Critical requirement of GABPalpha for normal T cell development

Abstract

GA binding protein (GABP) consists of GABPalpha and GABPbeta subunits. GABPalpha is a member of Ets family transcription factors and binds DNA via its conserved Ets domain, whereas GABPbeta does not bind DNA but possesses transactivation activity. In T cells, GABP has been demonstrated to regulate the gene expression of interleukin-7 receptor alpha chain (IL-7Ralpha) and postulated to be critical in T cell development. To directly investigate its function in early thymocyte development, we used GABPalpha conditional knock-out mice where the exons encoding the Ets DNA-binding domain are flanked with LoxP sites. Ablation of GABPalpha with the Lck-Cre transgene greatly diminished thymic cellularity, blocked thymocyte development at the double negative 3 (DN3) stage, and resulted in reduced expression of T cell receptor (TCR) beta chain in DN4 thymocytes. By chromatin immunoprecipitation, we demonstrated in DN thymocytes that GABPalpha is associated with transcription initiation sites of genes encoding key molecules in TCR rearrangements. Among these GABP-associated genes, knockdown of GABPalpha expression by RNA interference diminished expression of DNA ligase IV, Artemis, and Ku80 components in DNA-dependent protein kinase complex. Interestingly, forced expression of prearranged TCR but not IL-7Ralpha can alleviate the DN3 block in GABPalpha-targeted mice. Our observations collectively indicate that in addition to regulating IL-7Ralpha expression, GABP is critically required for TCR rearrangements and hence normal T cell development.

FIGURE 1.

Ablation of GABPα blocks T cell development at DN stage. A, GABPα targeting strategy. In the Gabpa gene, exons 8 and 9, which encode most of the Est DNA-binding domain, were flanked with 2 LoxP sites (open triangles). The relative locations of genotyping primers are indicated. With these primer combinations, WT, floxed, and deleted Gabpa alleles were detected at 153, 417, and 600 bp, respectively. Filled triangles, Frt sites. A paired of Frt sites were used to flank a neomycin-resistant gene, which was excised by a Flippase transgene to minimize the disturbance of natural gene structure by gene targeting. After the Flippase excision, only one Frt site was left in the targeted intron. B, representative flow cytometric profile of thymocytes. LckCre-GABPα^FL/+ and LckCre-GABPα^FL/− mice were analyzed at an age of 6–8 weeks. The thymocytes from mice of indicated genotypes were stained with anti-CD4 and anti-CD8 antibodies. The percentages of each population are shown. C, total thymic cellularity. D, DN thymocyte numbers. The data are pooled from more than five independent experiments. **, p < 0.01.

FIGURE 2.

Early T cell development in LckCre-GABPα^FL/− mice. A–C, ablation of GABPα blocks T cell development at DN3 stage. A, representative flow cytometric profile of DN thymocytes. DN thymocytes from mice of indicated genotypes were further fractionated based on CD25 and CD44 expression, and percentages of DN1-DN4 subsets in the DN population were shown. B, frequency of DN3 and DN4 thymocytes in DN population. C, absolute numbers of DN3 and DN4 thymocytes. **, p < 0.01. D, excision of the floxed GABPα allele in LckCre-GABPα^FL/+ and LckCre-GABPα^FL/− mice. DN3 and DN4 thymocytes were isolated by cell sorting, and genomic DNA extracted from these cells was analyzed for WT, floxed, and deleted GABPα allele by PCR using primers shown in Fig. 1A. The size of expected PCR products is marked in parentheses. The data are representative from three independent sorting and PCR assessments. E, the expression of IL-7Rα and TCRβ in DN3 and DN4 thymocytes. Cell surface expression of IL-7Rα and intracellular expression of TCRβ were measured in indicated mice, and the percentages of positive population are marked. For IL-7Rα staining, isotype controls are shown as shaded histograms. The data are representative of at least three independent experiments with more than 10 animals of each genotype examined.

FIGURE 3.

GABP directly regulates key genes involved in V(D)J recombination. A, GABP binding at selected gene loci inferred from ChIP-Seq. Sequence tags enriched by GABPα antibody in ChIP-Seq of Jurkat T cells were mapped to human genome on the UCSC genome browser. The raw data are displayed as accumulative tag numbers within a 200-bp-wide window as shown in the upper part of each panel, and the tag numbers are marked on the left. The ChIP-Seq data with GABPα and control IgG samples were further analyzed using the SISSR algorithm to calculate the relative enrichment in individual locations. The data are displayed as enrichment peaks in the lower part of each panel, with their heights (y axis) corresponding to fold enrichment of GABP sequence tags over control tags, as marked on the right of each panel. The gene symbols are as marked, and the gene structure and direction of transcription are shown under each panel, with vertical lines/boxes denoting exons. B, summary of GABPα binding at the TISs of genes involved in V(D)J recombination, including gene symbols, commonly used gene names, and fold enrichment of GABP binding at each gene locus based on the Jurkat ChIP-Seq data. The results from C and D were also summarized in the last two columns. C, validation of GABP binding in DN thymocytes. Chromatin fragments were prepared from murine DN thymocytes and immunoprecipitated with either anti-GABPα antibody or a control IgG. Gene regulatory regions overlapping with TISs of indicated genes were detected by quantitative PCR using primers designed based on the Jurkat ChIP-Seq results. The relative enrichment by the GABPα antibody versus control IgG was shown for each gene. D, effect of knocking down GABPα on the expression of GABP-bound genes. EL-4 cells were transfected with siGABPα or control pBS/U6 vector alone with pEYFP-N1, and EYFP-positive cells were sorted, RNA extracted, and reverse-transcribed. Expression of genes of interest was measured by quantitative PCR. The data are the means ± S.D. of four samples from two independent experiments. *, p < 0.05; **, p < 0.01 by t test.

FIGURE 4.

Forced expression of IL-7Rα or OT-I TCR transgene did not improve thymic cellularity in LckCre-GABPα^FL/− mice. A, total thymic cellularity in the presence of IL-7Rα or OT-I transgene. LckCre-GABPα^FL/+ and LckCre-GABPα^FL/− mice were crossed to IL-7Rα or OT-I TCR transgenic mice to acquire the transgenes. The data are pooled results from two independent experiments with three to eight animals analyzed. ns, not significant. B, representative flow cytometric profiles of thymocytes with CD4 and CD8 staining, and the percentages of each population are shown.

FIGURE 5.

Effect of IL-7Rα or OT-I TCR transgene on early thymocyte development. A, representative flow cytometric profiles of DN thymocytes based on CD25 and CD44 fractionation in mice of indicated genotypes. The percentages of each subset are shown. B, detection of cell surface IL-7Rα and intracellular TCRβ expression in the presence or absence of transgenes. Percentages of the positive populations are indicated. The data are representative from two independent experiments with three to eight animals analyzed.

FIGURE 6.

Frequency and numbers of DN3 and DN4 thymocytes in the presence of IL-7Rα or OT-I TCR transgene. DN3 frequency (A) and numbers (B) and DN4 frequency (C) and numbers (D) are shown. ns, not significant; **, p < 0.01. The data are the pooled results for two independent experiments with three to eight animals analyzed.