CAGE, RAMPAGE, & RNA Pol II - Washington University in St ...



1151255739140-762004230Module TSS3: CAGE, RAMPAGE, & RNA Pol II X-ChIP-Seq Meg Laakso & Wilson Leung TitleIdentifying transcription start sites for peaked promoters using experimental data from sequenced transcripts and RNA polymerase II occupancyObjectiveUse the placement of the Initiator motif; experimental data from CAGE, RAMPAGE, and RNA Pol II X-ChIP-Seq experiments; and TSS predictions from the Celniker group at modENCODE to identify putative transcription start sites (TSSs) and characterize promoter typePrerequisitesModule TSS1 OrderExplain the experimental techniques used to generate CAGE, RAMPAGE, and RNA Pol II X-ChIP-Seq data, and the Celniker TSS predictionsIdentify the Antennapedia TSS using CAGE, TSS predictions, RAMPAGE, and RNA Pol II X-ChIP-SeqAnalyze all of the evidence in order to identify the best TSS and characterize the promoter typeClass InstructionDiscuss the question: How can our understanding of transcription initiation be used to find a TSS?Conclude by challenging students to think about these questions:For each of the evidence tracks you have investigated, how might a broad promoter differ from a peaked promoter?If a gene has multiple isoforms, does it have to have multiple transcription start sites?Resources & ToolsRNA-Seq and TopHat VideoGEP UCSC Genome BrowserTable of Contents TOC \o "1-3" \h \z \u Identify the location of Transcription Start Site using CAGE, RAMPAGE and RNA Pol II X-ChIP-Seq data PAGEREF _Toc143263863 \h 2Summary of Cap Analysis of Gene Expression (CAGE) PAGEREF _Toc143263864 \h 2Exercise 1: Examining the CAGE Data for the Antennapedia Gene Using the UCSC Genome Browser Mirror PAGEREF _Toc143263865 \h 2RNA Annotation and Mapping of Promoters for the Analysis of Gene Expression (RAMPAGE) PAGEREF _Toc143263866 \h 5Exercise 2: Examining the RAMPAGE data for the Antennapedia gene PAGEREF _Toc143263867 \h 6RNA Pol II ChIP-Seq PAGEREF _Toc143263868 \h 9Exercise 3: Examining the X-ChIP-Seq data for the Antennapedia gene using the UCSC Genome Browser Mirror PAGEREF _Toc143263869 \h 9Wrapping up PAGEREF _Toc143263870 \h 10References PAGEREF _Toc143263871 \h 11Identify the location of Transcription Start Site using CAGE, RAMPAGE and RNA Pol II X-ChIP-Seq dataIn Module 1 we utilized Celniker data, DNase I hypersensitivity data and the 9-state model track to investigate the transcription start site region for the Antennapedia (Antp) gene in Drosophila melanogaster. This module will further explore the Antp gene in D. melanogaster to illustrate how the experimental techniques CAGE, RAMPAGE, and RNA Pol II X-ChIP-Seq can be used to identify or confirm transcription start sites (TSSs) and promoter type.Summary of Cap Analysis of Gene Expression (CAGE) The relative abundance of mRNA transcripts can be determined by many different methods such as RNA-Seq, quantitative PCR, or Serial Analysis of Gene Expression, which you may have studied in class. However, these methods tend to show a bias toward the body of the transcript. Hence, specialized techniques are needed to isolate sequences associated with the 5’ end (the “beginning”) of the transcript.One technique that can be used to identify a TSS is Cap Analysis of Gene Expression (CAGE).ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"abstract":"Transcript abundance can be determined by various methods, including reverse transcription (RT)-PCR, microarray analysis, sequencing of expressed sequence tags (ESTs), serial analysis of gene expression (SAGE) and massively parallel signature sequencing (MPSS), most of which rely on 3′ end–related sequences. But for the identification of transcription start sites (TSSs) and their associated promoters, 5′ end–specific signature sequences are required for higher annotations of expression profiles. Therefore, we and others began cloning of short sequence tags from the 5′ ends of cDNAs, using cap analysis of gene expression (CAGE) and 5′-SAGE. In these techniques linkers are attached to the 5′ ends of full-length enriched cDNAs to introduce a recognition site for the restriction endonuclease MmeI adjacent to the 5′ ends. MmeI cleaves cDNAs at a sequence 20 and 18 nucleotides away (3′) from its recognition site, creating a two-base overhang. After amplification, the sequencing tags are concatenated for highthroughput sequencing (Fig. 1). Here we present a CAGE protocol that has been used extensively for highthroughput analysis of mouse and human transcripts. The method includes new features for improved library construction, such as the use of random priming for gene discovery from nonpolyadenylated RNA, simplified