CSB Assignment Week 4

Assignments for Week 4

Note! This assignment is currently inactive. Major and minor unannounced changes may be made at any time.

 
 

Exercises for this week relate to this week's lecture.
Pre-reading for this week will prepare next week's lecture.
Exercises and pre-reading will be topics on next week's quiz.

Exercises

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Task:

• Work through the DAVID tutorial published in nature protocols:

Huang et al. (2009) Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc 4:44-57. (pmid: 19131956)

[ PubMed ] [ DOI ] DAVID bioinformatics resources consists of an integrated biological knowledgebase and analytic tools aimed at systematically extracting biological meaning from large gene/protein lists. This protocol explains how to use DAVID, a high-throughput and integrated data-mining environment, to analyze gene lists derived from high-throughput genomic experiments. The procedure first requires uploading a gene list containing any number of common gene identifiers followed by analysis using one or more text and pathway-mining tools such as gene functional classification, functional annotation chart or clustering and functional annotation table. By following this protocol, investigators are able to gain an in-depth understanding of the biological themes in lists of genes that are enriched in genome-scale studies.

• Access the Web version of the article, it conveniently contains the required links.

• Use Demo List 2, provided on the DAVID site for your analysis. Remember to read the description of the gene list.

• Do not use any of the Java tools. As of this writing Java applets in Web browsers are considered fundamentally insecure; Java should be disabled in your browser.

• For each of the analysis steps, think clearly about whether the results support od contradict your expectations about the data. Feel free to discuss your expectations and findings on the mailing list.

• If there are any problems with the assignment, contact me!

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January 30 2014 saw the publication of what may be the most important scientific advance published in our lifetimes. In two nature papers, Haruko Obokata described the creation of so-called STAP (stimulus-triggered acquisition of pluripotency) cells. These cells can be generated by simple stress-protocols applied to leukocytes, but also to brain-, skin-, muscle-, fat-, bone marrow, lung- and liver-derived cells. Successful protocols include bathing the cells in moderately acidic medium for half an hour, mechanically perturbing the cells, or inducing plasma cell membrane pores with streptolysin. Post stress, cells shrink, stop proliferating, downregulate their differentiation gene markers and begin expressing markers of pluripotent stem cells that include our friends OCT4, Nanog, and Sox-2, and others. Cell clusters that form after this apparent transformation can be propagated. Strikingly, not only are these clusters able to grow into entire embryos after blastocyst injection, and further into normal, adult mice, but these mice are fertile and demonstrate germline transmission of their genetic markers into their offspring.

Task:
1. Read ...

Obokata et al. (2014) Stimulus-triggered fate conversion of somatic cells into pluripotency. Nature 505:641-7. (pmid: 24476887)

[ PubMed ] [ DOI ] Here we report a unique cellular reprogramming phenomenon, called stimulus-triggered acquisition of pluripotency (STAP), which requires neither nuclear transfer nor the introduction of transcription factors. In STAP, strong external stimuli such as a transient low-pH stressor reprogrammed mammalian somatic cells, resulting in the generation of pluripotent cells. Through real-time imaging of STAP cells derived from purified lymphocytes, as well as gene rearrangement analysis, we found that committed somatic cells give rise to STAP cells by reprogramming rather than selection. STAP cells showed a substantial decrease in DNA methylation in the regulatory regions of pluripotency marker genes. Blastocyst injection showed that STAP cells efficiently contribute to chimaeric embryos and to offspring via germline transmission. We also demonstrate the derivation of robustly expandable pluripotent cell lines from STAP cells. Thus, our findings indicate that epigenetic fate determination of mammalian cells can be markedly converted in a context-dependent manner by strong environmental cues.

Among the obvious questions one has to ask are: can these mouse-findings be transferred to humans, what are the upstream signals that trigger the pluripotency response, and can this network be controlled? Obokata et al. describe the transformation as the release of an epigenetic differentiation state and this may point into the direction of genes that could possibly be involved.

How would you find them?

2. Think about what datasets you might need to pursue these questions and what comparisons you might want to undertake.

3. Navigate to GEO and search whether such datasets are available. Note down any that you find and bring your list to class.

Pre-reading

The Encode project is a current paradigm for the integration of multiple -omics datasets for comprehensive annotation of the human genome.

ENCODE Project Consortium (2011) A user's guide to the encyclopedia of DNA elements (ENCODE). PLoS Biol 9:e1001046. (pmid: 21526222)

[ PubMed ] [ DOI ] The mission of the Encyclopedia of DNA Elements (ENCODE) Project is to enable the scientific and medical communities to interpret the human genome sequence and apply it to understand human biology and improve health. The ENCODE Consortium is integrating multiple technologies and approaches in a collective effort to discover and define the functional elements encoded in the human genome, including genes, transcripts, and transcriptional regulatory regions, together with their attendant chromatin states and DNA methylation patterns. In the process, standards to ensure high-quality data have been implemented, and novel algorithms have been developed to facilitate analysis. Data and derived results are made available through a freely accessible database. Here we provide an overview of the project and the resources it is generating and illustrate the application of ENCODE data to interpret the human genome.

State-of-the-art proteome analysis. In your reading, make sure you understand the experimental principles applied, but focus on the computational techniques.

Beck et al. (2011) The quantitative proteome of a human cell line. Mol Syst Biol 7:549. (pmid: 22068332)

[ PubMed ] [ DOI ] The generation of mathematical models of biological processes, the simulation of these processes under different conditions, and the comparison and integration of multiple data sets are explicit goals of systems biology that require the knowledge of the absolute quantity of the system's components. To date, systematic estimates of cellular protein concentrations have been exceptionally scarce. Here, we provide a quantitative description of the proteome of a commonly used human cell line in two functional states, interphase and mitosis. We show that these human cultured cells express at least -10 000 proteins and that the quantified proteins span a concentration range of seven orders of magnitude up to 20 000 000 copies per cell. We discuss how protein abundance is linked to function and evolution.