复旦大学表观基因组学暑期国际讲习班Proposal投票
为期10天的表观基因组学暑期国际讲习班已经接近尾声，在课程的最后，我们希望学员们能够学以致用，同时检验学习成果。学员们需提一个Epigenetic 主题的proposal，其中包含研究背景、研究重要性、关键技术重点、实验流程等。
通过前期的征集和遴选，目前已经诞生了6份Proposal，并于12日下午进行集体展示。Proposal讨论将由Genome Research执行主编Hillary Sussman主持，美国NIH Roadmap epigenomics资深负责人Frederick (Fred) Tyson 博士、冷泉港实验室的Thomas Gingeras 教授、宾夕法尼亚州立大学的岳峰教授、复旦大学的于文强教授及倪挺教授共同点评，现场学员共同参与投票。
【文末有投票】
Proposal列表
| NO. | 姓名 | 单位 | 标题 |
| 1 | 刘发扬 | 西北农林科技大学 | 大鼠细胞在小鼠体内培养后染色体结构的变化 3D genomics analysis of Rat cells in Mouse by Interspecific Blastocyst Injection of Pluripotent Stem Cells |
| 2 | 夏燕 | 中南大学 | 性别差异的甲基化及其与精神疾病的关联 Sexually Dimorphic DNA Methylation in Human Brain and Relevance to Psychiatric Disorders |
| 3 | 金威 | 香港大学 | Exploring molecular mechanism of pogerin generation |
| 4 | 徐鹏 | 复旦大学 | 学术压力越大，头发越少？表观遗传在脱发中的作用 The More Academic Pressure, the Less Hair? Epigenetics matters in hair loss |
| 5 | 郝亚娟 | 中科院北京基因组研究所 | RNA m6A修饰与miRNA间的连接子是什么？ What’s the linker protein between RNA m6A modification and microRNAs? |
| 6 | 李晶 | 第二军医大学转化医学中心 | 组织特异性增强子失调介导肿瘤身份危机机制研究及临床应用 Cancer Cell Identity Crisis as a Result of Enhancer Epigenetic Dysregulation |
NO.1 大鼠细胞在小鼠体内培养后染色体结构的变化
刘发扬
西北农林科技大学
日本科学家发现将大鼠的多能干细胞注射到Pdx1-/-敲除的胰腺发育缺陷的小鼠囊胚中，可以使小鼠产生具有功能的大鼠胰腺；以猪为实验动物也发现同样现象。这为通过猪来产生人的胰腺组织提供了理论支持。但以这种方式产生的成体动物组织中含有两个物种的细胞。而从囊胚时期就和小鼠细胞存在互作的大鼠细胞是否已被改变，细胞核型和染色质的3D结构是否发生变化未见报道。因此，我们计划利用SCNT，染色体核型分析，Hi-C，ATAC-seq 等技术对以上问题进行研究。
3D genomics analysis of Rat cells in Mouse by
Interspecific Blastocyst Injection of Pluripotent Stem Cells
Liu Fayang
College of Veterinary Medicine, Northwest A&F University
Japanese scientists found that rat PSCs injected into Pdx1-/- mouse blastocysts will generate normally functioning rat pancreas in Pdx1-/- mice. The phenomenon were observed in pig as well. Experiments above provide the theoretical basis of obtaining human pancreas from pig. But they also found cells from both species in these functional organisms. Whether the rat cells that interacted with mouse cells from blastocyst to embryo have been transformed. And if the karyotype or 3D-Chromotin structure will be remodeled. Both of them have not been reported. To investigate these questions, SCNT, karyotype analysis, Hi-C, ATAC-seq are need to be carried out.
NO.2 性别差异的甲基化及其与精神疾病的关联
夏燕
中南大学
许多精神疾病如自闭症、抑郁等的发病率存在明显的性别差异，利用人脑组织研究甲基化水平的性别差异将有助于我们更好地理解精神疾病的发病机制。
通过系统的比较251个男性和446女性死亡后的脑组织的甲基化水平，寻找性别差异的甲基化位点和区域，构建共甲基化模块，并结合甲基化数量性状位点，探索单核苷酸多态对甲基化的影响，利用转录组测序的数据，研究性别差异甲基化位点是否调控基因表达，对以上结果进行精神疾病的候选基因的富集分析，深入探讨其在精神疾病的发生过程中所起的作用。
Sexually Dimorphic DNA Methylation in Human Brain and Relevance to
Psychiatric Disorders
Yan Xia
Central South University
Background: The prevalence of many psychiatric disorders, including autism spectrum disorders (ASDs), depression, intellectual disability (ID) and so on, has presented clear sex differences. Sex-associated biomarkers in the brain may help to reveal the mechanisms involved in biology and etiology of psychiatric disorders. DNA methylation is sexually dimorphic and thus is a candidate for such biomarkers. We investigated sex-associated differential methylation at individual CpG loci (differential methylation positions, DMPs) and genomic regions (differentially methylated regions, DMRs), their corresponding genetic regulators (methylation quantitative trait loci, meQTLs), and regulatory target genes. We also tested co-methylated CpG sites for association with sex. Lastly, we investigated whether SNPs and genes involved in sex-dimorphic methylation were implicated in psychiatric disorders.
