Pok Proteins in Ontogenesis, Lymphopoiesis, and Lymphomagenesis

Pandolfi, Pier

Abstract

POK (POZ and Kruppel) proteins are characterized by the presence of a C2H2 Zinc finger motif, typified by the Drosophila gap gene Kruppel, and the POZ (Poxvirus and Zinc finger) domain, initially identified in a Zinc finger protein named ZID where it was shown to facilitate protein- protein interactions. Two members of the POK family of transcription factors, BCL-6 (for B Cell Lymphoma 6) and PLZF, have already been found implicated in the pathogenesis of tumors affecting the lympho- hemopoietic compartment. The BCL6 gene was identified by virtue of its involvement in translocations associated with Non-Hodgkin's lymphoma. We have recently isolated a new member of the POK family, which we have named LRF1 (for Lymphoma Related Factor 1), that is coexpressed and physically interacts with BCL6. The focus of this proposal is to elucidate the role of the BCL6 and LRF1 POK proteins in development and lymphopoiesis, and to clarify how BCL6 and LRF1 functions relate to lymphomagenesis with the following Specific Aims: 1. To define, in knock out mice, the role of BCL6 and LRF1 in ontogenesis. Using homologous recombination technology, we have successfully disrupted the BCL6 gene in mouse Embryonic Stem (ES) cells, and mice homozygous for the mutation have been generated and are being characterized. Using a similar approach, we will disrupt the LRF1 gene. Mice or embryos homozygous for the LRF1 inactivating mutation will be generated and studied. We will define the developmental role of these genes by characterizing the embryonic or adult phenotype resulting from their inactivation. 2. To elucidate, in knock out mice and null ES cell lines, the role of BCL6 and LRF1 in lymphopoiesis. We will specifically analyze lymphopoiesis and B cell function in BCL6 and LRF1 mutants. Different experimental approaches will be undertaken depending on whether mice lacking the LRF1 gene are viable or on the stage of embryonic development at which they die. In parallel, we have produced BCL6 null ES cells and we will produce LRF1 null ES cells and study the capacity of these cells to differentiate towards lympho-hemopoietic precursors in vitro and in vivo in RAG-/- complementation assays. Finally, we will interbreed BCL-/- and LRF1-/- mice in order to generate and characterize the hemopoietic phenotype of the double mutants.