Enter the text here that is the new abstract information for your application. The organization of the nucleus and the regulated folding of the genome plays essential roles in regulating gene expression, chromosome segregation and chromosome structure. Long range interactions in chromatin are required for activation of gene transcription and repression of genes during the differentiation of eukaryotic cells. Long-range contacts between different chromosomal loci also regulate processes such as antibody diversity and mitotic chromosome condensation. Despite the wide range of processes that involve chromatin loops we know very little about the mechanisms that drive chromatin folding and stabilize long range interactions in the genome. This proposal is focused on developing new methods to measure the formation of looped domains dependent upon the activity of the chromatin protein CTCF. CTCF is known to be required for the stabilization of looped regions in the genome but how it generates or stabilizes looped domains is unknown. We propose to first characterize the dynamics of CTCF dependent looping using defined chromatin substrates in vitro and on chromatinized plasmids in cell extracts. Using mutagenesis and depletion we will alter the binding affinity and dimerization properties of CTCF and its interaction with the loop stabilizing protein cohesin to determine how these activities regulate the frequency of loop generation. Using the insight we gain from these in vitro experiments we will compare the dynamics of loop formation to the statistics of long range interactions at the human globin locus. By depleting CTCF and cohesin we will relate the cellular statistics of loop formation to the in vitro mechanics of loop stabilization. Our studies should provide unique and novel insight into the processes that regulate the formation of long range chromatin interactions and how they relate to essential developmental and cell biological processes.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM106005-02
Application #
8708160
Study Section
Special Emphasis Panel (ZGM1-CBB-0 (MI))
Program Officer
Janes, Daniel E
Project Start
2013-08-01
Project End
2017-05-31
Budget Start
2014-06-01
Budget End
2015-05-31
Support Year
2
Fiscal Year
2014
Total Cost
$272,037
Indirect Cost
$97,037
Name
Stanford University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94305