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

Giovannozzi, Simone; Lemmens, Veerle; Hendrix, Jelle; Gijsbers, Rik; Schrijvers, R.

doi:10.3389/fimmu.2020.01114

Cited by 11 publications

(25 citation statements)

References 57 publications

(100 reference statements)

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Section: Introductionmentioning

confidence: 96%

“…Recently, we and others ( 21 – 24 ) described that specific STAT1 GOF mutations are associated with different molecular mechanisms. We studied a subset of STAT1 GOF mutants (R274W, R321S, T419R, and N574I) that reside in different domains and at different interfaces of the STAT1 protein ( 24 ). At least three different mechanisms could be identified: (I) faster nuclear import was observed for the R274W mutation (antiparallel homodimer interface); (II) reduced nuclear mobility and delayed dephosphorylation for the R321S and the N574I mutants (distant from the homodimer or DNA interfaces); (III) slower diffusion in the nucleus, possibly due to enhanced affinity for chromatin, was observed for the T419R mutation (parallel homodimer-DNA interface).…”

Section: Introductionmentioning

confidence: 96%

“…For example, R274 is located at the interface of the antiparallel homodimer, in a coiled-coil domain. We hypothesized that mutating the positively charged aa R274 may destabilize the antiparallel, inactive, homodimer, resulting in a faster shift to the active parallel form ( 24 ). Alternatively, aa T419 is in close contact with the DNA backbone when STAT1 is in its active parallel conformation.…”

Section: Introductionmentioning

confidence: 99%

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Transcriptional Profiling of STAT1 Gain-of-Function Reveals Common and Mutation-Specific Fingerprints

et al. 2021

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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.

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Section: Introductionmentioning

confidence: 96%

Section: Introductionmentioning

confidence: 96%

Section: Introductionmentioning

confidence: 99%

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Transcriptional Profiling of STAT1 Gain-of-Function Reveals Common and Mutation-Specific Fingerprints

et al. 2021

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“…In regards to overexpression models, the majority of studies have employed the STAT1-null U3 fibrosarcoma cells (and more recently, HEK293 cells on a WT background). 14,[27][28][29][30][31] These models have been instrumental in studying STAT1 phosphorylation and de-phosphorylation kinetics, as well as migration of STAT1 between the cytoplasm and the nucleus. However, such models are characterized by expression of STAT1 under an exogenous promoter, and an inaccurate gene dosage.…”

Section: Discussionmentioning

confidence: 99%

“…Previous studies utilizing over-expression models generated in STAT1-null U3 fibrosarcoma cells (and more recently, HEK293 cells), have been instrumental in elucidating differences among mutations with respect to STAT1 phosphorylation kinetics, nuclear migration and accumulation. 14,[27][28][29][30][31] However, such models involve the expression of STAT1 under an exogenous promoter, and do not capture the heterozygous nature of the mutation. In the context of a delicately-regulated transcription factor, over-expression models may therefore be limited in their portrayal of gene expression patterns downstream of STAT1.…”

mentioning

confidence: 99%

Heterozygous Cell Models of STAT1 Gain-of-Function Reveal a Broad Spectrum of Interferon-Signature Gene Transcriptional Responses

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Lindsay

Erwood

et al. 2020

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Signal Transducer and Activator of Transcription 1 (STAT1) gain-of-function (GOF) is an autosomal dominant immune disorder marked by wide infectious predisposition, autoimmunity, vascular disease and malignancy. Its molecular hallmark, elevated phospho-STAT1 (pSTAT1) following interferon (IFN) stimulation, is seen consistently in all patients and may not fully account for the broad phenotypic spectrum associated with this disorder. While over 100 mutations have been implicated in STAT1 GOF, genotype-phenotype correlation remains limited, and current overexpression models may be of limited use in gene expression studies. We generated heterozygous mutants in diploid HAP1 cells using CRISPR/Cas9 base-editing, targeting the endogenous STAT1 gene. Our models recapitulated the molecular phenotype of elevated pSTAT1, and were used to characterize the expression of five IFN-stimulated genes under a number of conditions. At baseline, transcriptional polarization was evident among mutants compared with wild type, and this was maintained following prolonged serum starvation. This suggests a possible role for unphosphorylated STAT1 in the pathogenesis of STAT1 GOF. Following stimulation with IFNα or IFNγ, differential patterns of gene expression emerged among mutants, including both gain and loss of transcriptional function. This work highlights the importance of modelling heterozygous conditions, and in particular transcription factor-related disorders, in a manner which accurately reflects patient genotype and molecular signature. Furthermore, we propose a complex and multifactorial transcriptional profile associated with various STAT1 mutations, adding to global efforts in establishing STAT1 GOF genotype-phenotype correlation and enhancing our understanding of disease pathogenesis.

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JAKs and STATs from a Clinical Perspective: Loss-of-Function Mutations, Gain-of-Function Mutations, and Their Multidimensional Consequences

et al. 2023

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The JAK/STAT signaling pathway plays a key role in cytokine signaling and is involved in development, immunity, and tumorigenesis for nearly any cell. At first glance, the JAK/STAT signaling pathway appears to be straightforward. However, on closer examination, the factors influencing the JAK/STAT signaling activity, such as cytokine diversity, receptor profile, overlapping JAK and STAT specificity among non-redundant functions of the JAK/STAT complexes, positive regulators (e.g., cooperating transcription factors), and negative regulators (e.g., SOCS, PIAS, PTP), demonstrate the complexity of the pathway’s architecture, which can be quickly disturbed by mutations. The JAK/STAT signaling pathway has been, and still is, subject of basic research and offers an enormous potential for the development of new methods of personalized medicine and thus the translation of basic molecular research into clinical practice beyond the use of JAK inhibitors. Gain-of-function and loss-of-function mutations in the three immunologically particularly relevant signal transducers STAT1, STAT3, and STAT6 as well as JAK1 and JAK3 present themselves through individual phenotypic clinical pictures. The established, traditional paradigm of loss-of-function mutations leading to immunodeficiency and gain-of-function mutation leading to autoimmunity breaks down and a more differentiated picture of disease patterns evolve. This review is intended to provide an overview of these specific syndromes from a clinical perspective and to summarize current findings on pathomechanism, symptoms, immunological features, and therapeutic options of STAT1, STAT3, STAT6, JAK1, and JAK3 loss-of-function and gain-of-function diseases.

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

Cited by 11 publications

References 57 publications

Transcriptional Profiling of STAT1 Gain-of-Function Reveals Common and Mutation-Specific Fingerprints

Transcriptional Profiling of STAT1 Gain-of-Function Reveals Common and Mutation-Specific Fingerprints

Heterozygous Cell Models of STAT1 Gain-of-Function Reveal a Broad Spectrum of Interferon-Signature Gene Transcriptional Responses

JAKs and STATs from a Clinical Perspective: Loss-of-Function Mutations, Gain-of-Function Mutations, and Their Multidimensional Consequences

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