Research ArticleIMMUNODEFICIENCIES

ZNF341 controls STAT3 expression and thereby immunocompetence

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Science Immunology  15 Jun 2018:
Vol. 3, Issue 24, eaat4941
DOI: 10.1126/sciimmunol.aat4941
  • Fig. 1 Representative clinical and radiological manifestations in patients with homozygous ZNF341 nonsense mutations.

    (A) Severe eczema in patient B.II.4 on the upper arm and cubital. (B) Patient C.II.1 with eczema on the neck and a cold skin abscess in the lumbar region missing the typical inflammatory signs of rubor, calor, and dolor. (C) Oral thrush due to Candida in patient A.II.2. (D) Chest radiograph of patient A.II.1 showing bilateral pneumonia with positive air bronchogram, bronchiectasis, and pneumatoceles.

  • Fig. 2 Homozygous nonsense mutations in ZNF341 cause HIES with reduced TH17 cell numbers in patient PBMCs.

    (A to D) Pedigrees and genotypes with the nonsense mutated (mut) alleles g.32345116C>T (c.904C>T; p.Arg302*) for families A to C and g.32349795C>T (c.1156C>T; p.Arg386*) for family D. Heterozygous carriers are unaffected. Circles, female; squares, male; filled symbols, affected individuals with HIES; open symbols, healthy members; slash, deceased individual; double horizontal lines, consanguinity; black dot, miscarriage. (E) Both mutations predict premature termination of translation. (F) Flow cytometry of PBMCs demonstrate reduced TH17 cell counts on the basis of CD45RACCR6+CCR4+CXCR3 of CD3+CD4+ in patients (n = 6; triangles, family A; squares, family D) compared with HD controls (HD; n = 8; open circles) (left). In contrast to controls (n = 11), patient PBMCs (n = 6) fail to differentiate into IL-17+ cells (CD3+CD4+CD45RO+) upon in vitro stimulation (day 4) with TH17-polarizing cytokines IL-1β and IL-6 plus T cell activation/expansion (right). Significance was determined using Mann-Whitney test. (G) ZNF341 is an 854–amino acid “zinc finger–only” transcription factor with 12 C2H2 motifs (vertical boxes). R302* and R386* (arrows) delete zinc fingers 2 to 12 and 4 to 12, respectively. A putative NLS (blue) is retained in the R386* mutant. Numbers indicate amino acid positions in NP_001269862.

  • Fig. 3 Reduced STAT3 expression in patient-derived cells.

    (A) Full-length ZNF341 isoform 1 is undetectable in EBV cell lysates from patients with R302* (B.II.4 and B.II.1) and R386* (D.II.4 and D.II.2) mutations, respectively (top). Slightly increased relative ZNF341 mRNA expression in patients’ PBMCs (bottom; patients, n = 4; HD, n = 8). Data from independent experiments were normalized to mean of relative expression in controls. (B) Reduced STAT3 mRNA expression in patient-derived cells. For PBMCs, data from independent experiments were normalized to mean of relative expression in controls (patients, n = 4; HD, n = 10). EBV cell lines: combined data from two independent experiments were normalized to relative expression of one control (patient A.II.1; HD, n = 5). HVS-transformed T cell line or PSFs of patient A.II.3 compared with HD [mean values and SD of two (HVS) or three (PSF) independent experiments]. (C) Western blot and quantitative densitometry demonstrate reduced STAT3 expression in patient-derived PBMCs, EBV cells, and PSF. β-Actin and GAPDH were used as loading controls. TC, travel control; FC, freezing control. (D) ZNF341 knockout in Ramos cells by using CRISPR/Cas9 technology showed reduced STAT3 protein expression in clones 1 and 2 in comparison to wt Ramos cells. GAPDH was used as loading control.

  • Fig. 4 Patients’ primary T cells and EBV-transformed B cell lines showed reduced Y705 phosphorylation of STAT3.

    (A) Impaired IL-6 induced Y705 phosphorylation of STAT3 in patients’ PBMCs (gate CD3+) compared with healthy controls. (B) Reduced phospho-STAT3 (p-STAT3) in IFN-α–treated EBV cells from patients with R302* mutations (left). Bar graphs show SD of duplicates; MFI, mean fluorescence intensity. Representative histograms (middle) demonstrating reduced p-STAT3 in patient (solid line) compared with control (dashed line). Shaded area, unstimulated cells. Only marginal transient increase of p-STAT3 in patients 15 min after stimulation (right). Baseline p-STAT3 levels are reached within 150 min after stimulation. Mean values from independent experiments (HD1, HD2, and B.II.4, n ≥ 3; B.II.1, n = 2) are shown.

  • Fig. 5 ZNF341 binds to the STAT3 promoter.

    (A) Activation of a synthetic STAT3 promoter (with the −535/−33 upstream genomic sequence fused to the CMV minimal promoter) driving a red fluorescence (tdTomato) reporter upon cotransfection with EGFP-tagged wt or mutant ZNF341 in HEK293T cells (48 hours). Scale bar, 100 μm. (B) Relative reporter activity in two independent experiments in quadruplicate. Significance and P values were determined with Mann-Whitney test. n.s., not significant. (C) ChIP-seq analysis of ZNF341, performed with distinct antibodies, on EBV-transformed B cells reveals 1658 ZNF341 binding sites across various genomic regions and (D) shows high tag densities on the STAT3 promoter region (Chr17: 40,530,000 to 40,545,000, hg19 build; normalized tags). (E) Distribution of ZNF341 ChIP-seq signal across the 1658 binding sites. Green, 36 super-binding sites (SBS) with >200 normalized tags; blue, remaining binding sites (RBS). (F) Normalized tags in SBS and RBS as a fraction of total. (G) Cis-regulatory sequences associated with ZNF341 occupancy. P values (italics) reflect the significance of motif occurrence. (H) A 30-nt cis-regulatory sequence associated with ZNF341 occupancy (P value as above) and with ZNF341 occupancy in SBS. (I) Representative confocal images of transfected HEK293T (48 hours) showing nuclear localization of EGFP-tagged wt ZNF341 and R386*, whereas R302* remains cytoplasmic. Scale bar, 10 μm.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/3/24/eaat4941/DC1

