- Email: [email protected]
Elucidating the effects of disease-causing mutations on STAT3 function in autosomal-dominant hyper-IgE syndrome
Accepted Manuscript Elucidating the effects of disease-causing mutations on STAT3 function in autosomal dominant hyper-IgE syndrome Simon J. Pelham, MSc, Helen Lenthall, B Sc Hons, Elissa K. Deenick, PhD, Stuart G. Tangye, PhD PII:
S0091-6749(16)30282-2
DOI:
10.1016/j.jaci.2016.04.020
Reference:
YMAI 12119
To appear in:
Journal of Allergy and Clinical Immunology
Received Date: 30 November 2015 Revised Date:
24 March 2016
Accepted Date: 5 April 2016
Please cite this article as: Pelham SJ, Lenthall H, Deenick EK, Tangye SG, Elucidating the effects of disease-causing mutations on STAT3 function in autosomal dominant hyper-IgE syndrome, Journal of Allergy and Clinical Immunology (2016), doi: 10.1016/j.jaci.2016.04.020. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Elucidating the effects of disease-causing mutations on STAT3 function in autosomal
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dominant hyper-IgE syndrome
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Simon J Pelham MSc1,2,
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Helen Lenthall B Sc Hons2,
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Elissa K Deenick PhD1,2,
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Stuart G Tangye PhD1,2
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Immunology Research Program, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia;
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St Vincent’s Clinical School, University of New South Wales Australia, Darlinghurst, NSW, Australia
10 11 Corresponding authors
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Dr. Stuart Tangye; Dr Elissa Deenick
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Garvan Institute of Medical Research
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384 Victoria St. Darlinghurst. 2010. NSW. Australia
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e-mail: [email protected]; [email protected]
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This work was supported by project and program grants awarded by the National Health and
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Medical Research Council (NHMRC) of Australia (1016953, 1066694, and 1027400 to E.K.
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Deenick and S.G. Tangye). S.J.P. is supported by an Australian Postgraduate Award from the
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University of New South Wales. S.G. Tangye is a recipient of a Principal Research Fellowship
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(1042925) from the NHMRC of Australia.
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The authors declare no conflicts of interest
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Key words
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• autosomal dominant hyper-IgE syndrome; STAT3; transcription factors; cytokine signaling
29 Abbreviations
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AD-HIES: autosomal dominant hyper-IgE syndrome; DN: dominant negative; JAK: Janus
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activating kinase; STAT: signal transducer and activation of transcription; SH2: Src homology
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2; DNA BD: DNA binding domain; TA: transactivation; EBV-LCL: EBV-transformed
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lymphoblastoid cell line; WT: wild-type
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35 Capsule summary
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Analysis of germline mutations in STAT3 that cause AD-HIES revealed biochemical
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mechanisms of disease-causation. This has implications for developing therapeutics that
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could modulate the function of STAT3 in the settings of immunodeficiency, autoimmunity and
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malignancy.
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To the editor
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Signaling through STAT3 is an important process required for the function of a wide range of
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biological systems. This is demonstrated by embryonic lethality of germline Stat3 gene-
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targeted mice1. Furthermore, heterozygous dominant negative (DN) mutations in STAT3
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cause autosomal dominant hyper IgE syndrome (AD-HIES) in humans, a disease
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characterized by increased susceptibility to infection with Staphylococcus aureus,
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Streptococcus pneumonia and Candida albicans, eczema, extremely elevated levels of serum
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IgE, poor humoral immune responses, and non-immunological features including defects in
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musculoskeletal, dental, and vascular systems.2,
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been reported that affect all functional domains of STAT3.3,
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cytosolic transcription factor with a key role in signaling downstream of numerous cytokines,
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including IL-6, IL-10, IL-21 and IL-232. DN STAT3 mutations cause multiple cellular defects,
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including reduced Th17 cells, memory B cells, T follicular helper cells and MAIT cells, and
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impairing responsiveness of naïve B cells to IL-10 and IL-21 (Online Supplemental data Table
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1)2,
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production in AD-HIES. Despite this, our understanding of the molecular impact of various
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STAT3 mutations remains limited. Indeed, as there is no genotype/phenotype relationship7, it
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remains to be determined how diverse mutations impair STAT3 signaling to manifest the
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same clinical and cellular phenotype. Furthermore, while several studies have examined the
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effects of mutations in separate domains of STAT3 on signaling pathways8-10, few have
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systematically compared the impact of mutations located in different functional domains of
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STAT3.
More than 60 different mutations have STAT3 encodes a latent
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. These defects explain susceptibility to fungal infections and poor antibody
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To explore this, we examined the consequences of distinct disease-causing mutations in the
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DNA binding, SH2 and transactivation domains on each stage of STAT3 signaling. We
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selected 6 mutations previously identified in numerous cohorts of AD-HIES patients, including
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our own, that localize to the DNA binding domain (BD) (R382W, H437P, S465F), SH2 domain
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(Q644P, Y657N) or TA (L706M) domains5, 6 (Figure 1A; Fig S1A Online supplemental data).
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All mutations have a comparable deleterious effect on the generation of Th17, Tfh and
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memory B cells in affected individuals (Online Supplemental data Table S1). To test how
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each mutation affected tyrosine phosphorylation, HEK-293T cells were transfected with
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plasmids encoding WT or mutant STAT3 (Figure 1B, Fig S1B). IL-6 stimulation induced
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phosphorylation at tyrosine 705 (Y705) of WT STAT3 as well as STAT3 harboring mutations
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in the DNA BD, as well as the Q644P SH2 domain mutant (Figure 1B, Fig S1B). In contrast,
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the SH2 domain Y657N and TA domain L706M mutants failed to undergo significant
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phosphorylation in response to IL-6 (Figure 1B, Fig S1B). Reduced STAT3 phosphorylation
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due to Y657N SH2 and L706M TA domain mutants likely results from conformational
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alterations of STAT3 or steric hindrance that limit access of kinases to phosphorylate Y705.
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Thus, only mutations in the proximity of Y705 (Figure 1A) affected STAT3 phosphorylation.
