The y-axis indicates normalized interaction frequencies the x-axis shows the genomic position relative to the LCR. Interaction frequencies were measured by semiquantitative PCR in K562 (locus ON) and GM06990 (locus OFF) cells. ( B) 3C analysis of interactions between the LCR (HS5) and the rest of the β-globin locus. The location of a gene of unknown function (EST BU661736) is indicated by a green oval. Olfactory genes and pseudogenes are represented by black and yellow rectangles, respectively. The positions of HSs are indicated with black arrows. The β-globin genes and pseudogene are illustrated by red and yellow arrows, respectively. ( A) Schematic representation of the human β-globin locus. 5C should be widely applicable for large-scale mapping of cis- and trans- interaction networks of genomic elements and for the study of higher-order chromosome structure.Īnalysis of the human β-globin locus and development of 5C. Interestingly, this region has been implicated in the control of developmental globin gene switching. We also identified a new looping interaction in K562 cells between the beta-globin Locus Control Region and the gamma-beta-globin intergenic region. We validated 5C by detection of several previously identified looping interactions in the beta-globin locus. We applied 5C to analyze a 400-kb region containing the human beta-globin locus and a 100-kb conserved gene desert region. Here we present a high-throughput 3C approach, 3C-Carbon Copy (5C), that employs microarrays or quantitative DNA sequencing using 454-technology as detection methods. 3C converts physical chromatin interactions into specific ligation products, which are quantified individually by PCR. Physical interactions between genetic elements located throughout the genome play important roles in gene regulation and can be identified with the Chromosome Conformation Capture (3C) methodology.
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