Disruption of hSWI/SNF complexes in T cells by WAS mutations distinguishes X-linked thrombocytopenia from Wiskott-Aldrich syndrome

WASp, the protein mutated in Wiskott-Aldrich syndrome (WAS) supports actin polymerization in the cytoplasm by verprolin-homology/cofilin-homology/acidic(VCA)-domain using an actin-related-protein (ARP)2/3-dependent mechanism,¹  and transcription in the nucleus through a mechanism that is undefined, but appears to be ARP2/3-independent.^2,3 WAS mutations are causative of the human disease,⁴  and are linked to at least 3 clinical phenotypes (ie, gene pleiotropy).⁵  Loss-of-function mutations associate with X-linked thrombocytopenia (XLT), a platelet defect presenting with bleeding symptoms, or WAS, where, in addition to thrombocytopenia, immunodeficiency, autoimmunity, and cancer predominate.^6-9  In WAS, multiple cell-lineages of the immune system are affected.¹⁰  Gain-of-function mutations associate with X-linked neutropenia (XLN), a white-cell defect presenting with infections.¹¹  Loss-of-function missense mutations that still allow expression of mutant WASp are enigmatic in that the same mutation is associated with either XLT or XLT that progresses to WAS. Yet predicting which XLT patient will or will not develop WAS is impossible. Notably, WAS genotype-phenotype correlations show a predilection for missense mutations occurring in WASp-homology1(WH1)-domain but not VCA-domain to manifest XLT-to-WAS disease progression.^6-9  Such observations suggest novel, VCA:ARP2/3-independent function(s) of WASp in immunobiology, whose impairment we postulate contributes to the development of XLT-to-WAS cross-phenotype (CP) progression.⁵ 

Elucidating the underlying mechanism for the CP effect in WAS is a major challenge in the field. Currently, neither XLT nor XLT-to-WAS progression can be modeled in mice. Therefore, one approach to elucidate the pathophysiologic mechanism underlying CP effects is to perform functional studies of gene mutations directly in human cells that are relevant to WAS symptomatology.⁵  Because immune dysregulation is a primary manifestation of human WAS,^6,8  we investigated the molecular effects of WAS mutations on human CD4⁺T-helper(T_H) lymphocytes crucial for adaptive immunity.

We previously identified a nuclear location of WASp in T_H1 cells, where it is required for the “activating” histone H3K4me3 modification at the TBX21 promoter, a T_H1-network gene that is also a WASp-target gene.²  Furthermore, nuclear-WASp but not cytoplasmic-WASp associates with RBBP5 (a subunit of mixed lineage leukemia [MLL]-complex) and catalyzes H3K4me3 modification independently of its VCA-domain, and this domain, we show, is nonessential for T_H1 gene activation.³  In Xenopus oocytes, the gene activation function of Wave1, a WASp-family protein, is also independent of its VCA-like (verprolin-homology) domain.¹²  Although these findings align well with the observation that VCA-domain missense mutations generally do not result in XLT-to-WAS progression in humans,^6-9  the question remains: why are some WAS mutations more pathogenic to the immune system whereas others spare it completely? Answering this will require clarifying the mechanism(s) by which WASp functions in the nucleus of human T_H cells and other immune cells, and how individual WAS mutations differ in their proclivity to perturb this process.

Here, we addressed both issues, focusing mainly on the chromatin events of RNA polymerase II–dependent transcription, because the newfound nuclear roles of WASp, Neural-WASp, and Wave1 (all WASp family proteins) are linked to transcription.^2,12,13  Our study uncovers an unanticipated relationship between WASp and hSWI/SNF-complex in the T_H1 cell nucleus. It identifies 2 mechanistic, disease-related signaling cascades, WASp→BRM→Notch→NF-κB and WASp→EP400→H2A.Z, whose differential impairment by WH1-mutations, but not non–WH1-mutations, correlates with clinical severity grades in human XLT/WAS disease spectrum.

