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. 2012 May 1;188(9):4394-404.
doi: 10.4049/jimmunol.1103352. Epub 2012 Mar 30.

Differential transcription factor use by the KIR2DL4 promoter under constitutive and IL-2/15-treated conditions

Steven R Presnell  1 , Lei Zhang, Corrin N Chlebowy, Ahmad Al-Attar, Charles T Lutz
Affiliations

Differential transcription factor use by the KIR2DL4 promoter under constitutive and IL-2/15-treated conditions

Steven R Presnell et al. J Immunol. .

Abstract

KIR2DL4 is unique among human KIR genes in expression, cellular localization, structure, and function, yet the transcription factors required for its expression have not been identified. Using mutagenesis, EMSA, and cotransfection assays, we identified two redundant Runx binding sites in the 2DL4 promoter as essential for constitutive 2DL4 transcription, with contributions by a cyclic AMP response element (CRE) and initiator elements. IL-2- and IL-15-stimulated human NK cell lines increased 2DL4 promoter activity, which required functional Runx, CRE, and Ets sites. Chromatin immunoprecipitation experiments show that Runx3 and Ets1 bind the 2DL4 promoter in situ. 2DL4 promoter activity had similar transcription factor requirements in T cells. Runx, CRE, and Ets binding motifs are present in 2DL4 promoters from across primate species, but other postulated transcription factor binding sites are not preserved. Differences between 2DL4 and clonally restricted KIR promoters suggest a model that explains the unique 2DL4 expression pattern in human NK cells.