Methods: We systematically compared methylation in males and females in 697 human postmortem prefrontal cortex (PFC) samples, and another 210 samples to replicate the findings. Through DNA methylation profiling of 251 males and 446 females using Illumina 450K Methylation BeadChips, we applied an empirical Bayes method and DMRcate to investigate sexually DMPs and DMRs, respectively. We used weighted gene co-expression network analysis (WGCNA) adapted for methylation to examine co-methylation sites related to sex. Combining the data of meQTLs and genome-wide expression methylation correlation pairs, we explored the genetic regulators of DMPs and DMRs and the target genes regulated by DMPs and DMRs. By examining the significant overlap between DMP/DMR target genes and genes associated with psychiatric disorders, we investigated the potential roles of sexually dimorphic DNA methylation on psychiatric disorders.
Results: We identified 13,352 DMPs and 4,490 DMRs, 5148/5344 (96%) of which are located on the X chromosome. Those DMRs spanned genes enriched for calcium signaling pathway (adjusted p = 2.3e-10) and neuroactive ligand-receptor interaction (adjusted p = 1.5e-9), and enriched for ASDs (adjusted p = 2.4e-18) and depression (adjusted p value=4.9e-10) and so on.
There were 99,848 meQTLs pairs (involving 4,207 DMPs and 75,555 SNPs), 1,066 DMPs and gene expression pairs (involving 944 DMPs and 711 genes) related to sexually dimorphic methylation. Co-methylation analysis identified a sex-associated module containing 4,825 DMPs.
From 764 candidate genes with de novo mutations which have been reported in schizophrenia, ASDs, epilepsy and ID, we found 112 DMR target genes, and eleven of them were reported in at least two disorders above. Among the previously reported 108 genome-wide significant schizophrenia-associated loci, we also found seven loci which could regulate the DMPs by meQTL. One SNP, rs7085104, could regulate the DMP and then affect the expression of C10orf32.
Discussion: To our knowledge, this is a comprehensive analysis of sexually dimorphic methylation in the largest number of human brain tissues up to now. We identified 13,352 DMPs within 4,490 DMRs to be sexually dimorphic, and found one sex associated co-methylation module among 4,825 DMPs. Compared with previous studies; our results were more systematically investigated the DMPs which contained its upstream genetic regulators and downstream target genes’ expression. Furthermore, our findings of the association between the sexually dimorphic methylation and psychiatric disorders moved forward the understanding of the different prevalence of psychiatric disorders between males and females, and also provided a window to explain the GWAS signals.
NO.3 Exploring molecular mechanism of pogerin generation
Jin Wei
School of biomedical science, The University of Hong Kong
Aging is a progressive loss of physiological integrity caused by accumulation of genetic and epigenetic alterations, resulting in chronical function decline and increased susceptibility to disease and vulnerability to death.In higher vertebrate animals, aging process occurs at multiple but hierarchical levels, including molecule, cell, tissue and organism levels. Aging is characterized by genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, deregulated nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, and altered intercellular communication and all these aging-associated hallmarks are usually connected. Although the cause and consequence between aging and epigenetic alterations is controversial, chromatin structure, histone modification, DNA methylation, non-coding RNAs and transcriptional network changes are not only observed during aging but also profoundly affect cellular function and stress resistance, thereby contributing to the progression of aging.
Hutchinson–Gilford progeria syndrome (HGPS) is a genetic disorder in children characterized with osteoporosis, arthritis, cardiovascular dysfunction, lack of subcutaneous fat, hair loss, and premature death due to cardiovascular failure andstroke when they get 13.5 years old. HGPS is caused by a silent G608G (1824C>T) mutation withinthe LMNA gene that encodes lamin A/C. This point mutation exposes a crypticsplice site in exon 11 that leads to deletion of 50 amino acid required for normal lamin A processing, resulting in an aberrant and permanently farnesylated lamin A protein termed progerin. Previous studies reveal that progerin is involved in many signaling pathways (DNA repair defect, induce telomere dysfunction,somatic stem cell pool exhaustion and differentiation defect, decreased anti-oxidative stress capability etc.) which cause cellular dysfunction and accelerates aging process.
