A recessive form of hyper-IgE syndrome by disruption of ZNF341-dependent STAT3 transcription and activity

Abstract

Heterozygosity for human signal transducer and activator of transcription 3 (STAT3) dominant-negative (DN) mutations underlies an autosomal dominant form of hyper-immunoglobulin E syndrome (HIES). We describe patients with an autosomal recessive form of HIES due to loss-of-function mutations of a previously uncharacterized gene, ZNF341 ZNF341 is a transcription factor that resides in the nucleus, where it binds a specific DNA motif present in various genes, including the STAT3 promoter. The patients' cells have low basal levels of STAT3 mRNA and protein. The autoinduction of STAT3 production, activation, and function by STAT3-activating cytokines is strongly impaired. Like patients with STAT3 DN mutations, ZNF341-deficient patients lack T helper 17 (T_H17) cells, have an excess of T_H2 cells, and have low memory B cells due to the tight dependence of STAT3 activity on ZNF341 in lymphocytes. Their milder extra-hematopoietic manifestations and stronger inflammatory responses reflect the lower ZNF341 dependence of STAT3 activity in other cell types. Human ZNF341 is essential for the STAT3 transcription-dependent autoinduction and sustained activity of STAT3.

Fig. 1.. Autosomal recessive ZNF341 deficiency.

(A) Pedigrees of the six unrelated families, showing familial segregation of the c.904C>T (p.R302X) mutant ZNF341 allele in kindreds A-C, and of alleles c.1062delG (p.K355fs), c.1647C>G (p.Y542X), and c.583C>T (p.Q195X) in kindreds D-F, respectively. Generations are designated by a Roman numeral (I, II). P1–P8 are represented by black symbols; the probands are indicated by arrows. Individuals of unknown genotype are labeled with “E?” (B) Representative images of the cutaneous phenotypes of P2 and P4, with tongue and thumb candidiasis, eczematous lesions of the thighs, with excoriated and lichenified lesions (P2), and atrophic hypopigmented scars (P4). (C) Comparison of the patients’ haplotypes with ancestral alleles. MAF, minor allele frequency. Stars and hash tags indicate frequencies extracted from dbSNP and EXAC, respectively. (D) Frequency and Combined Annotation–Dependent Depletion score (CADD), for all homozygous variants reported in the ExAC database. The dotted line corresponds to the mutation significance cutoff (MSC). The CADD scores of 34, 35, 36 and 40 for the Q195X, K355fs/K362fs, Y542X/Y549X and R302X mutations, respectively, are well above the MSC of 3.31 for ZNF341 (34). The ZNF341 gene has intermediate gene damage index (GDI) and neutrality index (NI) scores, of 3.72 and 0.30, respectively, suggesting that ZNF341 is not under strong purifying selection, consistent with the segregation of ZNF341 deficiency as an AR disorder. (E) Schematic representation of the ZNF341 protein. ZNF341 has two main isoforms, isoform 1 and isoform 2, differing by 21 in-frame nucleotides at the 3’ end of exon 6 of the gene, resulting in proteins of 847 and 854 amino acids in length, respectively. Exons are designated by Roman numerals. Exon boundaries are depicted with dashed lines. Predicted zinc finger domains (C2H2) and proline-rich regions are shown as light red and blue boxes, respectively. Red arrows indicate the mutations. The predicted NLS sequences are indicated with thick black arrows.

Fig. 2.. Molecular characterization of ZNF341 mutations and their expression in leukocyte subsets.

