facilities. Images in schematics were created using Biorender.com.

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Authors and Affiliations

Program in Cancer and Cell Biology, Baylor College of Medicine, Houston, TX, USA
Hsiao-Chi Chen, Peihao He & Benjamin Deneen
Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX, USA
Hsiao-Chi Chen, Peihao He, Malcolm McDonald, Michael R. Williamson, Brittney Lozzi, Junsung Woo, Dong-Joo Choi, Debosmita Sardar, Emmet Huang-Hobbs & Benjamin Deneen
Center for Cancer Neuroscience, Baylor College of Medicine, Houston, TX, USA
Hsiao-Chi Chen, Peihao He, Malcolm McDonald, Michael R. Williamson, Brittney Lozzi, Junsung Woo, Dong-Joo Choi, Debosmita Sardar, Emmet Huang-Hobbs, Ganesh Rao & Benjamin Deneen
Integrative Molecular and Biomedical Sciences Graduate Program, Baylor College of Medicine, Houston, TX, USA
Hsiao-Chi Chen, Emmet Huang-Hobbs & Benjamin Deneen
Program in Development, Disease, Models and Therapeutics, Baylor College of Medicine, Houston, TX, USA
Malcolm McDonald & Benjamin Deneen
Center of Excellence in Neuro-Oncology Sciences (CENOS), St. Jude Children’s Research Hospital, Memphis, TN, USA
Srinidhi Varadharajan, Hua Sun, Siri M. Ippagunta & Stephen C. Mack
Program in Genetics and Genomics, Baylor College of Medicine, Houston, TX, USA
Brittney Lozzi
Mass Spectrometry Proteomics Core, Baylor College of Medicine, Houston, TX, USA
Antrix Jain
Department of Neurosurgery, Baylor College of Medicine, Houston, TX, USA
Ganesh Rao & Benjamin Deneen
Department of Radiation Oncology, St. Jude Children’s Research Hospital, Memphis, TN, USA
Thomas E. Merchant
Department of Pathology, St. Jude Children’s Research Hospital, Memphis, TN, USA
David W. Ellison
Department of Human and Molecular Genetics, Baylor College of Medicine, Houston, TX, USA
Jeffrey L. Noebels
Department of Neurology, Baylor College of Medicine, Houston, TX, USA
Jeffrey L. Noebels
Division of Neuro-Oncology, Department of Oncology, St. Jude Children’s Research Hospital, Memphis, TN, USA
Kelsey C. Bertrand
Department of Developmental Neurobiology, St Jude Children’s Research Hospital, Memphis, TN, USA
Stephen C. Mack
Neurobiology and Brain Tumor Program, St Jude Children’s Research Hospital, Memphis, TN, USA
Stephen C. Mack

Authors

Hsiao-Chi Chen
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Peihao He
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Malcolm McDonald
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Michael R. Williamson
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Srinidhi Varadharajan
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Brittney Lozzi
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Junsung Woo
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Dong-Joo Choi
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Debosmita Sardar
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Emmet Huang-Hobbs
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Hua Sun
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Siri M. Ippagunta
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Antrix Jain
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Ganesh Rao
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Thomas E. Merchant
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David W. Ellison
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Jeffrey L. Noebels
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Kelsey C. Bertrand
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Stephen C. Mack
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Benjamin Deneen
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Contributions

H.-C.C., S.C.M. and B.D. conceived the project and designed the experiments. H.-C.C., P.H., D.-J.C. and M.R.W. performed the mouse tumour experiments. H.-C.C., B.L. and E.H.-H. performed the barcoded screen. H.-C.C., D.S. and B.L. performed the RNA-seq and ChIP–seq experiments. H.-C.C. performed synaptic staining and immunostaining of tumours. A.J. performed the proteomic analysis. M.M. performed the electrophysiology studies. J.W. performed electrophysiology recording. J.L.N., S.I., G.R., T.E.M., D.W.E. and K.C.B. provided essential reagents. H.-C.C., S.V., H.S. and S.C.M. designed and performed the bioinformatics analyses. H.-C.C., S.C.M. and B.D. wrote the manuscript.

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Correspondence to Stephen C. Mack or Benjamin Deneen.

