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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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The ELF1-binding site within the UNC13D intron 1 regulatory element is important for the recruitment of STAT4, the chromatin remodeling complex BRG1 and high levels of H3K27ac. (a) Western blot analysis of STAT4 expression in isolated subsets of human peripheral blood lymphocytes from a healthy donor. STAT4 and β-actin for CD8+ T cell and NK cell subsets (columns 2 and 3) were run on the same Western blot gel as those shown for Munc13-4 in Fig. 1 a (rows 1 and 2 in columns 2 and 3), accounting for the shared β-actin image. (b) Cumulative STAT4 fold expression values relative to B cells, naive CD8+ T cells or CD56bright NK cells from four donors in three 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. (c) EMSA with probes for the UNC13D 5′ and 3′ STAT sites with nuclear lysates from NK92 cells. Data are representative of three independent experiments. (d) EMSA with longer probes spanning the wild-type or mutant ELF1 and 5′ or 3′ STAT sites within intron 1 of UNC13D with lysates from primary NK cells (left). EMSA with longer probes spanning the wild-type or mutant ELF1 and 5′ STAT sites with and without cold consensus ELF1- and STAT4-binding sequences or anti-ELF1 and anti-STAT4 antibodies with lysates from NK92 cells (right). All three sections in this figure are from one gel. Data are representative of two independent experiments. (e) ChIP analysis of STAT4 and RNA Pol II binding to intron 1 of UNC13D. Cumulative quantitative RT-PCR fold enrichment values relative to input for three separate experiments are shown. (f) Control PCRs showing the specificity of primers used to distinguish between the wild-type and mutant intron 1 regions of UNC13D in an individual heterozygous for the c.118-308C>T intron 1 mutation. ChIP analysis of allele-specific (g) STAT4 and (h) BRG1 binding, as well as (i) H3K27ac, in primary NK cells from an individual heterozygous for the c.118-308C>T intron 1 mutation. All data were generated in a single experiment with the heterozygous donor. (j) Western blot analysis of BRG1 expression in isolated subsets of human peripheral blood lymphocytes from a healthy donor. BRG1 and β-actin for CD8+ T cell and NK cell subsets (columns 2 and 3) were run on the same Western blot gel as those shown for Munc13-4 in Fig. 1 a (rows 1 and 2 in columns 2 and 3), accounting for the shared β-actin image. (k) Cumulative BRG1 fold expression values relative to B cells, naive CD8+ T cells, or CD56bright NK cells from four healthy donors in three independent experiments. All values are normalized to β-actin. Fold expression differences with P ≤ 0.01 are marked with a double asterisk.
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fig5: The ELF1-binding site within the UNC13D intron 1 regulatory element is important for the recruitment of STAT4, the chromatin remodeling complex BRG1 and high levels of H3K27ac. (a) Western blot analysis of STAT4 expression in isolated subsets of human peripheral blood lymphocytes from a healthy donor. STAT4 and β-actin for CD8+ T cell and NK cell subsets (columns 2 and 3) were run on the same Western blot gel as those shown for Munc13-4 in Fig. 1 a (rows 1 and 2 in columns 2 and 3), accounting for the shared β-actin image. (b) Cumulative STAT4 fold expression values relative to B cells, naive CD8+ T cells or CD56bright NK cells from four donors in three 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. (c) EMSA with probes for the UNC13D 5′ and 3′ STAT sites with nuclear lysates from NK92 cells. Data are representative of three independent experiments. (d) EMSA with longer probes spanning the wild-type or mutant ELF1 and 5′ or 3′ STAT sites within intron 1 of UNC13D with lysates from primary NK cells (left). EMSA with longer probes spanning the wild-type or mutant ELF1 and 5′ STAT sites with and without cold consensus ELF1- and STAT4-binding sequences or anti-ELF1 and anti-STAT4 antibodies with lysates from NK92 cells (right). All three sections in this figure are from one gel. Data are representative of two independent experiments. (e) ChIP analysis of STAT4 and RNA Pol II binding to intron 1 of UNC13D. Cumulative quantitative RT-PCR fold enrichment values relative to input for three separate experiments are shown. (f) Control PCRs showing the specificity of primers used to distinguish between the wild-type and mutant intron 1 regions of UNC13D in an individual heterozygous for the c.118-308C>T intron 1 mutation. ChIP analysis of allele-specific (g) STAT4 and (h) BRG1 binding, as well as (i) H3K27ac, in primary NK cells from an individual heterozygous for the c.118-308C>T intron 1 mutation. All data were generated in a single experiment with the heterozygous donor. (j) Western blot analysis of BRG1 expression in isolated subsets of human peripheral blood lymphocytes from a healthy donor. BRG1 and β-actin for CD8+ T cell and NK cell subsets (columns 2 and 3) were run on the same Western blot gel as those shown for Munc13-4 in Fig. 1 a (rows 1 and 2 in columns 2 and 3), accounting for the shared β-actin image. (k) Cumulative BRG1 fold expression values relative to B cells, naive CD8+ T cells, or CD56bright NK cells from four healthy donors in three independent experiments. All values are normalized to β-actin. Fold expression differences with P ≤ 0.01 are marked with a double asterisk.

