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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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CD3–CD28 signaling induces Munc13-4, STAT4, and BRG1 expression in naive CD8+ T cells. (a) Representative Western blots for Munc13-4, STAT4, pSTAT4, and BRG1 in naive CD8+ T cells from a healthy donor stimulated with IL-2 and IL-12, IFN-γ, type I IFN, or anti-CD3/CD28 microbeads. (b) Cumulative Munc13-4, STAT4, pSTAT4, and BRG1 fold expression values from each stimulation condition relative to unstimulated cells from five healthy donors in three independent experiments. All values are normalized to β-actin. (c) Representative Western blots for Munc13-4, STAT4 and BRG1 in naive CD8+ T cells from a healthy individual stimulated with anti-CD3/CD28 microbeads for 0, 2, 4, 8, and 16 h. (d) Cumulative Munc13-4, STAT4, and BRG1 fold expression values from each stimulation time point relative to unstimulated cells from three healthy individuals in two independent experiments. All values are normalized to β-actin. (e) Representative Western blots for total and phosphorylated (pY693) STAT4 expression in naive CD8+ T cells from a healthy donor at the indicated time points after stimulation with anti-CD3/CD28 microbeads. (f) Cumulative STAT4 and pSTAT4 fold expression values from each stimulation time point relative to unstimulated cells from three healthy donors in two independent experiments. All values are normalized to β-actin. Fold expression differences with P ≤ 0.05 are marked with a single asterisk, and with P ≤ 0.01 are marked with a double asterisk.
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fig7: CD3–CD28 signaling induces Munc13-4, STAT4, and BRG1 expression in naive CD8+ T cells. (a) Representative Western blots for Munc13-4, STAT4, pSTAT4, and BRG1 in naive CD8+ T cells from a healthy donor stimulated with IL-2 and IL-12, IFN-γ, type I IFN, or anti-CD3/CD28 microbeads. (b) Cumulative Munc13-4, STAT4, pSTAT4, and BRG1 fold expression values from each stimulation condition relative to unstimulated cells from five healthy donors in three independent experiments. All values are normalized to β-actin. (c) Representative Western blots for Munc13-4, STAT4 and BRG1 in naive CD8+ T cells from a healthy individual stimulated with anti-CD3/CD28 microbeads for 0, 2, 4, 8, and 16 h. (d) Cumulative Munc13-4, STAT4, and BRG1 fold expression values from each stimulation time point relative to unstimulated cells from three healthy individuals in two independent experiments. All values are normalized to β-actin. (e) Representative Western blots for total and phosphorylated (pY693) STAT4 expression in naive CD8+ T cells from a healthy donor at the indicated time points after stimulation with anti-CD3/CD28 microbeads. (f) Cumulative STAT4 and pSTAT4 fold expression values from each stimulation time point relative to unstimulated cells from three healthy donors in two independent experiments. All values are normalized to β-actin. Fold expression differences with P ≤ 0.05 are marked with a single asterisk, and with P ≤ 0.01 are marked with a double asterisk.

Mentions: Next, we sought to address the question of how signaling may induce Munc13-4 expression. We chose to focus on CD8+ T cells for these experiments due to low basal levels of STAT4 in naive CD8+ T cells and the fact that STAT4 is dramatically up-regulated in effector CD8+ T cells (Fig. 5, a and b), which exhibit strong degranulation, relative to naive CD8+ T cells (Chiang, et al., 2013). To this end, we stimulated naive CD8+ T cells for 16 h with IL-2 and IL-12, IFN-γ, IFN-α, or anti-CD3/CD28 beads and determined Munc13-4, STAT4, phosphorylated STAT4 (pY693; pSTAT4), and BRG1 protein expression levels by Western blot. Cytokine stimulation alone did not induce expression of any of these proteins. However, CD3/CD28 stimulation robustly increased the expression of all three proteins along with robust pSTAT4 (Fig. 7, a and b). Phosphorylation on tyrosine 693 is important for STAT4 transcriptional activity (Visconti et al., 2000). Of note, we observed an approximately threefold induction of both conventional and alternative intron 1 transcripts in CD3/CD28-stimulated cells (unpublished data).