83 Following phosphorylation, STAT3 forms dimers that are required for function11, 12. To test the
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ability of mutant STAT3 to form dimers with WT STAT3, and to recapitulate the setting of
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heterozygous STAT3 mutations in AD-HIES, HEK-293T cells were co-transfected with Myc-
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tagged WT and Flag-tagged mutant STAT3 plasmids (Figure 1C), and then stimulated with IL-
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6. STAT3 multimers were captured by co-immunoprecipitation using anti-Flag mAb. The
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levels of dimer formation between WT STAT3 and those with mutations in the DNA BD or TA
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domain were comparable to WT/WT STAT3 proteins (Figure 1C, D). This is consistent with
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initial observations by Minegishi et al.10 who reported that mutations in the DNA BD did not
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affect STAT3 dimerization. In contrast to DNA BD mutations, SH2 domain mutations reduced
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dimer formation by ~40-50%, with Y657N having the greatest effect (Figure 1C,D). This
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identifies these residues as being critical for intermolecular interactions between
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phosphorylated STAT3 (pSTAT3). It is likely that amino acid substitutions in the SH2 domain
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result in changes in charge, which affect assembly or stability of STAT3 dimers.
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Nuclear translocation of STAT3 is crucial for its role as a transcription factor11,
12
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investigate the impact of mutations in STAT3 on its nuclear translocation, nuclear extracts
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were prepared from IL-21-stimulated WT and STAT3 mutant EBV-LCLs and the levels of
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pSTAT3 determined. The accumulation of nuclear pSTAT3 in normal EBV-LCLs was maximal
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after 15 mins and declined at times thereafter (Figure 2A). Consistent with reductions in total
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pSTAT3 in transfected HEK-293T cells (Figure 1B; Fig S1B), the Y657N SH2 and L706M TA
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domain mutations resulted in significant reductions in levels of nuclear pSTAT3 in EBV-LCLs
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(Figure 2A). Remarkably, two of the three DNA BD mutations (R382W, H437P) significantly
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reduced nuclear translocation of pSTAT3 (Figure 2A). As several nuclear export sequences
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have been identified in the DNA BD of STAT313, reduced translocation due to these mutations
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this likely results from an imbalance between nuclear import and export. Interestingly, the
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S465F DNA BD mutation had no effect on nuclear pSTAT3 translocation (Figure 2A).
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. To
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Once in the nucleus STAT3 binds specific target sites to regulate gene transcription.11, 12 WT
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STAT3 exhibited strong binding to a canonical target sequence that was maximal after 15-30
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mins of IL-21 stimulation (Figure 2B). Conversely, all STAT3 mutant proteins showed
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significant decreases in their ability to bind DNA (Figure 2B). As some STAT3 mutations
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affected phosphorylation (Y657N, L706M) or nuclear translocation (R382W, H437P), the Page 4 of 8
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reduction in DNA binding reflects not only an impaired ability of mutant STAT3 to bind target
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sequences but also decreases in the amount of pSTAT3 in the nucleus. Consistent with these
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findings, induction of the STAT3 target genes SOCS3 (Figure 2C), PRDM1 (encoding BLIMP-
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1) and IL2RA (Fig S2A, B) were significantly reduced (50-90%) in response to IL-21 and IL-10
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in STAT3 mutant compared to normal EBV-LCLs, thereby indicating that the defect in DNA
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binding results in a functional impairment in the ability to induce gene transcription. Thus,
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impaired DNA binding of mutant STAT3 proteins manifests as reduced gene transcription.
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In conclusion our study gives insight into dysfunctional STAT3-dependent signaling in AD-
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HIES, and allowed for the dissection and identification of specific stages of STAT3 signaling
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affected by these disease-causing mutations (Fig S3; Table S1). This not only highlights
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which aspects of STAT3 signaling are impacted by these mutations but also identifies
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residues and regions of domains in STAT3 that are critical for normal function (Fig S3; Table
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S1). This reveals mechanisms not only for loss-of function of STAT3 in AD-HIES but reveals
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residues/regions of STAT3 that could be targeted in cases where STAT3 is overactive, such
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as gain-of function mutations in STAT3 resulting in multisystemic autoimmunity or
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hematological malignancies, respectively3.
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Simon J Pelham MSc1,2,
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Helen Lenthall B Sc Hons2,
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Elissa K Deenick PhD1,2,
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Stuart G Tangye PhD1,2
140 141 142 143 144 145
1
Immunology Research Program, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia;
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St Vincent’s Clinical School, University of New South Wales Australia, Darlinghurst, NSW, Australia
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Figure Legends
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Figure 1. Autosomal dominant hyper IgE syndrome-causing STAT3 mutations: effects
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on phosphorylation and dimerisation.
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(A) Schematic representation of STAT3, with the mutations studied indicated by black lines.
150
(B) HEK-293T cells were transfected with Flag-tagged WT or R382W, H437P, S465F,
151
Q644P, Y657N or L706M mutant STAT3, and either untreated or stimulated with IL-6 (30
152
mins). Phosphorylation of STAT3 (pSTAT3) was determined by fixing and permeabilising the
153
cells and labeling with Abs specific for Flag and pSTAT3 (Y705). Graph shows geometric MFI
154
of pSTAT3 (Two way ANOVA with Dunnett post-test, comparison between IL-6 stimulated WT
155
and mutant STAT3. ***p< 0.001. mean ± SEM, n=6). (C, D) HEK-293T cells were co-
156
transfected with plasmids containing Myc-tagged WT STAT3 and either Flag-tagged WT or
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mutant STAT3. After 48 hours, cells were harvested, rested for 2 hours then stimulated with
158
IL-6 (10 ng/ml, 30 mins). Cell lysates were immunoprecipitated with anti-Flag Ab, and
159
assessed for the presence of Myc-tagged WT or mutant STAT3 by Western blotting. (C)
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Diagram of the gel lanes (upper); Input whole cell lysates immunoblotted for Myc, Flag or
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GAPDH showing equivalent amounts of WT and mutant STAT3 (left; lower) and
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immunopreciptiation of lysates with anti-Flag beads, immunoblotted for Myc and Flag (right;
163
lower). (D) Quantification of ratio of WT:mutant STAT3, normalized to WT:mutant ratio of the
164
input. (One way ANOVA with Dunnett post-test, comparison between WT and various
165
mutants. **p < 0.01, *p < 0.05. mean ± SEM, n=5).
