Nuclear role of WASp in the pathogenesis of dysregulated TH1 immunity in human Wiskott-Aldrich syndrome

Abstract

The clinical symptomatology in the X-linked Wiskott-Aldrich syndrome (WAS), a combined immunodeficiency and autoimmune disease resulting from WAS protein (WASp) deficiency, reflects the underlying coexistence of an impaired T helper 1 (TH1) immunity alongside intact TH2 immunity. This suggests a role for WASp in patterning T(H) subtype immunity, yet the molecular basis for the TH1-TH2 imbalance in human WAS is unknown. We have discovered a nuclear role for WASp in the transcriptional regulation of the TH1 regulator gene TBX21 at the chromatin level. In primary TH1-differentiating cells, a fraction of WASp is found in the nucleus, where it is recruited to the proximal promoter locus of the TBX21 gene, but not to the core promoter of GATA3 (a TH2 regulator gene) or RORc (a TH17 regulator gene). Genome-wide mapping demonstrates association of WASp in vivo with the gene-regulatory network that orchestrates TH1 cell fate choice in the human TH cell genome. Functionally, nuclear WASp associates with H3K4 trimethyltransferase [RBBP5 (retinoblastoma-binding protein 5)] and H3K9/H3K36 tridemethylase [JMJD2A (Jumonji domain-containing protein 2A)] proteins, and their enzymatic activity in vitro and in vivo is required for achieving transcription-permissive chromatin dynamics at the TBX21 proximal promoter in primary differentiating TH1 cells. During TH1 differentiation, the loss of WASp accompanies decreased enrichment of RBBP5 and, in a subset of WAS patients, also of filamentous actin at the TBX21 proximal promoter locus. Accordingly, human WASp-deficient TH cells, from natural mutation or RNA interference-mediated depletion, demonstrate repressed TBX21 promoter dynamics when driven under TH1-differentiating conditions. These chromatin derangements accompany deficient T-BET messenger RNA and protein expression and impaired TH1 function, defects that are ameliorated by reintroducing WASp. Our findings reveal a previously unappreciated role of WASp in the epigenetic control of T-BET transcription and provide a new mechanism for the pathogenesis of WAS by linking aberrant histone methylation at the TBX21 promoter to dysregulated adaptive immunity.

Fig. 1

Nuclear and cytoplasmic dual-site localization of WASp in primary differentiating T_H1 cells. (A) Nomarski (differential interference contrast) and their corresponding immunofluorescence images of normal primary T_H cells, undifferentiated (T_H0) or T_H1-differentiating, dual- or triple-labeled with WASp (green) and DAPI (blue). All images were acquired by deconvolution microscope at ×63 magnification. The data are representative of at least 30 single cells analyzed. (B to E) Western blot analysis probed with the indicated antibodies performed on the cytoplasmic (cyt) and nuclear (nu) extracts generated from primary T_H cells [(B) and (E)] or T_H lines [(C) and (D)]. The purity of the two cell fractions was verified with histone H3 and LAMP1 probing, and an example in the T_H1 sample is shown. Data in (E) are from the nuclear extracts. (F) Representative immunofluorescence images of normal primary T_H1-differentiating cells, triple-labeled with WASp (green), DAPI (blue), and nucleolar mark B23 (red, top panel); hyperphosphorylated RNA polymerase II (Ser²) (red, middle panel); or TRAP220/MED1 (red, bottom panel). Also shown are images where the red or green color is removed with Photoshop (see figs. S2 and S3 for tiled images of the entire T cell 3D volume). (G) Coimmunoprecipitation with antibodies to WASp and IgG immunoblotted (IB) with the indicated antibodies performed on the nuclear extracts generated from the primary T_H1-differentiated cells from normal donors. The same blot was sequentially reprobed with the indicated antibodies. The use of TrueBlot reagent resulted in the reduction of signals from the denatured heavy and light chains.

