ChengyangWang的个人博客分享 http://blog.sciencenet.cn/u/ChengyangWang

博文

RNA-seq居然能挖出这么多机制!给你的文章加5分

已有 11076 次阅读 2018-4-25 16:39 |个人分类:生物信息|系统分类:科普集锦

 

本文转载自嘉因微信公众号,已获得授权。查看最新文章,敬请关注嘉因,微信ID:rainbow-genome

作者:小丫  来源: 嘉因


手里有RNA-seq数据,不甘心发5分,想加点机制,发10分。下面的思路,总有一款适合你:

  1. RNA-seq数据 + 别人的ChIP-seq数据,准确找到靶基因,详见《RNA-seq这样画图,国自然必得A》;

  2. RNA-seq数据,转换成ATAC-seq,挖出关键调控因子,详见《任意两组RNA-seq变身,国自然得AAA》;

  3. RNA-seq数据,找enhancer expression,挖明星分子的远距离调控机制,详见本文。




“enhancer expression”是啥?

2010年的Nature,Kim等人发现:基因转录时,不仅promoter附近转录出mRNA,enhancer区域也转录出RNA,也就是enhancer RNA(eRNA)。eRNA预示着active enhancer



Figure 1 : Enhancers near the c-fos gene with increased CBP/RNAPII/NPAS4 binding and eRNA production upon membrane depolarization.

出自Kim, T.K., Hemberg, M., Gray, J.M., Costa, A.M., Bear, D.M., Wu, J.,Harmin, D.A., Laptewicz, M., Barbara-Haley, K., Kuersten, S. and Markenscoff-Papadimitriou, E., 2010. Widespread transcription at neuronal activity-regulated enhancers. Nature, 465(7295), p.182.


同期刊载了任兵为这一发现写的VIEWS:




Figure 1 | Unsuspected sites of transcription. a, Kim et al. find that, before treatment with KCl, neuronal enhancers and promoters are in an open configuration but produce no RNA transcripts, or only low levels of them.b, On activation by membrane depolarization following KCl treatment, transcription factors and RNA polymerase (RNAP) bind to enhancers, and enhancer RNA (eRNA) is made. Simultaneously, RNAP and transcription machinery also bind to promoters and initiate mRNA transcription. c, If the promoter is truncated, the enhancer still binds transcription factors and RNAP but can no longer make eRNA.

出自Ren, B. (2010). Transcription: Enhancers make non-coding RNA.Nature 465, 173–174.


怎样找enhancer?

读Review,总结了1种预测方法和6种实验方法,优点、缺点和参考文献,见文末。

  1. Computational Analysis of Conservation and TF-Binding Motifs

  2. Regulator Binding. p300 ChIP-seq

  3. Chromatin Accessibility. DNase-seq, FAIRE, ATAC-seq

  4. Histone Modifications. H3K4me1, H3K27ac

  5. Chromosomal Interaction. ChIA-PET

  6. Functional Enhancer Screening by Reporter Assay

  7. eRNA-Based Detection


怎样找active enhancer?

用其中的第7种方法,特异性的找到active enhancer


这么多方法,小丫推荐哪个?

7种方法各有所长,我喜欢把目标细胞类型里所有已发表数据都找出来,放在一起看。

  1. 用H3K4me1和H3K27ac找到一大片结合信号;

  2. 用p300、PolII、DNase/ATAC-seq提高resolution;

  3. 用ChIA-PET/Hi-C找enhancer结合的是哪个基因的promoter;

  4. 用eRNA区分active和poised enhancer;

  5. 如果目标组织特别难获取,1-4步所需的数据有限,就用基于motif和保守性的纯分析方法做预测;

  6. 锁定几个目标,用3C、Reporter Assay验证。

最后画出他中了国自然,因为最后一周补了这张图》里的神图,放在文章里,作为key figure。


找到active enhancer有何用?

基因受哪些转录因子调控?有近距离的promoter调控,也有远距离enhancer调控。特别是组织特异性表达的基因,往往精准的受到众多转录因子的远距离调控。

找到了active enhancer,下一步还是用《他中了国自然,因为最后一周补了这张图》里的神图,找出哪些转录因子结合这个enhancer,从而远距离调控我们心仪的基因!



