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| BMC前沿视角 | 来自陈玲玲实验室的热点解读（一） |
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本期BMC前沿视角我们有幸邀请到了Genome Biology编委陈玲玲研究员及其实验室成员为我们解读最近发表在Genome Biology，Molecular Cancer，Nature biotechnology，Genome Medicine，BMC Biology的八篇重要文献，以飨读者。
该期内容涵盖基于CRISPRi的放疗筛选鉴定胶质瘤中潜在的可作为治疗靶点的长链非编码RNA；通过对表观基因组进行靶向去甲基化，成功建立表观遗传疾病模型小鼠; 对150个RNA结合蛋白的增强CLIP数据分析系统性地揭示了RNA结合蛋白调控RNA加工规律；2F1和EIF4A3介导的circRNA circSEPT9促进三阴性乳腺癌的发生和发展；基于CRISPR–Cas12的SARS-CoV-2检测方法；双相情感障碍患者大脑类器官的全转录组分析及功能研究；Hi-D：纳米水平勾勒单个活细胞的细胞核动力学；细胞分化过程中细胞核大小及DNA的可及性参与调控旁斑的形成。
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陈玲玲实验室简介
陈玲玲的实验室主要研究长非编码RNA（long noncoding RNAs，lncRNAs），这是一类长度大于 200 个核苷酸的非编码 RNA分子。她的团队通过创建无poly(A) 转录组纯化和分析体系，发现了小核仁RNA结尾的具有特殊结构的长非编码RNA家族，也和国际上其他实验室相继报道了环形RNA在哺乳动物细胞基因组中普遍存在。更重要的是，这些具有特殊结构的编码RNA在基因调控和人类疾病中有着密切的相关作用。她的团队将继续深入研究这些长非编码RNA的生成加工以及它们在人类健康和疾病中作用的机理，关于陈玲玲研究组的更多内容请见http://www.chenlab-ncrna.com/。
陈玲玲研究员目前是Cell、Cell Chem Cell、Genome Biol、RNA、Trends Cell Biol、Trends Genet 等期刊的编辑委员会的成员。她在2017年被选为霍华德·休斯医学研究所（Howard Hughes Medical Institute，HHMI）国际研究员。
感谢陈玲玲实验室以下成员对本期内容作出主要贡献：
前排：郭纯洁，陈玲玲，李响（PhD），刘楚霄（PhD）
后排：姚润文，王洋，蒋望（PhD）
点击链接 了解陈玲玲实验室更多介绍
本期文章解读
#1. Title: CRISPRi-based radiation modifier screen identifies long non-coding RNA therapeutic targets in glioma
First Author: S. John Liu
Correspondence Author: Erik M. Ullian & Daniel A. Lim
Research article|Genome Biology
Date: 2020-03-31
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人类基因组产生成千上万个lncRNA，其中一些lncRNA可通过不同的机制在癌症的发生过程中起到重要作用。很多lncRNA表达和功能因细胞类型而异，因此这些转录产物有可能成为癌症疗法的新靶点。Liu及团队利用CRISPRi进行了系统性的功能筛选，鉴定了在胶质瘤放射疗法中可能成为治疗靶点的lncRNA。这项筛选的文库中共含有56890个sgRNA，对应识别人体胶质瘤中表达的5689个lncRNA（每一个lncRNA 的转录起始位点有10个sgRNA靶向定位），同时还有1202个非特异靶向的对照sgRNA。研究人员通过8Gy分级辐射处理表达CRISPR/sgRNA的U87细胞，并对处理后的细胞进行12天的连续培养，最终筛选出了可能成为治疗靶点的467个lncRNA。排名第一的lncGRS-1，是一种在灵长类动物里保守且在细胞核内富集的lncRNA，它能够明显促进恶性胶质瘤的发生。在人类大脑类器官的3D模型中，用反义寡核苷酸靶向降解lncGRS-1可以选择性减缓肿瘤生长，并使得胶质瘤对放射疗法更加敏感。综合以上结果，这项研究表明通过高通量的CRISPRi筛选来鉴定各类癌症里可有效作为治疗靶点的lncRNA时代已经来临。
Human genome produces thousands of lncRNAs and certain lncRNAs play key roles in the pathogenesis of cancer via various mechanisms of actions. Expression and functions of many lncRNAs are cell-type dependent, making these transcripts attractive for targeted cancer therapy. Liu et al. performed systematic functional screens via CRISPRi to identify lncRNA candidates as potential therapeutic targets in glioma upon radiation treatments. This screen focused on 5,689 lncRNAs expressed in human glioma. The CRISPRi non-coding library contained 56,890 sgRNAs (10 sgRNAs targeting the transcription start site of each lncRNA) along with 1,202 non-targeting control sgRNAs. The potential therapeutic targets (467 “lncRNA hits”) were selected using sgRNA positive U87 cells under 8Gy fractionated radiation followed by 12 days of cell propagation. The top ranked lncGRS-1, which is a primate-conserved and nuclear-enriched lncRNA, significantly promoted the growth of malignant glioma. Further antisense oligonucleotides targeting lncGRS-1 selectively decreased tumor growth and sensitized glioma cells to radiation therapy in a three-dimensional human brain organoid model. Together, these studies highlight the advent of applying high throughput CRISPRi screening in identifying potent lncRNA therapeutic targets in various cancers for therapeutic potential.
