“Biochemical isolation and functional characteriza.. (InterPro)
“Biochemical isolation and functional characterization of the protein complexes that regulate interchromosomal interactions”
Start date: Jun 1, 2009,
End date: May 31, 2013
A Locus Control Region can act in cis, regulating the expression of cytokine genes on the same chromosome but can also act in trans to regulate the expression of several cytokine genes located on different chromosomes. We have recently shown that the physical interchromosomal association of loci located on three different mouse chromosomes precedes differentiation of CD4+ T cells and acts as a checkpoint for T cell fate determination. The functional significance of these interchromosomal associations is under investigation and preliminary results point towards a repressive role in non-differentiated naïve CD4+ cells and an activatory role in differentiated T cells. As a next step, to further understand how these interactions are established, I propose to use genetic and biochemical approaches to isolate, purify and characterize the protein complexes that generate and maintain such interchromosomal interactions. The model I currently envision integrates cell and tissue specific transcription factors that recruit more general protein complexes with the potency to relocalize whole loci or chromosomes. Biocomputing analysis will be performed to identify the conserved elements on the interacting loci, located on three different mouse chromosomes, which will subsequently be used as baits to perform yeast one hybrid screens, as well as for direct isolation of proteins using biotinylated DNA probes. The protein complexes will then be tested for their ability to remodel or relocalize chromatin using a combination of techniques such as RNA and DNA fluorescence in situ hybridization (FISH) and chromosome conformation capture assays (3C) in wild type T cells as well as in cells with the identified respective genes knocked down, applying RNA-interference or knockout methodologies. The proposed project will provide substantial novel information on how the genome is shaped and how the nuclear structure affects global gene expression.
Get Access to the 1st Network for European Cooperation