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Nature thanks Michelle Monje and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data figures and tables

Extended Data Fig. 1 DREADD-based activation of neurons by CNO treatment.

a, Schematic of DREADD-hM3Dq activation of ipsilateral cortical neurons in ZR^FUS EPN mice. b, Low-magnification image of EPN tumours and representative BrdU staining of EPN tumours after DREADD-hM3Dq activation of ipsilateral cortical neurons via CNO (scale bar = 50 μm). c, Quantification of BrdU staining in saline versus CNO treated EPN tumours (n = 3 per group, mean ± s.e.m., unpaired two-sided Student’s t-test, P_{SalinevsAAV_CNO} = 0.4522, P_{CNOvsAAV_CNO} = 0.1221, *P_{AAV_SalinevsAAV_CNO} = 0.0337). d, Schematic of DREADD-hM3Dq activation of ipsilateral inhibitory neurons in ZR^FUS EPN mice. e, Low-magnification image of EPN tumours and representative BrdU staining of EPN tumours after DREADD-based activation of ipsilateral inhibitory neurons via CNO (scale bar = 50 μm). f, Quantification of BrdU staining in saline versus CNO treated EPN tumours (Saline: n = 3, CNO: n = 3, AAV_Saline: n = 3, AAV_CNO: n = 4, mean ± s.e.m., unpaired two-sided Student’s t-test, P_{SalinevsAAV_CNO} = 0.0770, P_{CNOvsAAV_CNO} = 0.3896, P_{AAV_SalinevsAAV_CNO} = 0.8727). g, Representative BrdU staining in saline and CNO (0.5 and 5 mg per kg) treated EPN tumours (scale bar = 50 μm). h, Immunofluorescence staining of FOS in the ipsilateral cortical neurons in saline versus CNO treated EPN tumours (scale bar = 50 μm). i, Quantification of FOS positive neurons in the ipsilateral cortex in saline versus CNO treated EPN tumours (n = 3 per group, mean ± s.e.m., unpaired two-sided Student’s t-test, *P = 0.0155). j, Immunofluorescence staining of mCherry and TPH2 in the AAV injected dRN (scale bar = 30 μm). TPH2: tryptophan hydroxylase 2. k, Immunofluorescence staining of FOS in the dRN serotonergic neurons in saline versus CNO treated EPN tumours (scale bar = 50 μm). l, Quantification of FOS positive neurons in the dRN serotonergic neurons in saline versus CNO treated EPN tumours (n = 3 per group, mean ± s.e.m., unpaired two-sided Student’s t-test, ***P = 0.0005). a and d were created using Biorender.com.

Source Data

Extended Data Fig. 2 Expression of monoamine transporters in EPN.

a, Expression levels of genes under monoamine transporter GO term in human ZR^FUS versus non-ZR^FUS EPN tumours. b, Expression levels of genes under monoamine transporter GO term in mouse EPN tumours versus non-tumour tissues. c, Log₂(FC) of serotonin transporters in human ZR^FUS tumours versus non-ZR^FUS tumour tissues. d, Log₂(FC) of serotonin transporters in mouse EPN tumours versus non-tumour tissues. e, Normalized read counts of serotonin transporters in mouse EPN tumours versus non-tumour tissues (at least n = 10 per group, median ± upper and lower limits, box boundary states upper and lower quartiles). f, Immunofluorescence staining of SLC6A4 in mouse non-tumour cortex and EPN tumours (scale bar = 50 μm). g, Immunofluorescence staining of dopamine transporter (SLC6A3) in mouse substantia nigra, non-tumour cortex and EPN tumours (scale bar = 50 μm).

Source Data

Extended Data Fig. 3 Synaptic staining and H3-5HT staining after dRN manipulation.