Interestingly, the wild-type LMNA could also undergo progerin abnormal alternative splicing and express progerin in normal aging human cells. The number of cells expressing progerin and the protein level of progerin increase along with aging progress, and aging related telomere attrition correlates with progerin protein level. These findings implicate that normal aging might be, partially if not, attributed to the same molecular mechanism as HGPS.Previous studies mainly focus on how progerin works as aging-associated toxic protein, but how and when this protein is generated remains to be explored.Thereby, epigenetic alteration seems to be the only explanation for progerin generation.
From LMNA mutation database, G608 (1824C) is high-potentially methylated. Single base-resolution methylated DNA analysis at progerin splicing site shows that 1824C is methylated in human H1 embryonic stem cell and IMR90 cell lines, and MeCP2 can bind to this single methylated CpG pair. DNA methylation-MeCP2 regulating alternative splicing model matches well with progerin generation hypothesis. The genome globe DNA methylation level is decreased with aging. When methyl-CpG pair in the progerin splicing site loss DNA methylation progressively, there is no MeCP2 binding i which results in exon exclusion and potential progerin generation. Therefore, the possibility of MeCP2 mediating progerin generation through DNA methylation should be explored. If the results are positive, the study of progerin generation molecular mechanism could not only provide new insight for human aging process, drugs may also be designed to targeting this process to treat HGPS disease or even extend our healthy lifespan.
NO.4 学术压力越大，头发越少？表观遗传在脱发中的作用
徐鹏
复旦大学
统计结果显示，84.9%的中国研究者感受到了很大的科研压力，这种压力会严重损害身心健康，其中一个很重要且常被忽视的结果为脱发。脱发现象非常普遍，约1/3的女性会出现显著的脱发现象，约80%的白人会出现头发脱落的症状。脱发通常认为是一种激素和年龄依赖的症状，且部分脱发性状可遗传。但持续的科研压力是否会造成脱发？如果可以，那么表观遗传在学术压力介导的脱发中扮演什么角色？学术压力造成的脱发性状是否可以遗传？
本议题首先针对不同年龄段的科研工作者进行调研，建立学术压力与脱发之间的联系；其次，根据正在经历脱发的科研工作者提供的照片进行计算机模拟以验证头发的减少，同时利用他们提供的头发样本对脱发相关基因的启动子区域和增强子区域进行DNA甲基化检测，并鉴别异常高表达和低表达的基因；最后利用小鼠模型进行验证。本议题希望探究持续压力对脱发的影响以及表观遗传在该过程中的功能，并对那些正在经受脱发的人提供相应的建议。
The More Academic Pressure, the Less Hair? Epigenetics matters in hair loss
Xu Peng
Fudan University
It’s reported that 84.9% of Chinese researchers have felt great pressure from scientific researches, which would greatly impair their physical and mental health, one of the serious and easily neglected results of which is hair loss. Hair loss is a very common symptom. More than one third of women have clinically significant hair loss during their lifetime. At least 80% of Caucasian men show at least some signs of male pattern hair loss (MPHL). Hair loss is considered to be androgen- and age-dependent, some type of which could be inherited to next generation. However, could constant pressure from academics researches result in hair loss? If so, which role does epigenetics play in the hair loss mediated by academic pressure? Could this kind of hair loss be inherited to multi-generations?
Firstly, we design questionnaire for researchers to establish the correlation between academic pressure and hair loss. Secondly, we perform computer modeling of the pictures offered by those who are undergoing hair loss to calculate the number of hair, at the same time, we analyse the DNA methylation level in promoter and enhancer region of hair loss-related genes and identify high/low expression genes related with metabolism and hormonal regulation. Lastly, we contruct mouse model to confirm the result. This proposal focus on the effect of constantly academic pressure on hair loss and the role that epigenetics plays in this process, hoping to offer considerable advises for those who are troubled with hair loss.
NO.5 RNA m6A修饰与miRNA间的连接子是什么？
郝亚娟
中科院北京基因组研究所
N6-甲基腺嘌呤(m6A)修饰是真核生物mRNA中一种高丰度表观转录组学修饰。m6A能影响RNA剪切、出核、降解、翻译、细胞重编程等。m6A修饰由甲基转移酶复合物WTAP/METTL3/METTL14以SAM为甲基供体催化形成，由去甲基化酶ALKBH5和FTO在Fe2+、α-酮戊二酸辅助下催化去除，能被含有YTH结构域的m6A读码器YTHDF2、YTHDF1、YTHDC1识别，能被microRNA通过序列互补性机制调控生成。之前的研究发现microRNA和其生成酶Dicer通过影响METTL3与RNA的结合能力来影响m6A生成，但Dicer和METTL3之间的连接子蛋白并没有找到。本议题首先通过使用免疫沉淀-质谱技术找到Dicer和METTL3共有的相互作用蛋白protein A。其次分别在细胞内和细胞外证明其有相互作用。然后通过敲低或者过表达protein A，检测细胞中m6A水平和microRNA水平的变化。最终确认protein A为Dicer和METTL3的连接子蛋白。
What’s the linker protein between RNA m6A modification and microRNAs?
