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Human natural killer cell microRNA: differential expression of MIR181A1B1 and MIR181A2B2 genes encoding identical mature microRNAs.

Presnell SR, Al-Attar A, Cichocki F, Miller JS, Lutz CT - Genes Immun. (2014)

Bottom Line: We found that few microRNAs (miRNAs) differed significantly between human NK and T cells.TGF-β treatment increased NK-cell MIR181A2B2 transcription, whereas IL-2, IL-15 and IL-12/IL-18 treatments upregulated MIR181A1B1.The MIR181A2B2 promoter was strongly transactivated by SMAD3 and SMAD4 transcription factors, suggesting that TGF-β signaling upregulates MIR181A2B2 expression, at least in part, through SMAD-dependent promoter activation.

View Article: PubMed Central - PubMed

Affiliation: Departments of Pathology and Laboratory Medicine, University of Kentucky, Lexington, KY, USA.

ABSTRACT
Natural killer (NK) and T lymphocytes share many properties, yet only NK cells respond rapidly to infection and cancer without pre-activation. We found that few microRNAs (miRNAs) differed significantly between human NK and T cells. Among those miRNAs, miR-181a and miR-181b levels rose during NK cell differentiation. Prior studies indicate that miR-181a and miR-181b are critical for human NK cell development and are co-transcribed from genes on chromosome 1 (MIR181A1B1) and on chromosome 9 (MIR181A2B2). We mapped human MIR181A1B1 and MIR181A2B2 transcription start sites to 78.3 kb and 34.0 kb upstream of the mature miRNAs, generating predominantly unspliced transcripts of 80-127 kb and ~60 kb, respectively. Unlike mouse thymocytes, human T cells expressed both MIR181A1B1 and MIR181A2B2. We tested the hypothesis that NK cells differentially transcribe the two genes during development and in response to immune regulatory cytokines. During NK-cell differentiation, MIR181A2B2 expression rose markedly and exceeded that of MIR181A1B1. TGF-β treatment increased NK-cell MIR181A2B2 transcription, whereas IL-2, IL-15 and IL-12/IL-18 treatments upregulated MIR181A1B1. The MIR181A2B2 promoter was strongly transactivated by SMAD3 and SMAD4 transcription factors, suggesting that TGF-β signaling upregulates MIR181A2B2 expression, at least in part, through SMAD-dependent promoter activation.

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Characterizing the MIR181A1B1 and MIR181A2B2 promoters. A, B. Shown are schematic views of promoter truncation experiments. The arrow indicates the major TSS. Full length (I) and truncation fragments (II-V) of the 181-1 (A) and 181-2 (B) promoter were transfected into YT-HY NK cells and promoter activity was measured as described in Materials and Methods. The length of the promoter fragment 5’ of the major TSS and the length of the total promoter fragment are shown. pXPG-181-1-I-Luc or pXPG-181-2-I-Luc produced luciferase values that were 70-fold and 45-fold above empty vector background. Values represent averages from tests of 2-4 different plasmid preparations over at least two different experiments. “0” indicates background level produced by the empty pXPG-Luc basic vector. C. MIR181 promoters are active in T and NK cell lines. The white and back bars indicate the 181-1 and 181-2 promoters, respectively. Fold increase refers to normalized luciferase activity compared to the empty pXPGLuc basic vector. The experiment was done 3 times with two different plasmid preps each time; all Avg were significant above those of the empty vector (*p < 0.002). D. SMAD3/4 strongly transactivate the 181-2 promoter. HepG2 cells were transfected with pXPG-181-1-I-Luc and pXPG-181-2-I-Luc reporter plasmids, along with expression constructs for SMAD3 and 4, or empty vector. Fold increase refers to transactivation activity of SMAD3/4 divided transactivation activity by empty vector. Shown is Avg and SEM of 5 experiments.
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Figure 6: Characterizing the MIR181A1B1 and MIR181A2B2 promoters. A, B. Shown are schematic views of promoter truncation experiments. The arrow indicates the major TSS. Full length (I) and truncation fragments (II-V) of the 181-1 (A) and 181-2 (B) promoter were transfected into YT-HY NK cells and promoter activity was measured as described in Materials and Methods. The length of the promoter fragment 5’ of the major TSS and the length of the total promoter fragment are shown. pXPG-181-1-I-Luc or pXPG-181-2-I-Luc produced luciferase values that were 70-fold and 45-fold above empty vector background. Values represent averages from tests of 2-4 different plasmid preparations over at least two different experiments. “0” indicates background level produced by the empty pXPG-Luc basic vector. C. MIR181 promoters are active in T and NK cell lines. The white and back bars indicate the 181-1 and 181-2 promoters, respectively. Fold increase refers to normalized luciferase activity compared to the empty pXPGLuc basic vector. The experiment was done 3 times with two different plasmid preps each time; all Avg were significant above those of the empty vector (*p < 0.002). D. SMAD3/4 strongly transactivate the 181-2 promoter. HepG2 cells were transfected with pXPG-181-1-I-Luc and pXPG-181-2-I-Luc reporter plasmids, along with expression constructs for SMAD3 and 4, or empty vector. Fold increase refers to transactivation activity of SMAD3/4 divided transactivation activity by empty vector. Shown is Avg and SEM of 5 experiments.