a, Low-magnification image of tumour margin and representative higher-magnification images (derived from dashed box) of peritumoral excitatory synaptic staining in saline versus CNO treated tumours from Fig. 1f (scale bar = 10 μm). b, Quantification of excitatory synaptic staining in saline versus CNO treated tumours Fig. 1f (AAV_saline: n = 4, AAV_CNO: n = 3, mean ± s.e.m., two-sided Wilcoxon rank sum test, P = 0.6286). c, Low-magnification view of tumour margin and representative higher-magnification images (derived from dashed box) of peritumoral inhibitory synaptic staining in saline versus CNO treated tumours from Fig. 1f (scale bar = 10 μm). d, Quantification of inhibitory synaptic staining in saline versus CNO treated tumours from Fig. 1f (AAV_saline: n = 4, AAV_CNO: n = 3, mean ± s.e.m., unpaired two-sided Student’s t-test, P = 0.7481). e, Low-magnification image of tumour margin and representative higher-magnification images (derived from dashed box) of peritumoral excitatory synaptic staining in saline versus CNO treated tumours from Fig. 1i (scale bar = 10 μm). f, Quantification of excitatory synaptic staining in saline versus CNO treated tumours from Fig. 1i (n = 3 per group, mean ± s.e.m., unpaired two-sided Student’s t-test, P = 0.6460). g, Low-magnification image of tumour margin and representative higher-magnification images (derived from dashed box) of peritumoral inhibitory synaptic staining in saline versus CNO treated tumours from Fig. 1i (scale bar = 10 μm). h, Quantification of inhibitory synaptic staining in saline versus CNO treated tumours from Fig. 1i (n = 3 per group, mean ± s.e.m., unpaired two-sided Student’s t-test, P = 0.9864). i, Immunofluorescence staining of H3-5HT in saline versus CNO treated tumours from Fig. 1f (scale bar = 10 μm). j, Quantification of H3-5HT intensity in saline versus CNO treated tumours from Fig. 1f (n = 3 per group, mean ± s.e.m., unpaired two-sided Student’s t-test, P = 0.2894). k, Immunofluorescence staining of H3-5HT in saline versus CNO treated tumours from Fig. 1i (scale bar = 10 μm). l, Quantification of H3-5HT intensity in saline versus CNO treated tumours from Fig. 1i (n = 3 per group, mean ± s.e.m., unpaired two-sided Student’s t-test, *P = 0.0465).

Source Data

Extended Data Fig. 4 Tumour proliferation and ETV5 expression in H3.3-Q5A EPN tumours.

a, Immunofluorescence staining of H3-5HT in H3.3-WT and H3.3-Q5A EPN tumours (scale bar = 25 μm). b, Quantification of H3-5HT intensity in H3.3 control and H3.3-Q5A (n = 4 per group, mean ± s.e.m., two-sided Wilcoxon rank sum test, *P = 0.0286) c, Representative Ki67 staining of EPN control versus H3.3 wild-type and H3.3-Q5A tumours (scale bar = 50 μm). d, Quantification of Ki67 staining in EPN control versus H3.3 wild-type and H3.3-Q5A tumours (EPN control: n = 3, H3.3 wild-type: n = 5, H3.3-Q5A: n = 4, mean ± s.e.m., unpaired two-sided Student’s t-test, P_{EPNcontrolvsH3.3wild-type} = 0.9040, **P_{EPNcontrolvsH3.3-Q5A} = 0.0015, ****P_{H3.3wild-typevsH3.3-Q5A} = 1.59E-05). e, Representative ETV5 staining of EPN control versus H3.3 wild-type and H3.3-Q5A tumours (scale bar = 25 μm). f, Quantification of ETV5 staining in EPN control versus H3.3 wild-type and H3.3-Q5A tumours (EPN control: n = 3, H3.3 wild-type: n = 3, H3.3-Q5A: n = 4, mean ± s.e.m., unpaired two-sided Student’s t-test, P_{EPNcontrolvsH3.3wild-type} = 0.6180, **P_{EPNcontrolvsH3.3-Q5A} = 0.0053, *P_{H3.3wild-typevsH3.3-Q5A} = 0.0398).

