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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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High-resolution comparison of the UNC13D chromatin state in lymphocyte subsets. (a) Locations of primer pairs that were used for high-resolution analysis of UNC13D. The primers map a region spanning from –2,433 to +12,689 (relative to the conventional translational start site), and each primer pair was designed to amplify a 150–200-bp product. The location of the ELF1-binding site is indicated with an open circle. The alternative first exon is labeled in blue. (b) FAIRE analysis of open chromatin in primary B, NK, CD8+ naïve, and CD8+ effector T cells. Quantitative RT-PCR values averaged from two donors in two independent experiments are shown. (c–f) ChIP assays with antibodies against STAT4, BRG1, H3K4me3, and H3K27ac. Expression values normalized to an IgG-negative control and to input are shown. Quantitative RT-PCR values averaged from two donors in two independent experiments are shown.
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fig6: High-resolution comparison of the UNC13D chromatin state in lymphocyte subsets. (a) Locations of primer pairs that were used for high-resolution analysis of UNC13D. The primers map a region spanning from –2,433 to +12,689 (relative to the conventional translational start site), and each primer pair was designed to amplify a 150–200-bp product. The location of the ELF1-binding site is indicated with an open circle. The alternative first exon is labeled in blue. (b) FAIRE analysis of open chromatin in primary B, NK, CD8+ naïve, and CD8+ effector T cells. Quantitative RT-PCR values averaged from two donors in two independent experiments are shown. (c–f) ChIP assays with antibodies against STAT4, BRG1, H3K4me3, and H3K27ac. Expression values normalized to an IgG-negative control and to input are shown. Quantitative RT-PCR values averaged from two donors in two independent experiments are shown.

Mentions: To analyze UNC13D with high resolution, we designed 24 primer pairs covering a region extending from –2,433 to +12,689 nt relative to the translational start site (Fig. 6 a). These primers were used to identify nucleosome-depleted, open chromatin using the formaldehyde-assisted isolation of regulatory elements (FAIRE) approach (Simon et al., 2012). We observed an enrichment of open chromatin in the conventional and intron 1 promoters in NK cells relative to B cells (Fig. 6 b), which express very low levels of STAT4 (Fig. 5, a and b). Similarly, effector CD8+ T cells displayed an enrichment of open chromatin in the promoter and intron 1 regions relative to naive CD8+ T cells. High-resolution ChIP analysis with an antibody against STAT4 showed that STAT4 (Fig. 6 c) and BRG1 (Fig. 6 d) binding are enriched within intron 1 in NK cells and effector CD8+ T cells. The active histone marks (Karlić et al., 2010) H3K4me3 (Fig. 6 e) and H3K27ac (Fig. 6 f) were enriched throughout the conventional and intron 1 promoters in NK cells and effector CD8+ T cells. Collectively, the ChIP data indicate a poised environment surrounding the first exon of UNC13D in all lymphocyte subsets, with an increase in STAT4 and BRG1 binding in NK cells and effector T cells concomitant with increases in active histone marks.


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)

High-resolution comparison of the UNC13D chromatin state in lymphocyte subsets. (a) Locations of primer pairs that were used for high-resolution analysis of UNC13D. The primers map a region spanning from –2,433 to +12,689 (relative to the conventional translational start site), and each primer pair was designed to amplify a 150–200-bp product. The location of the ELF1-binding site is indicated with an open circle. The alternative first exon is labeled in blue. (b) FAIRE analysis of open chromatin in primary B, NK, CD8+ naïve, and CD8+ effector T cells. Quantitative RT-PCR values averaged from two donors in two independent experiments are shown. (c–f) ChIP assays with antibodies against STAT4, BRG1, H3K4me3, and H3K27ac. Expression values normalized to an IgG-negative control and to input are shown. Quantitative RT-PCR values averaged from two donors in two independent experiments are shown.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig6: High-resolution comparison of the UNC13D chromatin state in lymphocyte subsets. (a) Locations of primer pairs that were used for high-resolution analysis of UNC13D. The primers map a region spanning from –2,433 to +12,689 (relative to the conventional translational start site), and each primer pair was designed to amplify a 150–200-bp product. The location of the ELF1-binding site is indicated with an open circle. The alternative first exon is labeled in blue. (b) FAIRE analysis of open chromatin in primary B, NK, CD8+ naïve, and CD8+ effector T cells. Quantitative RT-PCR values averaged from two donors in two independent experiments are shown. (c–f) ChIP assays with antibodies against STAT4, BRG1, H3K4me3, and H3K27ac. Expression values normalized to an IgG-negative control and to input are shown. Quantitative RT-PCR values averaged from two donors in two independent experiments are shown.
Mentions: To analyze UNC13D with high resolution, we designed 24 primer pairs covering a region extending from –2,433 to +12,689 nt relative to the translational start site (Fig. 6 a). These primers were used to identify nucleosome-depleted, open chromatin using the formaldehyde-assisted isolation of regulatory elements (FAIRE) approach (Simon et al., 2012). We observed an enrichment of open chromatin in the conventional and intron 1 promoters in NK cells relative to B cells (Fig. 6 b), which express very low levels of STAT4 (Fig. 5, a and b). Similarly, effector CD8+ T cells displayed an enrichment of open chromatin in the promoter and intron 1 regions relative to naive CD8+ T cells. High-resolution ChIP analysis with an antibody against STAT4 showed that STAT4 (Fig. 6 c) and BRG1 (Fig. 6 d) binding are enriched within intron 1 in NK cells and effector CD8+ T cells. The active histone marks (Karlić et al., 2010) H3K4me3 (Fig. 6 e) and H3K27ac (Fig. 6 f) were enriched throughout the conventional and intron 1 promoters in NK cells and effector CD8+ T cells. Collectively, the ChIP data indicate a poised environment surrounding the first exon of UNC13D in all lymphocyte subsets, with an increase in STAT4 and BRG1 binding in NK cells and effector T cells concomitant with increases in active histone marks.

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