RNA-based Regulation of Transcription by RNA Binding Proteins

Increasing evidence suggests that transcriptional control and chromatin activities at large involve regulatory RNAs, which likely enlist specific RNA binding proteins (RBPs). Although multiple RBPs have been implicated in transcriptional control, it has remained unclear how extensively RBPs directly act on chromatin. We embarked on a large-scale RBP ChIP-seq analysis, revealing widespread RBP presence in active chromatin regions in the human genome. Like transcription factors (TFs), RBPs also showed strong preference for hotspots in the genome, particularly gene promoters, where their association is frequently linked to transcriptional output. Unsupervised clustering reveals extensive co-association between TFs and RBPs, as exemplified by YY1, a known RNA-dependent TF, and RBM25, an RBP involved in splicing regulation. Remarkably, RBM25 depletion attenuates all YY1-dependent activities, including chromatin binding, DNA looping and transcription. We propose that various RBPs may enhance network interaction through harnessing regulatory RNAs to control transcription.

癌症中细胞外囊泡miRNA的功能分析及其表达数据库

细胞外囊泡（EV）是细胞通讯和肿瘤发生、转移的重要媒介，也是肿瘤液体活检的重要手段。miRNA是EV中研究最为广泛的分子，在EV的功能调控中起重要作用。这里，我将介绍我们在EV相关miRNA中的功能研究和EVmiRNA表达数据库的生物信息学研究进展。基于已有的500多个EV miRNA测序数据集，我们分析了不同组织和疾病EV中的miRNA表达及其特异表达，构建了名为EVmiRNA的数据库，供全世界学者免费访问 http://bioinfo.life.hust.edu.cn/EVmiRNA/。我们的研究也发现白血病细胞来源的EV中的miRNA可以调控EV恶性转化正常造血干祖细胞，间充质干细胞MSC的EV中的miRNA可以作为MSC衰老的标志物分子，骨髓瘤细胞分泌的EV中的miRNA可以调控骨病进展等等，相关研究发表在Nucleic Acids Research，Cancer Research和Theranostics等杂志。

6mA-DNA-binding factor Jumu controls maternal-to-zygotic transition upstream of Zelda

A long-standing question in the field of embryogenesis is how the zygotic genome is precisely activated by maternal factors, allowing normal early embryonic development. We have previously shown that N6-methyladenine (6mA) DNA modification is highly dynamic in early Drosophila embryos and forms an epigenetic mark. However, little is known about how 6mA-formed epigenetic information is decoded. Here we report that the Fox-family protein Jumu binds 6mA-marked DNA and acts as a maternal factor to regulate the maternal-to- zygotic transition. We find that zelda encoding the pioneer factor Zelda is marked by 6 mA. Our genetic assays suggest that Jumu controls the proper zygotic genome activation (ZGA) in early embryos, at least in part, by regulating zelda expression. Thus, our findings not only support that the 6mA-formed epigenetic marks can be read by specific transcription factors, but also uncover a mechanism by which the Jumu regulates ZGA partially through Zelda in early embryos.

Detecting and mapping epitranscriptomic mark N⁶-methyladenosine

The epitranscriptomic mark N⁶-methyladenosine (m⁶A) is the most abundant post-transcriptional RNA modification in both eukaryotic mRNA and long non-coding RNA (lncRNA). These marks are commonly installed by an m⁶A writer complex and erased by AlkB family dioxygenases (e.g., FTO and ALKBH5 in human). m⁶A marks mediated by m⁶A-binding proteins can regulate RNA processing and metabolism. Owing largely to the inert reactivity of the methyl group of m⁶A, transcriptome-wide m⁶A detection methods have to date relied on m⁶A-antibody immunoprecipitation (m⁶A-IP). And the RNase H derived SCARLET method is the only one that can quantitatively detect the m⁶A status of a single mRNA or lncRNA locus, but its time-consuming and radioactive labeling requirements have limited its wider application. Thus it is desirable to develop new techniques/tools for detecting locus-specific m⁶A and transcriptional-wide m⁶A sites. Here we developed two tools: 1) an elongation- and ligation-based qPCR amplification method for the radiolabeling-free detection of m⁶A position in a single gene; and 2) antibody-free and chemical-labeling method for mapping m⁶A methylome.