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Figure 2. Impaired nuclear translocation, DNA binding and gene induction by mutant
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STAT3 proteins.
169
(A, B) EBV-LCLs from normal donors or AD-HIES patients were stimulated with IL-21 for 0,
170
15, 30 or 60 minutes, and nuclear fractions then isolated. (A) The abundance of pSTAT3 was
171
assessed by determining the ratio of intensity of mutant vs WT pSTAT3 stimulated with IL-21
172
for 15 minutes. (Two way ANOVA with Dunnett post-test, comparison between 15 minute time
173
points, ***p<0.001, **p< 0.01, *p< 0.05. mean ± SEM, n=4). (B) Binding of WT and mutant
174
STAT3 to consensus DNA binding sites was determined (TransAM kit, ActiveMotif). Values
175
were normalized to WT STAT3 at 15 mins. (Two way ANOVA with Dunnett post-test,
176
comparison between 15 minute time points, ***p< 0.001, **p< 0.01, *p< 0.05. mean ± SEM ,
177
n=3-4). The dashed line represents the basal response (i.e. time 0) of WT EBV LCLs to
178
cytokine stimulation. (C) EBV-LCLs from normal donors or AD-HIES patients were stimulated
179
with IL-21 or media only for 3 hours. Expression levels of SOCS3 were determined by qPCR.
180
(Two way ANOVA with Holm-Sidak post-test, comparison between IL-21 stimulated (SOCS3)
181
samples, **P < 0.01, *P < 0.05. mean ± SEM, n=5-9).
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182 Supplemental Online data
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Figure S1: Autosomal dominant hyper IgE syndrome-causing STAT3 mutations and
185
effects on cytokine-induced phosphorylation
186
(A) Crystallographic structure of a STAT3 dimer bound to DNA (Protein Data Bank, 1BG1).
187
Sites of the mutations are represented by red spheres, phosphorylated tyrosine 705 (Y705) is
188
indicated by black spheres.
189
(B) HEK-293T cells were transfected with Flag-tagged WT STAT3 or STAT3 harbouring the
190
following mutations: R382W, H437P, S465F, Q644P, Y657N or L706M. Cells were left
191
untreated or stimulated with IL-6 for 30 mins. Phosphorylation of STAT3 was determined by
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flow cytometry following fixing and permeabilising the cells and labeling with Abs specific for
193
Flag and pSTAT3 (Y705). Representative histograms of phosphorylated STAT3 induction
194
after IL-6 stimulation.
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Figure S2: Effect of STAT3 loss-of function mutations on induction of expression of
197
target genes.
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EBV-LCLs from normal donors or AD-HIES patients were unstimulated or stimulated with IL-
199
10 (A, B), IL-21 (C) for 3 hours. Expression of PRDM1 and IL2RA was then determined by
200
qPCR (Two way ANOVA with Holm-Sidak post-test, comparison between treated and
201
untreated (PRDM1 and IL2RA), *P < 0.05. mean ± SEM, n=5-9)
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Figure S3: Summary of the proximal stage of signaling disrupted by various AD-HIES
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STAT3 mutations. A diagram showing the stages of signaling affected by each STAT3
205
mutation. IL-21 bound to IL-21R (Protein Data Bank, 3TXG) used as an example of a STAT3
206
signaling cytokine receptor. Phosphorylation of tyrosine 705 (pY705) is represented in the
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STAT3 structure with black spheres (Protein Data Bank, 1BG1). Orange box surrounding the
208
mutation indicates TA domain, yellow box indicates SH2 domain and green box indicates
209
DNA binding domain.
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References
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8.
9. 10. 11. 12. 13.
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Takeda K, Noguchi K, Shi W, Tanaka T, Matsumoto M, Yoshida N, et al. Targeted disruption of the mouse Stat3 gene leads to early embryonic lethality. Proceedings of the National Academy of Sciences of the United States of America 1997; 94:3801-4. Kane A, Deenick EK, Ma CS, Cook MC, Uzel G, Tangye SG. STAT3 is a central regulator of lymphocyte differentiation and function. Current Opinion in Immunology 2014; 28:49-57. Vogel TP, Milner JD, Cooper MA. The Ying and Yang of STAT3 in Human Disease. J Clin Immunol 2015; 35:615-23. Woellner C, Gertz EM, Schaffer AA, Lagos M, Perro M, Glocker EO, et al. Mutations in STAT3 and diagnostic guidelines for hyper-IgE syndrome. Journal of Allergy and Clinical Immunology 2010; 125:424-32. Avery DT, Deenick EK, Ma CS, Suryani S, Simpson N, Chew GY, et al. B cell-intrinsic signaling through IL-21 receptor and STAT3 is required for establishing long-lived antibody responses in humans. Journal of Experimental Medicine 2010; 207:155-71. Ma CS, Chew GY, Simpson N, Priyadarshi A, Wong M, Grimbacher B, et al. Deficiency of Th17 cells in hyper IgE syndrome due to mutations in STAT3. Journal of Experimental Medicine 2008; 205:1551-7. Heimall J, Davis J, Shaw PA, Hsu AP, Gu W, Welch P, et al. Paucity of genotype-phenotype correlations in STAT3 mutation positive Hyper IgE Syndrome (HIES). Clin Immunol 2011; 139:75-84. Giacomelli M, Tamassia N, Moratto D, Bertolini P, Ricci G, Bertulli C, et al. SH2-domain mutations in STAT3 in hyper-IgE syndrome patients result in impairment of IL-10 function. Eur J Immunol 2011; 41:3075-84. He J, Shi J, Xu X, Zhang W, Wang Y, Chen X, et al. STAT3 mutations correlated with hyper-IgE syndrome lead to blockage of IL-6/STAT3 signalling pathway. J Biosci 2012; 37:243-57. Minegishi Y, Saito M, Tsuchiya S, Tsuge I, Takada H, Hara T, et al. Dominant-negative mutations in the DNA-binding domain of STAT3 cause hyper-IgE syndrome. Nature 2007; 448:1058-62. Shuai K, Liu B. Regulation of JAK-STAT signalling in the immune system. Nat Rev Immunol 2003; 3:900-11. Levy DE, Darnell JE, Jr. Stats: transcriptional control and biological impact. Nat Rev Mol Cell Biol 2002; 3:651-62. Bhattacharya S, Schindler C. Regulation of Stat3 nuclear export. Journal of Clinical Investigation 2003; 111:553-9.