Mentions: Next, we tested promoter function by cloning regions surrounding the MIR181A1B1 and MIR181A2B2 TSS. Sequences were placed into a Luciferase reporter vector and tested for promoter activity in NK and T cell lines by transient transfection. Both full length promoters (containing ~900 nt of MIR181A1B1 and MIR181A2B2 sequence, constructs 181-1 I and 181-2 I) produced strong transcriptional activity in YT NK cells and in other NK and T cell lines (Fig. 6A-C). To further characterize the promoters, we created a series of 5’ truncations (constructs II-V) and tested them for activity in YT NK cells. For both promoters, constructs I-III directed similar transcriptional activity, suggesting that the minimal promoter was contained within 201 nt and 301 nt regions upstream of the MIR181A1B1 and MIR181A2B2 major TSS, respectively. Further deletion of 5’ sequences decreased transcriptional activity, suggesting that the regions 51 to 201 nt and 150 to 301 nt upstream of the major TSS for the MIR181A1B1 and MIR181A2B2 genes, respectively, contained important transcription factor binding sites (Fig. 6A, B).


Human natural killer cell microRNA: differential expression of MIR181A1B1 and MIR181A2B2 genes encoding identical mature microRNAs.

Presnell SR, Al-Attar A, Cichocki F, Miller JS, Lutz CT - Genes Immun. (2014)

