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Transcriptional regulation of Munc13-4 expression in cytotoxic lymphocytes is disrupted by an intronic mutation associated with a primary immunodeficiency.

Cichocki F, Schlums H, Li H, Stache V, Holmes T, Lenvik TR, Chiang SC, Miller JS, Meeths M, Anderson SK, Bryceson YT - J. Exp. Med. (2014)

Bottom Line: The mechanisms regulating Munc13-4 expression are unknown.This mutation impairs UNC13D intron 1 recruitment of STAT4 and the chromatin remodeling complex component BRG1, diminishing active histone modifications at the locus.Thus, mutations associated with primary immunodeficiencies may cause disease by disrupting transcription factor binding.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre for Infectious Medicine, Department of Medicine; Clinical Genetics Unit, Department of Molecular Medicine and Surgery, and Center for Molecular Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, 141 86 Stockholm, Sweden Division of Hematology, Oncology and Transplantation, University of Minnesota Cancer Center, Minneapolis, MN 55455.

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STAT4 is necessary for Munc13-4 induction in response to CD3/CD28 stimulation. (a) Naive CD8+ T cells from a healthy donor were transfected with control siRNA or siRNA targeting STAT4 and stimulated overnight with anti-CD3/CD28 microbeads or left unstimulated. Western blots were performed with antibodies against Munc13-4, STAT4, and BRG1. (b) Cumulative Munc13-4, STAT4, and BRG1 fold expression values normalized to β-actin from control siRNA and siSTAT4 transfection and stimulation experiments from four healthy donors in two independent experiments. Fold expression differences between control and STAT4 knockdown conditions with P ≤ 0.05 are marked with an asterisk.
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fig8: STAT4 is necessary for Munc13-4 induction in response to CD3/CD28 stimulation. (a) Naive CD8+ T cells from a healthy donor were transfected with control siRNA or siRNA targeting STAT4 and stimulated overnight with anti-CD3/CD28 microbeads or left unstimulated. Western blots were performed with antibodies against Munc13-4, STAT4, and BRG1. (b) Cumulative Munc13-4, STAT4, and BRG1 fold expression values normalized to β-actin from control siRNA and siSTAT4 transfection and stimulation experiments from four healthy donors in two independent experiments. Fold expression differences between control and STAT4 knockdown conditions with P ≤ 0.05 are marked with an asterisk.

Mentions: To establish a direct link between STAT4 and the induction of Munc13-4 expression after TCR stimulation, naive CD8+ T cells were isolated from peripheral blood and transfected with either control siRNA or siRNA-targeting STAT4 (siSTAT4). Upon CD3/CD28 stimulation, STAT4 was highly induced in cells transfected with the control siRNA. No STAT4 induction was observed in cells transfected with siSTAT4, confirming the efficacy of the knockdown. As expected, Munc13-4 was induced upon stimulation in cells transfected with control siRNA. Notably, Munc13-4 protein levels were 76 ± 12% lower in siSTAT4-transfected cells compared with siRNA controls, supporting the hypothesis that STAT4 is important for Munc13-4 induction. BRG1 was comparably induced in both control and siSTAT4 transfected cells, suggesting that TCR signaling induces BRG1 expression independent of STAT4 (Fig. 8, a and b). These results provide conclusive evidence demonstrating a significant role for STAT4 in the regulation of Munc13-4 expression.


Transcriptional regulation of Munc13-4 expression in cytotoxic lymphocytes is disrupted by an intronic mutation associated with a primary immunodeficiency.

Cichocki F, Schlums H, Li H, Stache V, Holmes T, Lenvik TR, Chiang SC, Miller JS, Meeths M, Anderson SK, Bryceson YT - J. Exp. Med. (2014)

STAT4 is necessary for Munc13-4 induction in response to CD3/CD28 stimulation. (a) Naive CD8+ T cells from a healthy donor were transfected with control siRNA or siRNA targeting STAT4 and stimulated overnight with anti-CD3/CD28 microbeads or left unstimulated. Western blots were performed with antibodies against Munc13-4, STAT4, and BRG1. (b) Cumulative Munc13-4, STAT4, and BRG1 fold expression values normalized to β-actin from control siRNA and siSTAT4 transfection and stimulation experiments from four healthy donors in two independent experiments. Fold expression differences between control and STAT4 knockdown conditions with P ≤ 0.05 are marked with an asterisk.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4042637&req=5

fig8: STAT4 is necessary for Munc13-4 induction in response to CD3/CD28 stimulation. (a) Naive CD8+ T cells from a healthy donor were transfected with control siRNA or siRNA targeting STAT4 and stimulated overnight with anti-CD3/CD28 microbeads or left unstimulated. Western blots were performed with antibodies against Munc13-4, STAT4, and BRG1. (b) Cumulative Munc13-4, STAT4, and BRG1 fold expression values normalized to β-actin from control siRNA and siSTAT4 transfection and stimulation experiments from four healthy donors in two independent experiments. Fold expression differences between control and STAT4 knockdown conditions with P ≤ 0.05 are marked with an asterisk.
Mentions: To establish a direct link between STAT4 and the induction of Munc13-4 expression after TCR stimulation, naive CD8+ T cells were isolated from peripheral blood and transfected with either control siRNA or siRNA-targeting STAT4 (siSTAT4). Upon CD3/CD28 stimulation, STAT4 was highly induced in cells transfected with the control siRNA. No STAT4 induction was observed in cells transfected with siSTAT4, confirming the efficacy of the knockdown. As expected, Munc13-4 was induced upon stimulation in cells transfected with control siRNA. Notably, Munc13-4 protein levels were 76 ± 12% lower in siSTAT4-transfected cells compared with siRNA controls, supporting the hypothesis that STAT4 is important for Munc13-4 induction. BRG1 was comparably induced in both control and siSTAT4 transfected cells, suggesting that TCR signaling induces BRG1 expression independent of STAT4 (Fig. 8, a and b). These results provide conclusive evidence demonstrating a significant role for STAT4 in the regulation of Munc13-4 expression.

Bottom Line: The mechanisms regulating Munc13-4 expression are unknown.This mutation impairs UNC13D intron 1 recruitment of STAT4 and the chromatin remodeling complex component BRG1, diminishing active histone modifications at the locus.Thus, mutations associated with primary immunodeficiencies may cause disease by disrupting transcription factor binding.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre for Infectious Medicine, Department of Medicine; Clinical Genetics Unit, Department of Molecular Medicine and Surgery, and Center for Molecular Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, 141 86 Stockholm, Sweden Division of Hematology, Oncology and Transplantation, University of Minnesota Cancer Center, Minneapolis, MN 55455.

Show MeSH
Related in: MedlinePlus