full-length cDNA enrichment by multiwell filtration-based cap-trapping and a pooling strategy for high-throughput CAGE library preparation. The application of this CAGE protocol will contribute to genome annotation, gene discovery and expression profiling.","author":[{"dropping-particle":"","family":"Kodzius","given":"Rimantas","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Kojima","given":"Miki","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Nishiyori","given":"Hiromi","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Nakamura","given":"Mari","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Fukuda","given":"Shiro","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Tagami","given":"Michihira","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Sasaki","given":"Daisuke","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Imamura","given":"Kengo","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Kai","given":"Chikatoshi","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Harbers","given":"Matthias","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Hayashizaki","given":"Yoshihide","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Carninci","given":"Piero","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Nature Methods","id":"ITEM-1","issue":"3","issued":{"date-parts":[["2006"]]},"page":"211-222","title":"CAGE : cap analysis of gene expression","type":"article-journal","volume":"3"},"uris":[""]}],"mendeley":{"formattedCitation":"¹","plainTextFormattedCitation":"1","previouslyFormattedCitation":"¹"},"properties":{"noteIndex":0},"schema":""}1 This technique takes advantage of the fact that there is a unique nucleotide at the 5’ end of the newly synthesized eukaryotic mRNA, the “5’ cap”. This 5’ cap can be chemically modified, allowing the capped mRNAs to be purified and sequenced.In Exercise 1, we will use CAGE data to help identify the TSS and cross-reference with TSS predictions produced by the Celniker group at modENCODE to identify the TSSADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1101/gr.112466.110.Freely","abstract":"Core promoters are critical regions for gene regulation in higher eukaryotes. However, the boundaries of promoter regions, the relative rates of initiation at the transcription start sites (TSSs) distributed within them, and the functional significance of promoter architecture remain poorly understood. We produced a high-resolution map of promoters active in the Drosophila melanogaster embryo by integrating data from three independent and complementary methods: 21 million cap analysis of gene expression (CAGE) tags, 1.2 million RNA ligase mediated rapid amplification of cDNA ends (RLMRACE) reads, and 50,000 cap-trapped expressed sequence tags (ESTs). We defined 12,454 promoters of 8037 genes. Our analysis indicates that, due to non-promoter-associated RNA background signal, previous studies have likely overestimated the number of promoter-associated CAGE clusters by fivefold. We show that TSS distributions form a complex continuum of shapes, and that promoters active in the embryo and adult have highly similar shapes in 95% of cases. This suggests that these distributions are generally determined by static elements such as local DNA sequence and are not modulated by dynamic signals such as histone modifications. Transcription factor binding motifs are differentially enriched as a function of promoter shape, and peaked promoter shape is correlated with both temporal and spatial regulation of gene expression. Our results contribute to the emerging view that core promoters are functionally diverse and control patterning of gene expression in Drosophila and mammals.","author":[{"dropping-particle":"","family":"Hoskins","given":"Roger A","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Landolin","given":"Jane M","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Brown","given":"James B","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Sandler","given":"Jeremy E","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Takahashi","given":"Hazuki","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Lassmann","given":"Timo","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Yu","given":"Charles","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Booth","given":"Benjamin W","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Zhang","given":"Dayu","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Wan","given":"Kenneth H","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Yang","given":"Li","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Boley","given":"Nathan","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Andrews","given":"Justen","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Kaufman","given":"Thomas