    Section S1. Case reports for families A to D

    Section S2. Defective B cell development and TH17 cell differentiation in HIES patients with ZNF341 nonsense mutations

    Section S3. Exclusion of mutations in STAT3 in the etiology of the HIES phenotype in family A

    Section S4. Identification of ZNF341 as an AR-HIES–associated gene in a linkage region on chromosome 20 by WES

    Section S5. Expression of various ZNF341 mRNA variants in multiple cell lines and PBMCs

    Section S6. Supplementary Materials and Methods

    Fig. S1. B and T cell subsets in patient-derived and HD PBMCs.

    Fig. S2. Ubiquitous expression of ZNF341 mRNA in various cell types.

    Fig. S3. No association of the genomic region 20q11.22 spanning the ZNF341 locus with atopy.

    Fig. S4. Ectopic expression of ZNF341 variants in HEK293T cells.

    Fig. S5. Ectopically expressed wt ZNF341 binds to the STAT3 promoter leading to transcriptional activation.

    Fig. S6. Occupancy by ZNF341 across the STAT3 promoter as determined by ChIP.

    Fig. S7. ZNF341 R386* shows reduced binding to the STAT3 promoter.

    Fig. S8. Flow cytometry gating strategy for in vitro TH17 cell differentiation assay.

    Fig. S9. Flow cytometry gating strategy for Y705 phosphorylation of STAT3.

    Fig. S10. Flow cytometry gating strategy for immune phenotyping of PBMCs.

    Fig. S11. Flow cytometry gating strategy for IL-22+ T cells.

    Table S1. Clinical and immunological phenotype of HIES patients with ZNF341 mutations.

    Table S2. ZNF341 encodes three protein coding isoforms.

    Table S3. Transcriptome analysis of patient A.II.1 with gene-specific fold changes in comparison to healthy sibling A.II.5.

    Table S4. Raw data.

    References (4753)

  • Supplementary Materials

    Supplementary Material for:

    ZNF341 controls STAT3 expression and thereby immunocompetence

    Stefanie Frey-Jakobs, Julia M. Hartberger, Manfred Fliegauf, Claudia Bossen, Magdalena L. Wehmeyer, Johanna C. Neubauer, Alla Bulashevska, Michele Proietti, Philipp Fröbel, Christina Nöltner, Linlin Yang, Jessica Rojas-Restrepo, Niko Langer, Sandra Winzer, Karin R. Engelhardt, Cristina Glocker, Dietmar Pfeifer, Adi Klein, Alejandro A. Schäffer, Irina Lagovsky, Idit Lachover-Roth, Vivien Béziat, Anne Puel, Jean-Laurent Casanova, Bernhard Fleckenstein, Stephan Weidinger, Sara S. Kilic, Ben-Zion Garty, Amos Etzioni, Bodo Grimbacher*

    *Corresponding authors. Email: bodo.grimbacher{at}uniklinik-freiburg.de

    Published 15 June 2018, Sci. Immunol. 3, eaat4941 (2018)
    DOI: 10.1126/sciimmunol.aat4941

    This PDF file includes:

    • Section S1. Case reports for families A to D
    • Section S2. Defective B cell development and TH17 cell differentiation in HIES patients with ZNF341 nonsense mutations
    • Section S3. Exclusion of mutations in STAT3 in the etiology of the HIES phenotype in family A
    • Section S4. Identification of ZNF341 as an AR-HIES–associated gene in a linkage region on chromosome 20 by WES
    • Section S5. Expression of various ZNF341 mRNA variants in multiple cell lines and PBMCs
    • Section S6. Supplementary Materials and Methods
    • Fig. S1. B and T cell subsets in patient-derived and HD PBMCs.
    • Fig. S2. Ubiquitous expression of ZNF341 mRNA in various cell types.
    • Fig. S3. No association of the genomic region 20q11.22 spanning the ZNF341 locus with atopy.
    • Fig. S4. Ectopic expression of ZNF341 variants in HEK293T cells.
    • Fig. S5. Ectopically expressed wt ZNF341 binds to the STAT3 promoter leading to transcriptional activation.
    • Fig. S6. Occupancy by ZNF341 across the STAT3 promoter as determined by ChIP.
    • Fig. S7. ZNF341 R386* shows reduced binding to the STAT3 promoter.
    • Fig. S8. Flow cytometry gating strategy for in vitro TH17 cell differentiation assay.
    • Fig. S9. Flow cytometry gating strategy for Y705 phosphorylation of STAT3.
    • Fig. S10. Flow cytometry gating strategy for immune phenotyping of PBMCs.
    • Fig. S11. Flow cytometry gating strategy for IL-22+ T cells.
    • Table S1. Clinical and immunological phenotype of HIES patients with ZNF341 mutations.
    • Table S2. ZNF341 encodes three protein coding isoforms.
    • Table S3. Transcriptome analysis of patient A.II.1 with gene-specific fold changes in comparison to healthy sibling A.II.5.
    • References (47–53)

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    Other Supplementary Material for this manuscript includes the following:

    • Table S4 (Microsoft Excel format). Raw data.

    Files in this Data Supplement:

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