Identification of the interacting protein partners of the ThPOK transcription factor

Official URL: http://risc01.sabanciuniv.edu/record=b1301341 (Table of Contents)

T lymphocytes are an essential part of the adaptive immune system. In the course of T lymphocyte development, one crucial step is the decision to commit to one of two lineages: CD4 and CD8 single positive (SP) T cells. The cellular and molecular mechanisms underlying T cell lineage commitment has long been the subject of intense debate. ThPOK, T-helper-inducing POZ/Krüppel like factor, is a zinc finger transcription factor containing both a Krüppel-like zinc finger domain and a BTP/POZ domain, which has been linked to homodimerization and recruitment of other proteins. During the development of thymocytes, the ThPOK protein mediates the differentiation of MHC-II restricted thymocytes into the CD4 SP lineage, using its N-terminal BTB/POZ domain. We screened a human thymic cDNA library against the BTB/POZ domain of the ThPOK protein to identify its interacting protein partners by using a conventional yeast two hybrid system. We identified putative interacting proteins by performing DNA sequencing and bioinformatics analysis on relevant prey protein encoding plasmids. We re-confirmed these interactions in yeast cells by secondary yeast two hybrid screens. We confirmed the biological relevance of the identified interactions by performing co-immunoprecipitation experiments in transfected mammalian tissue culture cells. We demonstrated that seven proteins, named POMP, MEF2B, TCF7, ZNF384, DPP7, HINT2 and PARP12 can interact with the BTB/POZ domain of the ThPOK protein.