Abstract
Gain-of-function (GOF) mutations in the STAT1 gene are critical for the onset of chronic mucocutaneous candidiasis (CMC) disease. However, the molecular basis for the gain of STAT1 function remains largely unclear. Here, we investigated the structural features of STAT1 GOF residues to better understand the impact of these pathogenic mutations. We constructed STAT1 alanine mutants of the α3 helix residues of the coiled-coil domain, which are frequently found in CMC pathogenic mutations, and measured their transcriptional activities. Most of the identified GOF residues were located inside the coiled-coil domain stem structure or at the protein surface of the anti-parallel dimer interface. Unlike those, Arg-274 was adjacent to the DNA-binding domain. In addition, Arg-274 was found to functionally interact with Gln-441 in the DNA-binding domain. Because Gln-441 is located at the anti-parallel dimer contact site, Gln-441 reorientation by Arg-274 mutation probably impedes formation of the dimer. Further, the statistical analysis of RNA-seq data with STAT1-deficient epithelial cells and primary T cells from a CMC patient revealed that the R274Q mutation affected gene expression levels of 66 and 76 non-overlapping RefSeq genes, respectively. Because their transcription levels were only slightly modulated by wild-type STAT1, we concluded that the R274Q mutation increased transcriptional activity but did not change dramatically the repertoire of STAT1 targets. Hence, we provide a novel mechanism of STAT1 GOF triggered by a CMC pathogenic mutation.
Highlights
Gain-of-function (GOF) mutations in the Signal transducer and activator of transcription 1 (STAT1) gene are critical for the onset of chronic mucocutaneous candidiasis (CMC) disease
CMC hot spots were reported to converge on the residues around the third helix [3], implying that the local structure of this helix might be indispensable for the regulation of STAT1 activity (Fig. 1B)
If the identified mutation were registered as a STAT1 GOF mutation in the disease-related variation databases (Fig. 1A and supplemental Table S3), the carrier patient could be diagnosed with CMC
Summary
The STAT1 CCD is composed of four ␣-helices [11]. CMC hot spots were reported to converge on the residues around the third helix [3], implying that the local structure of this helix might be indispensable for the regulation of STAT1 activity (Fig. 1B). The TRANSFAC motif tool found putative IFN-␥-response elements at positions ϩ369 (TTCCCACAA) and ϩ132 (TTCCTCCAA) near the transcription start site of the GBP2 gene, but not in the IL6 gene promoter, implying that other DNA-binding factors could transiently regulate the IL6 gene before STAT1 recruitment Together, these data reinforce the model that R274Q magnified expression of the large part of the STAT1 target genes but exhibited behavior on the promoter similar to that of wild type. As shown in a scatter plot depicting the log-transformed FPKM values of wild-type and R274Q T cells, expression of the most significant RefSeq genes was increased in an IFN-␥-dependent manner (Fig. 8C). These data indicated that the R274Q mutation increased transcriptional activity but would not dramatically change the repertoire of the STAT1 target genes
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