最后,详细查看7种方法的优缺点:


MethodAdvantagesLimitations

Computational Analysis of Conservation and TF-Binding Motifs

Conservation analysis of noncoding elements between different speciesNovel conserved DNA segments or TF-binding motifs could be discovered.Many other regulatory elements could be included.

Not all enhancers are conserved.
Combinatorial analysis of TF-binding motif scan and conservationEasily applicable to many TFs with known motifs.High-false positive rate.

Regulator Binding

ChIP-seq of TFsOccupancy of key TFs predicts enhancers.Key TFs of the target cells must be known.

ChIP-grade antibodies are not available for all TFs.

Difficult to distinguish enhancers and promoters.

Do not cover all enhancers.
ChIP-seq of transcriptional coactivators such as p300p300-binding sites are reported to predict enhancers generally.Difficult to distinguish active and poised enhancers.
Chromatin Accessibility
DNase-seqExtensively used for various types of cells.Includes diverse types of regulatory elements such as promoters, insulators, and silencers besides enhancers.
Formaldehyde-assisted isolation of regulatory elements (FAIRE)-seq
ATAC-seqApplicable to small numbers of cells.

Short library preparation time.
Histone Modifications
ChIP-seq profiling of histone modifications (e.g., H3K4me1, H3K27ac)ChIP-grade antibodies are broadly available and applicable for many species.Broad peaks make it difficult to predict enhancers with high nucleotide resolution.
Chromosomal Interaction
Chromatin interaction analysis with paired-end tag sequencing (ChIA-PET)ChIA-PET of RNAPII predicts enhancers as well as enhancer–promoter interactions.Spatial proximity does not necessarily reflect functional regulatory relationships.
Functional Enhancer Screening by Reporter Assay
Massively parallel reporter assays (MPRAs)Directly assess enhancer activities in parallel by sequencing the transcribed reporter containing heterologous barcodes.Not optimal for genome-wide screening.
Self-transcribing active regulatory region (STARR)-seqDirectly assesses genome-wide enhancer activities with a high detection rate by sequencing the transcribed enhancers themselves.
eRNA-Based Detection
RNA-seqExpression levels of eRNAsand their nearby genes are quantified simultaneously.Low nucleotide resolution.

Difficult to detect eRNAs, which are typically lowly expressed.

Detects only transcribed enhancers (not all enhancers are transcribed).
Chromatin-associated RNA (ChromRNA)-seqDetects unstable transcripts including eRNA by enriching chromatin fraction.Detects only transcribed enhancers, which are more likely to be active.
GRO-seq

PRO-seq
Detects unstable transcripts including eRNAs.Requires elaborate in vitro experimental procedures.

Detects only transcribed enhancers.
NET-seqDetects unstable transcripts including eRNAs.

Detects 3′ ends of RNAPII-bound eRNAs at nucleotide resolution.
Detection of 3′ ends of eRNAs makes it difficult to pinpoint enhancer region.

Detects only transcribed enhancers.
CAGEDetects the TSSs of eRNAs at nucleotide resolution.

Expression levels of eRNAs and their nearby genes are quantified simultaneously.
Requires large sample size to detect lowly expressed eRNAs.

Detects only transcribed enhancers.

回复“enhancer”,直达表格网页

出自Murakawa, Y., Yoshihara, M., Kawaji, H., Nishikawa, M., Zayed, H., Suzuki, H., and Fantom Consortium, and Hayashizaki, Y. (2016). Enhanced Identification of Transcriptional Enhancers Provides Mechanistic Insights into Diseases. Trends Genet. 32, 76–88. 




Cell paper图多,多角度论证,结论可靠。为了读透这篇Cell paper,小丫做足了准备工作:

上期激发兴趣《Cell的模式图画反,反,反了

本期铺垫基础知识

下期学习正文思路,就是这篇:

Chen, H., Li, C., Peng, X., Zhou, Z., Weinstein, J.N., Liang, H. and Cancer Genome Atlas Research Network, 2018. A Pan-Cancer Analysis of Enhancer Expression in Nearly 9000 Patient Samples. Cell173(2), pp.386-399.