Peripheral blood mononuclear cells from a normal donor and a WAS patient were used to establish CD4⁺ T_H-cell lines by transformation with human T-lymphotropic virus type-1 (HTLV-1) as described.¹⁴  Immortalized WAS^null T_H cells were transfected to express different disease-causing mutations.³  Briefly, full-length (FL)-WASp cDNA was subcloned into mammalian expression vector pCMV6 containing Flag/Myc dual-tags (Origene). All disease-associated, amino acid substitutions in WASp cDNA were performed using QuickChange^IISite-Directed Mutagenesis (Stratagene) (supplemental Table 1, available on the Blood Web site), and mutant sequences were confirmed by DNA sequencing. FL-WASp and its mutants were transfected into Jurkat or WAS^null T_H cells by AmaxaNucleofector (Lonza). Stable expression was verified by immunoblotting and flow cytometry. Cells were cultured under T_H1- or T_H2-skewing or nonskewing T_H0 conditions as described.³ 

Multiple MS assays on immunoprecipitated WASp-enriched protein complexes were performed as described.³  Briefly, lysates from micrococcal nuclease (MNase)-treated nuclei of human primary or Jurkat T_H1-skewed cells expressing WASp-Flag/Myc dual-tagged protein were incubated with anti-WASp, or anti-Flag and anti-Myc, or their isotype-Ig antibodies. The recovered polypeptides were analyzed by nano-LC-mass spectrometry (MS)/MS on an LTQ-Orbitrap Velos mass spectrometer using the SEQUEST database.

Coimmunoprecipitation (coIP) and immunoblotting (IB) assays were performed as described^2,3  using the commercial reagents (supplemental Table 1).

Intracellular protein staining was performed as described.³  Briefly, cells were treated with protein transport inhibitors (GolgiPlug and GolgiStop), T-cell receptor (TCR)-activated, fixed, permeabilized, and labeled with fluorochrome-conjugated antibodies against cytokines and/or transcription factors. Cells were analyzed with a Becton-Dickinson LSRII using FACSDiva. Geometric mean fluorescence intensity was calculated using quadrant/histogram statistics.

The supernatant was harvested from untransfected and WASp mutant transfected T_H0 (nonskewed) or T_H1- or T_H2-skewed cells, and normal T_H cells in culture. Expression of granulocyte macrophage–colony-stimulating factor, interferon gamma (IFNG), and interleukin 4 (IL4) cytokines was quantitated by enzyme-linked immunosorbent assay (ELISA) (R&D) in 3 replicates.

All single- and sequential-ChIP assays were performed with MNase-digested chromatin isolated from ∼5000 cells after fixing protein-DNA interactions with 1% formaldehyde as described.^2,3  ChIP-grade antibodies and their isotype-Ig antibodies were used to pull-down DNA:Protein complexes (supplemental Table 1). ChIPed samples were used for quantitative polymerase chain reaction (qPCR) analysis and the derived C_t values converted to absolute copy numbers using cloned-DNA plasmid standard dilution curve. Nonspecific signals obtained with IgG-ChIP were subtracted from test samples. RNA prepared from ∼5000 nonskewed T_H0, T_H1-, or T_H2-skewed cells using Quick-RNA MiniPrep (Zymo Research) was used to synthesize cDNA, and the reverse transcriptase PCR (RT-PCR) analysis performed on a 7500HT RT-PCR System (Applied Biosystems) using primers/probes (detailed in supplemental Table 1). RT-PCR and ChIP-qPCR were performed from the same biological sample. The derived C_t-values were converted to absolute copy numbers with a cloned-DNA plasmid standard dilution curve^2,3 

The PCR-based DHS assay was performed as described.²  Briefly, T-cell nuclei were isolated with the Nuclei Isolation Kit (Sigma-Aldrich) and incubated with 6 U of DNase I enzyme and 1 μg of nuclei at 37°C for 10 minutes. This dose was selected as optimum from a serial dilution curve performed previously.²  Samples were analyzed by qRT-PCR to determine the number of amplicons remaining after nuclease treatment.