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Figures

Figure 1
Figure 1
3DL1 and 2DL4 promoters share overlapping but distinct sets of potential cis-acting elements. Shown are aligned 3DL1 (top) and 2DL4 (bottom) promoter sequences. Twenty-four contiguous 2DL4 10-bp segments (denoted S1–S24) were replaced with the linker sequence, GCAGATCCGC. Putative cis-acting elements are denoted by boxes. The sequences end at the ATG translational start site.
Figure 2
Figure 2
A. YT cells express high levels of 2DL4. YT cells were treated either with a PE-labeled mAb specific for 2DL4 (solid line) or with an isotype control (shaded) and surface staining was detected by flow cytometry. B. Linker-scanning mutagenesis identifies few 2DL4 promoter cis-acting sites. YT cells were transfected with the indicated 2DL4 substitution, numbered as in Fig. 1. WT, wild type. B, background level produced by the empty pGL3-basic vector. Values represent averages from tests of 3–8 different plasmid preparations (each measured in duplicate). Asterisks indicate significant differences from WT (p < 0.05).
Figure 3
Figure 3
An unmethylated CRE site contributes to 2DL4 promoter activity. A. 2DL4 promoter activity is reduced by mutations affecting the CRE site. Shown is 2DL4 promoter activity without (WT) and with CRE site point mutations (CRE1 and CRE2, see Table II). Promoter activity was measured as described in Fig. 2, and asterisks indicate significant differences from WT (p < 0.01). B. CpG methylation diminishes 2DL4 promoter DNA binding to CREB and ATF-1 in EMSA. YT nuclear extract was incubated with an unmethylated (Wt CRE, lanes 1–6) or a CpG methylated (Me CRE, lanes 7–12) probe encompassing the CRE site, either alone (lanes 1, 7) or with 150-fold excess of self-competitor (Wt, lanes 2, 8) or competitor with a mutated CRE site (M (CREm2, Table I), lanes 3, 9). Alternatively, nuclear extracts were pre-incubated with non-specific rabbit IgG (Ig, lanes 4, 10), or with Ab to CREB (Cb, lanes 5, 11) or to ATF-1 (A, lanes 6, 12) as indicated. The arrows indicate supershifted bands. The experiment shown is representative of three independent experiments with similar results.
Figure 4
Figure 4
2DL4 promoter activity depends on redundant activating sites. A. The activities of single site promoter mutations in Ets, distal Runx (dRx1, dRx2, dRx3), and proximal Runx (pRx) sites (described in Table II), and combinations of these mutations are shown, along with empty vector pGL3 Basic background activity (B). Promoter activity was measured as described in Fig. 2. Asterisks indicate significant differences from WT (*, p < 0.005; ** p < 0.0005). B. The 2DL4 Ets site is functional. HeLa cells were transfected with 1 ng of the control SV-40 Renilla luciferase construct, either 0, 200, or 400 ng Ets1 (solid square) or Ets2 (open square) expression plasmids and 1.5 ug 2DL4 promoter reporter that was either wild type (solid line) or mutated at the Ets site (dashed line). Empty pSG5 plasmid DNA was added as needed to equalize the total amount of transfected DNA. Each point represents the average of five tests; each test had a separate reporter plasmid preparation and one of three different Ets expression plasmid preparations. Shown is one representative experiment of three with similar results. Error bars (not always visible) show within-experiment SEM.
Figure 5
Figure 5
Runx transcription factor family members bind the 2DL4 promoter at two functional sites. A. Shown are sequences surrounding the 2DL4 distal (left) and proximal (right) Runx sites and the aligned 3DL1 promoter sequences. Boxes denote Runx motifs and the sequences shown denote EMSA probes. B. Runx2 and Runx3 bind to 2DL4 and 3DL1 distal Runx sites. YT nuclear extract was incubated with either 3DL1 (3DL1 A, lanes 1–7) or 2DL4 (2DL4 A, lanes 8–14) probes encompassing the distal Runx site either alone (lanes 1, 8) or in the presence of 150-fold excess of self-competitor (Wt, lanes 2, 9) or competitor with a mutated Runx site (M, lanes 3 and 10 (mut 3DL1 A, mut 2DL4 A, Table I)). Alternatively, probe was added to nuclear extracts that had been pre-incubated with non-specific rabbit IgG (Ig, lanes 4, 11), or with Ab to Runx1 (1, lanes 5, 12), Runx2 (2, lanes 6, 13) or Runx3 (3, lanes 7, 14) as indicated. Arrows indicate supershifted bands. C. Runx2 and Runx3 bind to the 2DL4 proximal Runx site but not to the 3DL1 aligned region. YT nuclear extract was incubated with either a 3DL1 (3DL1 B, lanes 1–6) or 2DL4 (2DL4 B, lanes 7–13) probe encompassing the proximal Runx site (2DL4) or the aligned 3DL1 sequence alone (lanes 1, 7) or in the presence of 150-fold excess of self-competitor (Wt, lanes 2, 8) or a competitor with a mutated proximal Runx site (M (mut 2DL4 B, Table I) lane 9). Alternatively, nuclear extracts were pre-incubated with IgG (Ig, lanes 3, 10), or with Ab to Runx1 (1, lanes 4, 11), Runx2 (2, lanes 5, 12) or Runx3 (3, lanes 6, 13) as indicated. For B. and C., the experiments shown are representative of three independent experiments with similar results. D. Both proximal and distal Runx sites are functional. HeLa cells were transfected with 1 ng of a control SV-40 Renilla luciferase construct, 500 ng CBFβ expression plasmid, 500 ng Runx1, Runx2, or Runx3 expression plasmids as indicated, and 1.5 ug 2DL4 promoter reporter that was either wild type (WT), mutated at the proximal Runx site (pRx), at the distal Runx site (dRx1), or at both Runx sites (pRx + dRx1, mutations described in Table II). Each group represents the average of five tests; each test had a separate reporter plasmid preparation and one of three different Runx1, Runx2 or Runx3 expression plasmid preparations. Error bars denote within-experiment SEM. Shown is one representative experiment of two with similar results.
