不同层次的表观

• Broadly, features at different levels of chromatin organization are generally associated with inactive (off) or active (on) transcription.
• From the top, genomic DNA is methylated(Me) on cytosine bases in specific contexts and is packaged into nucleosomes, which vary in histone composition and histone modifications (for example, histone H3 lysine 9 trimethylation (H3K9me3)); these features constitute the primary layer of chromatinstructure.
• Here, different histone modifications are indicated by coloured dots and histone variants such as H2A.Z are brown.
• DNA in chromatin may remain accessible to DNA-binding proteins such as transcription factors (TFs) and RNA polymerase II (RNAPII)or may be further compacted.
• Chromatin can also organize into higher-order structuressuch as nuclear lamina-associated domains and transcription factories.
• DOI:10.1038/nrg2905

表观实例

基因印迹

表观遗传定义

• A mitotically or meiotically heritable state of different gene activity and expression (phenotype) that is independent of differences in DNA sequence (genotype) – based on Conrad Waddington, 1942
• The sum of the alterations to the chromatin template that collectively establish and propagate different patterns of gene expression (transcription) and silencing from the same genome.
• Epigenetic changes influence the phenotype without altering the genotype.
• While epigenetics often refers to the study of single genes or sets of genes, epigenomics refers to more global analyses of epigenetic changes across the entire genome.

表观基因组 epigenome

• The complete number,location, and types ofepigenetic modificationsthat occur in a given cell

Epigenetic mechanisms

DNA methylation

• Normal cells — role in gene expression and chromosome stability
• Cancer cells — consequences of aberrant hypo- and hyper-methylation

Histone modification

• Normal cells — the histone code
• Cancer cells — consequences of altered histone modifying enzymes

Interaction between DNA methylation, histone modifications, and other players such as non-coding RNAs

Cell and tissue type specificity

Gene-environment interaction, disease susceptibility

DNA methylation

human 特征

• DNA methylation occurs mostly in CG context, but also non-CG sequences (mCHG, mCHH)
• DNA methylation is generally depleted from promoters, and enriched within gene bodies
• DNA methylation is partially depleted at regulatory element

植物中有特殊的 RdDM

bisulfite sequencing

基本原理

• DNA 的重亚硫酸盐转化来检测未甲基化的胞嘧啶
• 在文库制备过程中，重亚硫酸盐将未甲基化的胞嘧啶转化成尿嘧啶
• 转化后的碱基在测序中被识别为胸腺嘧啶（PCR 后），并通过序列计数来确定甲基化胞嘧啶的比例

比对的过程需要进行转换

常用 mapping 软件算法

• bismark

• bsmap

• Bisulfite seed table, using the original seed and bisulfite variants as keys and corresponding coordinates in the reference genome as values. Each read was looked up in the seed table for potential mapping positions.

• A positional specific mask of the corresponding reference sequence was generated by setting 01 to C(light blue) and 11 to A, G, T(black). The original read was masked by a bitwise AND operation with the positional specific mask.

• The reference sequence and the masked read were compared with a bitwise XOR operation. Non-zero XOR results were counted as mismatches (red). Bisulfite alignment is marked in green.

Methylated DNA immunoprecipitation (MeDIP)

• 甲基化 DNA 免疫共沉淀测序
• use 5-methylcytosine antibody
• 富集甲基化的 DNA 序列
• 有甲基化的地方会有 peak
• 不能精确到单碱基分辨率，这能知道一段区域的甲基化程度

Methyl-sensitive restriction enzymes (MREs)

• Unmethylated CpGs are identified by sequencing size selected fragments from parallel DNA digestions with the methyl-sensitive restriction enzymes (MREs)

• 没有甲基化的地方会有 peak

• 甲基化敏感性限制酶测序 (MRE-seq) 技术主要利用甲基化敏感性限制性酶 (methylation-sensitive restriction enzyme, MRE) 对基因组 DNA 进行切割，未甲基化位点可以被酶切而产生不同长度的片段，然后，结合高通量测序技术获得序列的甲基化信息

• 和 MeDIP 结合使用

  

• plant DOI:10.1146/annurev-arplant-043014-114633

• human DOI:10.1038/nature08514

组蛋白修饰

H3K4me3 含义

组蛋白修饰功能

• Promoters, gene bodies, an enhancer and a boundary element are indicated on a schematic genomic region.
• Active promoters are commonly marked by H3K4me2, H3K4me3, acetylation (ac), and H2A.Z.
• Transcribed regions are enriched for H3K36me3 and H3K79me2.
• Repressed genes may be located in large domains of H3K9me2 and/or H3K9me3 or H3K27me3.
• Enhancers are relatively enriched for H3K4me1, H3K4me2, H3K27ac and the histone acetyltransferase p300.
• CTCF binds many sites that may function as boundary elements, insulators or structural scaffolds

如何整合众多的 ChIP-seq 数据

半自动基因注释

两种常用算法

ChromHMM

输入参数

• 算法是 HMM，具体隐马尔可夫模型参考另一篇笔记

• 属于 HMM 中的 learning 类问题

• 使用迭代方法是 EM

• 200 bp resolution

• Bernoulli to model emission

• Posterior decoding

• Single model for all cell types

Segway

• 基于贝叶斯动态网络
• 1 bp resolution
• Gaussian to model emission
• Viterbi decoding
• One model per cell type

基因浏览器展示

具体学习地址

• ensembl

• Genome Segmentations from ENCODE

human 表观数据库

encode

• 网站 https://www.encodeproject.org/
• 数据下载地址http://genome.ucsc.edu/ENCODE/downloads.html

roadmap

• 网站 http://www.roadmapepigenomics.org/

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