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Figure 1
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A
N Terminal
320 Coiled coil
465
DNA binding
Linker
688
SH2
ACCEPTED MANUSCRIPT R382W
B
585
H437P S465F
TA
Q644P L706M Y657N
1000
*** 500
+
WT
-
+
-
R382W
+
H437P
-
+
S465F
Myc-tagged
1
2
3
4
-
-
WT
WT
Flag-tagged -
WT
-
+
-
Q644P
+
Y657N
2
3
4
5
6
7
8
WT
WT
WT
WT
IB: Anti Flag
7
8
9
10
1
2
3
4
5
1.25
Ratio WT/Mutant
1.00
0.75
*
**
Q644P
Y657N
0.50
0.25
0.00 WT
R382W
10
WT
WT
H437P
DNA BD
TA
IP: Anti-Flag
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IB: Anti GAPDH
6
9
SH2
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IB: Anti Myc
5
L706M
WT R382W H437P S465F Q644P Y657N L706M
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+
TA
DNA BD
Input
-
SH2
M AN U
DNA BD
C
-
SC
-
IL-6
***
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geometric MFI
1500
0
D
770
S465F
SH2
L706M
TA
6
7
8
9
10
Figure 2
A
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Nuclear Fraction WT
R382W
H437P
S465F
WT
Y675N
L706M
pSTAT3
Histone H3 GAPDH 15
30
60
0
15
30
60
0
15
30
60
0
15
30
60
0
15
30
1.50 1.25 1.00
SC
***
****
0.50 0.25 0.00 Control
R382W
H437P
S465F
DNA BD 1.25
TE D
*
***
0.75
0.50
AC C
0.25
WT
**
****
R382W
H437P
S465F
Y657N
L706M
SH2
TA
40
SOCS3 Expression Arbitrary Units
Minutes
***
DNA BD
C
0 15 30 60
EP
DNA binding (relative OD)
1.00
0.00
15
30
60
0
15
30
30
*
20
*
**
**
**
10 0 IL-21
-
+
WT
-
+
-
+
R382W H437P
DNA BD
-
+
S465F
-
+
-
+
Y657N
L706M
SH2
TA
60
Minutes
***
0.75
B
0
0 15 30 60
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1.75
60
RI PT
Minutes 0
****
Y657N
L706M
SH2
TA
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Table S1: Effects of STAT3 mutations on the generation of effector lymphocytes and on STAT3 signaling and function
H437P 10
5.3
3.5
3.8
3.5
Biochemical feature of STAT3 signaling Phosphorylation Dimerisation Nuclear localization DNA binding Induction of target genes
R382W Normal Normal
H437P Normal Normal
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R382W 3.55
L706M 7.5
4.2
2.1
7.8
4.5
3.7
2.5
2.5
Q644P Normal ND ND ND
Y657N
L706M Normal
S465F Normal Normal Normal
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1.3
SC
Lymphocyte population/function Th17 cells (%CD45RO-CCR6+ CD4+ T cells); Reference values: 23.0 ± 8.7% (mean ± sd; n=68) % Memory B cells (%CD27+ CD20+ cells) Reference values: 27.5 ± 14.5% (mean ± sd; n=64) Tfh cells (%CD45RA-CXCR5+ CD4+ T cells) Reference values: 8.2 ± 3.3% (mean ± sd; n=72)
STAT3 mutation S465F Q644P 3.3 5.5
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The proportions of Th17 cells, memory B cells, and T follicular helper (Tfh) cells in the peripheral blood of individual(s) with each mutation were determined by flow cytometry. For the R382W and L706M mutations, the values represent the mean from analysis of several patients with the same mutation. For the H437P, Q644P and Y657N mutations the values represent the mean of repeated analyses on the same individual. Reference ranges were from our previously published studies1-4. The effect of each of the mutations tested on the distinct stages of the STAT3 signaling pathway is also indicated.
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References 1. Avery DT, Deenick EK, Ma CS, Suryani S, Simpson N, Chew GY, et al. B cell-intrinsic signaling through IL-21 receptor and STAT3 is required for establishing long-lived antibody responses in humans. Journal of Experimental Medicine 2010; 207:155-71. 2. Deenick EK, Avery DT, Chan A, Berglund LJ, Ives ML, Moens L, et al. Naive and memory human B cells have distinct requirements for STAT3 activation to differentiate into antibody-secreting plasma cells. Journal of Experimental Medicine 2013; 210:2739-53. 3. Ma CS, Avery DT, Chan A, Batten M, Bustamante J, Boisson-Dupuis S, et al. Functional STAT3 deficiency compromises the generation of human T follicular helper cells. Blood 2012; 119:3997-4008. 4. Ma CS, Wong N, Rao G, Avery DT, Torpy J, Hambridge T, et al. Monogenic mutations differentially affect the quantity and quality of T follicular helper cells in patients with human primary immunodeficiencies. J Allergy Clin Immunol 2015; 136:993-1006 e1.