Fig. 2

Profile of WASp interaction in vivo with the human T_H cell genome. (A) Some examples of WASp-bound genomic regions in primary human T_H1-differentiating cells. Plots show ChIP enrichment ratios of WASp for all oligonucleotide probes within the indicated genomic loci analyzed on both forward and reverse chromatin strands. The data are for either forward or reverse chromatin strand. The exon-intron configuration of the indicated RefSeq genes (in red) derived from the Vista plot (in blue) is aligned with the corresponding chromosomal coordinates. The fold enrichment peaks for WASp (in gray) are derived from the raw data displayed in figs. S8 and S9. See table S1 for the list of WASp-binding and WASp-nonbinding target genes. (B) Conserved DNA sequence motif identified among WASp-bound genomic regions with the CEAS bioinformatic algorithm. (C) Coimmunoprecipitation assay demonstrating association of WASp with Sp1 in the nuclear fraction of primary T_H1 cells. (D) Pie chart demonstrating enrichment of WASp to specific genomic cis elements in primary human T_H1-differentiating cells.

Fig. 3

Endogenous WASp is recruited to the TBX21 proximal promoter in vivo. (A) Chromosomal coordinates of the WASp-bound cis elements at the human TBX21 promoter locus on chromosome 17. A, WBR1; B, WBR2; C, GAS (core motif: 5′-TTCAGGCAA-3′); TATA, TATA sequence (TCATAA). (B) Quantitative MNase-ChIP qPCR assay. Quantifying mononucleosomal enrichment of WASp and other trans-regulatory proteins at the TBX21 DNA, as well as the changes in the epigenetic profile at these sites in primary undifferentiated T_H cells and their T_H1-differentiating counterparts. For each primary antibody ChIP, a control ChIP performed with the corresponding isotype antibody was always included for specificity. IgG ChIP values are not shown to avoid crowding. The final results are displayed as percent amplicons normalized to nuclear input chromatin after subtracting background ChIP signals obtained with the corresponding isotype IgG antibody. Real-time PCR data were derived by converting the C_t values to absolute copy numbers with cloned DNA plasmid standard dilution curve. For each ChIP-qPCR assay, no DNA control samples were always included to negate potential human cell contamination. Data represent the average of triplicates from multiple experiments in purified T_H cells derived from at least seven normal donors, with bars indicating SE. See figs. S8, S10, and S14 for the complementary data mapping WASp-binding sites at the TBX21 locus with the ChIP-chip approach. The efficiency of chromatin digestion by MNase was >90% (fig. S14B). Asterisk denotes ChIP signals at or below the background obtained with the corresponding IgG control antibody. (C) DHS pattern at the three sampled cis regions of TBX21 promoter in primary undifferentiated T_H and differentiating T_H1 cells. T_H cell nuclei were treated with increasing doses of DNase I enzyme from 0 to 10 U, and the number of amplicons lost was quantified by real-time qPCR and is displayed as percent of DNase I–untreated control. Data represent the average of triplicates from four independent experiments, with bars indicating SE. (D) Quantitative sequential MNase-ChIP assay. Data for WASp/STAT1, WASp/RNAP2, WASp/RBBP5, and WASp/JMJD2A co-occupancy at the same cis elements of the TBX21 promoter locus in primary T_H1 cells. Sequential rounds of conventional ChIP assay was performed to obtain the absolute copy numbers for each immunoprecipitation (IP)/input ratio. This primary value was then converted to fold enrichment value, where the SeqChIP value obtained with the control IgG antibody was normalized to 1.0. Data represent the averages of triplicates in purified T_H cells from two normal donors, with bars indicating SE.

