ZNF341 controls STAT3 expression and thereby immunocompetence

Abstract

Signal transducer and activator of transcription 3 (STAT3) is a central regulator of immune homeostasis. STAT3 levels are strictly controlled, and STAT3 impairment contributes to several diseases including the monogenic autosomal-dominant hyper-immunoglobulin E (IgE) syndrome (AD-HIES). We investigated patients of four consanguineous families with an autosomal-recessive disorder resembling the phenotype of AD-HIES, with symptoms of immunodeficiency, recurrent infections, skeletal abnormalities, and elevated IgE. Patients presented with reduced STAT3 expression and diminished T helper 17 cell numbers, in absence of STAT3 mutations. We identified two distinct homozygous nonsense mutations in ZNF341, which encodes a zinc finger transcription factor. Wild-type ZNF341 bound to and activated the STAT3 promoter, whereas the mutant variants showed impaired transcriptional activation, partly due to nuclear translocation failure. In summary, nonsense mutations in ZNF341 account for the STAT3-like phenotype in four autosomal-recessive kindreds. Thus, ZNF341 is a previously unrecognized regulator of immune homeostasis.

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 sign of rubor, calor, 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-D) Pedigrees and genotypes with the nonsense mutated (mut) alleles g.32345116C>T (c.904C>T; p.Arg302*) for Families A-C and g.32349795C>T (c.1156C>T; p.Arg386*) for Family D. Heterozygous carriers are unaffected. Wt, wild-type. 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 CD45RA⁻CCR6⁺CCR4⁺CXCR3⁻ of CD3⁺CD4⁺ in patients (n=6; triangles, Family A; squares, Family D) compared to healthy donor controls (HD; n=8; open circles) (left). In contrast to controls (n=11), patient PBMCs (n=6) fail to differentiate into IL17⁺ cells (CD3⁺CD4⁺CD45RO⁺) upon in vitro stimulation (d4) 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 a 854 amino acids “zinc-finger-only” transcription factor with twelve C2H2 motifs (vertical boxes). R302* and R386* (arrows) delete zinc fingers 2–12 and 4–12, respectively. A putative nuclear localization sequence (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) ZNF341 isoform 1 is undetectable in EBV cell lysates from patient B.II.4 (upper panel). Slightly increased relative ZNF341 mRNA expression in patients´ PBMCs (lower panel; 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 primary skin fibroblasts (PSF) of patient A.II.3 compared to healthy donor (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. Beta 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 clone 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⁺). (B) Reduced phospho-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 patients (solid line) compared to controls (dotted line). Shaded area, unstimulated cells. Only marginal transient increase of p-STAT3 in patients 15 min post stimulation (right). Baseline p-STAT3 levels are reached within 150 min post stimulation. Mean values from independent experiments (HD1, 2 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 CMV minimal promoter) driving a red fluorescence (tdTomato) reporter upon co-transfection with EGFP-tagged wildtype ZNF341 in HEK293T cells (48h). Scale bar 100μM. (B) Relative reporter activity in two independent experiments in quadruplets. Significance and p-values were determined with Mann-Whitney test. (C) ChIP-Seq analysis of ZNF341, performed with distinct antibodies, on EBV-transformed B cells reveal 1658 ZNF341 binding sites across various genomic regions and (D) show high tag densities on the STAT3 promoter region (Chr17: 40,530,000–40,545,000, 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 (48h) showing nuclear localization of EGFP-tagged wild-type ZNF341 and R386*, whereas R302* remains cytoplasmic. Scale bar, 10 μM.