Lucia I. Ramirez Week 10: Difference between revisions

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Click [[BIOL398-04/S15:Week_10| here]] for assignment instructions.
Click [[BIOL398-04/S15:Week_10| here]] for assignment instructions.
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Lee, T. I., Rinaldi, N. J., Robert, F., Odom, D. T., Bar-Joseph, Z., Gerber, G. K., Hannett, N. M., Harbison, C. T., Thompson, C. M., Simon, I., Zeitlinger, J., Jennings, E. G., Murray, H.L ., Gordon, D. B., Ren, B., Wyrick, J. J., Tagne, J. B., Volkert, T. L., Fraenkel, E., Gifford, D. K. & Young, R. A. (2002). Transcriptional regulatory networks in ''Saccharomyces cerevisiae''. ''Science'', 298(5594), 799-804. DOI: 10.1126/science.1075090
[http://www.sciencemag.org/content/298/5594/799.full.pdf Link to PDF version of article]
==Biological Terms== <!-- provide 10 terms-->
#'''myc epitope tag''':provide a method to localize gene products in a variety of cell types, study the topology of proteins and protein complexes, identify associated proteins, and characterize newly identified, low abundance or poorly immunogenic proteins when protein specific antibodies are not available. Antibodies against c-myc epitopes recognize overexpressed proteins containing Myc epitope tag fused to either amino- or carboxy-termini of targeted proteins (http://www.pierce-antibodies.com/Myc-Epitope-Tag-antibody-clone-MycA7-Monoclonal--MA121316.html)
#'''physiologic''': of, or pertaining to physiology or normal functioning or state of an organism (http://www.biology-online.org/dictionary/Physiologic)
#'''immunoblot analysis''': is a rapid and sensitive assay for the detection and characterization of proteins that works by exploiting the specificity inherent in antigen-antibody recognition. It involves the solubilization and electrophoretic separation of proteins, glycoproteins, or lipopolysaccharides by gel electrophoresis, followed by quantitative transfer and irreversible binding to nitrocellulose, PVDF, or nylon (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2583035/)
#'''immunoprecipitated''': is the small-scale affinity purification of antigens using a specific antibody and is one of the most widely used methods for antigen purification and detection (https://www.lifetechnologies.com/us/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/immunoprecipitation-ip.html)
#'''stringency''':  reaction conditions, notably temperature, salt, and pH that dictate the annealing of single-stranded DNA/DNA, DNA/ rNA, and RNA/RNA hybrids. at high stringency, duplexes form only between strands with perfect one-to-one complementarity, lower stringency allows annealing between strands with some degree of mismatch between bases (http://www.biology-online.org/dictionary/Stringency)
#'''biosynthetic''': The production of a complex chemical compound from simpler precursors in a living organism, usually involving enzymes (to catalyze the reaction) and energy source (such as ATP) (http://www.biology-online.org/dictionary/Biosynthesis)
#'''pheromone''': chemical substances which, when secreted by an individual into the environment, cause specific reactions in other individuals, usually of the same species. The substances relate only to multicellular organisms (http://www.biology-online.org/dictionary/Pheromones)
#'''transient''': short-lived; passing; not permanent; said of a disease or an attack (http://www.biology-online.org/dictionary/Transient)
#'''FHL1''': Four and a half LIM domains protein 1 (http://amigo.geneontology.org/amigo/gene_product/UniProtKB:Q5JXI8)
#'''assaying''': analysis (as of an ore or drug) to determine the presence, absence, or quantity of one or more components (http://www.merriam-webster.com/dictionary/assay)
==Outline==
===Introduction===
*Cells, the product of specific gene expression programs involving regulated transcription of thousands of genes, move through the cell cycle, where transcriptional programs are modified
*Gene expression programs are dependent on the recognition of specific promoter sequences by transcriptional regulatory proteins
*Regulatory proteins recruit and regulate chromatin-modifying complexes and components of the transcriptional apparatus
'''Main result presented in this paper'''
*Knowing the sites bound by the transcriptional regulators encoded in a genome gives the information necessary to create models for transcriptional regulatory networks
*Capable of identifying set of target genes bound in vivo by all of the transcriptional regulators using the complete genome sequence and the development of a method for genome-wide binding analysis
===Methods===
Figure 1
*Genome-wide location analysis was used to see how yeast transcriptional regulators bind to promoter sequences throughout the genome
*Observed all 141 transcription factors listed in the Yeast Proteome Database that were reported to have DNA binding and transcriptional activity
*Myc epitope tagging (at COOH terminus of each regulator) was used to identify transcription factors in each yeast strain, might have affected the function of some transcriptional regulators
'''Analysis'''
*Immunoblot analysis showed 106 of the 124 tagged regulator proteins could be detected when yeast cells were grown in rich medium (yeast extract, peptone, and dextrose)
*Performed genome-wide location analysis experiment for the 106 yeast strains that expressed epitope-tagged regulators
*Genome-wide location data were subjected to quality control filters and normalized.
**Confidence value (p-value = 0.001) calculated for each spot from each array using error model
**Weighted average method used for combination of three samples of each data
**Average take after each ratio was weighted by p-value
**Calculate Final p-values for combined ratios
**Total number of protein-DNA interactions in the location analysis data was found
*Approximately 4000 interactions were observed between regulators and promoter regions
*The promoter regions of 2342 of 6270 yeast genes (37%) were bound by one or more of the 106 transcriptional regulators
*yeast genes are also frequently regulated through combinations of regulators


