STAT1 gain-of-function heterozygous cell models reveal diverse interferon-signature gene transcriptional responses

Abstract

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 modeling 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.

Systems Immunology Analyses ofSTAT1Gain-of-Function Immune Phenotypes Reveal Heterogeneous Response to IL-6 and Broad Immunometabolic Roles for STAT1

“…Although common in this patient population, neither patient here had evidence of current or past CMC, which may be attributed to the early identification of STAT1 GOF mutations, as well as timely prophylaxis. The IFN response of STAT1 GOF patients and distinct mutations can be heterogeneous and may reflect multifactorial transcriptional and biological regulation (48,49). The development of HLH is an infrequent manifestation of STAT1 GOF mutations (50).…”

DNA‐Binding domain mutations confer severe outcome at an early age among STAT1 gain‐of‐function patients

“…This increased disease severity caused by DBD mutations may be explained in part by prolonged nuclear presence and possibly DNA binding of STAT1, as recently demonstrated in cell models by our group and others. 11,12 Importantly, however, the above in vitro studies identified kinetic and transcriptional differences even among DBD mutations, suggesting that our understanding of the functional impact of such mutations remains incomplete.…”

“…To date, the genotype‐phenotype correlation in STAT1 GOF has remained elusive. While mutations in all STAT1 domains have been shown to result in the molecular hallmark of enhanced tyrosine‐701 phosphorylation, 9 the mechanisms leading to this GOF phenomenon may vary among different mutations 11‐15 . For instance, mutations in the DBD have been shown to result in enhanced nuclear accumulation, reduced nuclear mobility, and slower dephosphorylation of STAT1 in vitro in STAT1 GOF cell models 11,12 .…”

“…While mutations in all STAT1 domains have been shown to result in the molecular hallmark of enhanced tyrosine‐701 phosphorylation, 9 the mechanisms leading to this GOF phenomenon may vary among different mutations 11‐15 . For instance, mutations in the DBD have been shown to result in enhanced nuclear accumulation, reduced nuclear mobility, and slower dephosphorylation of STAT1 in vitro in STAT1 GOF cell models 11,12 . From a clinical standpoint, an accumulating body of evidence suggests that individuals harboring mutations in the DBD show a predisposition to early‐onset combined immunodeficiency (CID), manifesting as recurrent invasive and/or opportunistic infections and substantial impairment in T‐cell function 6,16‐22 .…”

“…[11][12][13][14][15] For instance, mutations in the DBD have been shown to result in enhanced nuclear accumulation, reduced nuclear mobility, and slower dephosphorylation of STAT1 in vitro in STAT1 GOF cell models. 11,12 From a clinical standpoint, an accumulating body of evidence suggests that individuals harboring mutations in the DBD show a predisposition to early-onset combined immunodeficiency (CID), manifesting as recurrent invasive and/or opportunistic infections and substantial impairment in T-cell function. 6,[16][17][18][19][20][21][22] However, the notion that DBD patients may be a unique subgroup within STAT1 GOF has not been formally explored.…”