Figure S1
A
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Y657
L706
phosphorylated Y705
Q644
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DNA
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S465
H437
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R382W (DNA BD)
H437P (DNA BD)
S465F (DNA BD) Unstimulated Mutant + IL-6
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R382
Q644P (SH2)
WT + IL-6
Y657N (SH2)
phosphorylated-STAT3
L706M (TA)
Figure S2
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* ns
ns
-
-
ns
ns
ns
2
1
0 IL-10
-
+
WT
+
+
R382W H437P
-
+
S465F
DNA BD
B
-
+
-
+
Y657N
L706M
SH2
TA
SC
1.5 1.0 0.5 0.0 IL-10
-
+
WT
-
+
-
+
R382W H437P
-
+
S465F
*
2.5
ns
2.0
-
+
L706M
SH2
TA
ns
ns
ns
EP
1.5
ns
1.0 0.5
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PRDM1 Expression Fold Change
C
+
Y657N
TE D
DNA BD
-
M AN U
PRDM1 Expression Fold Change
2.0
0.0
IL-21
RI PT
IL2RA Expression Fold Change
A
-
+
WT
-
+
-
+
R382W H437P
DNA BD
-
+
S465F
-
+
-
+
Y657N
L706M
SH2
TA
Figure S3
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RI PT
IL-21
Cytoplasm
Y657N
STAT3
M AN U
Y657N
SC
IL-21R
L706M pY705
Nucleus DNA
AC C
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H437P
R382W
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S0091-6749(16)30282-2
DOI:
10.1016/j.jaci.2016.04.020
Reference:
YMAI 12119
To appear in:
Journal of Allergy and Clinical Immunology
Received Date: 30 November 2015 Revised Date:
24 March 2016
Accepted Date: 5 April 2016
Please cite this article as: Pelham SJ, Lenthall H, Deenick EK, Tangye SG, Elucidating the effects of disease-causing mutations on STAT3 function in autosomal dominant hyper-IgE syndrome, Journal of Allergy and Clinical Immunology (2016), doi: 10.1016/j.jaci.2016.04.020. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Elucidating the effects of disease-causing mutations on STAT3 function in autosomal
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dominant hyper-IgE syndrome
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Simon J Pelham MSc1,2,
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Helen Lenthall B Sc Hons2,
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Elissa K Deenick PhD1,2,
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Stuart G Tangye PhD1,2
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Immunology Research Program, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia;
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St Vincent’s Clinical School, University of New South Wales Australia, Darlinghurst, NSW, Australia
10 11 Corresponding authors
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Dr. Stuart Tangye; Dr Elissa Deenick
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Garvan Institute of Medical Research
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384 Victoria St. Darlinghurst. 2010. NSW. Australia
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e-mail: [email protected]; [email protected]
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This work was supported by project and program grants awarded by the National Health and
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Medical Research Council (NHMRC) of Australia (1016953, 1066694, and 1027400 to E.K.
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Deenick and S.G. Tangye). S.J.P. is supported by an Australian Postgraduate Award from the
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University of New South Wales. S.G. Tangye is a recipient of a Principal Research Fellowship
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(1042925) from the NHMRC of Australia.
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The authors declare no conflicts of interest
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Key words
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• autosomal dominant hyper-IgE syndrome; STAT3; transcription factors; cytokine signaling
29 Abbreviations
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AD-HIES: autosomal dominant hyper-IgE syndrome; DN: dominant negative; JAK: Janus
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activating kinase; STAT: signal transducer and activation of transcription; SH2: Src homology
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2; DNA BD: DNA binding domain; TA: transactivation; EBV-LCL: EBV-transformed
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lymphoblastoid cell line; WT: wild-type
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35 Capsule summary
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Analysis of germline mutations in STAT3 that cause AD-HIES revealed biochemical
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mechanisms of disease-causation. This has implications for developing therapeutics that
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could modulate the function of STAT3 in the settings of immunodeficiency, autoimmunity and
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malignancy.
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To the editor
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Signaling through STAT3 is an important process required for the function of a wide range of
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biological systems. This is demonstrated by embryonic lethality of germline Stat3 gene-
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targeted mice1. Furthermore, heterozygous dominant negative (DN) mutations in STAT3
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cause autosomal dominant hyper IgE syndrome (AD-HIES) in humans, a disease
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characterized by increased susceptibility to infection with Staphylococcus aureus,
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Streptococcus pneumonia and Candida albicans, eczema, extremely elevated levels of serum
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IgE, poor humoral immune responses, and non-immunological features including defects in
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musculoskeletal, dental, and vascular systems.2,
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been reported that affect all functional domains of STAT3.3,
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cytosolic transcription factor with a key role in signaling downstream of numerous cytokines,
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including IL-6, IL-10, IL-21 and IL-232. DN STAT3 mutations cause multiple cellular defects,
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including reduced Th17 cells, memory B cells, T follicular helper cells and MAIT cells, and
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impairing responsiveness of naïve B cells to IL-10 and IL-21 (Online Supplemental data Table
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1)2,
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production in AD-HIES. Despite this, our understanding of the molecular impact of various
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STAT3 mutations remains limited. Indeed, as there is no genotype/phenotype relationship7, it
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remains to be determined how diverse mutations impair STAT3 signaling to manifest the
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same clinical and cellular phenotype. Furthermore, while several studies have examined the
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effects of mutations in separate domains of STAT3 on signaling pathways8-10, few have
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systematically compared the impact of mutations located in different functional domains of
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STAT3.
More than 60 different mutations have STAT3 encodes a latent
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. These defects explain susceptibility to fungal infections and poor antibody
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To explore this, we examined the consequences of distinct disease-causing mutations in the
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DNA binding, SH2 and transactivation domains on each stage of STAT3 signaling. We
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selected 6 mutations previously identified in numerous cohorts of AD-HIES patients, including
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our own, that localize to the DNA binding domain (BD) (R382W, H437P, S465F), SH2 domain
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(Q644P, Y657N) or TA (L706M) domains5, 6 (Figure 1A; Fig S1A Online supplemental data).
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All mutations have a comparable deleterious effect on the generation of Th17, Tfh and
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memory B cells in affected individuals (Online Supplemental data Table S1). To test how
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each mutation affected tyrosine phosphorylation, HEK-293T cells were transfected with
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plasmids encoding WT or mutant STAT3 (Figure 1B, Fig S1B). IL-6 stimulation induced
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phosphorylation at tyrosine 705 (Y705) of WT STAT3 as well as STAT3 harboring mutations
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in the DNA BD, as well as the Q644P SH2 domain mutant (Figure 1B, Fig S1B). In contrast,
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the SH2 domain Y657N and TA domain L706M mutants failed to undergo significant
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phosphorylation in response to IL-6 (Figure 1B, Fig S1B). Reduced STAT3 phosphorylation
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due to Y657N SH2 and L706M TA domain mutants likely results from conformational
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alterations of STAT3 or steric hindrance that limit access of kinases to phosphorylate Y705.
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Thus, only mutations in the proximity of Y705 (Figure 1A) affected STAT3 phosphorylation.