你可能还对下面两个问题的解决方案感兴趣:

转录因子调控了谁?》Plan ABCD

哪个蛋白质调控我感兴趣的基因?》Plan ABC


想用ChIP-seqATAC-seq研究感兴趣的基因?想整合ChIP-seq、ATAC-seq、eCLIP/RIP-seq、RNA-seq数据寻找线索?找嘉因生物吧!从实验、测序,到多种数据整合分析,为您一站式解决。(点击文中蓝字了解详情)




嘉因公众号定位:客户共性问题解答,生信学习资源导航,高通量实验导购 | 为您提供高通量实验-测序-分析-验证一站式解决方案

130225t2nzqrppn22nna6k.jpg



https://blog.sciencenet.cn/blog-3372875-1110860.html

上一篇:再次把Cell爆文的思路、方法用到自己的课题上
下一篇:Cell的模式图画反,反,反了
收藏 IP: 124.77.56.*| 热度|

0

该博文允许注册用户评论 请点击登录 评论 (0 个评论)

IP: 134.84.245.*   鍥炲 | 璧� 璧� +1 [18]crossing   2015-12-16 03:19
鍗氫富鏂囩瑪闅芥案
IP: 119.166.135.*   鍥炲 | 璧� 璧� +1 [17]鏉庝笘鏄�   2015-12-11 15:28
寰堝ソ鐨勬梾琛岋紝寰堝ソ鐨勬枃绔犮€�

鎹娉㈠叞鐨勬暟瀛﹀寰堝帀瀹筹紝鏄笉鏄紵
鍥炲  锛� 娉㈠叞澶氶洦锛屼汉浠墽鐫€涓€鏍圭瓔锛屼範鎯尗鍦ㄥ閲屾兂闂......
2015-12-12 02:241 妤硷紙鍥炲妤间富锛� 璧� 璧� +1 | 鍥炲
IP: 223.2.42.*   鍥炲 | 璧� 璧� +1 [16]鍛ㄦ床   2015-12-11 09:20
   绉戝涓庡熀鐫f暀鐩稿骞剁珛鏄澶氳タ鏂瑰浗瀹剁殑浼犵粺銆傚彧鏄垜浠ぉ鐪熷湴鐢ㄦ潵閾查櫎鑷繁鐨勬枃鍖栦紶缁熴€�
鍥炲  锛� 鐧惧勾鏉ユ垜浠厛鐢ㄧ瀛︾牬浜嗘棫鏂囧寲浼犵粺锛岀幇鍦ㄥ徆鎭浗浜烘病鏈夐亾寰疯蹇碉紝璇磋瀛︿汉瀹舵湁淇′话銆傚弽姝hタ鏂规病鏈夋垜浠湁鐨勬槸閿欙紝鎴戜滑娌′簡瑗挎柟杩樻湁锛屾垜浠篃閿欍€�
2015-12-12 02:451 妤硷紙鍥炲妤间富锛� 璧� 璧� +1 | 鍥炲
IP: 130.158.152.*   鍥炲 | 璧� 璧� +1 [15]鏌崇娴�   2015-12-10 11:26
鐜板湪鏈変簡鎵嬫満璋锋瓕鍦板浘鍘诲摢鍎块兘鐪熸柟渚垮晩锛�
鍥炲  锛� 鐜板湪璧拌矾蹇呭鎵嬫満鍦板浘瀵兼父銆�
2015-12-12 02:471 妤硷紙鍥炲妤间富锛� 璧� 璧� +1 | 鍥炲
IP: 183.92.210.*   鍥炲 | 璧� 璧� +1 [14]闄堣幑   2015-12-10 10:09
鏈熷緟鐫€
鍥炲  锛� 浠婂ぉ鍙戙€�
2015-12-12 02:471 妤硷紙鍥炲妤间富锛� 璧� 璧� +1 | 鍥炲
IP: 125.39.106.*   鍥炲 | 璧� 璧� +1 [13]lrx   2015-12-9 23:33
娉㈠叞浜烘暟瀛︿笉閿欙紝鐗瑰埆鏄瘑鐮侀鍩熴€備簩鎴樻湡闂达紝涓嶅皯娉㈠叞浜轰粠浜嬬牬瑙f硶瑗挎柉瀵嗙爜宸ヤ綔
鍥炲  锛� 娉㈠叞浜哄緢鎵х潃銆�
2015-12-10 03:121 妤硷紙鍥炲妤间富锛� 璧� 璧� +1 | 鍥炲
IP: 123.68.82.*   鍥炲 | 璧� 璧� +1 [12]浼嶅浗鍗�   2015-12-9 23:12
鍐欏緱鐪熷ソ鍟婏紒鎯宠捣鎴戠殑鍔犳嬁澶у甯堜簡锛屼粬鏄尝鍏颁汉锛岀澘鏅恒€佸嫟濂嬨€佺簿鍔涘厖娌涖€�
鍥炲  锛� 涓ヨ們涓撴敞鎵х潃銆�
2015-12-10 03:131 妤硷紙鍥炲妤间富锛� 璧� 璧� +1 | 鍥炲
IP: 115.205.130.*   鍥炲 | 璧� 璧� +1 [11]Hrrrli   2015-12-9 19:56
搴旇€佸笀杩滆冻澶╀笅锛屼簬涓滆タ浜ゆ眹涔嬪鎴樼暐鏁呭湴涓€瑙傦紝璇讳箣鍘嗗彶涔熸槸浠や汉鍢昏櫄銆傛尝鍏版湁鑲栭偊锛屽惉涔嬮挗鐞存洸锛屽惥鍥借瘧鍌呴浄涔嬪瓙浠ヨ瘎寮瑰叾涓烘渶銆備緷鍚惧剴瀹跺鍥箣瑙傦紝涔熸槸涓€鏂规按鍦熷吇涓€鏂逛汉銆備汉灞炰腑瑗匡紝涔熸槸闅惧彈锛岃嚜绔嬩负瀹跺洯涔熸槸鍘嗗彶涔嬫€ㄣ€�
鍥炲  锛� 璇诲彶浠や汉娓呴啋锛屼笉鑷充簬鐩蹭粠鍠у殻鐨勬祦淇椼€�
2015-12-10 03:191 妤硷紙鍥炲妤间富锛� 璧� 璧� +1 | 鍥炲
IP: 116.208.48.*   鍥炲 | 璧� 璧� +1 [10]榛勬案涔�   2015-12-9 19:54
鍗庢矙鏄釜濂藉湴鏂广€�
鍥炲  锛� 鍊煎緱涓€鍘汇€�
2015-12-10 03:201 妤硷紙鍥炲妤间富锛� 璧� 璧� +1 | 鍥炲
IP: 122.190.126.*   鍥炲 | 璧� 璧� +1 [9]闄堣幑   2015-12-9 19:35
鐪嬪埌杩欑瘒鏂囧拰鍥剧墖锛屾兂璧蜂竴娆$湅 鍩庡競涓€瀵逛竴 涓鍏嬫媺绉戝か鐨勬晠浜嬶紝涓€涓尝鍏拌瘲浜哄啓浜嗕竴棣栬瘲锛氬晩锛岀粰鎴戜竴鐩樺繀楂樻€�...
