Abstract
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 (Gfi1flox/-) or activation of ICN expression (Rosa26lox-stop-loxICN ires eGFP, “RosaICN”) 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<0.0001). To determine whether developmental context controlled this interaction, we used a series of temporally regulated T cell promoters to drive Cre expression. In addition to targeting thymocytes before TCRβ-selection with Lck-Cre, we also examined CD4-Cre (deleting after TCRβ-selection), as well as the distal Lck promoter-Cre (deleting after negative selection). Notably, CD4-Cre mediated activation of ICN and deletion of 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+RosaICNGfi1f/+ 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+RosaICNGfi1f/+, display an equivalent expression level of many Notch1 target genes as their Gfi1+/+ littermate controls (dLck-iCre+RosaICNGfi1+/+). 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.
Regulation of positive and negative selection and TCR signaling during thymic T cell development by capicua