Mentions: Munc13-4 expression levels correlate with lymphocyte cytotoxicity. (a) Western blot analysis of Munc13-4, Stx11, and Munc18-2 expression in eight different isolated subsets of human peripheral blood lymphocytes from a healthy peripheral blood donor. All cell types were isolated by negative magnetic bead selection except for CD56bright and CD56dim NK cells, which were FACS sorted. Munc13-4 was run on a separate Western blot gel from Stx11 and Munc18-2. Munc13-4 and β-actin for CD8+ T cell and NK cell subsets (rows 1 and 2 in columns 2 and 3) were run on the same Western blot gel as those shown for STAT4 in Figure 5 a (columns 2 and 3) and BRG1 in Figure 5 j (columns 2 and 3), accounting for the shared β-actin image. (b) Cumulative Munc13-4, Stx11, and Munc18-2 fold expression values relative to B cells, naive CD8+ T cells, or CD56bright NK cells from four healthy donors in three independent experiments. All values are normalized to β-actin. Fold expression differences with P ≤ 0.05 are marked with an asterisk.


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)

The ELF1-binding site within the UNC13D intron 1 regulatory element is important for the recruitment of STAT4, the chromatin remodeling complex BRG1 and high levels of H3K27ac. (a) Western blot analysis of STAT4 expression in isolated subsets of human peripheral blood lymphocytes from a healthy donor. STAT4 and β-actin for CD8+ T cell and NK cell subsets (columns 2 and 3) were run on the same Western blot gel as those shown for Munc13-4 in Fig. 1 a (rows 1 and 2 in columns 2 and 3), accounting for the shared β-actin image. (b) Cumulative STAT4 fold expression values relative to B cells, naive CD8+ T cells or CD56bright NK cells from four donors in three 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. (c) EMSA with probes for the UNC13D 5′ and 3′ STAT sites with nuclear lysates from NK92 cells. Data are representative of three independent experiments. (d) EMSA with longer probes spanning the wild-type or mutant ELF1 and 5′ or 3′ STAT sites within intron 1 of UNC13D with lysates from primary NK cells (left). EMSA with longer probes spanning the wild-type or mutant ELF1 and 5′ STAT sites with and without cold consensus ELF1- and STAT4-binding sequences or anti-ELF1 and anti-STAT4 antibodies with lysates from NK92 cells (right). All three sections in this figure are from one gel. Data are representative of two independent experiments. (e) ChIP analysis of STAT4 and RNA Pol II binding to intron 1 of UNC13D. Cumulative quantitative RT-PCR fold enrichment values relative to input for three separate experiments are shown. (f) Control PCRs showing the specificity of primers used to distinguish between the wild-type and mutant intron 1 regions of UNC13D in an individual heterozygous for the c.118-308C>T intron 1 mutation. ChIP analysis of allele-specific (g) STAT4 and (h) BRG1 binding, as well as (i) H3K27ac, in primary NK cells from an individual heterozygous for the c.118-308C>T intron 1 mutation. All data were generated in a single experiment with the heterozygous donor. (j) Western blot analysis of BRG1 expression in isolated subsets of human peripheral blood lymphocytes from a healthy donor. BRG1 and β-actin for CD8+ T cell and NK cell subsets (columns 2 and 3) were run on the same Western blot gel as those shown for Munc13-4 in Fig. 1 a (rows 1 and 2 in columns 2 and 3), accounting for the shared β-actin image. (k) Cumulative BRG1 fold expression values relative to B cells, naive CD8+ T cells, or CD56bright NK cells from four healthy donors in three independent experiments. All values are normalized to β-actin. Fold expression differences with P ≤ 0.01 are marked with a double asterisk.
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Related In: Results  -  Collection