#2. Title: Successful generation of epigenetic disease model mice by targeted demethylation of the epigenome
First Author: Takuro Horii
Correspondence Author: Izuho Hatada
Method|Genome Biology
Date: 2020-04-01
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表观遗传修饰的失调与许多表观遗传疾病，如癌症、肥胖症、自闭症和印记紊乱等有关。靶向特定的表观遗传修饰能帮助我们理解这些表观遗传疾病，并有可能成为这些疾病的潜在治疗方法。Horii 及同事通过利用dCas9-SunTag 和单链可变片段（scFv）-TET1催化域，成功地在H19甲基化有差异的区域（H19-DMR）进行了靶向DNA去甲基化。在这个技术中，研究者将dCas9与一个SunTag （多个GCN4）相融合，以此招募多个与抗体（scFv）融合的TET1催化域的拷贝，导致靶向目标中特定位点的羟基化，以此激活这些位点的去甲基化。这项技术在小鼠胚胎干细胞（ESCs）和受精卵中的应用成功地产生了患有原始侏儒症（SRS）的模型小鼠。SRS是一种印记紊乱疾病，其特征是父亲一方的H19-DMR等位基因上出现DNA低甲基化。所有用这个方式产生的SRS模型小鼠与SRS患者类似，都呈现出H19基因表达的上调和Igf2 基因表达的下调，导致严重的子宫内和出生后的生长延迟。这项研究报告了在受精卵中通过对表观基因组的特定位点进行靶向去甲基化，从而产生了第一只表观基因组编辑的SRS模型小鼠，并提示这是一种产生表观基因组编辑的小鼠用于表观遗传学疾病研究的简单可行的方法。
Dysregulation of epigenetic modifications has been linked with many epigenetic diseases such as cancer, obesity, autism, and imprinting disorders. Target-specific epigenetic modification offers one way to understand these epigenetic diseases with therapeutic potential. Horii et al. achieved target-specific DNA demethylation in the H19 differentially methylated region (H19-DMR) by using the dCas9-SunTag and single-chain variable fragment (scFv)-TET1 catalytic domain (TET1CD). In this technology, the dCas9 was fused to a SunTag (multiple GCN4) to recruit multiple copies of antibody (scFv)-fused TET1CD, resulting in hydroxylation at specific loci to activate site-specific demethylation in the target. Application of this technology into mouse embryonic stem cells (ESCs) and fertilized eggs allowed a successfully generation of model mice for Silver-Russell syndrome (SRS), an imprinting disorder characterized by DNA hypomethylation of H19-DMR in the paternal allele. All generated SRS model mice showed H19 upregulation and Igf2 downregulation, leading to severe intrauterine and postnatal growth retardation like SRS patients. This study reports the first epigenome-edited mice for SRS using targeted demethylation of specific loci of the epigenome in fertilized eggs, revealing a simple and feasible way to produce epigenome-edited mice to study epigenetic diseases.
