טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentSharon Ehelich
SubjectCharacterization of the Interaction between ICSBP and Miz-1
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Full Professor Levi Ben-Zion
Full Thesis text - in Hebrew Full thesis text - Hebrew Version


Abstract

ICSBP (Interferon Consensus Sequence Binding Protein), also known as IRF-8 (Interferon Regulatory Factor-8) is a member of the IRF (Interferon Regulatory Factor) family of transcription factors. Its expression is restricted mainly to cells of the immune system where ICSBP is constitutively expressed in B cells, monocyte cells and at low levels in resting T cells. Its expression can be further induced by  IFN-g (Interferon-g) by combined exposure of macrophages to IFN-g, LPS (lipopolysaccharide), and by antigenic stimulation of T cells. ICSBP has a pivotal role in the differentiation of myeloid progenitor cells toward the macrophage lineage and production of IL-12 (Interleukin-12) by activated macrophages in response to pathogen exposure. Since ICSBP lacks the ability to bind by it self to interferon stimulating response elements (ISRE) in promoters of genes regulated by IFN signaling, its diverse activity in cells of the immune system  is mediated by complexing  with other transcription factors from the IRF family (like IRF1/2), and foreign transcription factors like PU.1 and E47.

Using yeast two-hybrid screen we identified a truncated version of a gene coding for Myc interacting zinc finger protein (Miz-1). While human full Miz-1 sequence encompasses 803 amino acids, the truncated clone identified in the screen was missing its first 357 amino acids. Miz-1 was originally identified in Y2H screens thanks to its association with c-Myc, a well known oncoprotein with pivotal role in cellular proliferation, growth and apoptosis. Miz-1 binds to Inr (Initiator) sites of Adml (Adenovirus major late) and cycline D1 promoters and activates transcription from both promoters. Miz-1 induces genes like p15INK4b and p21Cip1, mediators of cell-cycle arrest at G1, through direct binding to Inr within their promoters. By using coimmunoprecipitation assays in COS7 cells and mammalian two-hybrid assays in NIH3T3 and U937 cells, we were able to confirm the interaction between ICSBP and MIz-1, in-vivo, and to show that this interaction is hematopoietic specific.  Furthermore, we discovered that Miz-1 acts as a strong activator in reporter gene assay using a synthetic promoter.

 Since no biological target promoter harboring both ICSBP and Miz-1 binding sites was reported at the time the experiments took place, we used a synthetic promoter consisted of firefly luciferase, as a reporter gene, driven by  5 repeats of  GAL4 DBD  (DNA binding domain). Using this synthetic promoter in transient transfection in NIH3T3 cells along with GAL4-ICSBP and GAL4-Miz-1 we were able to show that ICSBP exerts synergetic repression on Miz-1.