Genome Browsers

Introduction

Large scale genome sequencing and annotation has made a wealth of information available that is all related to the same biological objects: the DNA. The information however can be of very different types, it includes: * the actual sequence * sequence variants (SNPs and CNVs) * conservation between related species * genes (with introns and exons) * mRNAs * expression levels * regulatory features such as transcription factor bindings sites and much more.

Since all of this information relates to specific positions or ranges on the chromosome, displaying it alongside the chromosomal coordinates is a useful way to integrate and visualize it. We call such strips of annotation tracts and display them in genome browsers. Quite a number of such browsers exist and most work on the same principle: server hosted databases are queried through a Web interface; the resulting data is displayed graphically in a Web browser window. The large data centres each have their own browsers, but arguably the best engineered, most informative and most widely used one is provided by the University of California Santa Cruz (UCSC) Genome Browser Project.

Compiling the data requires a massive annotation effort, which has not been completed for all genome-sequenced species. In particular, not all of our MYSPEs have been included in the major model-organism annotation efforts. The general strategy for analysis of a gene in MYSPE is thus to map it to homologous genes in model organisms(W). In this assignment you will explore the UCSC genome browser and we will go through an exercise that relates fungal replication genes to human genes. We have previously focused a lot on Mbp1 homologs, but these have no clear equivalences in “higher” eukaryotes. However one of the key target genes of Mbp1 is the cell cycle protein Cdc6(W), which is well conserved in fungi and other eukaryotes eukaryotes and has a human homolog(W). Since generally speaking the annotation level for human genes is the highest, we will have a closer look at that gene.

The UCSC genome browser

The University of California Santa Cruz (UCSC) Genome Browser Project has the largest offering of annotation information. However it is strictly model-organism oriented and you will probably not find MYSPE among its curated genomes. Nevertheless, if you are studying eg. human genes, or yeast, the UCSC browser will probably be your first choice.

Task…

In this task you will access the UCSC genome browser view of the yeast Cdc6 gene and its human orthologue, the human Cdc6 gene. You will explore some of the very large number of tracks that are available and study the transcription factor binding region.

Navigate to the UCSC Genome Bioinformatics entry page and follow the link to the Genome Browser in the “Our tools” section.
To view genomes, you need to select a species. Note the scrollable species overview on the left hand side. The species are arranged by their position in the universal Tree of Life for eukaryotes.
- Find and click on “Saccharomyces cerevisiae”. This loads the genome data overview for the sequence. Enter Cdc6 as the Position search term. Click on Go.
Click on the link to the Cdc6 gene on chromosome X.
Click on the button to zoom out 3x - we want to see the upstream regulatory region.
In the subsection for Expression and Regulation, find the menu for Regulatory Code and select full; select hide for all other expression tracks. Click refresh.

This tracts show you the ChIP-chip validated TF-binding sites in the upstream regulatory region of yeast Cdc6. Note that there are several Mbp1 binding sites. Curiously, Swi6 is also listed there - but you know that Swi6 does not actually bind DNA directly, but forms a complex with either of the APSES domain transcription factors Mbp1 and Swi4 which form the MBF complex. However, crosslinking of the complex, and immunoprecipitation with anti-Swi6, would certainly identify this region. You should be aware that an annotation of a protein in a ChIP-chip experiment is not the same as demonstrating a protein’s physical interaction with DNA.

Zoom in, for better resolution, and shift-drag the view to keep the regulatory region of the Cdc6 gene centred. Then note that both stretches of DNA that have demostrated TF binding sites are also listed as conserved regions (dark red bars). As with all track elements, clicking on the bars will expand the display density, and clicking again will take you to an information page about this experimental tract, with further download options.
Zoom in until you can see the individual nucleotides for the Mbp1 binding sites. Then click on one of the Mbp1 bars to get information about the specific binding site. Note that the canoncial binding sequence corresponds to a regular expression of [AT]CGCG[AT] … a pattern with a probability of occurrence of about 1/1000 in random sequence. This is not very stringent - but here we have three such motifs within 200 bp - two of them adjacent.