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Show All Figures
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fig5: The ELF1-binding site within the UNC13D intron 1 regulatory element is important for the recruitment of STAT4, the chromatin remodeling complex BRG1 and high levels of H3K27ac. (a) Western blot analysis of STAT4 expression in isolated subsets of human peripheral blood lymphocytes from a healthy donor. STAT4 and β-actin for CD8+ T cell and NK cell subsets (columns 2 and 3) were run on the same Western blot gel as those shown for Munc13-4 in Fig. 1 a (rows 1 and 2 in columns 2 and 3), accounting for the shared β-actin image. (b) Cumulative STAT4 fold expression values relative to B cells, naive CD8+ T cells or CD56bright NK cells from four donors in three 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. (c) EMSA with probes for the UNC13D 5′ and 3′ STAT sites with nuclear lysates from NK92 cells. Data are representative of three independent experiments. (d) EMSA with longer probes spanning the wild-type or mutant ELF1 and 5′ or 3′ STAT sites within intron 1 of UNC13D with lysates from primary NK cells (left). EMSA with longer probes spanning the wild-type or mutant ELF1 and 5′ STAT sites with and without cold consensus ELF1- and STAT4-binding sequences or anti-ELF1 and anti-STAT4 antibodies with lysates from NK92 cells (right). All three sections in this figure are from one gel. Data are representative of two independent experiments. (e) ChIP analysis of STAT4 and RNA Pol II binding to intron 1 of UNC13D. Cumulative quantitative RT-PCR fold enrichment values relative to input for three separate experiments are shown. (f) Control PCRs showing the specificity of primers used to distinguish between the wild-type and mutant intron 1 regions of UNC13D in an individual heterozygous for the c.118-308C>T intron 1 mutation. ChIP analysis of allele-specific (g) STAT4 and (h) BRG1 binding, as well as (i) H3K27ac, in primary NK cells from an individual heterozygous for the c.118-308C>T intron 1 mutation. All data were generated in a single experiment with the heterozygous donor. (j) Western blot analysis of BRG1 expression in isolated subsets of human peripheral blood lymphocytes from a healthy donor. BRG1 and β-actin for CD8+ T cell and NK cell subsets (columns 2 and 3) were run on the same Western blot gel as those shown for Munc13-4 in Fig. 1 a (rows 1 and 2 in columns 2 and 3), accounting for the shared β-actin image. (k) Cumulative BRG1 fold expression values relative to B cells, naive CD8+ T cells, or CD56bright NK cells from four healthy donors in three independent experiments. All values are normalized to β-actin. Fold expression differences with P ≤ 0.01 are marked with a double asterisk.
Mentions: Munc13-4 expression levels correlate with lymphocyte cytotoxicity. (a) Western blot analysis of Munc13-4, Stx11, and Munc18-2 expression in eight different isolated subsets of human peripheral blood lymphocytes from a healthy peripheral blood donor. All cell types were isolated by negative magnetic bead selection except for CD56bright and CD56dim NK cells, which were FACS sorted. Munc13-4 was run on a separate Western blot gel from Stx11 and Munc18-2. Munc13-4 and β-actin for CD8+ T cell and NK cell subsets (rows 1 and 2 in columns 2 and 3) were run on the same Western blot gel as those shown for STAT4 in Figure 5 a (columns 2 and 3) and BRG1 in Figure 5 j (columns 2 and 3), accounting for the shared β-actin image. (b) Cumulative Munc13-4, Stx11, and Munc18-2 fold expression values relative to B cells, naive CD8+ T cells, or CD56bright NK cells from four healthy donors in three independent experiments. All values are normalized to β-actin. Fold expression differences with P ≤ 0.05 are marked with an asterisk.

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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Related in: MedlinePlus