C","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Graveley","given":"Brenton R","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Bickel","given":"Peter J","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Carninci","given":"Piero","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Carlson","given":"Joseph W","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Celniker","given":"Susan E","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Genome Research","id":"ITEM-1","issued":{"date-parts":[["2011"]]},"page":"182-192","title":"Genome-wide analysis of promoter architecture in Drosophila melanogaster","type":"article-journal","volume":"21"},"uris":[""]}],"mendeley":{"formattedCitation":"²","plainTextFormattedCitation":"2","previouslyFormattedCitation":"²"},"properties":{"noteIndex":0},"schema":""}2. The Celniker TSS predictions were based on the analysis of experimental data from three methods: CAGEADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1093/nar/gkv054","abstract":"Cap analysis of gene expression (CAGE) is a highthroughput method for transcriptome analysis that provides a single base-pair resolution map of transcription start sites (TSS) and their relative usage. Despite their high resolution and functional significance, published CAGE data are still underused in promoter analysis due to the absence of tools that enable its efficient manipulation and integration with other genome data types. Here we present CAGEr, an R implementation of novel methods for the analysis of differential TSS usage and promoter dynamics, integrated with CAGE data processing and promoterome mining into a first comprehensive CAGE toolbox on a common analysis platform. Crucially, we provide collections of TSSs derived from most published CAGE datasets, as well as direct access to FANTOM5 resource of TSSs for numerous human and mouse cell/tissue types from within R, greatly increasing the accessibility of precise context-specific TSS data for integrative analyses. The CAGEr package is freely available from Bioconductor at packages/release/bioc/html/CAGEr.html.","author":[{"dropping-particle":"","family":"Haberle","given":"Vanja","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Forrest","given":"Alistair R R","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Hayashizaki","given":"Yoshihide","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Carninci","given":"Piero","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Lenhard","given":"Boris","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Nucleic Acids Research","id":"ITEM-1","issue":"8","issued":{"date-parts":[["2015"]]},"page":"e51","title":"CAGEr : precise TSS data retrieval and high-resolution promoterome mining for integrative analyses","type":"article-journal","volume":"43"},"uris":[""]}],"mendeley":{"formattedCitation":"³","plainTextFormattedCitation":"3","previouslyFormattedCitation":"³"},"properties":{"noteIndex":0},"schema":""}3, 5’ Rapid amplification of cDNA ends (5’ RACE), and 5’ Expressed Sequence Tags (ESTs).Exercise 1: Examining the CAGE Data for the Antennapedia Gene Using the UCSC Genome Browser Mirror Open a new web browser window and go to the Genomics Education Partnership (GEP) UCSC Genome Browser Mirror ( REF _Ref111904234 \h Figure 1).Figure 1 Access the GEP UCSC Genome Browser Gateway page using the “Genome Browser” link.To navigate to the genomic region surrounding the Antennapedia (Antp) gene in Drosophila melanogaster, select “D. melanogaster” and “Aug. 2014 (BDGP Release 6 + ISO1 MT/dm6)” under the “D. melanogaster Assembly” field, and then enter “Antp” under the “Position/Search Term” field. Click on the “GO” button ( REF _Ref111904247 \h Figure 2).Figure 2 Change the settings on the Genome Browser Gateway page to navigate to the Antp gene in the D. melanogaster release 6 assembly.Select "Antp-RM at chr3R:6896253-6999228" from the list of FlyBase Protein-Coding Genes to investigate the M isoform of the Antp gene.The Antp gene is on the minus strand. Click on the “reverse” button located in the display controls below the Genome Browser image. Click on the “hide all” button located below the Genome Browser image. Then, configure the display modes as follows:Under “Mapping and Sequencing Tracks”Base Position: “full”Click on the blue “Short Match” linkEnter “TCAKTY” (i.e., the Initiator motif) into the “Short (2-30 base) sequence” text boxChange the “Display mode” to “pack”Click on the “Submit” buttonUnder “Genes and Gene Prediction Tracks”FlyBase Genes: “pack”Under “Expression and Regulation”TSS (Celniker) (R5): “pack”Click on the blue “Combined modENCODE CAGE TSS” linkChange the “Maximum display mode” to “full”Scroll down to the “Select views” section, and change the “Peaks” display mode to “pack”In the “List subtracks” section, uncheck the box for “modENCODE CAGE (Plus)” Scroll up to the top of the page, and then click on the “Submit” button to update the display. Enter “chr3R:6,999,222-6,999,236” into the “chromosome range, or search terms, see examples” text box, and then click on the “go” button.We will examine the region near exon 1 of Antp-RM to determine where transcription begins ( REF _Ref111904263 \h Figure 3). This is a non-coding exon and does not contain a start codon for translation. Q1. Based on the “FlyBase Protein-Coding Genes” track, what is the coordinate of the transcription start site?Q2. Based on the Transcription Start Sites (Celniker Group) (R5) track, what is the coordinate of the transcription start site?Q3. Is the coordinate of the Transcription Start Sites (Celniker Group) (R5) prediction the same as the Combined modENCODE CAGE Read Density prediction? Remember that the Combined modENCODE CAGE Read Density track shows the density of the first base of the CAGE reads, which corresponds to the 5’ end of the mRNA.Figure 3 Genome Browser view of the TSS predicted by the Celniker Group (red arrow) and Combined modENCODE Read Density.Q4. Is there an Inr motif that agrees with the other TSS predictions?Q5. Zoom out 10x, and then zoom out another 3x. Compare the CAGE read density for the TSS that you just identified for Antp-RM, and the CAGE read densities for other positions in the region. How many positions within this region have CAGE read densities that are similar to the CAGE read density for the TSS of Antp-RM? Are there any Inr motifs within this 450bp region?RNA Annotation and Mapping of Promoters for the Analysis of Gene Expression (RAMPAGE)Additional evidence for the TSS location(s) can be gathered from RAMPAGE (RNA Annotation and Mapping of Promoters for the Analysis of Gene Expression) data. This technique allows for the identification of the putative TSS with base-pair resolution, and it generally has higher specificity (i.e., signal to noise ratio) than CAGEADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1101/gr.139618.112.23","abstract":"Many eukaryotic genes possess multiple alternative promoters with distinct expression specificities. Therefore, comprehensively annotating promoters and deciphering their individual regulatory dynamics is critical for gene expression profiling applications and for our understanding of regulatory complexity. We introduce RAMPAGE, a novel promoter activity profiling approach that combines extremely specific 59-complete cDNA sequencing with an integrated data analysis workflow, to address the limitations of current techniques. RAMPAGE features a streamlined protocol for fast and easy generation of highly multiplexed sequencing libraries, offers very high transcription start site specificity, generates accurate and reproducible promoter expression measurements, and yields extensive transcript connectivity information through paired-end cDNA sequencing. We used RAMPAGE in a genome-wide study of promoter activity throughout 36 stages of the life cycle of Drosophila melanogaster, and describe here a comprehensive data set that represents the first available developmental time-course of promoter usage. We found that >40% of developmentally expressed genes have at least two promoters and that alternative promoters generally implement distinct regulatory programs. Transposable elements, long proposed to play a central role in the evolution of their host genomes through their ability to regulate gene expression, contribute at least 1300 promoters shaping the developmental transcriptome of D. melanogaster. Hundreds of these promoters drive the expression of annotated genes, and transposons often impart their own expression specificity upon the genes they regulate. These observations provide support for the theory that transposons may drive regulatory innovation through the distribution of stereotyped cis-regulatory modules throughout their host genomes.","author":[{"dropping-particle":"","family":"Batut","given":"Philippe","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Dobin","given":"Alexander","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Plessy","given":"Charles","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Carninci","given":"Piero","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Gingeras","given":"Thomas R","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Genome