Nuclear and cytosolic fractions were isolated from T_H cells. Electrophoretic mobility shift assay (EMSA) was carried out in 5 g nuclear extract and oligonucleotide-containing consensus for NF-κB binding site. Oligonucleotide was end-labeled to a specific activity of 10⁵ CPM using γ-[³²P]-ATP and T4-polynucleotide kinase and purified on a Nick column. Reaction mixtures with radiolabeled oligonucleotides were incubated for 20 minutes and then resolved on 6% nondenaturing polyacrylamide gels after adding 0.1% bromophenol blue, and then subjected to autoradiography. For competition assays, 30-fold excess unlabeled oligonucleotide (competitor) or unlabeled mutated oligonucleotide (Mutator) was added for 20 minutes after incubation with oligonucleotides. For super-shift assays, specific antibody was added for 20 minutes after the reaction with oligonucleotide was completed.

Gene promoters that lack WASp or Wave1 show impaired H3K4me3 enrichment,^2,12  which in the case of WASp occurs consequent to MLL (RBBP5) perturbation.²  Because SWI/SNF-like chromatin-remodeling complexes (CRCs) favor binding H3K4me3-marked promoters,¹⁵  we hypothesized that WASp effect on transcription is linked to MLL-SWI/SNF chromatin-signaling module. To this end, we queried our mass-spectrometry (MS) proteomic dataset of nuclear and cytoplasmic WASp complexes previously generated from human T_H1-skewed cells, both primary and Jurkat T cells, the latter transfected with Flag/Myc-tagged WASp.³  Using a candidate, hypothesis-driven approach, we captured polypeptides of hSWI/SNF-complexes (hBRM, BRG1, BAF47, BAF53a, BAF57, BAF60, BAF155, BAF170, SMARCA1, and SMARCA5), hSWR1 (EP400, SRCAP), and β-actin from both endogenous WASp and Flag/Myc-tagged transfected WASp (Figure 1A and supplemental Figure 1). The presence of hBRM and the absence of BAF180 from the WASp-proteome reported by MS (Figure 1A) suggest that nuclear-WASp associates predominantly with BAF but not PBAF complexes in human T_H1-skewed cells.^16-18  Notably, only nuclear-WASp but not cytoplasmic-WASp associates with CRCs. Although the binding of some of the CRCs to nuclear-WASp could be nonspecific, the persistence of these associations after MNase digestion favors the capture of specific chromatin-located, WASp:SWI/SNF-complexes, as previously demonstrated for other chromatin-bound proteins.^19,20  Nonetheless, the WASp:SWI/SNF association yielded by MS was validated by sequential coIP (1stIP:anti-Flag; 2ndIP:anti-Myc) in MNase-treated Jurkat T_H1-skewed cells transfected with Flag/Myc doubly-tagged WASp (Figure 1B). The collective presence of these SWI/SNF subunits suggests a role for nuclear-WASp in remodeling the gene locus with which it interacts.

To test the aforementioned hypothesis, we focused mainly on “core” T_H1-genes (IFNG, TBX21), because nuclear localization of WASp and its targeting to “core” T_H2 genes (IL4, GATA3) are negligible in T_H2-skewed cells (supplemental Figure 2A).³  Full-length (FL), Flag/Myc-tagged WASp was stably expressed in a patient-derived T_H-line lacking endogenous WASp (previously described).³  Upon T_H1-skewing, ChIP demonstrates that endogenous CRCs (BAF47, BAF53, BAF170, hBRM, BRG1, EP400) and transfected Flag-WASp (or endogenous WASp) are both enriched at the promoters of IFNG and TBX21 (Figure 1C). WASp (or Flag) is not enriched at the promoter of CSF2, a WASp-nontarget gene in T_H1-skewed cells, or at COL8A2-TRAPPC3 intergenic locus that is devoid of protein-coding genes (supplemental Figure 2A), which together denote targeting specificity of WASp in T_H1-cell genome. Significantly, sequential-ChIP showed that WASp and CRCs co-occupy the same promoter regions of IFNG and TBX21 genes (Figure 1C).