Return to the genome browser entry page to access the genome browser for the human genome.
Click on the link to humans. Note that this is the hg38 assembly.
Enter CDC6 into the “Position/Search Term” field and click “Go”. You should get a list of entries, click on the top link, the Homo sapiens cell division cycle 6 (CDC6), mRNA gene on chromosome 17.`
Zoom out 1.5x to view the upstream regulatory region: the end of the adjacent WIPF2 gene should have just come into view on the left.
Study the Genome Browser view of the human CDC6 homolog.
- In particular, note the extensive functional annotations of DNA and the alignments of vertebrate syntenic regions that allow detailed genomic comparisons.
- Distinguish between exon and intron sequence.
- Note that the mammal Conservation track has high values for all of the exons, but not only for exons.
- Find more information on the “Layered H3K27Ac” tract.
Note the large number of available tracks that have been integrated into this view. Most of them are switched off. Find the Regulation section, and follow the link to the “ORegAnno” information to see what that is about. Note that you can switch individual annotations on or off on this page, as well as set the display format for all of the results. Select the check-box only for “transcription factor binding site” to be on, select the “Display mode” to full and click submit.
Study this information and note:
- There is a cluster of TFBS just upstream of the transcription initiation site.
- This cluster coincides with the highest H3K27Ac density.
- If you <control>-click (right-click?) on the top orange bar of this cluster, a contextual menu opens from which you can access the details page for OREG1791811 in a new window. Follow the link to the RBL2 transcription factor via ENST00000379935 … from where you can access transcript and gene and expression and protein family and GO and all other information.
Go back to the Genome Browser and set the ORegAnno tract to “pack” and click “refresh”.
Slide the SNP track to just beneath the RefSeq genes track that contains the introns and exons. You will notice that one of the SNPs is green, and two are red. Why? Set the “Common SNPs” track display mode to “pack” and click “refresh”.

Based on this kind of information, it should be straightforward to identify human transcription factors that potentially regulate human Cdc6 and determine - via sequence comparisons - whether any of them are homologous to any of the yeast transcription factors or factors in MYSPE. Through a detailed analysis of existing systems, their regulatory components and the conservation of regulation, one can in principle establish functional equivalences across large evolutionary distances.

Alternatives

Task…

Visit the following three alternatives to UCSC:

Genome browser provides a graphical interface for users to browse, search, retrieve and analyze genomic sequence and annotation data. Web-based genome browsers can be classified into general genome browsers with multiple species and species-specific genome browsers. In this review, we attempt to give an overview for the main functions and features of web-based genome browsers, covering data visualization, retrieval, analysis and customization. To give a brief introduction to the multiple-species genome browser, we describe the user interface and main functions of the Ensembl and UCSC genome browsers using the human alpha-globin gene cluster as an example. We further use the MSU and the Rice-Map genome browsers to show some special features of species-specific genome browser, taking a rice transcription factor gene OsSPL14 as an example.

Sloan, Cricket A et al.. (2016). “ENCODE data at the ENCODE portal”. Nucleic Acids Research 44(D1):D726–32 .
[PMID: 26527727] [DOI: 10.1093/nar/gkv1160]

Abstract …

The Encyclopedia of DNA Elements (ENCODE) Project is in its third phase of creating a comprehensive catalog of functional elements in the human genome. This phase of the project includes an expansion of assays that measure diverse RNA populations, identify proteins that interact with RNA and DNA, probe regions of DNA hypersensitivity, and measure levels of DNA methylation in a wide range of cell and tissue types to identify putative regulatory elements. To date, results for almost 5000 experiments have been released for use by the scientific community. These data are available for searching, visualization and download at the new ENCODE Portal (www.encodeproject.org). The revamped ENCODE Portal provides new ways to browse and search the ENCODE data based on the metadata that describe the assays as well as summaries of the assays that focus on data provenance. In addition, it is a flexible platform that allows integration of genomic data from multiple projects. The portal experience was designed to improve access to ENCODE data by relying on metadata that allow reusability and reproducibility of the experiments.

Pazin, Michael J. (2015). “Using the ENCODE Resource for Functional Annotation of Genetic Variants”. Cold Spring Harbor Protocols 2015(6):522–36 .
[PMID: 25762420] [DOI: 10.1101/pdb.top084988]

Abstract …

This article illustrates the use of the Encyclopedia of DNA Elements (ENCODE) resource to generate or refine hypotheses from genomic data on disease and other phenotypic traits. First, the goals and history of ENCODE and related epigenomics projects are reviewed. Second, the rationale for ENCODE and the major data types used by ENCODE are briefly described, as are some standard heuristics for their interpretation. Third, the use of the ENCODE resource is examined. Standard use cases for ENCODE, accessing the ENCODE resource, and accessing data from related projects are discussed. Although the focus of this article is the use of ENCODE data, some of the same approaches can be used with data from other projects.

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Genome Browsers

Boris Steipe

Contents

Introduction

The UCSC genome browser

Alternatives

Further Reading

Questions, comments

References