Characterizing the MIR181A1B1 and MIR181A2B2 promoters. A, B. Shown are schematic views of promoter truncation experiments. The arrow indicates the major TSS. Full length (I) and truncation fragments (II-V) of the 181-1 (A) and 181-2 (B) promoter were transfected into YT-HY NK cells and promoter activity was measured as described in Materials and Methods. The length of the promoter fragment 5’ of the major TSS and the length of the total promoter fragment are shown. pXPG-181-1-I-Luc or pXPG-181-2-I-Luc produced luciferase values that were 70-fold and 45-fold above empty vector background. Values represent averages from tests of 2-4 different plasmid preparations over at least two different experiments. “0” indicates background level produced by the empty pXPG-Luc basic vector. C. MIR181 promoters are active in T and NK cell lines. The white and back bars indicate the 181-1 and 181-2 promoters, respectively. Fold increase refers to normalized luciferase activity compared to the empty pXPGLuc basic vector. The experiment was done 3 times with two different plasmid preps each time; all Avg were significant above those of the empty vector (*p < 0.002). D. SMAD3/4 strongly transactivate the 181-2 promoter. HepG2 cells were transfected with pXPG-181-1-I-Luc and pXPG-181-2-I-Luc reporter plasmids, along with expression constructs for SMAD3 and 4, or empty vector. Fold increase refers to transactivation activity of SMAD3/4 divided transactivation activity by empty vector. Shown is Avg and SEM of 5 experiments.
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Figure 6: Characterizing the MIR181A1B1 and MIR181A2B2 promoters. A, B. Shown are schematic views of promoter truncation experiments. The arrow indicates the major TSS. Full length (I) and truncation fragments (II-V) of the 181-1 (A) and 181-2 (B) promoter were transfected into YT-HY NK cells and promoter activity was measured as described in Materials and Methods. The length of the promoter fragment 5’ of the major TSS and the length of the total promoter fragment are shown. pXPG-181-1-I-Luc or pXPG-181-2-I-Luc produced luciferase values that were 70-fold and 45-fold above empty vector background. Values represent averages from tests of 2-4 different plasmid preparations over at least two different experiments. “0” indicates background level produced by the empty pXPG-Luc basic vector. C. MIR181 promoters are active in T and NK cell lines. The white and back bars indicate the 181-1 and 181-2 promoters, respectively. Fold increase refers to normalized luciferase activity compared to the empty pXPGLuc basic vector. The experiment was done 3 times with two different plasmid preps each time; all Avg were significant above those of the empty vector (*p < 0.002). D. SMAD3/4 strongly transactivate the 181-2 promoter. HepG2 cells were transfected with pXPG-181-1-I-Luc and pXPG-181-2-I-Luc reporter plasmids, along with expression constructs for SMAD3 and 4, or empty vector. Fold increase refers to transactivation activity of SMAD3/4 divided transactivation activity by empty vector. Shown is Avg and SEM of 5 experiments.
Mentions: Next, we tested promoter function by cloning regions surrounding the MIR181A1B1 and MIR181A2B2 TSS. Sequences were placed into a Luciferase reporter vector and tested for promoter activity in NK and T cell lines by transient transfection. Both full length promoters (containing ~900 nt of MIR181A1B1 and MIR181A2B2 sequence, constructs 181-1 I and 181-2 I) produced strong transcriptional activity in YT NK cells and in other NK and T cell lines (Fig. 6A-C). To further characterize the promoters, we created a series of 5’ truncations (constructs II-V) and tested them for activity in YT NK cells. For both promoters, constructs I-III directed similar transcriptional activity, suggesting that the minimal promoter was contained within 201 nt and 301 nt regions upstream of the MIR181A1B1 and MIR181A2B2 major TSS, respectively. Further deletion of 5’ sequences decreased transcriptional activity, suggesting that the regions 51 to 201 nt and 150 to 301 nt upstream of the major TSS for the MIR181A1B1 and MIR181A2B2 genes, respectively, contained important transcription factor binding sites (Fig. 6A, B).

Bottom Line: We found that few microRNAs (miRNAs) differed significantly between human NK and T cells.TGF-β treatment increased NK-cell MIR181A2B2 transcription, whereas IL-2, IL-15 and IL-12/IL-18 treatments upregulated MIR181A1B1.The MIR181A2B2 promoter was strongly transactivated by SMAD3 and SMAD4 transcription factors, suggesting that TGF-β signaling upregulates MIR181A2B2 expression, at least in part, through SMAD-dependent promoter activation.

View Article: PubMed Central - PubMed

Affiliation: Departments of Pathology and Laboratory Medicine, University of Kentucky, Lexington, KY, USA.

ABSTRACT
Natural killer (NK) and T lymphocytes share many properties, yet only NK cells respond rapidly to infection and cancer without pre-activation. We found that few microRNAs (miRNAs) differed significantly between human NK and T cells. Among those miRNAs, miR-181a and miR-181b levels rose during NK cell differentiation. Prior studies indicate that miR-181a and miR-181b are critical for human NK cell development and are co-transcribed from genes on chromosome 1 (MIR181A1B1) and on chromosome 9 (MIR181A2B2). We mapped human MIR181A1B1 and MIR181A2B2 transcription start sites to 78.3 kb and 34.0 kb upstream of the mature miRNAs, generating predominantly unspliced transcripts of 80-127 kb and ~60 kb, respectively. Unlike mouse thymocytes, human T cells expressed both MIR181A1B1 and MIR181A2B2. We tested the hypothesis that NK cells differentially transcribe the two genes during development and in response to immune regulatory cytokines. During NK-cell differentiation, MIR181A2B2 expression rose markedly and exceeded that of MIR181A1B1. TGF-β treatment increased NK-cell MIR181A2B2 transcription, whereas IL-2, IL-15 and IL-12/IL-18 treatments upregulated MIR181A1B1. The MIR181A2B2 promoter was strongly transactivated by SMAD3 and SMAD4 transcription factors, suggesting that TGF-β signaling upregulates MIR181A2B2 expression, at least in part, through SMAD-dependent promoter activation.

Show MeSH
Related in: MedlinePlus