{{Template: Lucia I. Ramirez}}
{{Template: Lucia I. Ramirez}}
[[Category: BIOL398-04/S15]]
[[Category: BIOL398-04/S15]]

Revision as of 20:52, 23 March 2015

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Lee, T. I., Rinaldi, N. J., Robert, F., Odom, D. T., Bar-Joseph, Z., Gerber, G. K., Hannett, N. M., Harbison, C. T., Thompson, C. M., Simon, I., Zeitlinger, J., Jennings, E. G., Murray, H.L ., Gordon, D. B., Ren, B., Wyrick, J. J., Tagne, J. B., Volkert, T. L., Fraenkel, E., Gifford, D. K. & Young, R. A. (2002). Transcriptional regulatory networks in Saccharomyces cerevisiae. Science, 298(5594), 799-804. DOI: 10.1126/science.1075090

Link to PDF version of article

Biological Terms

  1. myc epitope tag:provide a method to localize gene products in a variety of cell types, study the topology of proteins and protein complexes, identify associated proteins, and characterize newly identified, low abundance or poorly immunogenic proteins when protein specific antibodies are not available. Antibodies against c-myc epitopes recognize overexpressed proteins containing Myc epitope tag fused to either amino- or carboxy-termini of targeted proteins (http://www.pierce-antibodies.com/Myc-Epitope-Tag-antibody-clone-MycA7-Monoclonal--MA121316.html)
  2. physiologic: of, or pertaining to physiology or normal functioning or state of an organism (http://www.biology-online.org/dictionary/Physiologic)
  3. immunoblot analysis: is a rapid and sensitive assay for the detection and characterization of proteins that works by exploiting the specificity inherent in antigen-antibody recognition. It involves the solubilization and electrophoretic separation of proteins, glycoproteins, or lipopolysaccharides by gel electrophoresis, followed by quantitative transfer and irreversible binding to nitrocellulose, PVDF, or nylon (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2583035/)
  4. immunoprecipitated: is the small-scale affinity purification of antigens using a specific antibody and is one of the most widely used methods for antigen purification and detection (https://www.lifetechnologies.com/us/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/immunoprecipitation-ip.html)
  5. stringency: reaction conditions, notably temperature, salt, and pH that dictate the annealing of single-stranded DNA/DNA, DNA/ rNA, and RNA/RNA hybrids. at high stringency, duplexes form only between strands with perfect one-to-one complementarity, lower stringency allows annealing between strands with some degree of mismatch between bases (http://www.biology-online.org/dictionary/Stringency)
  6. biosynthetic: The production of a complex chemical compound from simpler precursors in a living organism, usually involving enzymes (to catalyze the reaction) and energy source (such as ATP) (http://www.biology-online.org/dictionary/Biosynthesis)
  7. pheromone: chemical substances which, when secreted by an individual into the environment, cause specific reactions in other individuals, usually of the same species. The substances relate only to multicellular organisms (http://www.biology-online.org/dictionary/Pheromones)
  8. transient: short-lived; passing; not permanent; said of a disease or an attack (http://www.biology-online.org/dictionary/Transient)
  9. FHL1: Four and a half LIM domains protein 1 (http://amigo.geneontology.org/amigo/gene_product/UniProtKB:Q5JXI8)
  10. assaying: analysis (as of an ore or drug) to determine the presence, absence, or quantity of one or more components (http://www.merriam-webster.com/dictionary/assay)

Outline

Introduction

  • Cells, the product of specific gene expression programs involving regulated transcription of thousands of genes, move through the cell cycle, where transcriptional programs are modified
  • Gene expression programs are dependent on the recognition of specific promoter sequences by transcriptional regulatory proteins
  • Regulatory proteins recruit and regulate chromatin-modifying complexes and components of the transcriptional apparatus

Main result presented in this paper

  • Knowing the sites bound by the transcriptional regulators encoded in a genome gives the information necessary to create models for transcriptional regulatory networks
  • Capable of identifying set of target genes bound in vivo by all of the transcriptional regulators using the complete genome sequence and the development of a method for genome-wide binding analysis

Methods

Figure 1

  • Genome-wide location analysis was used to see how yeast transcriptional regulators bind to promoter sequences throughout the genome
  • Observed all 141 transcription factors listed in the Yeast Proteome Database that were reported to have DNA binding and transcriptional activity
  • Myc epitope tagging (at COOH terminus of each regulator) was used to identify transcription factors in each yeast strain, might have affected the function of some transcriptional regulators

Analysis

  • Immunoblot analysis showed 106 of the 124 tagged regulator proteins could be detected when yeast cells were grown in rich medium (yeast extract, peptone, and dextrose)
  • Performed genome-wide location analysis experiment for the 106 yeast strains that expressed epitope-tagged regulators
  • Genome-wide location data were subjected to quality control filters and normalized.
    • Confidence value (p-value = 0.001) calculated for each spot from each array using error model
    • Weighted average method used for combination of three samples of each data
    • Average take after each ratio was weighted by p-value
    • Calculate Final p-values for combined ratios
    • Total number of protein-DNA interactions in the location analysis data was found
  • Approximately 4000 interactions were observed between regulators and promoter regions
  • The promoter regions of 2342 of 6270 yeast genes (37%) were bound by one or more of the 106 transcriptional regulators
  • yeast genes are also frequently regulated through combinations of regulators



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