Research","id":"ITEM-1","issued":{"date-parts":[["2013"]]},"page":"169-180","title":"High-fidelity promoter profiling reveals widespread alternative promoter usage and transposon-driven developmental gene expression","type":"article-journal","volume":"23"},"uris":[""]}],"mendeley":{"formattedCitation":"⁴","plainTextFormattedCitation":"4","previouslyFormattedCitation":"⁴"},"properties":{"noteIndex":0},"schema":""}4.Importantly, if there are multiple TSSs for a gene (characteristic of genes with an Intermediate or Broad promoter), RAMPAGE will quantify how frequently each TSS is used. Exercise 2: Examining the RAMPAGE data for the Antennapedia gene To examine the RAMPAGE evidence tracks, we will make the following changes to the display modes of the Genome Browser:Under “Mapping and Sequencing Tracks”, change the “Short Match” track to “hide”Under “Expression and Regulation”TSS (Celniker) (R5): hideCombined modENCODE CAGE TSS: hideClick on the blue “Combined RAMPAGE TSS (R5)” linkChange the “Maximum display mode” to “full”Scroll down to the “Select views” sectionChange the “Peaks” display mode to “pack”In the “List subtracks” section, uncheck the box for “RAMPAGE (Plus)”Scroll up to the top of the page, and then click on the “Submit” buttonClick on the blue “RAMPAGE TSS Read Density (R5)” linkChange the “Display mode” field to ““full”Scroll down to the “Select subtracks by strand and stage” sectionClick on the “-” button at the top left corner to unselect all the datasets ( REF _Ref111904281 \h Figure 4)Select the “Minus” strand checkboxes for the following developmental stages: Embryos 10hr, Embryos 24hr, L1, L2, Adult Females 5d, Adult Males 5dScroll up to the top of the page, and then click on the “Submit” button to update the Genome Browser display2540018542000right200660Figure 4 Configure the “RAMPAGE TSS Read Density (R5)” track to show only the data for six developmental stages on the minus strand. Click on the “-” button at the top left corner to unselect all the evidence tracks (red arrow). Select the checkboxes for the developmental stages of interests underneath the “Minus” column label. (The “…” denotes additional stages that were omitted from the screenshot.)00Figure 4 Configure the “RAMPAGE TSS Read Density (R5)” track to show only the data for six developmental stages on the minus strand. Click on the “-” button at the top left corner to unselect all the evidence tracks (red arrow). Select the checkboxes for the developmental stages of interests underneath the “Minus” column label. (The “…” denotes additional stages that were omitted from the screenshot.)Enter “chr3R:6,999,101-6,999,400” into the “chromosome range, or search terms” text box and then click on the “go” button.Q6. How many TSSs are supported by the RAMPAGE evidence track ( REF _Ref111904309 \h \* MERGEFORMAT Figure 5)?Figure 5 The RAMPAGE read density for all samples combined and for six developmental stages of Antp-RM.Q7. Do all of the developmental stages shown use the same TSS?To ascertain whether RAMPAGE identified additional TSS in the other developmental stages, we will reconfigure the “RAMPAGE TSS Read Density (R5)” track to show the minus strand RAMPAGE data for all of the available developmental stages.Under “Expression and Regulation”Click on the blue “RAMPAGE TSS Read Density (R5)” linkScroll down to the “Select subtracks by strand and stage” sectionClick on the “+” button underneath the “Minus” column label ( REF _Ref111904324 \h Figure 6)At the top of the page, click on the “Submit” button to update the display32988254445Figure 6 Click on the “+” button underneath the “Minus” column label in the “Select subtracks by strand and stage” section (red arrow) to show the minus strand RAMPAGE data for all of the available developmental stages.Figure 6 Click on the “+” button underneath the “Minus” column label in the “Select subtracks by strand and stage” section (red arrow) to show the minus strand RAMPAGE data for all of the available developmental stages.