TRF2: A new target for telomere dysregulation in human lymphoid cells
Date de publication2018
LMP1 is an oncogenic protein that is observed in tumor cells of Epstein-Barr virus (EBV)-associated Hodgkin’s lymphoma. LMP1 is responsible for interacting and/or interfering with multiple signaling pathways. At least some of those pathways will eventually affect transcriptional activity on many genes by activating and repressing their promoters. In EBV- negative as well as EBV-positive Hodgkin lymphoma cells, previous experiments uncovered chromosomal end-fusions, translocations, chromosomal duplications and telomere dysfunction. The Shelterin complex, consisting of the six subunits TRF1, TRF2, RAP1, TIN2, POT1 and TPP1, is associated with the formation of the T-loop structure at telomeres, thus forming an end cap for chromosomes. Our lab showed that expression of LMP1 induces a downregulation of the Shelterin components TRF1, TRF2 and POT1 at the transcriptional level. In the Shelterin complex, TRF2 was found to be the major protein affected by the induction of LMP1. It was shown that when a myc-driven TRF2 is reintegrated into a cell line expressing LMP1 oncogenic protein, that LMP1 had no longer effect on its regulation. My master’s project focused on understanding how LMP1 regulates the TRF2 at its promoter level. Our hypothesis is that LMP1 interferes with TRF2 at the promoter level since TRF2 RNA levels are reversible when LMP1 is suppressed. Several TRF2 promoter constructs fused with a GFP reporter gene were introduced into a LMP1-tet-OFF cell line and reporter gene expression level were monitored using Western blot. In that way, a LMP1-induced transcriptional shutdown could be documented. By appropriate mapping of the promoter region, a 30bp fragment of the TRF2 promoter was identified to play a pivotal role in this regulation. Using this 30bp as a dsDNA radio-labelled probe, EMSA was performed with nuclear extracts from cells that were induced or uninduced to express LMP1. The results reveal distinct and specific bands for LMP1-induced and LMP1-uninduced nuclear extracts in vitro. An RNA seq on the total cell extract was performed to obtain data of the genes that are downregulated and upregulated in the prescence of LMP1. This was done to determine the cellular dynamic composition in the absence and presence of LMP1-induction. These new data outline a specific region on the promoter of TRF2 for regulation that was analyzed by DNA affinity chromatography and mass spectrometry. The finding of regulator proteins of the TRF2 promoter will have great impact in understanding the regulatory mechanisms at play for TRF2 abundance in vivo.
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