IDENTIFICATION OF NOVEL PROTEINS IN TRF1 COMPLEX

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Telomeres form a protein-DNA complex that protects chromosome ends from degradation or end-to-end fusion. One of the major telomeric DNA binding proteins in mammals is TRF1, which binds specifically to duplex TTAGGG repeats. TRF1 is thought to interact with tankyrase and TIN2 to regulate telomere length maintenance. Tankyrase is a telomeric poly(ADP-ribose)polymerase (PARP) that binds to the acidic N-terminus of TRF1, and TIN2 is a TRF1-interacting nuclear protein. Using co-immunoprecipitation coupled with mass spectrometry, we attempted to identify novel proteins in the TRF1 complex. A cell line expressing FLAG-HA-tagged TRF1 as well as a cell line containing the empty vector as a control was created in human BJ fibroblast cells that also express hTERT. Using these cells, small-scale FLAG-HA IPs were done to verify that FLAG-HA-TRF1 is interacting with its known binding partners, tankyrase and TIN2, using Western blot analysis and immunofluorescent co-localization experimen ts. The IP was scaled up, and the FLAG-HA-peptide elutions from both cell populations (FLAG-HA-tagged TRF1 and vector control) were run out on a polyacrylamide gel and stained by negative zinc staining. Unique bands seen in the FLAG-HA-TRF1 elution lane were excised from the gel and sent for mass spectrometry analysis. The results of this experiment suggested that TRF1 might be interacting with proteins such as hnRNP-U, hnRNP-D, and nucleolin. Further experiments are being done to investigate the validity of these interactions.