(A) HEK293T cells were transfected with an empty pCDNA plasmid or with pCDNA plasmids encoding the wild-type (WT), R302X, K355fs, or Y542X ZNF341 isoform 1. Cytoplasmic and nuclear fractions were separated and subjected to immunoblotting with a polyclonal Ab (pAb) against the N-terminal segment of ZNF341 (HPA024607). α-tubulin and lamin A/C were used as controls for the cytoplasmic and nuclear fractions, respectively. Western blots representative of three different experiments are shown. (B) SV40-fibroblasts from P4 were transfected with constructs encoding the two WT ZNF341 isoforms or the R302X mutant isoform, with a V5 tag at their C-terminus. Cells were analyzed by confocal microscopy 24 hours after transfection. WT and mutant ZNF341 isoforms were detected with an anti-V5 antibody. The cytoplasm and nucleus were identified by phalloidin and DAPI staining, respectively. An example of staining representative of three independent experiments is shown. (C) HEK293T cells were cotransfected with empty plasmid or plasmids containing cDNAs encoding WT isoform 1 and/or 2 of ZNF341, each tagged C-terminally with either V5 or Myc/DDK. Whole-cell lysates (left panel) or anti-V5 immunoprecipitates (right panel) are shown. Vinculin was used as a loading control. Results representative of three independent experiments are shown. (D-E) RNA extracted from (D) the Epstein-Barr virus-immortalized B (EBV-B) cells of four controls and P3, P4, P6 and P7 and (E) Saimiri-transformed T (HVS-T) cells from five controls, P3 and P4, were subjected to reverse transcription-quantitative polymerase chain reaction for total ZNF341. Data are displayed as 2^-ΔCt after normalization relative to GUS (endogenous control) expression (ΔCt). Results representative of three independent experiments are shown. Bars represent the mean and the standard deviation. Dots represent the mean of technical duplicates. (F-G) Western blot of nuclear protein extracts (50 μg) obtained from (F) EBV-B cells from P4 stably transduced with an empty vector (EV) or WT ZNF341, three controls and four patients (P3, P4, P6, P7) and (G) HVS-T cells from P3 stably transduced with an empty vector (EV) or WT ZNF341, two controls and two patients (P3 and P4), with a mouse monoclonal anti-ZNF341 antibody (8B3.1, raised against ZNF341 C-terminal residues 366–468). Lamin A/C was used as a loading control. Results representative of three independent western blots are shown. (H) RNA was extracted from the indicated leukocyte subsets from two healthy controls and subjected to RT-qPCR for total ZNF341. Data are displayed as 2^-ΔCt after normalization relative to GUS (endogenous control) expression (ΔCt). Results representative of three independent experiments are shown. Bars represent the mean and the standard deviation. Dots represent the mean of technical triplicates. (I) Nuclear protein extracts (50 μg) were obtained from the indicated leukocyte subsets from a healthy control and subjected to western blotting with a mouse anti-ZNF341 mAb (8B3.1). Lamin A/C was used as a loading control. Results representative of three independent experiments are shown. (J-L) Nuclear protein extracts were obtained from monocytes (J), CD3⁺ T cells (K), total dendritic cells (K), and basophils (L) from a healthy control, and were subjected to western blotting with the mouse anti-ZNF341 mAb (8B3.1). We used 100 μg, 50 μg and 25 μg of nuclear protein extracts in J, K and L, respectively. Lamin B1 was used as a loading control. Nuclear extracts from P4 EBV-B cells stably transduced with an empty vector (EV) or WT ZNF341 served as negative and positive controls, respectively, for ZNF341 expression in I-L. Results representative of three independent experiments are shown.

Fig. 3.. NK-, ILC-, B- and T-cell subpopulation immunophenotyping.