83 Following phosphorylation, STAT3 forms dimers that are required for function11, 12. To test the
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ability of mutant STAT3 to form dimers with WT STAT3, and to recapitulate the setting of
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heterozygous STAT3 mutations in AD-HIES, HEK-293T cells were co-transfected with Myc-
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tagged WT and Flag-tagged mutant STAT3 plasmids (Figure 1C), and then stimulated with IL-
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6. STAT3 multimers were captured by co-immunoprecipitation using anti-Flag mAb. The
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levels of dimer formation between WT STAT3 and those with mutations in the DNA BD or TA
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domain were comparable to WT/WT STAT3 proteins (Figure 1C, D). This is consistent with
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initial observations by Minegishi et al.10 who reported that mutations in the DNA BD did not
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affect STAT3 dimerization. In contrast to DNA BD mutations, SH2 domain mutations reduced
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dimer formation by ~40-50%, with Y657N having the greatest effect (Figure 1C,D). This
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identifies these residues as being critical for intermolecular interactions between
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phosphorylated STAT3 (pSTAT3). It is likely that amino acid substitutions in the SH2 domain
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result in changes in charge, which affect assembly or stability of STAT3 dimers.
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Nuclear translocation of STAT3 is crucial for its role as a transcription factor11,
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investigate the impact of mutations in STAT3 on its nuclear translocation, nuclear extracts
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were prepared from IL-21-stimulated WT and STAT3 mutant EBV-LCLs and the levels of
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pSTAT3 determined. The accumulation of nuclear pSTAT3 in normal EBV-LCLs was maximal
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after 15 mins and declined at times thereafter (Figure 2A). Consistent with reductions in total
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pSTAT3 in transfected HEK-293T cells (Figure 1B; Fig S1B), the Y657N SH2 and L706M TA
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domain mutations resulted in significant reductions in levels of nuclear pSTAT3 in EBV-LCLs
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(Figure 2A). Remarkably, two of the three DNA BD mutations (R382W, H437P) significantly
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reduced nuclear translocation of pSTAT3 (Figure 2A). As several nuclear export sequences
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have been identified in the DNA BD of STAT313, reduced translocation due to these mutations
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this likely results from an imbalance between nuclear import and export. Interestingly, the
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S465F DNA BD mutation had no effect on nuclear pSTAT3 translocation (Figure 2A).
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. To
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Once in the nucleus STAT3 binds specific target sites to regulate gene transcription.11, 12 WT
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STAT3 exhibited strong binding to a canonical target sequence that was maximal after 15-30
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mins of IL-21 stimulation (Figure 2B). Conversely, all STAT3 mutant proteins showed
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significant decreases in their ability to bind DNA (Figure 2B). As some STAT3 mutations
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affected phosphorylation (Y657N, L706M) or nuclear translocation (R382W, H437P), the Page 4 of 8
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reduction in DNA binding reflects not only an impaired ability of mutant STAT3 to bind target
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sequences but also decreases in the amount of pSTAT3 in the nucleus. Consistent with these
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findings, induction of the STAT3 target genes SOCS3 (Figure 2C), PRDM1 (encoding BLIMP-
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1) and IL2RA (Fig S2A, B) were significantly reduced (50-90%) in response to IL-21 and IL-10
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in STAT3 mutant compared to normal EBV-LCLs, thereby indicating that the defect in DNA
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binding results in a functional impairment in the ability to induce gene transcription. Thus,
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impaired DNA binding of mutant STAT3 proteins manifests as reduced gene transcription.
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In conclusion our study gives insight into dysfunctional STAT3-dependent signaling in AD-
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HIES, and allowed for the dissection and identification of specific stages of STAT3 signaling
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affected by these disease-causing mutations (Fig S3; Table S1). This not only highlights
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which aspects of STAT3 signaling are impacted by these mutations but also identifies
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residues and regions of domains in STAT3 that are critical for normal function (Fig S3; Table
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S1). This reveals mechanisms not only for loss-of function of STAT3 in AD-HIES but reveals
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residues/regions of STAT3 that could be targeted in cases where STAT3 is overactive, such
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as gain-of function mutations in STAT3 resulting in multisystemic autoimmunity or
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hematological malignancies, respectively3.
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Simon J Pelham MSc1,2,
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Helen Lenthall B Sc Hons2,
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Elissa K Deenick PhD1,2,
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Stuart G Tangye PhD1,2
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Immunology Research Program, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia;
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St Vincent’s Clinical School, University of New South Wales Australia, Darlinghurst, NSW, Australia
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Figure Legends
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Figure 1. Autosomal dominant hyper IgE syndrome-causing STAT3 mutations: effects
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on phosphorylation and dimerisation.
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(A) Schematic representation of STAT3, with the mutations studied indicated by black lines.
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(B) HEK-293T cells were transfected with Flag-tagged WT or R382W, H437P, S465F,
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Q644P, Y657N or L706M mutant STAT3, and either untreated or stimulated with IL-6 (30
152
mins). Phosphorylation of STAT3 (pSTAT3) was determined by fixing and permeabilising the
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cells and labeling with Abs specific for Flag and pSTAT3 (Y705). Graph shows geometric MFI
154
of pSTAT3 (Two way ANOVA with Dunnett post-test, comparison between IL-6 stimulated WT
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and mutant STAT3. ***p< 0.001. mean ± SEM, n=6). (C, D) HEK-293T cells were co-
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transfected with plasmids containing Myc-tagged WT STAT3 and either Flag-tagged WT or
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mutant STAT3. After 48 hours, cells were harvested, rested for 2 hours then stimulated with
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IL-6 (10 ng/ml, 30 mins). Cell lysates were immunoprecipitated with anti-Flag Ab, and
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assessed for the presence of Myc-tagged WT or mutant STAT3 by Western blotting. (C)
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Diagram of the gel lanes (upper); Input whole cell lysates immunoblotted for Myc, Flag or
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GAPDH showing equivalent amounts of WT and mutant STAT3 (left; lower) and
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immunopreciptiation of lysates with anti-Flag beads, immunoblotted for Myc and Flag (right;
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lower). (D) Quantification of ratio of WT:mutant STAT3, normalized to WT:mutant ratio of the
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input. (One way ANOVA with Dunnett post-test, comparison between WT and various
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mutants. **p < 0.01, *p < 0.05. mean ± SEM, n=5).