基因组标签计划（Genome Tagging Project，GTP）: tag every protein in mice through “artificial spermatids”

哺乳动物单倍体胚胎干细胞的建立为生命科学研究提供了新的工具。2012年，我们建立了只携带精子遗传物质的小鼠孤雄单倍体胚胎干细胞，并证明这一细胞能代替精子在注入卵母细胞后支持胚胎发育产生健康的半克隆小鼠，即半克隆技术。2015年，我们通过将调控雄性印记基因H19和Gtl2表达的H19-DMR和IG-DMR敲除后获得了能稳定产生半克隆小鼠的单倍体干细胞（又称为“人造精子细胞”），并证明它们能用于稳定高效产生遗传修饰的半克隆小鼠。与CRISPR-Cas9技术结合，“人造精子细胞”介导的半克隆技术可以实现：（1）一步获得携带多基因突变的杂合小鼠模型，用于模拟人类多基因介导的复杂疾病；（2）快速获得携带人类疾病相关点突变小鼠用于研究疾病发生的分子机制；（3）一步获得针对不同基因的突变小鼠，实现小鼠个体水平的遗传筛选，便于从大量候选基因中快速筛选出重要基因进行深入研究；（4）一步获得针对特定蛋白质不同碱基突变的小鼠，实现蛋白质关键氨基酸的在体遗传筛选；（5）建立携带蛋白质标签敲入的“人造精子细胞”库，进而获得携带蛋白质标签的小鼠库，为实现全基因组蛋白质标签计划（genome tagging project, GTP）提供技术保障。这些研究显示“人造精子细胞”介导的半克隆技术为研究人类疾病和发育，以及开展蛋白质功能的在体、实时、动态、网络研究提供了新的手段。

丝素水凝胶包裹的外泌体递送miR-675改善小鼠后腿血管功能障碍

衰老诱导的血管功能障碍是目前亟待解决的疾病难题，但是该过程的分子机制并不清楚。本研究的目的是鉴定出介导衰老诱导的血管功能障碍的信号通路，并且开发基于外泌体的治疗方法从而抑制衰老引发的血管病变。我们探索了衰老早期的分子机制，并选择了参与其中变化的上游分子miR-675进行干预治疗。

我们选择11月龄的 C57BL/6小鼠作为衰老前期体内模型，及H₂O₂模拟氧化应激诱导的H9C2心肌细胞衰老作为体外模型研究 miR-675的潜在治疗作用。结果表明，跟两周龄小鼠相比，11月龄小鼠的miR-675在骨骼肌组织中明显下调。在H₂O₂诱导的衰老细胞模型中，β-gal染色阳性细胞数明显增高，同时细胞增殖数量明显下降，伴随p21，TGF-β1的蛋白水平和RNA的显著上调及miR-675的表达明显下调。miR-675 mimic预处理可以上调细胞内miR-675的表达，下调衰老相关的β-gal染色，同时上调细胞增殖水平。提示miR-675可以在细胞衰老模型中抑制控衰老相关的表型。为了探索miR-675在衰老过程中的分子机制，我们使用RNA22对miR-675和TGF-β1的相互作用关系进行了预测，结果表明TGF-β1是miR-675潜在的靶基因。随后使用双荧光素酶实验来验证该位点和miR-675的结合情况，结果表明miR-675可以和TGF-β1上的3′UTR位点结合，并且下调荧光素酶的表达，从而证明了TGF-β1是miR-675的靶基因。我们进而探索了在外泌体中装载miR-675的方法，同时检测了富含miR-675的外泌体在抑制衰老过程中的作用。在UMSC细胞中转入miR-675 mimic获得了富含miR-675的外泌体，将该外泌体和心肌细胞共孵育后导致心肌细胞中的miR-675明显上调，表明外泌体可以递送miR-675。随后将普通外泌体和富含miR-675的外泌体预处理衰老的心肌细胞，结果显示富含miR-675的外泌体比普通外泌体更有效的抑制了衰老，表现为衰老相关的β-gal染色的降低，细胞增殖的增高和衰老相关蛋白p21和TGF-β1表达的降低。原位注射外泌体具有驻留时间短的缺点。为了解决这一难题，我们选择了丝素水凝胶来对外泌体进行包封。傅里叶红外检测结果显示丝素水凝胶对外泌体进行了成功的包封。激光多普勒灌注成像显示用丝素水凝胶包封的富含miR-675的外泌体能够有效提高小鼠后腿缺血损伤后的血液灌流。体外释放实验和体内不同时间点组织切片的红色荧光PKH26-外泌体信号表明丝素水凝胶可以在体外和体内提高外泌体的驻留时间。