(A) Natural killer cell immunophenotyping for controls (n=44) and patients (n=6, P2–P7), showing the total NK cell (CD3^-CD56⁺) frequency in lymphocytes (left panel), the frequency of CD56^bright cells within the NK-cell compartment (middle panel) and the terminal differentiation profile of the CD56^dim compartment. (B) ILC phenotyping, showing the frequencies of total ILCs (Lin^-CD7⁺CD56^-CD127⁺), ILC1 (EOMES^-IFNγ⁺), ILC2 (GATA3⁺IL13⁺) and ILCP (CD117⁺) among the CD45⁺ PBMCs of controls (n=21), ZNF341-deficient patients (n=3, P2–P4) and patients with STAT3 DN mutations (S3^DN, n=3). All analyses were conducted after the exclusion of dead cells. Mann-Whitney tests were used for comparisons. (C) Frequency of CD27⁺ memory cells within the B-cell compartment of patients (n=6) and controls (n=47). Mann-Whitney tests were used for comparisons. (D) Frequency of IgM⁺, IgA⁺ and IgG⁺ cells within the memory B-cell compartment of patients (n=6), and controls (n=26–47). Mann-Whitney tests were used for comparisons. (E-F) Frequency of naïve (CD45RA⁺CCR7⁺), central memory (CD45RA^-CCR7⁺), effector memory (CD45RA^-CCR7^-) and T_EMRA (CD45RA⁺CCR7^-) cells among the CD4⁺ (E) and CD8⁺ (F) T cells of patients (P2–P7) and controls (n=51). Mann-Whitney tests were used for comparisons. (G). T-cell subset immunophenotyping. Frequency of Treg (CD3⁺CD4⁺CD25^hiFoxP3⁺) cells in the CD4⁺ T-cell compartment, and frequency of γδ T cells (CD3⁺TCR-γδ⁺), MAIT (CD3⁺CD161⁺TCR-vα7.2⁺) and iNKT (CD3⁺TCR-iNKT⁺) cells among the T cells of patients (n=5 or n=6, P2–P7) and controls (n=18–46). Mann-Whitney tests were used for comparisons.

Fig. 4.. Transcriptional activity of ZNF341.

(A) Motif analysis for the top 500 ZNF341 binding sites in P4 EBV-B cells transduced with WT ZNF341 isoform 1, as compared with empty vector. (B) ChIP-Seq profile of IgG, EV, and ZNF341 (isoform 1 or isoform 2) or control CD3⁺ T cells after 2 days of stimulation with plate-bound anti-CD3 and soluble anti-CD28 mAb for the genomic loci corresponding to STAT3, STAT1 and ZNF341. The scale of the y-axis, corresponding to the number of reads, is indicated at the top right corner of each plot. (C) Models tested to determine the orientation and spacing preferences of the ZNF-like core motif and the Sp1-like motif. (D) Distribution of the observed spacing counts between the Sp1-like motif and the ZNF-like core motif in EBV-B cells from P4 stably transduced with WT ZNF341. Counts are displayed relative to the model tested (left) and p-values are calculated for a given spacing (right). (E) Electrophoretic mobility shift assay (EMSA) of nuclear extracts of HEK293T cells transfected with an