Notch Signaling Requires Gfi1 for T Cell Development

Abstract 2174 Growth factor independent-1 (Gfi1) is a zinc finger transcriptional repressor protein originally identified in a rodent model of T-cell leukemia. Gfi1 deficient mice have defects in T cell development and a moderate loss of thymic cellularity. In Drosophila , orthologs of Notch1 and Gfi1 cooperate to specify embryo sensory organ precursors. Given the established requirement for Notch1 in T cell specification and development as well as the functional relationship of Notch and Gfi1 orthologs in Drosophila genetics, we investigated the ability of Gfi1 and Notch to cooperate in T-cell development. Utilizing transgenic mice in which the expression of Cre recombinase is controlled by the proximal Lck promoter ( LckCre) to both activate intracellular Notch1 (ICN) while simultaneously deleting Gfi1 , we demonstrate that T cells overexpressing ICN require Gfi1 for their survival and proper integration of ICN signaling. First, we validated our approach by showing that Lck-Cre-mediated deletion of Gfi1 alleles ( Gfi1 flox/ - ) or activation of ICN expression ( Rosa26 lox-stop-lox ICN ires eGFP, “Rosa ICN ” ) lead to expected phenotypes. We next examined the consequences of ICN activation with simultaneous deletion of Gfi1 . Whereas inducible deletion of Gfi1 alone decreases thymic cellularity by ∼4-fold, Gfi1 deletion coupled with ICN activation leads to complete thymic involution with a 14-fold reduction in total T cell numbers ( p Gfi1 results in an ∼9-fold reduction in thymocyte numbers, similar to proximal Lck-Cre. However, the requirement for Gfi1 in ICN-expressing cells is not global, in that distal Lck-Cre mediated deletion in post-negative selection thymocytes revealed normal cell numbers. Variation in Notch signaling defects may explain the profound differences in cellularity observed between deleting Gfi1 early verses late in T cell development. We limited one allele of Gfi1 and examined the transcriptional effect upon ICN target genes. First, FACS sorted DN3 thymocytes (CD4 − , CD8 − , CD44 − , CD25 + ) from proximal LckCre + Rosa ICN Gfi1 f/ + transgenic mice, showed that a full one-third of all ICN-activated genes are differentially regulated upon the loss of a single copy of Gfi1 . In contrast, splenic T cells from distal Lck-iCre + Rosa ICN Gfi1 f/ + , display an equivalent expression level of many Notch1 target genes as their Gfi1 +/+ littermate controls ( dLck-iCre + Rosa ICN Gfi1 +/ + ). Moreover, these Notch signaling defects do not appear to require supraphysiological levels of activated ICN as evidenced by dysregulated endogenous Notch1 target gene activation in Gfi1 −/− mice, including FACS sorted DN1 thymocytes and early bone marrow progenitors. Finally, this defect is cell autonomous in that Gfi1 −/− early thymic progenitors do not develop on OP9-DL1 stroma cells whereas their WT littermates develop into DN3 T cells within 6 days. Therefore, our data both confirms and extends a functional genetic relationship between Notch1 and Gfi1 from fruit fly to mammalian lymphocyte development. Furthermore, our data suggests that Gfi1 −/− developing thymocytes are incapable of correctly interpreting Notch signals, which ultimately leads to their death. Disclosures: No relevant conflicts of interest to declare.