Abstract The transcriptional repressor Gfi1 is a nuclear zinc‐finger protein that is expressed in T cell precursors in the thymus, but is down‐regulated in mature, resting T cells. Gfi1 expression rises transiently to levels seen in thymocytes upon antigenic activation. We show here that lack of Gfi1 causes delayed cell cycle entry and apoptosis after antigenic stimulation in both mature CD4 + and CD8 + T cells ex vivo . DNA micro‐array analysis demonstrated that this correlated with an up‐regulation of the death receptor CD95, the proapoptotic factors Bad and Apaf1 and the cell cycle inhibitor p21, and a down‐regulation of Bcl‐2 expression in Gfi1 –/– T cells. Surprisingly, while Gfi1‐deficient CD4 + T cells showed the same defective behavior in vivo , Gfi1‐deficient CD8 + T cells showed no aberration in vivo and were fully able to mount an anti‐viral immune response. This indicates that Gfi1 exerts different functions in CD4 + and CD8 + T cells very likely by maintaining different genetic programs in both cell types, and appears to be essential for the CD4 helper T cell immune response but dispensable for the function of cytotoxic CD8 + T cells.
In the thymus, several steps of proliferative expansion and selection coordinate the maturation of precursors into antigen-specific T cells. Here we identify the transcriptional repressor Gfi1 as an important regulator of this maturation process. Mice lacking Gfi1 show reduced thymic cellularity due to an increased cell death rate, lack of proliferation, and a differentiation block in the very early uncommitted CD4−/CD8−/c-Kit+ cytokine-dependent T cell progenitors that have not yet initiated VDJ recombination. In addition, Gfi1-deficient mice show increased major histocompatibility complex class I–restricted positive selection and develop significantly more CD8+ cells suggesting a requirement of Gfi1 for a correct CD4/CD8 lineage decision. Absence of Gfi1 correlates with high level expression of the genes for lung Krüppel-like factor (LKLF), inhibitor of DNA binding (Id)1 and Id2, suggesting the existence of new regulatory pathways in pre-T cell development and thymic selection in which Gfi1 acts upstream of LKLF as well as the E-proteins, which are negatively regulated by Id1 and Id2.
ABSTRACT Like many other bacteria, Corynebacterium glutamicum possesses two types of l -malate dehydrogenase, a membrane-associated malate:quinone oxidoreductase (MQO; EC 1.1.99.16 ) and a cytoplasmic malate dehydrogenase (MDH; EC 1.1.1.37 ) The regulation of MDH and of the three membrane-associated dehydrogenases MQO, succinate dehydrogenase (SDH), and NADH dehydrogenase was investigated. MQO, MDH, and SDH activities are regulated coordinately in response to the carbon and energy source for growth. Compared to growth on glucose, these activities are increased during growth on lactate, pyruvate, or acetate, substrates which require high citric acid cycle activity to sustain growth. The simultaneous presence of high activities of both malate dehydrogenases is puzzling. MQO is the most important malate dehydrogenase in the physiology of C. glutamicum . A mutant with a site-directed deletion in the mqo gene does not grow on minimal medium. Growth can be partially restored in this mutant by addition of the vitamin nicotinamide. In contrast, a double mutant lacking MQO and MDH does not grow even in the presence of nicotinamide. Apparently, MDH is able to take over the function of MQO in an mqo mutant, but this requires the presence of nicotinamide in the growth medium. It is shown that addition of nicotinamide leads to a higher intracellular pyridine nucleotide concentration, which probably enables MDH to catalyze malate oxidation. Purified MDH from C. glutamicum catalyzes oxaloacetate reduction much more readily than malate oxidation at physiological pH. In a reconstituted system with isolated membranes and purified MDH, MQO and MDH catalyze the cyclic conversion of malate and oxaloacetate, leading to a net oxidation of NADH. Evidence is presented that this cyclic reaction also takes place in vivo. As yet, no phenotype of an mdh deletion alone was observed, which leaves a physiological function for MDH in C. glutamicum obscure.
Pou4f3 (Brn3.1, Brn3c) is a class IV POU domain transcription factor that has a central function in the development of all hair cells in the human and mouse inner ear sensory epithelia. A mutation of POU4F3 underlies human autosomal dominant non-syndromic progressive hearing loss DFNA15. Through a comparison of inner ear gene expression profiles of E16.5 wild-type and Pou4f3 mutant deaf mice using a high density oligonucleotide microarray, we identified the gene encoding growth factor independence 1 ( Gfi1 ) as a likely in vivo target gene regulated by Pou4f3. To validate this result, we performed semi-quantitative RT–PCR and in situ hybridizations for Gfi1 on wild-type and Pou4f3 mutant mice. Our results demonstrate that a deficiency of Pou4f3 leads to a statistically significant reduction in Gfi1 expression levels and that the dynamics of Gfi1 mRNA abundance closely follow the pattern of expression for Pou4f3. To examine the role of Gfi1 in the pathogenesis of Pou4f3-related deafness, we performed comparative analyses of the embryonic inner ears of Pou4f3 and Gfi1 mouse mutants using immunohistochemistry and scanning electron microscopy. The loss of Gfi1 results in outer hair cell degeneration, which appears comparable to that observed in Pou4f3 mutants. These results identify Gfi1 as the first downstream target of a hair cell specific transcription factor and suggest that outer hair cell degeneration in Pou4f3 mutants is largely or entirely a result of the loss of expression of Gfi1 .
