Líffræðifélag Íslands
Líffræðiráðstefnan 2015
Erindi/veggspjald / Talk/poster V79
Kristján Hólm Grétarsson (1), Dilixiati Remina (1), Eiríkur Steingrímsson (1), Erna Magnúsdóttir (1)
University of Iceland, Department of Biochemistry and Molecular Biology
Kynnir / Presenter: Kristján Hólm Grétarsson
Tengiliður / Corresponding author: Erna Magnúsdóttir (erna@hi.is)
Gene expression is heavily dependent on the 3D conformation of the genome as genetic elements loop out to interact with each other. As chromatin organization is highly dynamic, varying both during the cell cycle and between different cell types it is interesting to look at differences in chromatin interactions to study the underlying control mechanism of individual genes. The goal of this project is to look at the long range interactions of transcriptional elements responsible for the transcription of IRF4 in myeloma and melanoma cells with the use of the chromosome conformation capture (3C) technique. IRF4 expression leads to B-cell heavy chain class switch recombination (CSR) and the generation of plasma cells from germinal center and memory B cells. Additionally, IRF4 holds an important role in melanoma and pigmentation. Chromosome conformation capture: The 3D organization of the genome is fixed in point with a fixate agent. The fixed chromatin is digested with a restriction enzyme and the sticky ends of the cross-linked fragments are ligated under conditions to promote intramolecular ligations. These ligated fragments, which reflect interaction between two genomic loci are then studied with quantitative methods (such as qPCR) to measure the number of ligation events, using primers located near the ligation junctions. Applying the 3C method on the human Waldenström macroglobulinemia cell line, RPCI-WM1 a 10kbp region upstream of the IRF4 promoter that forms chromatin loop to the IRF4 promoter was identified. This interaction was not seen in other cell lines (HEK293T and 501mel) indicating a role in the regulation of IRF4 expression. Further, chromatin immunoprecipitation of the IRF4 protein in RPCI-WM1 showed strong binding of IRF4 in a more defined region within the looping region, previously predicted to bind IRF4 and a luciferase reporter assay indicate a degree of enhancer activity of this very same region.