#3. Title: Principles of RNA processing from analysis of enhanced CLIP maps for 150 RNA binding proteins
First Author: Eric L. Van Nostrand
Correspondence Author: Brenton R. Graveley, Gene W. Yeo
Research article|Genome Biology
Date: 2020-04-06
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RNA结合蛋白（RBPs） 通过识别RNA的各种一级序列基序和RNA二级结构基序与各类RNA相互作用 ，从而调控RNA的加工。交联免疫沉淀（CLIP）法被用于鉴定RBP直接结合的靶标RNA。在本研究中，Van Nostrand及同事采取了增强CLIP法（eCLIPs）和生物信息学分析系统性地鉴定了150 个RBPs 在K562 和HepG2 细胞系中结合的靶标RNA及结合在靶标RNA的位置，获得了一个独特且无偏好性的RBPs与靶标RNA相互作用的资源库。通过定量分析RNA重复序列和多拷贝原件，eCLIPs的结果显示，70%的RBPs会富集结合在非mRNA区域。他们还鉴定出了新型核糖体RNA加工因子和结合位点，以及重复序列与一部分重要的RBPs的相关性。更进一步地，通过分析剪接体中RBPs的eCLIP数据， 例如结合在RNA 3′端剪切位点的U2AF1和U2AF2， 结合在RNA分支位点的 SF3B4和SF3A3, 结合在RNA 5′端剪切位点的PRPF8，以及结合在RNA近端内含子的RBFOX2和PTBP1等，他们在全转录组水平证实了一个基于分支位点，用于识别3′ 端的剪切位点的扫描模型，同时也加深了人们对剪接体及核心剪切调控的了解。总的来说，这类研究提供了一个众多RBPs结合靶标RNA情况的蓝图，加深了人们对于RBPs在RNA加工过程中调控功能的认知。
RNA binding proteins (RBPs) interact with all types of RNAs through a variety of primary sequence motifs and RNA structural elements to control all processing steps. The crosslinking and immunoprecipitation (CLIP) methods are frequently used to identify direct RNA targets of RBPs. In this study, Van Nostrand et al. performed enhanced CLIPs (eCLIPs) and integrative computational analyses to map diverse RNA targets for 150 RBPs in K562 and HepG2 cells. These efforts have allowed them to create a unique and unbiased resource of RBP interactomes. Quantification of eCLIPs for repetitive and multicopy elements revealed that 70% of RBPs have enrichments for non-mRNA elements. They also identified novel ribosomal RNA processing factors and binding sites, and the association of repetitive elements with a significant fraction of RBPs. Further, the analysis of spliceosomal RBPs such as U2AF1 and U2AF2 at the 3′ splice site, SF3B4 and SF3A3 at the branch point, PRPF8 at the 5′ splice site, and RBFOX2 and PTBP1 at proximal introns by eCLIPs has provided a genome-wide validation for a branch point-based scanning model for the 3′ splice site recognition and has also revealed additional insights into spliceosomal association and core splicing regulation. All in all, these studies provide a plentiful resource for RBP binding landscapes, which has enabled the identification of novel insights into RBPs in RNA processing regulation.
#4. Title: The circRNA circSEPT9 mediated by E2F1 and EIF4A3 facilitates the carcinogenesis and development of triple-negative breast cancer
First Author: Xiaying Zheng
Correspondence Author: Junxia Chen
Research article |Molecular Cancer
Date: 2020-04-07
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三阴性乳腺癌（TNBC），是雌性激素受体（ER）、孕酮受体（PR）、人表皮生长因子受体2（HER2）的表达均为阴性的类别， 是乳腺癌中侵袭性最强的一个子分类。环形RNA（circRNA）是一类非编码RNA；目前尚不清楚它是否与TNBC有关或在其中扮演什么角色。在最近的一项研究中，Zheng及同事对四对TNBC组织和癌旁组织进行了RNA测序，发现环形RNA- circSEPT9 - 在TNBC组织中表达明显升高。他们进一步确认，circSEPT9表达高的TNBC患者的预后情况不良。这些数据说明circSEPT9是TNBC的一个新型预后标记。进一步利用体内和体外试验研究了circSEPT9的功能显示circSEPT9可以通过结合与LIF/Stat3信号转导相关的miR-637 来促进TNBC的增殖和入侵。有趣的是，他们同时还发现转录因子E2F1和EIF4A3能够通过与SEPT9启动子和前体mRNA的结合来增强circSEPT9的表达。综上，这个研究揭示了circSEPT9是一个新型肿瘤启动因子，因此它也有可能成为一个TNBC治疗靶点。
Triple-negative breast cancer (TNBC), defined as lack of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2), is the most aggressive subtypes of breast cancer. Circular RNA (circRNA) is one type of non-coding RNAs while its role in TNBC has remained elusive, if any. In a recent study, using RNA-seq of four paired TNBC tissues and paracancerous tissues, Zheng et al. found that the circular RNA, circSEPT9, was upregulated in TNBC tissues. They further confirmed that TNBC patients with high circSEPT9 expression had poor prognosis. These data suggest that circSEPT9 is a novel prognostic marker in TNBC. Next, to investigate the function of circSEPT9, the authors performed in vitro and in vivo assays. They found that circSEPT9 was able to promote TNBC proliferation and invasion via sponging miR-637 related to the LIF/Stat3 signaling. Interestingly, they also identified that transcription factors E2F1 and EIF4A3 could enhance the expression of circSEPT9 via binding to the SEPT9 promoter and pre-mRNA. Together, this study has suggested that circSEPT9 is a novel tumor-driven factor, which may serve as a potential therapeutic target in TNBC.
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（来源：科学网）
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