The information stored in the DNA of every living thing must be read and interpreted, and this is accomplished chiefly by proteins. One class of regulatory proteins control the transcription of DNA into messenger RNAs, which are then translated into structural proteins and enzymes. Defects in the ability to properly regulate transcription are at the foundation of many human diseases, with some, such as cancer and many aging-related maladies, very clearly rooted in genomic dysfunction. To take part in development and to respond to their environment, cells respond extremely rapidly to their surroundings, in part by enacting specific transcriptional responses. Therefore transcriptional regulation is by necessity a fundamentally dynamic process. However, almost everything we know about the mechanisms underlying transcriptional regulation are derived from static assays like footprinting or Chromatin Immunoprecipitation (ChIP). The major thrust of this grant is to combine elements from distinct disciplines to explore in vivo binding dynamics, a fundamental parameter that is lost completely in standard ChIP experiments.
We aim to (1) measure transcription factor binding dynamics for nearly every transcription factor in yeast, each at every position the genome simultaneously, (2) to create experimental systems in yeast amenable to both FRAP and sequential ChIP experiments, so that we and other expert laboratories can use their methods on the exact same system, and (3) to measure purified transcription factor targeting and dynamics on reconstituted chromatin templates. We can then use these systems to test specific hypotheses regarding competition between chromatin components and transcription factors, to test the biological function of turnover in regulating transcription, and to determine the cellular components required for proper regulation of turnover dynamics.

Public Health Relevance

Human health relevance Defects in transcriptional regulation are at the foundation of many human diseases, with some, such as cancer and many aging-related maladies, very clearly rooted in genomic dysfunction. We propose to combine elements from distinct disciplines to explore in vivo transcription factor- DNA binding dynamics, a fundamental parameter that is lost completely in standard ChIP-chip or ChIP-seq experiments. We will conduct experiments that test specific hypotheses regarding competition between chromatin components and transcription factors, and that determine the cellular components required for proper regulation of binding dynamics. Due to the powerful genetic tools available in yeast, these experiments can only be conducted in yeast at this time, but the results we obtain and perhaps just as importantly the approaches and technologies we develop will be fundamental in nature and applicable to more complex genomes, including humans.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
7R01GM072518-08
Application #
8514628
Study Section
Molecular Genetics B Study Section (MGB)
Program Officer
Carter, Anthony D
Project Start
2005-08-01
Project End
2015-07-31
Budget Start
2013-08-01
Budget End
2014-07-31
Support Year
8
Fiscal Year
2013
Total Cost
$236,487
Indirect Cost
$38,662
Name
Princeton University
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
002484665
City
Princeton
State
NJ
Country
United States
Zip Code
08544
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Lickwar, Colin R; Mueller, Florian; Lieb, Jason D (2013) Genome-wide measurement of protein-DNA binding dynamics using competition ChIP. Nat Protoc 8:1337-53
Lickwar, Colin R; Mueller, Florian; Hanlon, Sean E et al. (2012) Genome-wide protein-DNA binding dynamics suggest a molecular clutch for transcription factor function. Nature 484:251-5
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Berchowitz, Luke E; Hanlon, Sean E; Lieb, Jason D et al. (2009) A positive but complex association between meiotic double-strand break hotspots and open chromatin in Saccharomyces cerevisiae. Genome Res 19:2245-57
Lickwar, Colin R; Rao, Bhargavi; Shabalin, Andrey A et al. (2009) The Set2/Rpd3S pathway suppresses cryptic transcription without regard to gene length or transcription frequency. PLoS One 4:e4886
Giresi, Paul G; Lieb, Jason D (2009) Isolation of active regulatory elements from eukaryotic chromatin using FAIRE (Formaldehyde Assisted Isolation of Regulatory Elements). Methods 48:233-9

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