Because CRC activity is essential for remodeling T_H1-gene loci,^16,21-23  we asked whether pathogenic WAS missense mutations impair T_H1 gene induction and, if so, whether they do so by disrupting CRC function at T_H1 gene promoters. We introduced disease-causing, point mutations in full-length WASp and stably expressed each mutant in patient-derived T_H cells lacking endogenous WASp. Mutations in PH-domain (S24F), WH1-domain (T45M, R86C), basic-domain (A236G, D224G), and VCA-domain (K476E, R477K) all reported to result in at least XLT were selected (Figure 2A and supplemental Figure 3A).^6-9  Similar to FL-WASp, all mutants translocate to the nucleus in T_H1- and TCR-activation–dependent manner (reported by pTyr319 ZAP70) because their association with importin-β1 remains intact (supplemental Figure 3D-E).³  Because WASp-deficient T_H cells demonstrate defect in T_H0 > T_H1 gene activation that is corrected by re-expressing FL-WASp,^2,3  we asked whether re-expression of pathogenic WASp-mutants similarly restores this deficiency. Upon T_H1-skewing, both WH1 mutants fail to restore the deficient expression of IFNG and TBX21 mRNA and protein, latter determined by ELISA and intracellular protein staining with flow cytometry (Figure 2B and supplemental Figure 4A-C). Dissimilarly, all non–WH1-domain mutants, including a PH-domain mutant, restore T_H1-gene (mRNA and/or protein) expression to the level achieved with FL-WASp. Because these WH1 mutants clinically manifest XLT-to-WAS disease progression, whereas non–WH1 mutants (except PH-mutant S24F) do not,^6-9  the observed mRNA and protein expression profiles of T_H1 genes correlate with their respective disease severity grades. Notably, none of the mutants impaired T_H2-driven upregulation of IL4 or GATA3 mRNA and protein (Figure 2B and supplemental Figure 4A-C), suggesting that the deleterious effect of WH1-domain mutations is context-specific.

To clarify the molecular basis for impaired T_H1 but intact T_H2 gene activation, we investigated chromatin-signaling events at T_H1 and T_H2 gene loci in WAS-null T_H cells reconstituted with the representative WH1 mutants (T45M, R86C) or non–WH1 mutants (A236G, R477K, S24F). Despite intact nuclear localization of all mutants under T_H1 skewing (supplemental Figure 3D-E), locus-specific recruitment of WH1 mutants (determined with both anti-WASp and anti-Flag antibodies in ChIP assays) at TBX21 and IFNG promoters is impaired compared with that of non–WH1 mutants (Figure 2C, first row; supplemental Figure 5C). Correspondingly, only WH1 mutants show a coordinate reduction in the promoter enrichment of BAF47 and BAF170, the 2 SWI/SNF subunits critical for optimal nucleosomal remodeling (Figure 2C, second row; supplemental Figure 5C).²⁴  These WH1 mutants, however, do not impair promoter enrichment of BAF53 (ARP4) or β-actin, the 2 SWI/SNF subunits critical for BRG1 recruitment to chromatin and for its maximum adenosine triphosphatase (ATPase) activity (Figure 2C, third row).²⁵  Consequently, recruitment of BRG1 to T_H1-gene promoters is unaffected, whereas that of hBRM is impaired by WH1 mutants (Figure 2C, fourth row). WH1 mutants, however, do not disrupt promoter recruitment of hBRM/BAFs to CSF2 in T_H1-skewed cells (Figure 2C) or to IL4 and GATA3 in T_H2-skewed cells (supplemental Figure 4), implying that the deleterious effect of WH1 mutants is both gene- and T_H1/T_H2-differentiation–specific. In support of ChIP findings, sequential coIP (1stFlag>2ndMyc) on MNase-treated T_H1-skewed cells also shows disrupted binding of WH1 mutants to BAF47, BAF170, and hBRM, whereas binding to BAF53, β-actin, or BRG1 is unaffected (Figure 2D). Because BRG1 and hBRM are present in separate CRCs classified as BAF (hBRM-containing) or PBAF (hBRM-devoid, BRG1-containing),^16-18  our data propose a model wherein the deleterious effect of WH1 mutations on T_H1 gene promoters is linked to impaired BAF but not PBAF chromatin-remodeling activity. A more direct evidence to support this model, however, will require RNAi-mediated depletion of BRG1 and hBRM in human T cells.

