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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 UNC13D intron 1 regulatory element acts as an enhancer and lymphocyte-specific alternative promoter. Quantitative RT-PCR using primers and probes specific for the (a) conventional and (b) intron 1 UNC13D transcripts in sorted B cells, CD4+ T cells, CD8+ T cells, NK cells, and monocytes from a fresh, healthy buffy coat donor, healthy donor cells that were previously frozen, healthy donor cells that were frozen and transported, and a homozygous c.118-308C>T patient that were frozen and transported. All PCR data are normalized to 18S RNA levels, and fold expression values were calculated for each donor relative to B cells. All PCRs were performed in triplicate and averaged. All data were generated together in a single experiment with sorted cells from the homozygous donor.
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fig3: The UNC13D intron 1 regulatory element acts as an enhancer and lymphocyte-specific alternative promoter. Quantitative RT-PCR using primers and probes specific for the (a) conventional and (b) intron 1 UNC13D transcripts in sorted B cells, CD4+ T cells, CD8+ T cells, NK cells, and monocytes from a fresh, healthy buffy coat donor, healthy donor cells that were previously frozen, healthy donor cells that were frozen and transported, and a homozygous c.118-308C>T patient that were frozen and transported. All PCR data are normalized to 18S RNA levels, and fold expression values were calculated for each donor relative to B cells. All PCRs were performed in triplicate and averaged. All data were generated together in a single experiment with sorted cells from the homozygous donor.

Mentions: To further elucidate the nature of the UNC13D intron 1 regulatory element, we designed primers and probes specific for the conventional and intron 1 UNC13D transcripts and performed qRT-PCR on sorted CD19+ B cells, CD4+ T cells, CD8+ T cells, CD3−CD56+ NK cells, and monocytes from healthy donors and an individual homozygous for the UNC13D c.118-308C>T mutation. Conventional UNC13D transcripts were expressed at comparable levels between subsets in healthy donors and were significantly lower, but still readily detectible, in all analyzed cellular subsets from the individual with the UNC13D c.118-308C>T mutation (Fig. 3 a). UNC13D transcripts originating from intron 1 were expressed in a lymphocyte–specific pattern in healthy donors, with the highest levels observed in CD8+ T cells and CD3−CD56+ NK cells. Intron 1 transcripts were observed at the limit of detection in B cells and were not detectable in CD4+ T cells, CD8+ T cells CD3−CD56+ NK cells, or monocytes from the homozygous UNC13D c.118-308C>T patient (Fig. 3 b).


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 UNC13D intron 1 regulatory element acts as an enhancer and lymphocyte-specific alternative promoter. Quantitative RT-PCR using primers and probes specific for the (a) conventional and (b) intron 1 UNC13D transcripts in sorted B cells, CD4+ T cells, CD8+ T cells, NK cells, and monocytes from a fresh, healthy buffy coat donor, healthy donor cells that were previously frozen, healthy donor cells that were frozen and transported, and a homozygous c.118-308C>T patient that were frozen and transported. All PCR data are normalized to 18S RNA levels, and fold expression values were calculated for each donor relative to B cells. All PCRs were performed in triplicate and averaged. All data were generated together in a single experiment with sorted cells from the homozygous donor.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig3: The UNC13D intron 1 regulatory element acts as an enhancer and lymphocyte-specific alternative promoter. Quantitative RT-PCR using primers and probes specific for the (a) conventional and (b) intron 1 UNC13D transcripts in sorted B cells, CD4+ T cells, CD8+ T cells, NK cells, and monocytes from a fresh, healthy buffy coat donor, healthy donor cells that were previously frozen, healthy donor cells that were frozen and transported, and a homozygous c.118-308C>T patient that were frozen and transported. All PCR data are normalized to 18S RNA levels, and fold expression values were calculated for each donor relative to B cells. All PCRs were performed in triplicate and averaged. All data were generated together in a single experiment with sorted cells from the homozygous donor.
Mentions: To further elucidate the nature of the UNC13D intron 1 regulatory element, we designed primers and probes specific for the conventional and intron 1 UNC13D transcripts and performed qRT-PCR on sorted CD19+ B cells, CD4+ T cells, CD8+ T cells, CD3−CD56+ NK cells, and monocytes from healthy donors and an individual homozygous for the UNC13D c.118-308C>T mutation. Conventional UNC13D transcripts were expressed at comparable levels between subsets in healthy donors and were significantly lower, but still readily detectible, in all analyzed cellular subsets from the individual with the UNC13D c.118-308C>T mutation (Fig. 3 a). UNC13D transcripts originating from intron 1 were expressed in a lymphocyte–specific pattern in healthy donors, with the highest levels observed in CD8+ T cells and CD3−CD56+ NK cells. Intron 1 transcripts were observed at the limit of detection in B cells and were not detectable in CD4+ T cells, CD8+ T cells CD3−CD56+ NK cells, or monocytes from the homozygous UNC13D c.118-308C>T patient (Fig. 3 b).

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