empty vector (EV), the C-terminal DDK-tagged WT, R302X, K355fs, or Y542X ZNF341 alleles. Extracts were incubated with a 5’ fluorescent DNA probe containing the putative bipartite ZNF341-binding motif from the STAT3 promoter in the presence or absence of a control isotype (IgG2b) or an anti-ZNF341 mAb (8B3.1) for supershift experiments. An untagged competitor probe (CP) of similar sequence but with a concentration ten times higher was used to test binding specificity. Results representative of three independent experiments are shown. (F) Pulldown of the 5’ biotinylated DNA probe containing the putative bipartite ZNF341-binding motif from the STAT3 promoter after incubation with nuclear extracts of HEK293T cells transfected with an empty vector (EV), the C-terminal DDK-tagged WT, R302X, K355fs, or Y542X ZNF341 alleles. Extracts were incubated in the presence or absence of various doses of a competitor probe (CP) to test binding specificity (1:0, 1:10 and 1:100 are the ratios of specific biotinylated probe to CP). The presence or absence of ZNF341 constructs in the pulldown fraction (left panel), or in the input (right panel) was assessed by immunoblotting for the C-terminal DDK tag. Results representative of three independent experiments are shown. (G-H) Pulldown of the 5’ biotinylated DNA probe containing the putative bipartite ZNF341-binding motif from the STAT3 promoter after incubation with nuclear extracts of negatively sorted primary CD3⁺ T cells of one control (G) or of EBV-B cell lines from two controls, P4, P6 and P7 (H). Extracts were incubated in the presence or absence of a competitor probe (CP) at a concentration ten times higher than that of the biotinylated DNA probe, to test binding specificity. The presence or absence of ZNF341 in the pulldown fraction was assessed by immunoblotting with an anti-ZNF341 mAb (8B3.1). Lamin A/C was used as a loading control. Results representative of three independent experiments are shown. (I) Luciferase activity of HEK293T cells cotransfected with WT or mutant ZNF341 isoform 1, plus a pGL4.10 reporter plasmid encoding the luciferase cDNA downstream from the bipartite ZNF341 binding motif of the promoters of STAT1 and STAT3. The results shown are the mean and standard deviation of three independent experiments. (J) Total RNA sequencing data for the EBV-B cells of P4 stably transduced with WT ZNF341 isoform 1 (Iso 1), isoform 2 (Iso 2) or an empty vector (EV). Scatter dot plots comparing mRNA levels in cells transduced with ZNF341 isoform 1 or 2 with those in cells transduced with the empty vector. (K) STAT1 and STAT3 mRNA levels extracted from total RNA sequencing data.