-25400000 Examination of the RAMPAGE data for all of the available developmental stages shows a more complex picture of transcription initiation for Antp-RM. Examine REF _Ref111904336 \h Figure 7 and your genome browser and note that some developmental stages have a RAMPAGE signal at a different genomic position or have an additional RAMPAGE signal besides the TSS annotated by FlyBase. For other isoforms of Antp (or other genes) there may be no RAMPAGE signal for some developmental stages.Figure 7 RAMPAGE read density for Antp-RM for different stages of development.Q8. Based on the RAMPAGE data, do you think that RNA polymerase II initiates transcription at a single site, or at multiple sites for Antp-RM (e.g., examine the RAMPAGE signal for L1 vs. L2, and 1 hr embryo vs. 2 hr embryo)?To compare the TSS identified by RAMPAGE with the mRNA expression levels:Under the “RNA Seq Tracks” section, click on the blue “modENCODE RNA-Seq (Development) (R5)” link Scroll down to the “Select subtracks by strand and stage” sectionClick on the “-” button at the top left corner to unselect all the datasetsClick on the “+” button underneath the “Minus” column labelScroll up to the top of the page, and then click on the “Submit” button to update the Genome Browser display Q9. For each developmental stage that shows RAMPAGE signal, is there a corresponding RNA-Seq signal?RNA Pol II ChIP-SeqChromatin immunoprecipitation followed by high throughput sequencing (ChIP-Seq) is a valuable method that can be used to identify DNA binding sites for proteins such as transcription factors. More recently, X-ChIP-Seq has been used to identify where RNA polymerase II (RNA Pol II) is enriched along the geneADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.7554/eLife.09225","author":[{"dropping-particle":"","family":"Skene","given":"Peter J","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Henikoff","given":"Steven","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Elife","id":"ITEM-1","issued":{"date-parts":[["2015"]]},"page":"e09225","title":"A simple method for generating high- resolution maps of genome-wide protein binding","type":"article-journal","volume":"4"},"uris":[""]}],"mendeley":{"formattedCitation":"⁵","plainTextFormattedCitation":"5","previouslyFormattedCitation":"⁵"},"properties":{"noteIndex":0},"schema":""}5. DNA is crosslinked to histone proteins, then nuclease is added to cleave the linker DNA between nucleosomes. An antibody against one of the proteins in the RNA Pol II complex is used to immunoprecipitate DNA bound by the polymerase. The DNA is then sequenced and mapped to the genome, as with other types of ChIP-Seq.The results show the genomic regions that are enriched in RNA Pol II, which usually corresponds to the approximate location of TSSs. The RNA Pol II X-ChIP-Seq data can also be used to identify genes whose expression is regulated by RNA Pol II pausing, where RNA Pol II is paused between 30–60nt downstream of the TSS after the initiation of transcription.Exercise 3: Examining the X-ChIP-Seq data for the Antennapedia gene using the UCSC Genome Browser Mirror To examine the X-ChIP-Seq data for the Antp-RM isoform more closely:Change the “chromosome range, or search terms” field to “chr3R:6,999,151-6,999,320” and then click “go”.Click on the “hide all” button to hide all the evidence tracksUnder “Mapping and Sequencing Tracks”Base Position: “full”Under “Genes and Gene Prediction Tracks”FlyBase Genes: “pack”Under “ChIP Seq Tracks”RNA PolII X-ChIP-Seq: “full”Under "Expression and Regulation"TSS (Celniker) (R5): “full”Under “RNA Seq Tracks”Click on the blue “Combined modENCODE RNA-Seq (Development) (R5)” linkChange the “Display mode” field to “full”Scroll down to the “List subtracks” sectionUncheck the box for the “modENCODE RNA-Seq (Plus)” trackScroll up to the top of the page, and then click on the “Submit” button to update the Genome Browser displayFigure 8 Enrichment of RNA Pol II near the 5’ end of Antp-RM, and the combined RNA-Seq data on the minus strand.Q10. According to the X-ChIP-Seq Log Likelihood Enrichment track ( REF _Ref111904363 \h \* MERGEFORMAT Figure 8), is the region immediately upstream of exon 1 enriched in RNA Pol II?Wrapping upMultiple evidence tracks can be used to identify the TSS for a given isoform of a gene. Examining CAGE, RAMPAGE, and/or X-ChIP-Seq data for genes in D. melanogaster may provide additional insight when annotating orthologs in other Drosophila species.ReferencesKodzius, R. et al. CAGE: cap analysis of gene expression. Nat. Methods 3, 211–222 (2006).Hoskins, R. A. et al. Genome-wide analysis of promoter architecture in Drosophila melanogaster. Genome Res. 21, 182–192 (2011).Haberle, V., Forrest, A. R. R., Hayashizaki, Y., Carninci, P. & Lenhard, B. CAGEr: precise TSS data retrieval and high-resolution promoterome mining for integrative analyses. Nucleic Acids Res. 43, e51 (2015).Batut, P., Dobin, A., Plessy, C., Carninci, P. & Gingeras, T. R. High-fidelity promoter profiling reveals widespread alternative promoter usage and transposon-driven developmental gene expression. Genome Res. 23, 169–180 (2013).Skene, P. J. & Henikoff, S. A simple method for generating high-resolution maps of genome-wide protein binding. eLife 4, e09225 (2015). ................
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