טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentMararo David
SubjectThe Role of Protein-Protein and DNA-Protein Interactions
in Modulating Biological Activities of IRFs
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Professor Ben-Zion Levi


Abstract

IFN regulatory factors (IRFs) are a growing family of transcription factors that are involved in regulation of immune responses, cytokine signaling and oncogenesis. The IRF members are characterized by their well-conserved DNA Binding Domain (DBD) at the N-terminal region, which explains their ability to bind a similar DNA motif, IFN- Stimulated Regulatory Element (ISRE).

IFN consensus sequence binding protein (ICSBP) and IRF-4, are highly homologous IRF members that are expressed exclusively in hematopoietic cells. Both factors bind target DNA with greater efficiency following interaction with other transcription factors. These interactions are mediated by a conserved domain located at their carboxyl terminus region and termed IRF Association Domain (IAD).

In this research we have further characterized the interactions between  ICSBP, IRF-4, IRF-1 and IRF-2. We show that the DNA binding domain of IRF-2 and ICSBP is essential for the formation of DNA binding heterocomplexes and transcriptional activity. In addition, The DBD and the IAD of ICSBP are independent modules that are essential for the formation of DNA binding heterocomplexes. The IAD also mediates the interaction and transcriptional synergy of ICSBP with non-IRF members such as PU.1 and E47. Moreover, the same conserved IAD in IRF-4 is also essential for its transcriptional synergy with these two factors. Finally, we used the chimeric protein GAL4-ICSBP to develop a dominant negative approach that will allow us to negate the activities of ICSBP-interacting partners. In order to gain a more comprehensive insight to the mode of interaction among IRFs, the association domain of IRF-2, which is different from IAD, was mapped. The IAD of IRF-2 (IAD2), was located between residues 210-265, and found also to be conserved in IRF-1. Computer analysis indicates that IAD2 is a PEST domain, with similar characteristics as the PEST domain of PU.1 that enables PU.1 association with IRF-4 and ICSBP. Our results identified the molecular mechanisms of protein-protein and DNA-protein interactions among some of IRF members. Understanding the ternary structure of IRFs complex assembly should provide insight into the biological activities of combinatorial interactions of IRFs in mediating gene expression.