ChIP-seq实战 | 染色质免疫共沉淀技术 | ATAC-seq | 染色质开放性测序技术

参考：生信技能树

ChIP-Seq综述

一些简单的copy，纯属个人笔记。

ChIP-seq的原理

用于在全基因组范围中研究DNA结合蛋白（相互反应）、组蛋白修饰（表观遗传标记）和核小体的技术，研究这三个主题可有助于了解基因之间的相互调控以及染色体的功能结构。

在生理状态下，把细胞内的DNA与蛋白质交联（Crosslink）后裂解细胞，分离染色体，通过超声或酶处理将染色质随机切割，利用抗原抗体的特异性识别反应，将与目的蛋白相结合的DNA片段沉淀下来，再通过反交联（Reverse crosslink）释放结合蛋白的DNA片段，最后测序获得DNA片段的序列。

先锁定DNA和蛋白质，裂解，抗原富集特定DNA，解锁DNA和蛋白质，就得到了我们想要的DNA片段，测序分析。

Paper: RYBP and Cbx7 Define Specific Biological Functions of Polycomb Complexes in Mouse Embryonic Stem Cells

实验设计方面：

ChIP-seq of RYBP, Cbx7, Ring1B, Suz12, and H2AK119Ub （knock down了一些基因，然后测ChIP-seq）

文章中的分析方法：

ChIP-Seq Data Analyses
The millions of reads produced by ChIP-seq of RYBP, Cbx7, Ring1B, Suz12,and H2AK119Ub were aligned with the mouse genome (version NCBIM37)using the Bowtie (Langmead et al., 2009) tool version 0.12.7; two mismatcheswere allowed within the seed alignment. Sequence tags were aligned to thegenome and then subsequently analyzed by MACS software version 1.4.1(Zhang et al., 2008) to detect genomic regions enriched for multiple overlappingDNA fragments (peaks) that we considered to be putative binding sites.MACS estimated the FDR by comparing the peaks obtained from the sampleswith those from the control samples, using the same p value cutoff (1*10-5).Peaks with a FDR lower than 5% from Cbx7, Ring1B, Suz12, and H2AK119ub,and lower than 1% from RYBP, were combined to detect chromosomalregions for further analyses. Genes with a peak within the gene body, or within2.5 kb from the TSS, were considered to be target genes. An area of 5 kbsurrounding each TSS that was associated to one or more ChIP-seq (RYBP,Ring1B, Cbx7, Suz12, or H2AK119ub) was used to calculate the ChIP-seqprofile and the whole ChIP-seq coverage. ChIP-seq profiles around theTSSs were generated for each IP by calculating the average coverage ineach position normalized for the total number of mapped reads with theBED tools package (Quinlan and Hall, 2010).

Figure 1. RYBP and Cbx7 Target Genes Are Not Mutually Exclusive in Mouse ESCs

Figure 2. Cbx7 Target Genes Contain More H2AK119ub and Have Different Biological Functions than RYBP Target Genes

Figure 3. RYBP/Ring1B Target Genes Are More Likely to Be Expressed than Cbx7/Ring1B Targets

Figure 4. Interdependency of RYBP and Cbx7 Recruitment to Chromatin

基本概念：

Polycomb Complexes：一种蛋白复合体，Polycomb repressive complex 1 (PRC1)，Polycomb-group proteins are a family of proteins first discovered in fruit flies that can remodel chromatin（染色质重塑） such that epigenetic silencing of genes takes place. Polycomb-group proteins are well known for silencing Hox genes through modulation of chromatin structure during embryonic development in fruit flies (Drosophila melanogaster). 一些重要的蛋白，可以重塑染色体结构，从而实现一些重要过程（胚胎发育）的表观调控，最终决定干细胞的命运。

RYBP and Cbx7： mutually exclusive presence of Cbx7 or RYBP，结合到PRC1上的物质，从而决定不同的PRC1变种，它们在基因的表观调控上有一些差异。虽然有些target gene有overlap，但它们还是相互排斥。 Cbx7 is necessary for recruitment of Ring1B to chromatin；RYBP enhances the PRC1 enzymatic activity。the canonical PRC1 consists of one of the Cbx proteins, polyhomeotic (PHC), PCGF, RYBP/YAF2, and a Ring1A/B E3 ligase subunit that monoubiquitinates histone H2A at lysine 119 (H2AK119ub)。

Cbx7 is the predominant PRC1-associated Cbx subunit in proliferating ESCs and requires the H3K27me3 mark to localize to chromatin and thereby silence the expression of lineage commitment genes. Once ESCs differentiate, other Cbx proteins, including Cbx2 and Cbx4, replace Cbx7 within PRC1 to mediate fate choices along the three germ layers

RYBP-PRC1 does not seem to require the H3K27me3 mark to bind to chromatin in ESCs

Although both complexes are biochemically distinct, they can cooperatively establish epigenetic gene repression.

最终结论：different PRC1 subtypes establish a complex pattern of gene regulation that regulates common and nonoverlapping aspects of ESC pluripotency and differentiation.

一些问题：

Here, we investigated the following questions about the PRC1 complexes that contain either Cbx7 or RYBP:
- What is the genome-wide localization of these two types of PCR1 complexes?
- Is the expression of specific sets of genes differentially regulated by both complexes?
- Do they exert common and/or unique biological functions?
- Do they show any interdependency for localizing to chromatin?

ATAC-seq | 染色质开放性测序技术

参考：ATAC-seq：染色质开放性测序技术

ATAC-seq全称Assay for Transposase Accessible Chromatin with high-throughput sequencing，即利用转座酶研究染色质可进入性的高通量测序技术。

DNA转座，是一种把DNA序列从染色体的一个区域搬运到另外一个区域的现象，由DNA转座酶来实现。这种转座插入DNA，也是需要插入位点的染色质是开放的，否则就会被一大坨高级结构给卡住。

ATAC-seq出来的结果，和传统方法出来的结果具有很强的一致性，同时也和基于组蛋白修饰marker的ChIP-seq有较高的吻合程度。也就是说，ATAC-seq中的peak，往往是启动子、增强子序列，以及一些反式调控因子结合的位点。

请问转座酶Tn5对DNA序列有选择性吗？我想应该是没有，不然这个方法就只对特定的序列敏感了。

ATAC-seq能干啥？

The landscape of accessible chromatin in mammalian preimplantation embryos

promoters, enhancers, insulators and the locus-control regions

promoters：启动子，启动子是一段提供RNA聚合酶识别和结合位点的DNA序列，位于基因上游。

enhancers：增强子，位于结构基因附近，能够增强该基因转录活性的一段DNA顺序称为增强子（enhancer）。

insulators：绝缘子，在基因组内建立独立的转录活性结构域的边界DNA序列称为绝缘子/隔离子（insulator）。绝缘子能够阻止邻近的增强子或沉默子对其界定的基因的启动子发挥调控作用。

locus-control regions：基因座控制区域

TSS：transcription start site

TES：transcription end site

待续~

参考：

增强子、终止子、沉默子、绝缘子