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Live Cell Imaging Demonstrates Multiple Routes Toward a STAT1 Gain-of-Function Phenotype.

ABSTRACT: Signal transducer and activator of transcription 1 (STAT1) gain-of-function (GOF) mutations result in a primary immunodeficiency (PID) characterized typically by chronic mucocutaneous candidiasis (CMC), but a wider phenotypic range is reported and remains unexplained from a pathophysiological point-of-view. We hypothesized that different STAT1 GOF mutations may result in distinct molecular mechanisms, possibly explaining the variable phenotypes observed in patients. We selected STAT1 GOF mutants (R274W, R321S, T419R, and N574I) that are spread over the protein and studied their dynamic behavior in vitro in U3A and HeLa cell lines. All GOF mutants showed increased STAT1 phosphorylation compared to STAT1 WT. Real-time imaging demonstrated three underlying mechanisms for STAT1 GOF: (i) R274W showed a faster nuclear accumulation, (ii) both R321S and N574I showed a reduced nuclear mobility and slower dephosphorylation, whereas (iii) T419R was near-immobile in the nucleus, potentially due to enhanced binding to chromatin.

SUBMITTER: Giovannozzi S

PROVIDER: S-EPMC7296103 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Live Cell Imaging Demonstrates Multiple Routes Toward a STAT1 Gain-of-Function Phenotype.

Giovannozzi Simone S Lemmens Veerle V Hendrix Jelle J Gijsbers Rik R Schrijvers Rik R

Frontiers in immunology 20200609

Signal transducer and activator of transcription 1 (STAT1) gain-of-function (GOF) mutations result in a primary immunodeficiency (PID) characterized typically by chronic mucocutaneous candidiasis (CMC), but a wider phenotypic range is reported and remains unexplained from a pathophysiological point-of-view. We hypothesized that different STAT1 GOF mutations may result in distinct molecular mechanisms, possibly explaining the variable phenotypes observed in patients. We selected STAT1 GOF mutants ...[more]

PMID: 32582194

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Project description:Signal transducer and activator of transcription (STAT1)1 gain of function (GOF) pathogenic variants have been associated with increased levels of phosphorylated STAT1 and STAT1-dependent cellular responses. Delayed dephosphorylation was proposed as the underlying mechanism leading to the characteristically raised pSTAT1 levels. We examined the levels of STAT1 protein and message as well as rates of STAT1 phosphorylation, dephosphorylation, and degradation associated with STAT1 GOF pathogenic variants. Fresh peripheral blood mononuclear cells (PBMC) from 14 STAT1 GOF patients carrying 10 different pathogenic variants in the coiled-coil, DNA binding, and SH2 domains and healthy donors were used to study STAT1 levels and phosphorylation (pSTAT1) following IFNγ and IFNα stimulation. STAT1 protein levels were measured by flow cytometry and immunoblot. STAT1 mRNA levels were measured using quantitative reverse transcription PCR. STAT1 protein degradation was studied using cycloheximide. Patient IFNγ and IFNα induced peak pSTAT1 was higher than in healthy controls. The velocity of pSTAT1 dephosphorylation after treatment of IFNγ stimulated CD14+ monocytes with the Janus Kinase (JAK)-inhibitor ruxolitinib was significantly faster in patient cells. STAT1 protein levels in patient CD14+ monocytes and CD3+ T cells were higher than in healthy donors. There was a strong and positive correlation between CD14+ STAT1 protein levels and peak pSTAT1 levels. Patient fresh PBMC STAT1 mRNA levels were increased at rest and after 16 h of incubation. STAT1 protein degradation was similar in patient and healthy volunteer cells. Patient IFNγ receptors 1 and 2 and JAK2 levels were normal. One patient in our cohort was treated with the oral JAK inhibitor ruxolitinib. Treatment was associated with normalization of both STAT1 protein and peak pSTAT1 levels. After JAK inhibitor treatment was stopped the patient's CD14+ monocyte STAT1 protein and peak phosphorylation levels increased proportionally. These findings suggest that patients with STAT1 GOF mutations have higher levels of total STAT1 protein, leading to high levels of pSTAT1 after stimulation, despite rapid STAT1 dephosphorylation and normal degradation.

Project description:STAT1 gain-of-function (GOF) is a primary immunodeficiency typically characterized by chronic mucocutaneous candidiasis (CMC), recurrent respiratory infections, and autoimmunity. Less commonly, also immunodysregulation polyendocrinopathy enteropathy X-linked (IPEX)-like syndromes with CMC, and combined immunodeficiency without CMC have been described. Recently, our group and others have shown that different mutation-specific mechanisms underlie STAT1 GOF in vitro, including faster nuclear accumulation (R274W), and reduced mobility (R321, N574I) to near immobility in the nucleus (T419R) upon IFNγ stimulation. In this work, we evaluated the transcriptomic fingerprint of the aforementioned STAT1 GOF mutants (R274W, R321S, T419R, and N574I) relative to STAT1 wild-type upon IFNγ stimulation in an otherwise isogenic cell model. The majority of genes up-regulated in wild-type STAT1 cells were significantly more up-regulated in cells expressing GOF mutants, except for T419R. In addition to the common interferon regulated genes (IRG), STAT1 GOF mutants up-regulated an additional set of genes, that were in part shared with other GOF mutants or mutation-specific. Overall, R274W and R321S transcriptomes clustered with STAT1 WT, while T419R and N574I had a more distinct fingerprint. We observed reduced frequency of canonical IFNγ activation site (GAS) sequences in promoters of genes up-regulated by all the STAT1 GOF mutants, suggesting loss of DNA binding specificity for the canonical GAS consensus. Interestingly, the T419R mutation, expected to directly increase the affinity for DNA, showed the most pronounced effects on the transcriptome. T419R STAT1 dysregulated more non-IRG than the other GOF mutants and fewer GAS or degenerate GAS promotor sequences could be found in the promoter regions of these genes. In conclusion, our work confirms hyperactivation of common sets of IFNγ-induced genes in STAT1 GOF with additional dysregulation of mutation-specific genes, in line with the earlier observed mutation-specific mechanisms. Binding to more degenerate GAS sequences is proposed as a mechanism toward transcriptional dysregulation in R274W, R321S, and N574I. For T419R, an increased interaction with the DNA is suggested to result in a broader and less GAS-specific response. Our work indicates that multiple routes leading to STAT1 GOF are associated with common and private transcriptomic fingerprints, which may contribute to the phenotypic variation observed in vivo.

Dataset Information

Live Cell Imaging Demonstrates Multiple Routes Toward a STAT1 Gain-of-Function Phenotype.

Publications

Live Cell Imaging Demonstrates Multiple Routes Toward a STAT1 Gain-of-Function Phenotype.

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