Source Data

Extended Data Fig. 5 SLC6A4-LOF EPN analyses, expression of serotonin synthetase and SSRI treatment in EPN.

a, Kaplan–Meier survival curve of EPN control (n = 19, median = 74 days) and SLC6A4-LOF (n = 14, median = 95 days, log-rank test, P = 0.1177). b, Representative Ki67 staining of EPN control versus SLC6A4-LOF tumours (scale bar = 50 μm). c, Quantification of Ki67 staining in EPN control versus SLC6A4-LOF tumours (n = 3 per group, mean ± s.e.m., unpaired two-sided Student’s t-test, **P = 0.0055). d, Representative H3-5HT staining of EPN control versus SLC6A4-LOF tumours (scale bar = 25 μm). e, Quantification of H3-5HT intensity in EPN control versus Slc6a4-LOF tumours (n = 4 per group, mean ± s.e.m., two-sided Wilcoxon rank sum test, *P = 0.0286). f, Immunofluorescent staining of TPH2 in mouse dRN (positive control), non-tumour cortex, and EPN tumour (scale bar = 50 μm) g, Quantification of TPH2 intensity in mouse dRN, non-tumour cortex, and EPN tumour (n = 3 per group, mean ± s.e.m., unpaired two-sided Student’s t-test, ****P_{dRNvsnon-tumourcortex} = 3.76E-05, **P_{dRNvsEPNtumour} = 3.73E-05, P_{non-tumourcortexvsEPNtumour} = 0.7262). h, Schematic of DMSO or SSRI treatment in EPN i, Representative H3-5HT staining of DMSO versus sertraline-HCl treated tumours (scale bar = 25 μm). j, Quantification of H3-5HT intensity in DMSO versus sertraline-HCl treated tumours (n = 3 per group, mean ± s.e.m., unpaired two-sided Student’s t-test, P = 0.0743). h was created using Biorender.com.

Source Data

Extended Data Fig. 6 H3-5HT ChIP–seq and ETV5 ChIP–seq analyses in EPN.

a, Venn diagram depicting core TFs with H3-5HT peaks in mouse EPN tumours. b, ETV5 ChIP–seq heat map profiles in mouse EPN tumours and Venn diagram depicting genes overlapped between downregulated DEGs acquiring H3K27me3 peaks in ETV5-GOF tumours and ETV5 annotated genes.

Extended Data Fig. 7 Validation of candidates from functional screen.

a, Immunoblots of LHX2, LHX4, ETV5, and KLF12 in mouse non-tumour cortex versus EPN tumours (n = 3 per group). b, Kaplan–Meier survival curves of EPN control (n = 51, median = 70 days), LHX2-GOF (n = 23, median = 82 days, log-rank test, P = 0.9640), LHX4-GOF (n = 9, median=114 days, log-rank test, P = 0.3532), and KLF12-GOF (n = 23, median = 77 days, log-rank test, P = 0.7588). c, Immunoblots of LHX2, LHX4, and KLF12 in control versus corresponding GOF tumours. d, Immunoblots of ETV5 in control versus ETV5-GOF and ETV5-LOF tumours. e, RT–qPCR fold enrichment of ETV5 and Etv5 transcript (ddCt) in control versus ETV5-GOF and ETV5-LOF tumours (n = 3 per group, mean ± s.e.m., unpaired two-sided Student’s t-test, GOF: **P = 0.0068, LOF: ***P = 0.0007). f, Surveyor Nuclease Digestion assay of Etv5 gRNA efficiency. Mouse non-tumour cortex: negative control. Two primer sets were used, and gel images are presented in left and right panel. Asterisks label the nuclease digested bands. g, Representative BrdU staining of control versus ETV5-GOF and ETV5-LOF tumours (scale bar = 50 μm). h, Quantification of BrdU staining in control versus ETV5-GOF and ETV5-LOF tumours (n = 3 per group, mean ± s.e.m., unpaired two-sided Student’s t-test, GOF: **P = 0.00258, LOF: ***P = 0.000976). i, Immunoblots of H3K27ac and H3K27me3 in control versus ETV5-GOF tumours. Total H3: loading control. j, Ring chart showing percentage of H3K27me3 sites in ETV5-GOF tumours carrying ETS motif allowing 0 mismatch at 1,000 bp from peak centre. k, GO-terms analysis of ETV5 binding partners in mouse EPN tumours performed using Enrichr (two-sided Fisher’s exact test). l, RT–qPCR fold enrichment of gene transcript (ddCt) in control versus ETV5-GOF tumours (n = 3 per group, mean ± s.e.m., unpaired two-sided Student’s t-test, Npy: ***P = 0.00024, Gabra5: P = 0.4342, Chrm4: *P = 0.0239, Kcnmb4: *P = 0.0109, Nptx2: P = 0.2549). m, H3K27me3 ChIP–seq peaks at Npy and Chrm4 locus in control and ETV5-GOF tumours.

