Transcriptional regulation of Munc13-4 expression in cytotoxic lymphocytes is disrupted by an intronic mutation associated with a primary immunodeficiency.
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.
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
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Mentions: The marked increase in Munc13-4 expression upon differentiation indicated that lineage-specific mechanisms promote high levels of Munc13-4 expression. Recently, we identified a c.118-308C>T mutation in an evolutionarily conserved region deep within intron 1 of UNC13D (Fig. 2 a) in patients with FHL3 that leads to a lymphocyte-specific loss of Munc13-4 expression (Meeths et al., 2011). NK cells from these individuals exhibit defective degranulation (Fig. 2 b). These results led us to hypothesize that a regulatory region critical for Munc13-4 expression in cytotoxic lymphocytes is located within intron 1 of UNC13D. We used transcription factor binding prediction software (Heinemeyer et al., 1998) to identify potential binding sites within intron 1 of UNC13D that might be disrupted by the mutation and found that a consensus ETS site overlaps with the location of the c.118-308C>T mutation (Fig. 2 c). To determine whether an ETS family member binds to this site, we synthesized wild-type and mutant oligonucleotide probes corresponding to the sequences shown in Fig. 2 a for use in EMSAs with nuclear lysates from primary NK cells and the NK92 cell line. A clearly discernable protein complex bound to the wild-type UNC13D probe that was absent from the probe with the c.118-308C>T mutation. This protein complex migrated at the same size as a probe containing the consensus sequence for the ETS family member ELF1, and addition of an antibody against ELF1 resulted in a super-shift (Fig. 2 d). No super-shifts were observed with antibodies against other ETS family members, including ETS1 and ELF4 (unpublished data).
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.