综上所述，miR-675在调控细胞衰老中发挥重要的作用。丝素水凝胶包封外泌体的策略为治疗衰老诱导的血管功能障碍开辟了一个新的途径。

N6甲基腺嘌呤的精准检测和功能研究

N6甲基腺嘌呤（m6A）是mRNA上最丰富的一种修饰，同时也可能发生在DNA上。最近的研究表明，m6A参与了RNA相关的多种核心通路，在干细胞多能性维持、肿瘤发生、免疫应答等过程中发挥了重要功能。然而，现在针对m6A的检测基本依赖于免疫共沉淀技术，数据的质量和可重复性难以得到保证。甚至某些相互矛盾的结论也引起了学界的担忧。我们实验室致力于发展新型m6A检测技术，并与高通量测序和多组学分析结合，以求在更加精准的前提下去研究m6A的作用机制和生物学功能。

Gene expression regulation by RNA Methylation

Over 100 types of chemical modifications have been identified in various types of RNAs including non-coding RNA and mRNA, among which methylation is the most common modification. The N6- methyl-adenosine (m6A) and N5-methyl-cytosine (m5C) are the most common and abundant internal modifications on mRNA molecules. The recent identification of methyltransferases METTL3/METTL14/WTAP and NSUN2, and m6A demethylases ALKBH5 and FTO, supports the reversibility of RNA methylation. Several YTH-domain-containing proteins YTHDF1-3 and YTHDC1-2 specifically binding to m6A and ALYREF recognizing m5C have been identified to regulate various mRNA processing, suggesting vital roles of RNA modifications in gene expression control. Our recent works revealed indispensable roles of m6A in mRNA translation, spermatogonial differentiation, and haematopoietic stem and progenitor cell specification, and 5-methylcytosine promotes mRNA export. We have further performed RNA-BisSeq to map transcriptomic profiles of m5C in human cancers. We will discuss the recent progress in RNA modifications and their potential biological significance in this seminar.

m6A RNA modification: mechanism, function and social implications

As the most abundant modification on mRNAs, N6-methyladenosine (m6A) has been recently identified as an important regulators for many essential biological processes. m6A modification usually occurs on adenosine within RRACH motifs, but usually only a small proportion of adenosine within the RRACH motif are methylated at certain cell stage. We have identified that the selectivity of m6A modification sites are regulated by miRNAs via sequencing pairing, revealing a novel function of miRNAs in regulating mRNA epigenetic modification. We also systematically characterized the function of m6A modification in regulating mouse cerebellum development and functions. Intriguingly, we found that the formation of m6A modification can enhance the efficacy of hippocampus-dependent memory consolidation by regulating early-response genes, yet excessive training can somehow compensate the function of m6A in regulating long term memory formation.

A microRNA-inducible CRISPR-Cas9 platform serves as microRNA sensors and cell type specific genome regulation tools

microRNAs (miRNAs) are an important class of small regulatory noncoding RNAs expressed by plants, animals and viruses. However, measuring miRNA activity in vivo remains a big challenge. Using a miRNA-mediated sgRNA releasing strategy and dCas9-VPR to drive a transgene RFP expression, we create a miRNA sensor that can faithfully measure miRNA activity at cellular levels and show that it can be used to monitor the differentiation status of stem cells. When sgRNAs are designed to target endogenous locus, we show this system can be adopted to achieve cell type specific activation of endogenous genes. Furthermore, when dCas9 is fused with a transcriptional repressor or a base editor, we show this system can be used to repress the expression of endogenous genes or mutate specific DNA bases of chromosome depending on the expression of cell type-specific miRNAs. Finally, by a miRNA sensor library screening, we discovered a previously undefined layer of heterogeneity associated with miR-21a activity in mouse embryonic stem cells. Together, these results highlight the utility of a miRNA-induced CRISPR-Cas9 system as miRNA sensors and cell type-specific genome regulation tools.