Source Data

Extended Data Fig. 8 Comparison of Kaplan–Meier survival curves between sexes.

a–c, All Kaplan–Meier survival curves (a), table of median survival (b) and comparison of log-rank test results (c) between groups divided by sex.

Source Data

Extended Data Fig. 9 Analysis of NPY-GOF EPN tumours.

a, RT–qPCR fold enrichment of NPY transcript (ddCt) in control versus NPY-GOF tumours (n = 3 per group, mean ± s.e.m., unpaired two-sided Student’s t-test, *P = 0.0255). b, RT–qPCR fold enrichment of Npy2r transcript (ddCt) in mouse non-tumour cortex and EPN tumours (n = 3 per group, as mean ± s.e.m., unpaired two-sided Student’s t-test, *P = 0.0215). c, Immunofluorescence staining of NPY2R in mouse cortex. NeuN: neuronal marker, Aldh1l1: astrocytic marker (scale bar = 50 μm). d, Low-magnification image of the tumour margin and representative images of NPY2R staining in mouse non-tumour and EPN tumours (scale bar = 50 μm). Top right panel: Quantification of NPY2R intensity normalized to DAPI in mouse non-tumour cortex versus EPN tumours. (n = 4 per group, mean ± s.e.m., unpaired two-sided Student’s t-test, **P = 0.0014). e, GO-terms analysis of the downregulated DEGs in NPY-GOF tumours versus control performed using Enrichr (two-sided Fisher’s exact test). f, Zoom-out EEG traces from mice bearing control and NPY-GOF tumours.

Source Data

Supplementary information

Uncropped immunoblots associated with Figs. 2, 3 and Extended Data Fig. 7

Reporting Summary

Peer Review file

Supplementary Tables 1-17

Supplementary Table 1. DEGs from RNA-seq data of patients with ZR^FUS versus non-ZR^FUS EPN; associated with Fig. 1. Supplementary Table 2. Motif analysis of genes with ZR^FUS-HA and H3-5HT overlapped genes; associated with Fig. 2. Supplementary Table 3. H3-5HT annotated genes and core TF lists; associated with Fig. 2. Supplementary Table 4. TF lists in barcode screen; associated with Fig. 3. Supplementary Table 5. EPNCtrl_H3K27ac annotated genes; associated with Fig. 3. Supplementary Table 6. EPNETV5GOF_H3K27ac annotated genes; associated with Fig. 3. Supplementary Table 7. EPNCtrl_H3K27me3 annotated genes; associated with Fig. 3. Supplementary Table 8. EPNETV5GOF_H3K27me3 annotated genes; associated with Fig. 3. Supplementary Table 9. ETV5 IP–MS list; associated with Fig. 3 Supplementary Table 10. GO analysis of ETV5 interacting proteins in EPN tumour exclusively; analysed and acquired table from Enrichr datasets, associated with Extended Data Fig. 7. Supplementary Table 11. DEGs from RNA-seq data of mouse ETV5-GOF versus EPN Ctrl tumours; associated with Fig.3. Supplementary Table 12. EPN_ETV5 annotated genes; associated with Extended Data Fig. 6. Supplementary Table 13. Overlapped genes between downregulated DEGs which gain H3K27me3 peaks in mouse ETV5-GOF tumours; associated with Fig. 3. Supplementary Table 14. GO analysis of downregulated DEGs which gain H3K27me3 peaks in mouse ETV5-GOF tumours; analysed and acquired table from Enrichr datasets, associated with Fig. 3. Supplementary Table 15. DEGs from RNA-seq data of mouse NPY-GOF versus EPN Ctrl tumours; associated with Fig.4. Supplementary Table 16. GO analysis of downregulated DEGs in NPY-GOF versus EPN Ctrl tumours; analysed and acquired table from Enrichr datasets, associated with Extended Data Fig. Supplementary Table 17. Primer, antibody, and plasmid tables

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Chen, HC., He, P., McDonald, M. et al. Histone serotonylation regulates ependymoma tumorigenesis. Nature

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(2024). https://doi.org/10.1038/s41586-024-07751-zIF: 50.5 Q1

