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. 2011 May;31(9):1894-904.
doi: 10.1128/MCB.05065-11. Epub 2011 Mar 7.

An NK and T cell enhancer lies 280 kilobase pairs 3' to the gata3 structural gene

Sakie Hosoya-Ohmura  1 , Yu-Hsuan Lin, Mary Herrmann, Takashi Kuroha, Arvind Rao, Takashi Moriguchi, Kim-Chew Lim, Tomonori Hosoya, James Douglas Engel
Affiliations

An NK and T cell enhancer lies 280 kilobase pairs 3' to the gata3 structural gene

Sakie Hosoya-Ohmura et al. Mol Cell Biol. 2011 May.

Abstract

Transcription factor GATA-3 is vital for multiple stages of T cell and natural killer (NK) cell development, and yet the factors that directly regulate Gata3 transcription during hematopoiesis are only marginally defined. Here, we show that neither of the Gata3 promoters, previously implicated in its tissue-specific regulation, is alone capable of directing Gata3 transcription in T lymphocytes. In contrast, by surveying large swaths of DNA surrounding the Gata3 locus, we located a cis element that can recapitulate aspects of the Gata3-dependent T cell regulatory program in vivo. This element, located 280 kbp 3' to the structural gene, directs both T cell- and NK cell-specific transcription in vivo but harbors no other tissue activity. This novel, distant element regulates multiple major developmental stages that require GATA-3 activity.