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Figure 2. Impaired nuclear translocation, DNA binding and gene induction by mutant
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STAT3 proteins.
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(A, B) EBV-LCLs from normal donors or AD-HIES patients were stimulated with IL-21 for 0,
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15, 30 or 60 minutes, and nuclear fractions then isolated. (A) The abundance of pSTAT3 was
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assessed by determining the ratio of intensity of mutant vs WT pSTAT3 stimulated with IL-21
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for 15 minutes. (Two way ANOVA with Dunnett post-test, comparison between 15 minute time
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points, ***p<0.001, **p< 0.01, *p< 0.05. mean ± SEM, n=4). (B) Binding of WT and mutant
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STAT3 to consensus DNA binding sites was determined (TransAM kit, ActiveMotif). Values
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were normalized to WT STAT3 at 15 mins. (Two way ANOVA with Dunnett post-test,
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comparison between 15 minute time points, ***p< 0.001, **p< 0.01, *p< 0.05. mean ± SEM ,
177
n=3-4). The dashed line represents the basal response (i.e. time 0) of WT EBV LCLs to
178
cytokine stimulation. (C) EBV-LCLs from normal donors or AD-HIES patients were stimulated
179
with IL-21 or media only for 3 hours. Expression levels of SOCS3 were determined by qPCR.
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(Two way ANOVA with Holm-Sidak post-test, comparison between IL-21 stimulated (SOCS3)
181
samples, **P < 0.01, *P < 0.05. mean ± SEM, n=5-9).
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182 Supplemental Online data
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Figure S1: Autosomal dominant hyper IgE syndrome-causing STAT3 mutations and
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effects on cytokine-induced phosphorylation
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(A) Crystallographic structure of a STAT3 dimer bound to DNA (Protein Data Bank, 1BG1).
187
Sites of the mutations are represented by red spheres, phosphorylated tyrosine 705 (Y705) is
188
indicated by black spheres.
189
(B) HEK-293T cells were transfected with Flag-tagged WT STAT3 or STAT3 harbouring the
190
following mutations: R382W, H437P, S465F, Q644P, Y657N or L706M. Cells were left
191
untreated or stimulated with IL-6 for 30 mins. Phosphorylation of STAT3 was determined by
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flow cytometry following fixing and permeabilising the cells and labeling with Abs specific for
193
Flag and pSTAT3 (Y705). Representative histograms of phosphorylated STAT3 induction
194
after IL-6 stimulation.
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Figure S2: Effect of STAT3 loss-of function mutations on induction of expression of
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target genes.
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EBV-LCLs from normal donors or AD-HIES patients were unstimulated or stimulated with IL-
199
10 (A, B), IL-21 (C) for 3 hours. Expression of PRDM1 and IL2RA was then determined by
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qPCR (Two way ANOVA with Holm-Sidak post-test, comparison between treated and
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untreated (PRDM1 and IL2RA), *P < 0.05. mean ± SEM, n=5-9)
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Figure S3: Summary of the proximal stage of signaling disrupted by various AD-HIES
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STAT3 mutations. A diagram showing the stages of signaling affected by each STAT3
205
mutation. IL-21 bound to IL-21R (Protein Data Bank, 3TXG) used as an example of a STAT3
206
signaling cytokine receptor. Phosphorylation of tyrosine 705 (pY705) is represented in the
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STAT3 structure with black spheres (Protein Data Bank, 1BG1). Orange box surrounding the
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mutation indicates TA domain, yellow box indicates SH2 domain and green box indicates
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DNA binding domain.
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References
213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244
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8.
9. 10. 11. 12. 13.
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Takeda K, Noguchi K, Shi W, Tanaka T, Matsumoto M, Yoshida N, et al. Targeted disruption of the mouse Stat3 gene leads to early embryonic lethality. Proceedings of the National Academy of Sciences of the United States of America 1997; 94:3801-4. Kane A, Deenick EK, Ma CS, Cook MC, Uzel G, Tangye SG. STAT3 is a central regulator of lymphocyte differentiation and function. Current Opinion in Immunology 2014; 28:49-57. Vogel TP, Milner JD, Cooper MA. The Ying and Yang of STAT3 in Human Disease. J Clin Immunol 2015; 35:615-23. Woellner C, Gertz EM, Schaffer AA, Lagos M, Perro M, Glocker EO, et al. Mutations in STAT3 and diagnostic guidelines for hyper-IgE syndrome. Journal of Allergy and Clinical Immunology 2010; 125:424-32. Avery DT, Deenick EK, Ma CS, Suryani S, Simpson N, Chew GY, et al. B cell-intrinsic signaling through IL-21 receptor and STAT3 is required for establishing long-lived antibody responses in humans. Journal of Experimental Medicine 2010; 207:155-71. Ma CS, Chew GY, Simpson N, Priyadarshi A, Wong M, Grimbacher B, et al. Deficiency of Th17 cells in hyper IgE syndrome due to mutations in STAT3. Journal of Experimental Medicine 2008; 205:1551-7. Heimall J, Davis J, Shaw PA, Hsu AP, Gu W, Welch P, et al. Paucity of genotype-phenotype correlations in STAT3 mutation positive Hyper IgE Syndrome (HIES). Clin Immunol 2011; 139:75-84. Giacomelli M, Tamassia N, Moratto D, Bertolini P, Ricci G, Bertulli C, et al. SH2-domain mutations in STAT3 in hyper-IgE syndrome patients result in impairment of IL-10 function. Eur J Immunol 2011; 41:3075-84. He J, Shi J, Xu X, Zhang W, Wang Y, Chen X, et al. STAT3 mutations correlated with hyper-IgE syndrome lead to blockage of IL-6/STAT3 signalling pathway. J Biosci 2012; 37:243-57. Minegishi Y, Saito M, Tsuchiya S, Tsuge I, Takada H, Hara T, et al. Dominant-negative mutations in the DNA-binding domain of STAT3 cause hyper-IgE syndrome. Nature 2007; 448:1058-62. Shuai K, Liu B. Regulation of JAK-STAT signalling in the immune system. Nat Rev Immunol 2003; 3:900-11. Levy DE, Darnell JE, Jr. Stats: transcriptional control and biological impact. Nat Rev Mol Cell Biol 2002; 3:651-62. Bhattacharya S, Schindler C. Regulation of Stat3 nuclear export. Journal of Clinical Investigation 2003; 111:553-9.