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)

CD3–CD28 signaling induces Munc13-4, STAT4, and BRG1 expression in naive CD8+ T cells. (a) Representative Western blots for Munc13-4, STAT4, pSTAT4, and BRG1 in naive CD8+ T cells from a healthy donor stimulated with IL-2 and IL-12, IFN-γ, type I IFN, or anti-CD3/CD28 microbeads. (b) Cumulative Munc13-4, STAT4, pSTAT4, and BRG1 fold expression values from each stimulation condition relative to unstimulated cells from five healthy donors in three independent experiments. All values are normalized to β-actin. (c) Representative Western blots for Munc13-4, STAT4 and BRG1 in naive CD8+ T cells from a healthy individual stimulated with anti-CD3/CD28 microbeads for 0, 2, 4, 8, and 16 h. (d) Cumulative Munc13-4, STAT4, and BRG1 fold expression values from each stimulation time point relative to unstimulated cells from three healthy individuals in two independent experiments. All values are normalized to β-actin. (e) Representative Western blots for total and phosphorylated (pY693) STAT4 expression in naive CD8+ T cells from a healthy donor at the indicated time points after stimulation with anti-CD3/CD28 microbeads. (f) Cumulative STAT4 and pSTAT4 fold expression values from each stimulation time point relative to unstimulated cells from three healthy donors in two independent experiments. All values are normalized to β-actin. Fold expression differences with P ≤ 0.05 are marked with a single asterisk, and with P ≤ 0.01 are marked with a double asterisk.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4042637&req=5

fig7: CD3–CD28 signaling induces Munc13-4, STAT4, and BRG1 expression in naive CD8+ T cells. (a) Representative Western blots for Munc13-4, STAT4, pSTAT4, and BRG1 in naive CD8+ T cells from a healthy donor stimulated with IL-2 and IL-12, IFN-γ, type I IFN, or anti-CD3/CD28 microbeads. (b) Cumulative Munc13-4, STAT4, pSTAT4, and BRG1 fold expression values from each stimulation condition relative to unstimulated cells from five healthy donors in three independent experiments. All values are normalized to β-actin. (c) Representative Western blots for Munc13-4, STAT4 and BRG1 in naive CD8+ T cells from a healthy individual stimulated with anti-CD3/CD28 microbeads for 0, 2, 4, 8, and 16 h. (d) Cumulative Munc13-4, STAT4, and BRG1 fold expression values from each stimulation time point relative to unstimulated cells from three healthy individuals in two independent experiments. All values are normalized to β-actin. (e) Representative Western blots for total and phosphorylated (pY693) STAT4 expression in naive CD8+ T cells from a healthy donor at the indicated time points after stimulation with anti-CD3/CD28 microbeads. (f) Cumulative STAT4 and pSTAT4 fold expression values from each stimulation time point relative to unstimulated cells from three healthy donors in two independent experiments. All values are normalized to β-actin. Fold expression differences with P ≤ 0.05 are marked with a single asterisk, and with P ≤ 0.01 are marked with a double asterisk.
Mentions: Next, we sought to address the question of how signaling may induce Munc13-4 expression. We chose to focus on CD8+ T cells for these experiments due to low basal levels of STAT4 in naive CD8+ T cells and the fact that STAT4 is dramatically up-regulated in effector CD8+ T cells (Fig. 5, a and b), which exhibit strong degranulation, relative to naive CD8+ T cells (Chiang, et al., 2013). To this end, we stimulated naive CD8+ T cells for 16 h with IL-2 and IL-12, IFN-γ, IFN-α, or anti-CD3/CD28 beads and determined Munc13-4, STAT4, phosphorylated STAT4 (pY693; pSTAT4), and BRG1 protein expression levels by Western blot. Cytokine stimulation alone did not induce expression of any of these proteins. However, CD3/CD28 stimulation robustly increased the expression of all three proteins along with robust pSTAT4 (Fig. 7, a and b). Phosphorylation on tyrosine 693 is important for STAT4 transcriptional activity (Visconti et al., 2000). Of note, we observed an approximately threefold induction of both conventional and alternative intron 1 transcripts in CD3/CD28-stimulated cells (unpublished data).

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