Hao Yajuan
Beijing Institute of Genomics, Chinese Academy of Sciences
N6-methyladenosine (m6A) has been identified as the most prevalent epitranscriptomic modification on eukaryotic mRNAs. m6A can affect RNA splicing, exporting, degradation, translation, cell reprogramming, etc. RNA m6A modification is catalyzed by a multicomponent methytransferase complex composed of at least three subunits, METTL3, METTL14 and WTAP. Dioxygenases FTO and ALKBH5 are the two known m6A demethylases. m6A modification is recongnized by YTH domain containing family of proteins namely YTHDF1, YTHDF2 and YTHDC1. m6A is regulated by microRNAs through a sequence pairing mechanism. Our previous study shows microRNAs and Dicer modulate the binding ability of METTL3 to a subset of mRNAs targeted by microRNAs, but the linker protein between Dicer and METTL3 hasn’t been identified. In this proposal, I will use IP-MS to find out the potential partners both of METTL3 and Dicer and overlap them to figure out a potential linker named protein A. I will confirm the interaction of protein A with METTL3 and Dicer both in vivo and in vitro. I will check the m6A level and microRNAs level upon protein A knock down or over expression. Finally, I get the conclusion that protein A is the linker between Dicer and METTL3, between microRNAs and m6A.
NO.6 组织特异性增强子失调介导肿瘤身份危机机制研究及临床应用
李晶
第二军医大学转化医学中心
肿瘤的发生是一种自身属性丢失的过程。正是这种“身份危机”赋予肿瘤细胞无序生长、转移甚至遍布全身的能力。调控这种自身属性的重要机制就是细胞/组织特异性基因的表达。在DNA基础上，对于调控这些细胞/组织特异性基因表达的重要功能元件即增强子。
在该研究中，我们提出了肿瘤初步的形成（原发病灶）就是肿瘤丢失重要的维持组织特异性的增强子的过程，从而导致组织特异性基因表达的失调，最终启动肿瘤生成。而对于肿瘤转移入其他部位（转移病灶）的研究，将进一步揭示转移过程中肿瘤细胞进一步获取其他细胞/组织特异性增强子进而调控基因表达获取生存优势的机制。本课题的研究将解释肿瘤转移偏好性，并通过在血液中监控这些组织特异性增强子的获得/丢失，将为监控/预测肿瘤转移或复发提供新的液体活检方案，并早期示警临床关注甚至是干预。
Cancer Cell Identity Crisis as a Result of Enhancer Epigenetic Dysregulation
Jing Li
Center for Translational Medcine
Second Military Medical University
Cancer cells suffer an identity crisis. Arising from a background of normal tissues and cells, cancer cells are often confused about where they come from, what they are, and where they should go – am I a brain cell? Am I a stem cell? In such crisis and confusion they grow in an uncontrolled fashion, wonder around, and eventually take over the body. One important determinant of cellular identity is cell-type specific gene expression, which is orchestrated by DNA regulatory elements called enhancer elements.
The loss of cell fate commitment and gain in ability to proliferate are key features of carcinogenesis. We hypothesize that during carcinogenesis, cancer cells undergo epigenetic dysregulation of tissue-specific enhancers. Enhancers specific to cancer cells’ tissue of origin are epigenetically repressed, leading to loss of tissue specificity; furthermore and possibly during metastasis, enhancers specific to other tissues are epigenetically activated, enhancing cancer cells’ ability to adapt to new tissue environment and evade immune surveillance. These processes are likely orchestrated by interaction between specific transcription factors and chromatin landscape, both are dysregulated in cancer but maintain functional interactions that allow cancer cells to explore specific niche to grow. Thus, exploring cancer cells’ abnormal and dynamic enhancer landscape in the context of these enhancers’ normal function would allow us to understand target organ preference of metastasis. Importantly, the tracing tissue-specific enhancer markers in cancer could potentially reveal the cancer cells’ tissue provenance. We propose that tracking tissue specific enhancers by liquid biopsy is a novel and paradigm shifting concept for detection of metastasis at the very early stages and provides a guideline for clinicians as to where to look for a tumor.
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