Figure 6
Figure 6
Distinct 2DL4 promoter transcription factor requirements in constitutive and cytokine-treated cells. A. IL-2 treatment activates Segment 5 and 6 sites. YT cells transfected with 2DL4 segment substitution mutant reporter plasmids were either IL-2 treated or not treated (Nil), as described in Methods. Promoter activity was measured as described in Fig. 2. Asterisks indicate significant differences between the indicated groups (*, p < 0.0005). WT and B are described in Fig. B. IL-2 and IL-15 specifically activate the compound Ets/Runx site. YT cells transfected with 2DL4 promoter reporter plasmids with mutations in either the proximal Runx (pRx), Ets (Ets), proximal Runx and Ets site (pRx + Ets), or in the distal Runx site (dRx1, dRx2, dRx3, mutations described in Table II) were treated with IL-2, IL-15, or not treated (Nil) as described in Methods. Promoter activity was measured as described in Fig. 2. Asterisks indicate significant differences between the indicated groups (*, p < 0.05; ** p < 0.0005). WT and B are described in Fig. 2.
Figure 7
Figure 7
CRE, Ets, and Runx sites are required for full 2DL4 promoter activity in NK and T cells. YT (black), LNK (dark gray), NKL (light gray), and Hut-78 (white) cell lines were transfected with 2DL4 reporter plasmids with mutations to either the CRE (CRE2), Ets (Ets), proximal Runx (pRx), distal Runx (dRx1), or combined distal and proximal Runx mutations (dRx1 + pRx, mutations described in Table II), and were cultured with IL-2 (with the exception of Hut-78) as described in Methods. Promoter activity was measured as described in Fig. 2. With the exception of CRE2 and dRx1 for NKL, all mutations had significant declines in activity compared to wild type (p < 0.05). WT and B are described in Fig. 2.
Figure 8
Figure 8
Runx3 and Ets1 specifically bind to the endogenous 2DL4 promoter. Cross-linked chromatin was purified from IL-2-cultured NK92.26.5 (A, B) or freshly-isolated primary NK (C) cells and immunoprecipitated with Ab to Runx3, Ets1, or with negative control Ab. DNA from immunoprecipitates was purified and qPCR amplified using primers specific either to the 2DL4 promoter (P) or 2DL4 intron 4 (I), as indicated. A. ChIP was performed with anti-Runx3 mAb 5G4 (Rx-A) and 6E9 (Rx-B), and negative control mAb (anti-FLAG). Values represent the average enrichment of DNA bound to specific Ab (Runx/FLAG) from tests of five different chromatin preparations. B. ChIP was performed with anti-Ets1 Ab and negative control Ab, either rabbit anti-FLAG or normal rabbit IgG. Values represent the average enrichment of DNA bound to Ab (Ets/FLAG or Ets/IgG) from tests of at least three different chromatin preparations. C. ChIP was performed with anti-Runx3 mAb 5G4 and a negative control mAb (anti-FLAG). Values represent the enrichment of DNA bound to specific Ab (Runx 5G4/FLAG) using chromatin preparations from three different human donors. Averages and p values are shown.
Figure 9
Figure 9
CRE, Ets, and distal Runx promoter sites are present in 2DL4 primate orthologs. Chimpanzee (C), orangutan (O), and rhesus monkey (R) promoter sequences were aligned to the human (H) sequence (−225 to +3) using CLUSTALW2. Putative cis-acting elements are denoted by boxes. Chimpanzee, orangutan, and rhesus monkey sequences had 98%, 95%, and 69% identity, respectively, to the human sequence.
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References

    1. Lanier LL. NK cell recognition. Annu Rev Immunol. 2005;23:225–274. - PubMed
    1. Long EO. Negative signaling by inhibitory receptors: the NK cell paradigm. Immunol Rev. 2008;224:70–84. - PMC - PubMed
    1. Rajalingam R, Parham P, Abi-Rached L. Domain shuffling has been the main mechanism forming new hominoid killer cell Ig-like receptors. J Immunol. 2004;172:356–369. - PubMed
    1. Santourlidis S, Graffmann N, Christ J, Uhrberg M. Lineage-specific transition of histone signatures in the killer cell Ig-like receptor locus from hematopoietic progenitor to NK cells. J Immunol. 2008;180:418–425. - PubMed
    1. Santourlidis S, Trompeter HI, Weinhold S, Eisermann B, Meyer KL, Wernet P, Uhrberg M. Crucial role of DNA methylation in determination of clonally distributed killer cell Ig-like receptor expression patterns in NK cells. J Immunol. 2002;169:4253–4261. - PubMed

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