Fig. 5:. STAT3 production and function in primary cells.

(A-D) STAT3 production and function in primary fibroblasts. STAT3 mRNA levels (A) as evaluated by RT-qPCR after RNA extraction from the primary fibroblasts of three controls and three patients (P2–P4). Data are displayed as 2^-ΔCt after normalization relative to GUS (endogenous control) expression (ΔCt). Data representative of two independent experiments are shown. (B) STAT3 levels, as evaluated by flow cytometry, in primary fibroblasts. Left panel, representative image of STAT3 expression in fibroblasts from P3 and a healthy control, as well as the isotypic control. Right panel, recapitulative graph showing the mean fluorescence intensity (MFI) of STAT3, as measured by flow cytometry, in three controls, and three patients (P2–P4). Data representative of two independent experiments are shown. (C) Recapitulative graph of the mean fluorescence intensity (MFI) of pY705-STAT3, as evaluated by flow cytometry, in primary fibroblasts from three controls, and three patients (P2–P4), left unstimulated or after 30 minutes of stimulation with IL6/IL-6Rα. (D) SOCS3 mRNA levels, as evaluated by RT-qPCR after RNA extraction from the primary fibroblasts of three controls and three patients (P2–P4), with or without two hours of stimulation with IL6/IL-6Rα. Data are displayed as 2^-ΔCt after normalization relative to GUS (endogenous control) expression (ΔCt). Data representative of two independent experiments are shown. (E-I) STAT3 production and function in monocytes. STAT3 mRNA levels (E), as evaluated by RT-qPCR after RNA extraction from the primary monocytes of three controls, P4 and P5. Data are displayed as 2^-ΔCt after normalization relative to GUS (endogenous control) expression (ΔCt). (F) STAT3 expression, as measured by flow cytometry in primary monocytes. Recapitulative graph of the mean fluorescence intensity (MFI) of STAT3, as measured by flow cytometry, in three controls, P4 and P5. (G) STAT3 phosphorylation (pY705), evaluated by flow cytometry, in monocytes from P5 and a healthy control, after 30 minutes of stimulation with IL-10. Representative image for two patients tested. (H) SOCS3 mRNA levels, as evaluated by RT-qPCR after RNA extraction from the primary monocytes of five controls and two patients (P4 and P5), with or without two hours of stimulation with IL-10. Data are displayed as 2^-ΔCt after normalization relative to GUS (endogenous control) expression (ΔCt). (I) Percentage of TNF⁺ monocytes for nine controls, P4 (tested twice) and three STAT3-DN patients, after four hours of stimulation with LPS in the presence or absence of IL-10, as evaluated by flow cytometry. (J) Flow cytometry quantification of STAT3 levels in primary NK cells. Recapitulative graph of the mean fluorescence intensity (MFI) of STAT3 measured in four controls and P4. (K) Graph of the mean fluorescence intensity (MFI) of pY705-STAT3, as evaluated by flow cytometry, in primary CD56^dim NK cells from four controls and P4, left unstimulated or after 30 minutes of stimulation with IL-21. (L) Graph of the mean fluorescence intensity (MFI) of STAT3 in primary naïve CD8⁺ T cells, measured in four controls and P4. (M) Representation of the phosphorylation of STAT3 (pY705) in primary naïve CD8⁺ T cells from one control and P4, evaluated by flow cytometry, without stimulation or after 30 minutes of stimulation with IL-21 (left panel). Right panel: graph of the mean fluorescence intensity (MFI) of pY705-STAT3, as evaluated by flow cytometry, in primary naïve CD8⁺ T cells from four controls and P4, left unstimulated or after 20 minutes of stimulation with IL-21. (J-M) Data are representative of two independent experiments (P4 and P8). (N) Flow cytometry quantification of STAT3 levels in primary B cells. Recapitulative graph of the mean fluorescence intensity (MFI) of STAT3, as measured by flow cytometry, in six controls, P4 and P6. (O) Quantification of the phosphorylation of STAT3 (pY705) in primary B cells from three controls, P4 and P6, evaluated by flow cytometry, with the cells left unstimulated or after 30 minutes of stimulation with IL-21. (P) Levels of STAT3 mRNA in naïve B cells from one healthy individual, one STAT3 DN patient and one ZNF341 deficient patient (P8) after 0, 4, 48 and 96 hours of stimulation with the indicated combinations of CD40L and IL-21. Data are displayed as 2^-ΔΔCt after normalization relative to GUS (endogenous control) expression (ΔCt) and non-stimulated cells of the control (ΔΔCt). (Q) Sorted naïve B cells from controls (n=14), one STAT3-DN patient, and six ZNF341 deficient patients (P2–P4, P6, P7 and P8) were cultured in the presence of CD40L (200 ng/mL), with or without IL-21 (50 ng/mL) for 7 days. The production (pg/ml) of IgM, IgG and IgA was then assessed by Ig heavy chain-specific ELISA on cell culture supernatants. (R) Sorted memory B cells from controls (n=12) and five ZNF341 deficient patients (P2–P4, P6 and P7) were cultured in the presence of CD40L (200 ng/mL), with or without IL-21 (50 ng/mL), for 7 days. The production (pg/ml) of IgM, IgG and IgA was then assessed by Ig heavy chain-specific ELISA on cell culture supernatants.