CRISPR screening for the functional identification of lncRNAs

We have previously developed a high-throughput screening (HTS) method using CRISPR/Cas9 system, which has been broadly applied in the functional studies of coding genes. We have also established the first high-throughput screening strategy for functional investigation of long non-coding RNAs (lncRNAs) using a lentiviral paired-guide RNA (pgRNA) library. We have recently developed a novel approach for genome-wide screening of lncRNAs, a high-throughput method to study the function of topologically associating domains and active chromatin hubs, a re-designed sgRNA scaffold that greatly boosts the efficiency and data quality for HTS, and, a new approach for mapping of functional sites of protein of interest at single amino acid resolution. These high-throughput strategies will facilitate the accurate and rapid identification of functional genomic elements in various settings, and further expand CRISPR toolbox for mammalian genetics.

Exploring small RNA world by single cell sequencing

Single cell sequencing methods has greatly expanded our understanding of the cell heterogeneity and the complexity of biological processes. Small non-coding RNAs (sncRNAs) have  important biological functions. However, their expression dynamics and modifications are poorly understood at the single cell level because of the scarcity of RNA that is obtainable for sequencing analysis. Herein we have developed a CAS-seq method, which greatly improves the sensitivity and quality of small RNA sequencing, especially useful for detecting small RNAs expressed at very low level. Using CAS-seq, we profiled small RNAs in single oocytes of human and monkey and identified a novel class of ~20-nucleotide Piwi-interacting RNAs (piRNAs), which are the most highly expressed small RNAs in human oocytes and are specifically associated with Hiwi3 (Piwil3), a Piwi family protein lacked in mouse and rat. These new class of 20-nt piRNAs is significantly shorter than the classic piRNAs (26- to 30-nt), and is named as oocyte short piRNAs (os-piRNAs). More surprisingly, the os-piRNAs lacks 2’-O-methylation at their 3’ end, a hallmark of classical piRNAs. Such type of 20-nt os-piRNAs are absent in mouse oocytes, which are likely due to lack of Piwil3 gene on mice genome. In contrast, endo-siRNAs, the most abundant sncRNAs present in mice oocytes, are not expressed in human oocytes. It’s also noteworthy that less than 30% of os-piRNA clusters are overlapped with testis-piRNA clusters, indicating the male- and female- germ cells contain two different classes of piRNAs and their expression appears to be regulated by distinct transcription factors. The os-piRNAs are enriched on the antisense strand of recently evolved transposable elements (TEs) with 10A/10-nt 5’ overlap ping-pong signatures, consistent with their potential functions in silencing active TEs. Therefore, this study provide a valuable dataset for studying the function and evolution of small RNAs in primate oocytes and early embryos.

精准且可预测的CRISPR非编码片段编辑揭示成串绝缘子CTCF位点的三维架构规律

传统的第三代基因编辑CRISPR/Cas9系统通常被认为是随机和不可预测的，但我们最近发现在一对sgRNA造成双切口的情况下有很多是精准的，进一步研究发现可以通过干扰基因修复通路的方法提高基因编辑精准度，Cas9通过核酸内切酶活性可以造成突出末端切口，引起可预测的CRISPR基因编辑，对于众多人类遗传性疾病的基因治疗产生了广泛而深远的影响。我将汇报通过DNA片段编辑发现成串CTCF位点在三维基因组拓扑绝缘子架构以及复杂基因组单细胞特异性调控的规律。CTCF is a key insulator-binding protein and mammalian genomes contain numerous CTCF-binding sites (CBSs), many of which are organized in tandem arrays. Here we provide direct evidence that CBSs, if located between enhancers and promoters in the Pcdh and -globin clusters, function as an enhancer-blocking insulator by forming distinct directional chromatin loops, regardless whether enhancers contain CBS or not. Moreover, computational simulation and experimental capture revealed balanced promoter usage in cell populations and stochastic monoallelic expression in single cells by large arrays of tandem variable CBSs. Finally, gene expression levels are negatively correlated with CBS insulators located between enhancers and promoters on a genome-wide scale. Thus, single CBS insulators ensure proper enhancer insulation and promoter activation while tandem-arrayed CBS insulators determine balanced promoter usage. This finding has interesting implications on the role of topological insulators in 3D genome folding and developmental gene regulation.

