2 EpigeneticsHeritable changes in gene function that occur without a change in the DNA sequenceHow come not all the motif sites are bound by the factor?How come TF binding only regulate some of the nearby genes?
3 EpigeneticsThe study of heritable (transgenerational) changes in gene activity that are not caused by changes in the DNA sequenceThe study of stable, long-term alterations in the transcriptional potential of a cell that are not necessarily heritableFunctionally relevant changes to the genome that do not involve a change in the nucleotide sequence
4 In Human Nature vs nurture Zygotic twins: same DNA different epigenome North American Ice Storm of 1998
6 The Making of a QueenLarvaeQueenWorkerFrom Ting Wang, Wash U
7 Epigenetic Landscape Conrad Hal Waddington (1905–1975) Developmental biologistPaleontologistGeneticistEmbryologistPhilosopherFounder for systems biology
8 Components DNA-methylation Nucleosome position and histone modificationsChromatin accessibilityHigher order chromatin interactionsAnalogy
9 DNA Methylation Distribution in Mammalian Genomes In human somatic cells, 60%-80% of all CpGs(~1% of total DNA bases) are methylatedMost methylation is found in “repetitive” elements“CpG islands”, GC-rich regions that possess a high density of CpGs, remain methylation-freeThe promoter regions of ~70% of genes have CpG islandsFrom Ting Wang, Wash U
10 Two classes of DNA methyltransferases (DNMTs) The basis of this model is that DNA methylation patterns are established in germ cells and in developing embryos by the activity of the de novo DNA methyltransferases (DNMTs)Methylation patterns are copied by DNMT1. copying a pattern that is present on DNA onto newly synthesized strandJones and Liang, 2009 Nature Review Genetics
11 Inheritance of DNA Methylation From Ting Wang, Wash U
12 DNA Methylation Detection Bisulfite sequencingUnmethyl C THigh resolution, quantitative, but expensive!
14 BS-seq Methylation Call Most regions are either mostly methylated or mostly unmethylated (dichotomy)Methylation level within a short distance is consistentACGGGCTTACTTGCTTTCCTACGGGCTTACTTGCTTTCCTACGGGCTTACTTGCTTTCCTACGGGCTTACTTGCCGGGTTTATTTGCTTTTTTATGGGCTGGGTTTATTTGCTTTTTTATGGGCTGGGTTTATTTGCTTTCCTATGGGCCGGGCTTATTTGCTTTCCTATGGGCCGGGCTTATTTGCTTTCCTATGGGC3/50/560% methylated0% methylatedFrom Ting Wang, Wash U
15 DNA Methylation Controls Gene Expression Methylation at CpG islands often repress nearby gene expressionMany highly expressed genes have CpG methylation on their exonsSome genes could be imprinted, so maternal and paternal copies have different DNA methylationFrom Ting Wang, Wash U
16 DNA Methylation in Cancer Prevalent misregulation of DNA methylation in cancer: global hypomethylation and CpG island hypermethylationMethylation variable regions in cancer
17 DNA DemethylationRecently, another type of DNA methylation called hydroxyl methylation (hmC) is foundhmC is an intermediate step between mC and C.TET family of proteins are important for DNA demethylationMutation in TET is linked to many cancers
18 Components DNA-methylation Nucleosome position and histone modificationsChromatin accessibilityHigher order chromatin interactionsAnalogy
19 Nucleosome Occupancy & Histone Modification Influence Factor Binding TFLast year, Keji Zhao’s group published a pioneering studying using Solexa to conduct ChIP-seq of 21 histone marks in human CD4 T cells. In the study, chromatin was digested with MNase and mononucleosomes carrying specific histone mark was enriched using IP. Solexa sequencing was used to read 25-mer from either end of the nucleosomal DNA. Although an unprecedented 185 million nucleosome tags were sequenced, no analysis to-date aims to use them to study nucleosome position at specific locations in the genome.
20 Histone Modifications Different modifications at different locations by different enzymes
21 Histone Modifications in Relation to Gene Transcription From Ting Wang, Wash U
22 Histone Modifications Gene body mark: H3K36me3, H3K79me3Active promoter (TSS) mark: H3K4me3Active enhancer (TF binding) mark: H3K4me1, H3K27acBoth enhancers and promoters: H3K4me2, H3/H4ac, H2AZRepressive promoter mark: H3K27me3Repressive mark for DNA methylation: H3K9me3
23 lncRNA Identification H3K4me3 active promotersH3K36me3 transcription elongationGuttman et al, Nat 2009
26 Nucleosome Occupancy & Histone Modification Influence Factor Binding Antibody forMNase digestTFLast year, Keji Zhao’s group published a pioneering studying using Solexa to conduct ChIP-seq of 21 histone marks in human CD4 T cells. In the study, chromatin was digested with MNase and mononucleosomes carrying specific histone mark was enriched using IP. Solexa sequencing was used to read 25-mer from either end of the nucleosomal DNA. Although an unprecedented 185 million nucleosome tags were sequenced, no analysis to-date aims to use them to study nucleosome position at specific locations in the genome.
27 Combine Tags From All ChIP-Seq So we went about looking at the data more. For each specific region, we combined all the tags from all marks, because they are all nucleosome ends.
28 Extend Tags 3’ to 146 nt Check Tag Count Across Genome Then at each position, we counted the number of extended tags that landed there.The sequenced nucleosomes were not always 150bp. To better locate the center of positioned nuclesomes, we took the center 75 bp of each extended tag.
30 Use H3K4me2 / H3K27ac Nucleosome Dynamics to Infer TF Binding Events Nucleosome Stabilization-Destabilization (NSD) ScoreCondition 1Condition 2In a proof of concept study we collaborated with Myles Brown’s lab, and found that very often, a cell will anticipate the stimulus it might get, and mark the potential TF binding sites before the TF is stably bound. Upon….We devised a simple scoring function to measure how much the middle down vs flanking up, and we call it NSD.Positive NSD is nucleosome depletion, negative NSD is nucleosome moving back, NSD of 0 means no nucleosome moving.He et al, Nat Genet, 2010; Meyer et al, Bioinfo 2011
31 Condition-Specific Binding, Epigenetics and Gene Expression Condition-specific TF bindings are associated with epigenetic signaturesCan we use the epigenetic profile and TF motif analysis to simultaneous guess the binding of many TFs together?Genes TF1 TF2 TF3 Epigenetics
32 Predict Driving TFs and Bindings for Gut Differentiation
33 Identify Major TF Modules Regulating Gut Differentiation and Function GATA6Cdx2Embryonic and organ development genesHNF4Metabolic and digestive genesCdx2Nucleosome dynamics now applied to hematopoiesis and cancer cell reprogrammingVerzi et al, Dev Cell, 2010
34 Components DNA-methylation Nucleosome position and histone modificationsChromatin accessibilityHigher order chromatin interactionsAnalogy
35 DNase Hypersensitive (HS) Mapping DNase randomly cuts genome (more often in open chromatin region)Select short fragments (two nearby cuts) to sequenceMap toactivepromotersandenhancersLing et al, MCB 2010
36 DHS Peaks Capture Most TF Binding Sites Motif occurrence in the DHS peaks suggest TF bindingQuantitative signal strength also suggest binding stabilityThurman et al, Nat 2012