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RNA-seq居然能挖出这么多机制！给你的文章加5分

已有 10874 次阅读 2018-4-25 16:39 |个人分类:生物信息|系统分类:科普集锦

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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：

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》；
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》；
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。

Computational Analysis of Conservation and TF-Binding Motifs
Regulator Binding. p300 ChIP-seq
Chromatin Accessibility. DNase-seq, FAIRE, ATAC-seq
Histone Modifications. H3K4me1, H3K27ac
Chromosomal Interaction. ChIA-PET
Functional Enhancer Screening by Reporter Assay
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。

用H3K4me1和H3K27ac找到一大片结合信号；
用p300、PolII、DNase/ATAC-seq提高resolution；
用ChIA-PET/Hi-C找enhancer结合的是哪个基因的promoter；
用eRNA区分active和poised enhancer；
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；
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：

Method	Advantages	Limitations
Computational Analysis of Conservation and TF-Binding Motifs
Conservation analysis of noncoding elements between different species	Novel 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 conservation	Easily applicable to many TFs with known motifs.	High-false positive rate.
Regulator Binding
ChIP-seq of TFs	Occupancy 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 p300	p300-binding sites are reported to predict enhancers generally.	Difficult to distinguish active and poised enhancers.
Chromatin Accessibility
DNase-seq	Extensively 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-seq	Applicable 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)-seq	Directly assesses genome-wide enhancer activities with a high detection rate by sequencing the transcribed enhancers themselves.
eRNA-Based Detection
RNA-seq	Expression 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)-seq	Detects 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-seq	Detects 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.
CAGE	Detects 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. Cell, 173(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

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