RFLAT2 co-activates RANTES transcription: Building the RANTES promoter enhanceosome

The chemokine RANTES was initially identified as part of a general screen for molecules expressed “late” after activation in T lymphocytes. RANTES is rapidly upregulated in a variety of cell types though uniquely expressed late, 3–5 days, after activation in T cells. This pattern of expression is significant with respect to function, as RANTES regulates leukocyte transendothelial migration, generates a cellular infiltrate at a site of inflammation, and sustains a prolonged state of inflammation as it is further expressed by the activated infiltrating lymphocytes. RANTES is involved in autoimmune diseases, AIDS, and transplant rejection. Therefore, RANTES and the regulation of its gene expression are of great interest as targets for the development of potential therapeutics.; Previous studies defined the RANTES promoter as having five distinct regions, A, B, E, C, and D, regulated by rel proteins, as well as three novel factors. It was expected that these novel factors likely accounted for the late pattern of RANTES expression in activated T lymphocytes.; This thesis dissertation describes the work of characterizing the second novel factor, named RANTES Factor of Late Activated T cells (RFLAT)-2. RFLAT2 was isolated as partial 1.7 kB cDNA through expression cloning by virtue of its interaction with “E” region of the RANTES promoter and is referred to as the “E clone” in early chapters.; Subsequent chapters describe the identification of the gene intron/exon structure, determination of the 7.5kB full length gene and the observation that an RNA message that is present throughout the time course of activation in peripheral blood lymphocytes (PBL).; Later chapters describe reagents generated for the determination that RFLAT2 protein expression is delayed 3–5 days after activation in PBL, that protein expression correlates to the occupation of the E site of the RANTES promoter, and that the “E clone” was indeed determined to be RFLAT2 acting as a co-activator of RANTES transcription.; Given these findings in addition to the finding that RFLAT2 shares homology with CBP/p300 co-activator proteins, I propose that RFLAT2 is a central mediator of the RANTES promoter enhanceosome regulating the unique late-expression of RANTES in T lymphocytes.