Fig. 6.. Impaired STAT3 production and function in naïve CD4⁺ cells from ZNF341-deficient patients.

(A) STAT3 mRNA levels, as evaluated by RT-qPCR after RNA extraction from the naïve CD4⁺ T cells of five controls, P4, P5 and P7. Data are displayed as 2^-ΔCt after normalization relative to GUS (endogenous control) expression (ΔCt). (B) STAT3 protein levels, as evaluated by flow cytometry, in naïve CD4⁺ T cells. Left panel, representative histogram of STAT3 levels in P4, one healthy control and the isotypic control. Right panel, recapitulative graph of the mean fluorescence intensity (MFI) of STAT3, as evaluated by flow cytometry, in naïve primary CD4⁺ T cells from five controls, P2, P3 and P4, left unstimulated or after 30, 120 and 240 minutes of stimulation with IL-6/IL-6Rα. (C) Flow cytometry quantification of the phosphorylation of STAT3 (pY705) in naïve CD4⁺ T cells after stimulation with IL-6/IL-6Rα. The left panel shows a representative flow cytometry histogram for STAT3 (pY705) in the naïve CD4⁺ T cells of P4 and in those of one healthy control, left unstimulated or after 30 minutes of stimulation with IL-6/IL-6Rα. The right panel shows a recapitulative graph of the mean fluorescence intensity (MFI) of STAT3 (pY705) for five controls, P2, P3 and P4, after 0, 30, 120 and 240 minutes of IL-6/IL-6Rα stimulation. Dots and error bars show the means and standard deviations, respectively. (D) Levels of STAT3 mRNA in thawed naïve CD4⁺ T cells from healthy individuals (n=5), two STAT3 DN (S3^DN) patients and three ZNF341-deficient patients (P4, P5 and P7), after two hours of stimulation with the indicated combinations of IL-6/IL-6Rα and beads (anti-CD2/CD3/CD28 mAb-coated beads). Data are displayed as 2^-ΔCt after normalization relative to GUS (endogenous control) expression (ΔCt). (E) Western blot of cytoplasmic and nuclear protein extracts of naïve CD4⁺ T cells obtained from one healthy control after four hours of stimulation with the indicated combinations of IL-6/IL-6Rα and beads (anti-CD2/CD3/CD28 mAb-coated beads) in the presence or absence of 10 μg/mL cycloheximide. GAPDH and lamin B1 were used as loading controls for the cytoplasmic and nuclear fractions, respectively. Data representative of three independent experiments are shown. (F) Induction of SOCS3 mRNA in control naïve CD4⁺ T cells after stimulation with IL-6/IL-6Rα and/or beads (anti-CD2/CD3/CD28 mAb-coated beads) for 0, 2, 4, 8, 12 and 24 hours. Data are displayed as 2^-ΔCt after normalization relative to GUS (endogenous control) expression (ΔCt). Dots and error bars represent the mean and standard deviation for two donors. (G-H) Levels of SOCS3 (G) or ZNF341 (H) mRNA in thawed naïve CD4⁺ T cells from healthy individuals (n=5), two STAT3 DN patients (S3^DN) and two (P4 and P7) or three (P4, P5 and P7) ZNF341-deficient patients after two hours of stimulation with the indicated combinations of IL-6/IL-6Rα and beads (anti-CD2/CD3/CD28 mAb-coated beads). Data are displayed as 2^-ΔCt after normalization relative to GUS (endogenous control) expression (ΔCt). Bar graphs and error bars represent the mean and standard deviation.

Fig. 7.. Impaired Th17 differentiation in ZNF341-deficient patients.

(A) Frequency of T-helper (Th) subsets within the CD4⁺ memory compartments of controls (n=49) and patients (n=5). Subsets were defined as follows: Tfh (CXCR5⁺), Th1 (CXCR5^-CXCR3⁺CCR4^-CCR6^-), Th2 (CXCR5^-CXCR3^-CCR4⁺CCR6^-), Th1* (CXCR5^-CXCR3⁺CCR4^-CCR6⁺) and Th17 (CXCR5^-CXCR3^-CCR4⁺CCR6⁺). Mann-Whitney tests were used for comparisons. (B) Secretion (pg/ml) of Th1 (IFN-γ, TNF), Th2 (IL-4, IL-5, IL-13) and Th17 (IL-17A, IL-17F, IL-22) cytokines by memory CD4⁺ T cells after five days of culture under Th0 conditions (anti-CD2/CD3/CD28 mAb-coated beads). Mann-Whitney tests were used for comparisons. (C) Secretion (pg/ml) of Th17 (IL-17A, IL-17F) cytokines by naïve CD4⁺ T cells after five days of culture under Th0 (anti-CD2/CD3/CD28 mAb-coated beads) or Th17 (anti-CD2/CD3/CD28 mAb-coated beads together with IL-1β, IL-6, IL-21, IL-23 and TGF-β) polarizing conditions. Mann-Whitney tests were used for comparisons. (D) Expression of RORC, TBX21 and GATA3 by naïve CD4⁺ T cells after five days of culture under Th0, Th17 or Th1 (anti-CD2/CD3/CD28 mAb-coated beads together with IL-12) or Th2 (anti-CD2/CD3/CD28 antibody-coated beads together with IL-4) polarizing conditions, or by memory (Mem.) CD4⁺ T cells, as determined by RT-qPCR, relative to GAPDH. Results are shown for four controls and three ZNF341-deficient patients (P2–P4).