鍥炲  锛� 涓嬬瘒鍐欏厠鎷夌澶€�
2015-12-10 03:201 妤硷紙鍥炲妤间富锛� 璧� 璧� +1 | 鍥炲
IP: 58.254.4.*   鍥炲 | 璧� 璧� +1 [8]姹檽鍐�   2015-12-9 18:47
鐪嬫潵鍗庢矙鍊煎緱涓€鍘伙紒
鍥炲  锛� 鏄殑銆�
2015-12-10 03:211 妤硷紙鍥炲妤间富锛� 璧� 璧� +1 | 鍥炲
IP: 115.224.131.*   鍥炲 | 璧� 璧� +1 [7]zjzhaokeqin   2015-12-9 17:51
娉㈡緶璧蜂紡
鍥炲  锛� 鍘嗗彶鏈韩涓嶅钩闈欙紝鍙槸涔︿笂缁忓父鐣ヨ繃涓€閮ㄥ垎銆�
2015-12-10 03:231 妤硷紙鍥炲妤间富锛� 璧� 璧� +1 | 鍥炲
IP: 137.132.250.*   鍥炲 | 璧� 璧� +1 [6]寮犳礇娆�   2015-12-9 17:47
鍐欑殑绮惧僵銆備粖骞�4鏈堝湪鍗庢矙寮€浼氾紝浜嗚В浜嗕竴浜涘崕娌欏巻鍙层€傜浉褰撻渿鎾笺€傞偅涓皬鍩庯紝鏈夊ソ鍑犱釜鍘嗗彶鍚嶄汉锛� 鍖呮嫭灞呴噷澶汉锛岃倴閭︺€�
鍥炲  锛� 娉㈠叞浠庝腑涓栫邯鐩村埌杩戜唬锛岃澶氭柟闈㈣繕鏄蛋鍦ㄦ娲插ぇ澶氬浗瀹跺墠闈㈢殑銆�
2015-12-10 03:271 妤硷紙鍥炲妤间富锛� 璧� 璧� +1 | 鍥炲
IP: 212.128.158.*   鍥炲 | 璧� 璧� +1 [5]鏉庡ぉ鎴�   2015-12-9 17:42
鍐欑殑绮惧僵锛�
鍥炲  锛�   
2015-12-10 03:271 妤硷紙鍥炲妤间富锛� 璧� 璧� +1 | 鍥炲
IP: 183.157.160.*   鍥炲 | 璧� 璧� +1 [4]璧电編濞�   2015-12-9 15:49
搴旇€佸笀濂戒箙娌¤锛屽師鏉ュ幓涓浜唦~
鍥炲  锛�   
2015-12-10 03:281 妤硷紙鍥炲妤间富锛� 璧� 璧� +1 | 鍥炲
IP: 124.207.8.*   鍥炲 | 璧� 璧� +1 [3]鍒橀攱   2015-12-9 13:39
浜虹湡灏�
鍥炲  锛�   
2015-12-9 14:301 妤硷紙鍥炲妤间富锛� 璧� 璧� +1 | 鍥炲
IP: 223.104.9.*   鍥炲 | 璧� 璧� +1 [2]鏉庤交鑸�   2015-12-9 13:14
涓€鐪嬪埌姣曡嫃鏂熀锛屽氨鎯冲埌鈥滅孩鑹叉嬁鐮翠粦鈥濆浘鍝堝垏澶柉鍩�(澶ф竻娲楋紝鏂ぇ鏋楅€佷粬鐨勫附瀛愭濂芥槸鈥滄尝鎷垮反涓讳箟鈥濓紝鎯虫潵璁藉埡)鎶樻垷鍗庢矙鍩庝笅锛屾湁鍔ㄧ瑪鐨勫啿鍔ㄤ簡
鍥炲  锛� 鍏佃€呰閬擄紝鍥惧搱鍒囧か鏂熀杈撳拰姝婚兘鏈夌偣鍐ゃ€傛瘯鑻忔柉鍩轰笌濂戝崱澶村瓙鎹峰皵浠绘柉鍩烘槸鍚屽锛屾椂鍔块€犺嫳闆勶紝閭f椂澶╂墠涔熺湡澶氾紝涔熻涓嶈繃鏄儐澶ф暍璧屻€�
2015-12-9 14:291 妤硷紙鍥炲妤间富锛� 璧� 璧� +1 | 鍥炲
IP: 101.231.92.*   鍥炲 | 璧� 璧� +1 [1]yangb919   2015-12-9 12:07
浠庢尝鍏板埌涓滃痉,闈㈠鑻忚仈,
棰嗗湡鍙兘鍚戝乏杞�,璧锋璧�

1/1 | 鎬昏:18 | 棣栭〉 | 涓婁竴椤� | 涓嬩竴椤� | 鏈〉 | 璺宠浆

Archiver|手机版|科学网 ( 京ICP备07017567号-12 )

GMT+8, 2025-3-2 03:03

Powered by ScienceNet.cn

Copyright © 2007-2025 中国科学报社

返回顶部