基于表观转录组和表观基因组数据解析组蛋白修饰指导m6A修饰生成的调控机制

N6-methyladenosine (m6A) is the most prevalent internal post-transcriptional modification in human transcriptomes and has been shown to have important roles in various normal and pathological processes. However, the process by which m6A is deposited on mRNAs is largely unknown. Here we developed a serial of computational methods to decode the regulation of m6A methylation from epigenomic and epitranscriptomic data and demonstrated that histone H3 trimethylation at Lys36 (H3K36me3), a marker for transcription elongation, guides m6A deposition globally. Comparative analyses of ChIP-seq data for H3K36me3 and m6A-seq data revealed that majorities of m6A peaks overlapped with H3K36me3 sites and that the overlapping sites were enriched near stop codons. We also found that m6A sites identified from miCLIP-seq are enriched in the vicinity of H3K36me3 peaks and are reduced globally when cellular H3K36me3 is depleted.  Furthermore, we show that a significant genome-wide correlation between chromatin binding of METTL14 to H3K36me3. Mechanistically, H3K36me3 is recognized and bound directly by METTL14, a crucial component of the m6A methyltransferase complex (MTC), which in turn facilitates the binding of the m6A MTC to adjacent RNA polymerase II, thereby delivering the m6A MTC to actively transcribed nascent RNAs to deposit m6A co-transcriptionally. The discovery of interplay between modified histones and RNA methylation represents a new regulatory layer, and an additional level of complexity, in the control of gene expression.

In vivo genome editing for gene therapy

基因编辑工具 (比如CRISPR系统) 可以在细胞和组织内精确的修改基因，因而其存在治疗甚至治愈很多无药可治的疾病的可能性, 比如遗传病，感染性疾病和癌症等。尽管在临床上以及其他应用上有巨大的潜力，但CRISPR系统作为蛋白和RNA复合体，其有效低脱靶的体内传输 (in vivo delivery) 是最大的难点之一。我们综合应用分子生物学，生物信息学，病毒学，模式动物，纳米科学和化学的手段来解决此难点。

miRNA in Nucleus， A New Network of NamiRNAs and Enhancers

miRNA广泛存在于细胞核内，其功能成谜。 我们发现miRNA在细胞核中可以通过增强子在全基因组水平激活基因的表达，我们将其定义为NamiRNA（Nuclear Activating miRNA）。据此我们提出了miRNA-增强子-基因激活全新的miRNA调控理论，miRNA通过增强子介导的基因激活为miRNA研究提供新的视角。

A Pandas complex adapted for piRNA-guided transposon silencing

The repression of transposons by the Piwi-interacting RNA (piRNA) pathway is essential to protect germ cells. In Drosophila ovaries, Panoramix enforces transcriptional silencing by binding to the target-engaged Piwi-piRNA complex, although the precise mechanisms by which this occur remain elusive. Here, we show that a germline specific paralogue of a nuclear export factor, dNxf2, functions together with Panoramix and dNxt1 (p15) as a ternary complex to suppress transposon expression. Structural and functional analysis demonstrate a critical role of the UBA domain of dNxf2 in Panoramix association and transposon silencing. Moreover, dNxf2 binds to transposon transcripts directly, probably via its essential RRM domain. Because dNxf1/TAP interacts with dNxf2 and is required for Panx-mediated silencing, we propose that dNxf2 functions as a Pandas (Panoramix-dNxf2 dependent TAP silencing) complex, which may counteract the canonical RNA exporting machinery (TAP/p15) and restrict transposons to nuclear peripheries.

RNA-directed DNA methylation in plants

In plants RNA-directed DNA methylation (RdDM) is the only pathway required for de novo DNA methylation. DNA methylation in plants happens in three sequence contexts:  CG, CHG and CHH (H is A, C, T). RdDM play important roles in in biological processes such as viral defense, genome imprinting, maintenance of genome stability etc. Using a reporter gene based genetic screen system, we identified several factors that functionally interact RNA Polymerase IV and V, two plant-specific RNA polymerases that are core components of RdDM. Possible mechanisms of how these factors regulate the function of Pol IV and Pol V will be presented.