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Figure 1
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A
N Terminal
320 Coiled coil
465
DNA binding
Linker
688
SH2
ACCEPTED MANUSCRIPT R382W
B
585
H437P S465F
TA
Q644P L706M Y657N
1000
*** 500
+
WT
-
+
-
R382W
+
H437P
-
+
S465F
Myc-tagged
1
2
3
4
-
-
WT
WT
Flag-tagged -
WT
-
+
-
Q644P
+
Y657N
2
3
4
5
6
7
8
WT
WT
WT
WT
IB: Anti Flag
7
8
9
10
1
2
3
4
5
1.25
Ratio WT/Mutant
1.00
0.75
*
**
Q644P
Y657N
0.50
0.25
0.00 WT
R382W
10
WT
WT
H437P
DNA BD
TA
IP: Anti-Flag
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6
9
SH2
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IB: Anti Myc
5
L706M
WT R382W H437P S465F Q644P Y657N L706M
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+
TA
DNA BD
Input
-
SH2
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DNA BD
C
-
SC
-
IL-6
***
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geometric MFI
1500
0
D
770
S465F
SH2
L706M
TA
6
7
8
9
10
Figure 2
A
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Nuclear Fraction WT
R382W
H437P
S465F
WT
Y675N
L706M
pSTAT3
Histone H3 GAPDH 15
30
60
0
15
30
60
0
15
30
60
0
15
30
60
0
15
30
1.50 1.25 1.00
SC
***
****
0.50 0.25 0.00 Control
R382W
H437P
S465F
DNA BD 1.25
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*
***
0.75
0.50
AC C
0.25
WT
**
****
R382W
H437P
S465F
Y657N
L706M
SH2
TA
40
SOCS3 Expression Arbitrary Units
Minutes
***
DNA BD
C
0 15 30 60
EP
DNA binding (relative OD)
1.00
0.00
15
30
60
0
15
30
30
*
20
*
**
**
**
10 0 IL-21
-
+
WT
-
+
-
+
R382W H437P
DNA BD
-
+
S465F
-
+
-
+
Y657N
L706M
SH2
TA
60
Minutes
***
0.75
B
0
0 15 30 60
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1.75
60
RI PT
Minutes 0
****
Y657N
L706M
SH2
TA
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Table S1: Effects of STAT3 mutations on the generation of effector lymphocytes and on STAT3 signaling and function
H437P 10
5.3
3.5
3.8
3.5
Biochemical feature of STAT3 signaling Phosphorylation Dimerisation Nuclear localization DNA binding Induction of target genes
R382W Normal Normal
H437P Normal Normal
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R382W 3.55
L706M 7.5
4.2
2.1
7.8
4.5
3.7
2.5
2.5
Q644P Normal ND ND ND
Y657N
L706M Normal
S465F Normal Normal Normal
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1.3
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Lymphocyte population/function Th17 cells (%CD45RO-CCR6+ CD4+ T cells); Reference values: 23.0 ± 8.7% (mean ± sd; n=68) % Memory B cells (%CD27+ CD20+ cells) Reference values: 27.5 ± 14.5% (mean ± sd; n=64) Tfh cells (%CD45RA-CXCR5+ CD4+ T cells) Reference values: 8.2 ± 3.3% (mean ± sd; n=72)
STAT3 mutation S465F Q644P 3.3 5.5
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The proportions of Th17 cells, memory B cells, and T follicular helper (Tfh) cells in the peripheral blood of individual(s) with each mutation were determined by flow cytometry. For the R382W and L706M mutations, the values represent the mean from analysis of several patients with the same mutation. For the H437P, Q644P and Y657N mutations the values represent the mean of repeated analyses on the same individual. Reference ranges were from our previously published studies1-4. The effect of each of the mutations tested on the distinct stages of the STAT3 signaling pathway is also indicated.
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References 1. Avery DT, Deenick EK, Ma CS, Suryani S, Simpson N, Chew GY, et al. B cell-intrinsic signaling through IL-21 receptor and STAT3 is required for establishing long-lived antibody responses in humans. Journal of Experimental Medicine 2010; 207:155-71. 2. Deenick EK, Avery DT, Chan A, Berglund LJ, Ives ML, Moens L, et al. Naive and memory human B cells have distinct requirements for STAT3 activation to differentiate into antibody-secreting plasma cells. Journal of Experimental Medicine 2013; 210:2739-53. 3. Ma CS, Avery DT, Chan A, Batten M, Bustamante J, Boisson-Dupuis S, et al. Functional STAT3 deficiency compromises the generation of human T follicular helper cells. Blood 2012; 119:3997-4008. 4. Ma CS, Wong N, Rao G, Avery DT, Torpy J, Hambridge T, et al. Monogenic mutations differentially affect the quantity and quality of T follicular helper cells in patients with human primary immunodeficiencies. J Allergy Clin Immunol 2015; 136:993-1006 e1.
Figure S1
A
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Y657
L706
phosphorylated Y705
Q644
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DNA
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S465
H437
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R382W (DNA BD)
H437P (DNA BD)
S465F (DNA BD) Unstimulated Mutant + IL-6
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Y657N (SH2)
phosphorylated-STAT3
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Figure S2
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* ns
ns
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ns
ns
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1
0 IL-10
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R382W H437P
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S465F
DNA BD
B
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Y657N
L706M
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SC
1.5 1.0 0.5 0.0 IL-10
-
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WT
-
+
-
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R382W H437P
-
+
S465F
*
2.5
ns
2.0
-
+
L706M
SH2
TA
ns
ns
ns
EP
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ns
1.0 0.5
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PRDM1 Expression Fold Change
C
+
Y657N
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DNA BD
-
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PRDM1 Expression Fold Change
2.0
0.0
IL-21
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IL2RA Expression Fold Change
A
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WT
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+
-
+
R382W H437P
DNA BD
-
+
S465F
-
+
-
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Y657N
L706M
SH2
TA
Figure S3
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IL-21
Cytoplasm
Y657N
STAT3
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IL-21R
L706M pY705
Nucleus DNA
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