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Figures

Fig. 1.
Fig. 1.
Neither the Gata3-1b promoter alone nor a 662-kbp Gata3/LacZ YAC containing both 1a and 1b promoters recapitulates GATA-3 activity in thymocytes. (A) eGFP expression in CD4 SP thymocytes from Gata3g/+ mice (left, gray shaded histogram) or Tg1b.eGFP mice (right, gray shaded histogram). The black-line (open) histograms indicate eGFP fluorescence in wild-type thymocytes. Data represent at least three mice of each genotype. (B) LacZ expression in thymocytes stained with anti-CD4 and anti-CD8 antibodies was examined by flow cytometry. Each population was gated as depicted. The gray shaded histograms indicate fluorescein di-β-d-galactopyranoside fluorescence (13) resulting from hydrolysis due to β-galactosidase expression in either Gata3-lacZ knock-in (Gata3z/+) (17) or B125-lacZ YAC transgenic (27) mice, while the black-line (open) histograms indicate expression in wild-type mice. These individual data are representative of results from at least three mice of each genotype.
Fig. 2.
Fig. 2.
A candidate T lymphocyte enhancer element is located far 3′ to the Gata3 gene. (A) The Gata3 gene and adjacent genes on mouse chromosome 2. The relative genomic positions of the BAC and YAC clones examined in this study are depicted graphically. (B) Schematic diagram of the targeting cassette used to generate modified BACs. H1 and H2, homology arms; Neo, neomycin resistance gene; G3P, Gata3 promoter; SacBII, SacBII gene present in the vector backbone of the RPCI-23 mouse BAC library; CmR, chloramphenicol acetyltransferase gene C, founder screening of BAC-trap Tg embryos. eGFP expression in total thymocytes recovered from E18.5 F0 Tg embryos was analyzed by flow cytometry. The results of two independent F0 Tg embryos for each BAC clone are shown; in each case, a fraction of the thymocytes expressed eGFP, except from wild-type embryos.
Fig. 3.
Fig. 3.
Mapping conserved noncoding sequences (CNS) and DNase I hypersensitive sites (DHS) in the overlap between two BAC clones. (A) The region of overlap (approximately 25 kbp) between BACs 43G17 and 263A8 is depicted. (B) Genomic sequences within the overlap (mouse chromosome 2, 9,530,005 to 9,504,884) were compared with the human, dog, and rat genomes, respectively. CNSs are colored pink. (C) The DHS homologies corresponding to human CD4+ T cells are depicted as gray rectangles. Open arrows in panel B indicate CNSs that were predicted to be potential regulatory elements (i.e., high ESPERR scores).
Fig. 4.
Fig. 4.
A 7.1-kbp fragment (TCE-7.1) within the BAC overlap directs αβ T cell reporter gene transcription. (A) eGFP expression in CD69+ CD4 SP thymocytes from Tg43G17, Tg43G17Δ7.1, Tg7.1-1b.eGFP, Tg1b.eGFP, and wild-type mice. Data are representative of results from at least three individual mice of each genotype. (B and C) Normalized mean fluorescence intensity (MFI) of eGFP in each population of thymocytes (B) and splenocytes (C). MFI was normalized using the LinearFlow green flow cytometry intensity calibration kit and presented as percentage of relative fluorescence of calibration beads. Error bars (B and C) denote means ± standard deviations (SD). Two lines of each construct-derived Tg mouse were examined (data not shown), and at least three individual mice of each Tg line were analyzed. Data represent a single line from each construct-derived Tg mouse. Note that all MFI expression data are presented on a log scale. ETP, lineage-negative (Lin−) CD25low c-Kithigh; DN2, Lin− CD25high c-Kithigh; DN3, Lin− CD25high c-Kitlow; DN4, Lin− CD25low c-Kitlow; ISP, TCRbetalow CD8 SP; CD69− DP, TCRbetalow CD69− DP; CD69+ DP, TCRbeta+ CD69+ DP; CD4 SP CD69+, TCRbeta+ CD69+ CD4 SP; CD4 SP CD69−, TCRbeta+ CD69− CD4 SP; CD8 SP CD69+, TCRbeta+ CD69+ CD8 SP; CD8 SP CD69−, TCRbeta+ CD69− CD8 SP; Naïve CD4+, CD4+ CD62Lhigh CD44low; Activated/Memory CD4+, CD4+ CD62Llow CD44high; Naïve CD8+, CD8+ CD62Lhigh CD44low; Activated/Memory CD8+, CD8+ CD62Llow CD44high.
Fig. 5.
Fig. 5.
TCE-7.1 directs reporter gene transcription in stimulated CD4+ cells. (A) Histograms of eGFP in naïve CD4+ splenocytes and stimulated CD4+ splenocytes under various conditions. Data are representative of results from at least three individual mice of each genotype. (B) MFI of eGFP in naïve and stimulated CD4+ splenocytes under various conditions. Note that MFI was not normalized using calibration beads in this experiment. Error bars denote means ± SD. Two lines of each construct-derived Tg mouse were examined (data not shown), and at least three individual mice of each Tg line were analyzed. Data represent a single line from each construct-derived Tg mouse. Note that MFI expression data are presented on a log scale.
Fig. 6.
Fig. 6.
Among hematopoietic cells, TCE-7.1 confers only NK cell and αβ and γδ T cell enhancer activity. (A) Normalized MFI of eGFP in erythroid cells (TER119+), B cells (CD19+ B220+ CD3−), and myeloid cells (Gr1+ Mac1+) in the bone marrow, as well as γδ T cells (TCRγδ+) and NK cells (CD3− CD19− DX5+) in the thymus are shown. Error bars denote means ± SD. Note that MFI data are presented on a log scale. (B) eGFP expression in bone marrow hematopoietic progenitors (Lin− Sca1+ c-Kithi). The shaded histograms indicate each Tg mouse, while dashed lines indicate wild-type mice. For both panels A and B, two lines of each construct-derived Tg mouse were examined (data not shown), and at least three individual mice of each Tg line were analyzed. Data represent a single line from each construct-derived Tg mouse.
Fig. 7.
Fig. 7.
The TCE-7.1 enhancer is T cell specific. (A) eGFP expression in living P4 mice. Data are representative of multiple pups examined in two independent experiments. (B) eGFP expression in various organs from each genotype of adult mice. Three individual mice of each genotype were analyzed. B, brain; Li, liver; H, heart; K, kidney; Lu, lung; St, stomach; T, thymus; Sp, spleen. In both panels A and B, two lines of both Tg7.1-1b.eGFP and Tg1b.eGFP mouse were examined (data not shown).
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