Fig. 4

Endogenous nuclear WASp in primary T_H1-differentiating cells associates with H3K4 trimethylase and H3K9 tridemethylase activities in vitro. (A) Colorimetric methylation or demethylation assay. Purified nuclear lysates from primary differentiating T_H1 cells were enriched for WASp, IgG, and the respective positive control histone methyltransferase or histone demethylase enzyme by immunoprecipitation with specific antibodies. The resultant eluates were then tested for the indicated histone methylation or demethylation activity as per the manufacturer’s specifications. Additional controls in LFA-1– and N-WASp–enriched eluates were also included in the assay. The primary values acquired as optical density per hour per milliliter (OD hour⁻¹ ml⁻¹) were converted to the displayed percent methylase or demethylase activity normalized to the activity of their respective positive control (histone-modifying) enzymes provided in the kit. The data represent an average of at least five biological replicates, with bars indicating the SE, reflecting the interindividual variation. (B) The coimmunoprecipitation aliquots obtained from the above assay were separately coincubated with either the recombinant human unmodified H3 (for the methylation assay) or the endogenous mix of methylated histones derived from HeLa cells (for the demethylase assay) as substrates. The reaction was assessed by Western blot analysis probing with the specified antibodies to detect the formation of the indicated histone-modified products. Blots were stripped and reprobed for total H3 as a loading control. Immunocomplexes enriched with antibodies to RBBP5 and JMJD2A served as positive controls. A representative Western blot from two independent experiments is shown. The signal density was quantified, and results were graphically displayed as fold change in relation to total H3. (C) Nuclear lysates from normal T_H1-differentiating primary cells derived from two representative normal donors were subjected to reciprocal coimmunoprecipitation assay with the indicated antibodies along with the corresponding IgG controls. The data are representative of at least three independent experiments in different donor samples. (D) 3D immunofluorescence images of primary T_H1 cells triple-labeled with WASp (green), DAPI (blue), and RBBP5 (red, top panel); JMJD6 (red, middle panel); or JMJD2A (red, bottom panel). The degree of overlap between any two fluorochromes was quantified for the entire 3D volume of the cell (z stack) and is shown as Pearson’s r_p values (mean ± SEM). Calculations to quantify protein colocalization were performed on the merged three-color images. The data are representative of at least 30 single T_H1 cells analyzed.

Fig. 5

Critical role of WASp in the epigenetic reprogramming of the TBX21 proximal promoter during T_H1 differentiation in primary T_H cells. (A, C, and D) Histone modification and protein binding patterns inscribed at the TBX21 promoter in the indicated primary T_H cells (normal and three WAS patients), undifferentiated T_H0 and T_H1-differentiating, resolved at the mononucleosomal level by MNase-μChIP assay with between 1000 and 5000 T_H cells. In all experiments, three to five independent ChIPs were performed for the three cis sites (WBR1, WBR2, and GAS) with each antibody (plus the corresponding isotype IgG antibody), and the data were analyzed by triplicate qPCR. Background enrichment obtained with the isotype IgG antibodies was subtracted from the final values shown. Data are expressed as percent immunoprecipitation relative to nuclear input chromatin (mean ± SEM) averaged from all three cis sites within the TBX21 promoter. μChIP assay precipitates more DNA relative to input than does conventional ChIP, as reflected by enhanced precipitation values when compared with the corresponding values depicted in Fig. 3. Only those primary antibody μChIP enrichment values that were above the background obtained with the concomitantly performed IgG antibody μChIP were included in the analysis. (B) The stacked column representation of the primary μChIP values compares the percentage contribution of each histone modification to the total combinatorial histone profile at the TBX21 promoter locus spanning <1 kb from TSS.