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DOI: https://doi.org/10.1038/s41586-024-07751-zIF: 50.5 Q1 B1IF: 50.5 Q1 B1

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Abstract 摘要

Similar content being viewed by others他人正在浏览的类似内容

Main 主

Serotonergic neurons suppress EPN growth血清素能神经元抑制嗜睡症结节增长

Histone serotonylation governs EPN组蛋白色氨酸羟化调控内吞蛋白

ETV5 regulates EPN tumour progressionETV5 调控 EPN 肿瘤进展

ETV5 suppresses NPY gene expressionETV5 抑制 NPY 基因表达

NPY suppresses brain hyperactivityNPY 抑制大脑过度活跃

Discussion 讨论

Methods 方法

Analysis of human RNA-seq data人类 RNA 测序数据分析

Acquisition of samples from patients with EPN从 EPN 患者中获取样本

IUE mouse ZRFUS EPN modelIUE 小鼠 ZR FUS EPN 模型

AAV generation, AAV-DREADD delivery and CNO treatmentAAV 制备、AAV-DREADD 递送及 CNO 处理

Mouse survival analyses, bromodeoxyuridine labelling and tumour collection小鼠生存分析、溴脱氧尿苷标记及肿瘤收集

Immunofluorescence staining免疫荧光染色

Confocal imaging 共聚焦成像

SSRI treatment SSRI 治疗

Preparation of protein lysates and histone extracts, and western blotting蛋白质裂解液与组蛋白提取物的制备，以及蛋白质印迹法

ChIP 染色质免疫沉淀

ChIP–seq library preparation, sequencing and bioinformatic analysisChIP-seq 文库制备、测序及生物信息学分析

In vivo barcoded screen 体内条形码筛选

IP–MS and co-immunoprecipitationIP-MS 与共免疫沉淀

RNA purification and RT–qPCRRNA 纯化与 RT-qPCR

RNA-seq library preparation, sequencing and bioinformatic analysisRNA-seq 文库制备、测序及生物信息学分析

Slice recording for EPSCs and IPSCsEPSCs 和 IPSCs 的切片记录

EEGs 脑电图

Quantification and statistical analysis定量与统计分析

Reporting summary 报告摘要

Data availability 数据可用性

References 参考文献

Acknowledgements

Author information

Authors and Affiliations

Contributions

Corresponding authors

Ethics declarations

Competing interests

Peer review

Peer review information

Additional information

Extended data figures and tables

Supplementary information

Source data

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Similar content being viewed by others
他人正在浏览的类似内容

Serotonergic neurons suppress EPN growth
血清素能神经元抑制嗜睡症结节增长

Histone serotonylation governs EPN
组蛋白色氨酸羟化调控内吞蛋白

ETV5 regulates EPN tumour progression
ETV5 调控 EPN 肿瘤进展

ETV5 suppresses NPY gene expression
ETV5 抑制 NPY 基因表达

NPY suppresses brain hyperactivity
NPY 抑制大脑过度活跃

Analysis of human RNA-seq data
人类 RNA 测序数据分析

Acquisition of samples from patients with EPN
从 EPN 患者中获取样本

IUE mouse ZR^FUS EPN model
IUE 小鼠 ZR ^FUS EPN 模型

AAV generation, AAV-DREADD delivery and CNO treatment
AAV 制备、AAV-DREADD 递送及 CNO 处理

Mouse survival analyses, bromodeoxyuridine labelling and tumour collection
小鼠生存分析、溴脱氧尿苷标记及肿瘤收集

Immunofluorescence staining
免疫荧光染色

Preparation of protein lysates and histone extracts, and western blotting
蛋白质裂解液与组蛋白提取物的制备，以及蛋白质印迹法

ChIP–seq library preparation, sequencing and bioinformatic analysis
ChIP-seq 文库制备、测序及生物信息学分析

IP–MS and co-immunoprecipitation
IP-MS 与共免疫沉淀

RNA purification and RT–qPCR
RNA 纯化与 RT-qPCR

RNA-seq library preparation, sequencing and bioinformatic analysis
RNA-seq 文库制备、测序及生物信息学分析

Slice recording for EPSCs and IPSCs
EPSCs 和 IPSCs 的切片记录

Quantification and statistical analysis
定量与统计分析