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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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An intact ELF1-binding site is not necessary for active transcription from the regulatory element within intron 1 of UNC13D. (a) Luciferase assays showing the relative transcriptional activity of the full UNC13D promoter region, the intron 1 promoter region and the intron 1 promoter region with the c.118-308C>T mutation in the YT-Indy NK cell line. Cumulative results from three independent experiments are shown. (b) Western blot analysis of ELF1 expression in isolated subsets of human peripheral blood lymphocytes from a healthy donor. (c) Cumulative ELF1 fold expression values relative to B cells from three donors in two independent experiments. All values are normalized to β-actin.
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fig4: An intact ELF1-binding site is not necessary for active transcription from the regulatory element within intron 1 of UNC13D. (a) Luciferase assays showing the relative transcriptional activity of the full UNC13D promoter region, the intron 1 promoter region and the intron 1 promoter region with the c.118-308C>T mutation in the YT-Indy NK cell line. Cumulative results from three independent experiments are shown. (b) Western blot analysis of ELF1 expression in isolated subsets of human peripheral blood lymphocytes from a healthy donor. (c) Cumulative ELF1 fold expression values relative to B cells from three donors in two independent experiments. All values are normalized to β-actin.

Mentions: To test the transcriptional activity of the UNC13D intron 1 promoter, we created a luciferase reporter vector and tested its activity in the readily transfectable YT-Indy NK cell line. Luciferase expression levels from the intron 1 promoter were comparable to those of the full-length UNC13D promoter. We also created a version of the intron 1 promoter reporter vector with the UNC13D c.118-308C>T mutation. Remarkably, luciferase levels were not statistically different between the wild-type and mutated versions (Fig. 4 a), suggesting that the ELF1-binding site is not essential for promoter transactivation. Moreover, CD19+ B cells, CD4+ T cells, CD8+ T cells, and CD3–CD56+ NK cells all expressed similar levels of ELF1 (Fig. 4, b and c). Together, the results suggest that the UNC13D intron 1 regulatory element serves as both an enhancer for the conventional promoter and a lymphocyte-specific alternative promoter, and the intronic ELF1 site alone may not explain the observed increase in Munc13-4 expression upon cytotoxic lymphocyte differentiation.


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)

An intact ELF1-binding site is not necessary for active transcription from the regulatory element within intron 1 of UNC13D. (a) Luciferase assays showing the relative transcriptional activity of the full UNC13D promoter region, the intron 1 promoter region and the intron 1 promoter region with the c.118-308C>T mutation in the YT-Indy NK cell line. Cumulative results from three independent experiments are shown. (b) Western blot analysis of ELF1 expression in isolated subsets of human peripheral blood lymphocytes from a healthy donor. (c) Cumulative ELF1 fold expression values relative to B cells from three donors in two independent experiments. All values are normalized to β-actin.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig4: An intact ELF1-binding site is not necessary for active transcription from the regulatory element within intron 1 of UNC13D. (a) Luciferase assays showing the relative transcriptional activity of the full UNC13D promoter region, the intron 1 promoter region and the intron 1 promoter region with the c.118-308C>T mutation in the YT-Indy NK cell line. Cumulative results from three independent experiments are shown. (b) Western blot analysis of ELF1 expression in isolated subsets of human peripheral blood lymphocytes from a healthy donor. (c) Cumulative ELF1 fold expression values relative to B cells from three donors in two independent experiments. All values are normalized to β-actin.
Mentions: To test the transcriptional activity of the UNC13D intron 1 promoter, we created a luciferase reporter vector and tested its activity in the readily transfectable YT-Indy NK cell line. Luciferase expression levels from the intron 1 promoter were comparable to those of the full-length UNC13D promoter. We also created a version of the intron 1 promoter reporter vector with the UNC13D c.118-308C>T mutation. Remarkably, luciferase levels were not statistically different between the wild-type and mutated versions (Fig. 4 a), suggesting that the ELF1-binding site is not essential for promoter transactivation. Moreover, CD19+ B cells, CD4+ T cells, CD8+ T cells, and CD3–CD56+ NK cells all expressed similar levels of ELF1 (Fig. 4, b and c). Together, the results suggest that the UNC13D intron 1 regulatory element serves as both an enhancer for the conventional promoter and a lymphocyte-specific alternative promoter, and the intronic ELF1 site alone may not explain the observed increase in Munc13-4 expression upon cytotoxic lymphocyte differentiation.

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