Fig. 6

Histone profile, DHS pattern, and transcription factor expression profile in T_H lines lacking WASp from natural mutation or RNAi-mediated depletion. (A) FACS profile depicting the transfection efficiency of control and WASp-shRNA-GFP knockdown (k/d) constructs in Jurkat T cells with the BTX electroporator. (B) Western blot analysis of control and WASp knockdown Jurkat T cells enriched after sorting GFP⁺ T cells on days 2 to 4 (d2/d3/d4) after electroporation. (C) Corresponding densitometric quantification for day 2 or 3 blots. Individual immunoblot images were obtained by sequential reprobing of a single blot with the indicated antibodies. The displayed blot is representative of two independent experiments. (D) Absolute quantification of mRNA expression in the sorted GFP⁺ (control or WASp knockdown) Jurkat T cells (~95% purity) performed with real-time qPCR. “T_H0” denotes Jurkat T cells propagated in T_H-neutral culture conditions (IL-2 alone), and “T_H1” denotes activation under complete T_H1-polarizing conditions. The displayed data are an average of triplicates from at least five experiments, with bars indicating SE. P values reporting on the significance of the observed differences in the mRNA copy numbers between control and WASp knockdown T cells were calculated with Wilcoxon nonparametric statistical test. **P ≤ 0.01. Unmarked bars denote non-significant differences. (F) Corresponding epigenetic profiles at the TBX21 locus in the control and WASp knockdown Jurkat T cells. (E and F) Conventional MNase-ChIP analysis performed on the indicated HTLV-immortalized T_H lines [normal, WAS-1, WAS-5, and WAS-5 T_H line reconstituted with normal WASp (WAS-5 corr)] or Jurkat T_H cells depleted of WASp by RNAi that were activated under T_H1-polarizing conditions. Primary ChIP values for the individual histone modifications (E) and the relative percent enrichment depicting the combinatorial histone profile (F) at the TBX21 promoter are both shown. The background ChIP values obtained with the corresponding isotype IgG antibody were deducted from the final values displayed in the figure. Data are expressed as percent immunoprecipitation relative to nuclear input chromatin (mean ± SEM) and represent an average of five to seven experiments for the HTLV T_H lines and two experiments for Jurkat T cells each performed in triplicates. (G) DHS pattern at the TBX21 promoter in the indicated T_H lines activated under T_H1-polarizing conditions. The amplicon calculations are as described in the legend to Fig. 3C. The nuclease-insensitive DNA in WAS-5 T cells was rendered nuclease-sensitive at a DNase I dose of 20 U (data not shown). (H and I) Protein (H) and mRNA (I) expression profile in the nuclear and cytoplasmic fractions in normal, WAS-5, and WAS-5 corrected (WAS-5 corr) T_H lines resolved by Western blot analysis (H) and real-time PCR assays (I). The RT-PCR values are average of triplicates, with bars indicating SE. ***P < 0.001, Wilcoxon nonparametric statistical analysis that compares values in normal with those in WAS T_H cells. See fig. S19 to contrast this result with the result demonstrating an intact ability of the same WAS T_H lines to differentiate down the T_H2 pathway.

Fig. 7

T_H cells from WAS-1 patients display functional characteristics similar to those in normal T_H cells. (A) Left panel: Western analysis performed on the nuclear lysates of T_H1-activated WAS-1 T_H cells using the indicated antibodies. This mutant WASp was only detected with the rabbit polyclonal antibody but not with the mouse monoclonal antibody. Middle panel: corresponding immunofluorescence images of WAS-1 T_H cells triple-labeled with antibodies to WASp (green), T-BET (red), and DAPI (blue). Right panel: mRNA expression profile of the same WAS-1 T_H cells, either T_H1-activated (T_H1) or undifferentiated (T_H0), is shown for the indicated genes. The primary values in T_H1 cells were adjusted for the β-actin mRNA expression and displayed as fold change over the steady-state T_H0 values (y axis). (B) Coimmunoprecipitation assay performed with the antibodies to WASp and isotype IgG on the nuclear lysate derived from T_H line generated from a patient with clinical grade 1 (WAS-1). The same gel was sequentially reprobed with the indicated antibodies. The use of TrueBlot reagent resulted in the reduction of signals from the denatured heavy and light chains. The blot is representative of two independent experiments in different donors. (C and D) MNase-μChIP assay was performed on the primary T_H cells derived from another WAS-1 patient, and the ChIP enrichment results averaged from three experiments are shown along with SEM (C). In (D), the stacked column representation of the primary ChIP values compares the percentage contribution of each histone modification to the total combinatorial histone profile at the TBX21 promoter locus. The asterisk denotes undetectable ChIP enrichment (or enrichment at or below that obtained with control IgG antibody) for H3K27me3.