To determine whether WASp:hBRM association is functionally relevant in vivo, we tested the hypothesis that WASp effect on T_H1-transcriptional reprogramming is linked with Notch, because hBRM but not BRG1 recruits Notch to gene promoters.²⁶  Two-step coIP (1stFlag>2ndMyc) on MNase-treated T_H1-skewed cells showed that the transfected FL-WASp binds endogenous Notch1-intracellular-domain (NICD1), CBF1/RBP-Jκ, and Mastermind-like1 (MAML1) (Figure 3A), a ternary complex that converts CBF1 from a repressor to an activator.^27,28  Accordingly, single and sequential MNase-ChIP assays demonstrate FL-WASp coenriching with endogenous NICD1, CBF1, or MAML1 at the same promoter loci of IFNG or TBX21 (Figure 3B), which are both Notch-target genes.^29-31  These data, suggestive of WASp:Notch alliance in T_H1 activation, raised the possibility that WH1 mutations that impair BRM activity would contemporaneously impair Notch signaling at T_H1-gene promoters.²⁶  CoIP demonstrates that WH1 mutants (T45M, R86C) do not bind these Notch components, whereas non–WH1 mutants (A236G, R477K, S24F) bind them normally (Figure 3A and supplemental Figure 5B). Correspondingly, promoter coenrichment of Notch components is diminished in T_H1-skewed cells expressing WH1 mutants, but not non–WH1 mutants, demonstrated by single and sequential ChIP assay (Figure 3B and supplemental Figure 5C). In T_H2-skewed cells, however, chromatin enrichment of SWI/SNF- and Notch-complexes at IL4 and GATA3 promoters is unaffected in both WH1- and non-WH1 mutant–expressing cells (supplemental Figure 2B), implying context-dependent WASp specificity. We conclude that nuclear-WASp is required for both hBRM- and Notch-driven chromatin-signaling cascade(s) during T_H1 but not T_H2 differentiation, and that the “hot spot” missense mutations in the WH1 domain of WASp disrupt this signaling cascade.

To further consolidate the WASp:BRM:Notch alliance in T_H1 differentiation, we investigated the functional consequences of WH1 mutations on a downstream target of this signaling module. Because Notch1 and BRM both regulate NF-κB activity^32,33  and both transcription factors (Notch, NF-κB) are critical in T-BET and IFN-γ production,^29-31,34,35  we hypothesized that the deleterious effect of WH1 mutations on T_H1 gene activation is linked to impaired NF-κB signaling. EMSA demonstrates increased DNA-binding activity of NF-κB in T_H1-skewed compared with that in T_H2-skewed or nonskewed human, primary T_H0 cells (Figure 3C, left panel), which reinforces the role of NF-κB in T_H1 differentiation.^34,35  Significantly, EMSA/Supershift assays demonstrate that WASp is part of NF-κB:DNA ternary complex in T_H1-skewed cells (Figure 3C).

WASp:NF-κB:DNA association in vitro reported by EMSA was validated in vivo by single and sequential ChIP. These demonstrate that WASp and NF-κB co-occupy the same promoter regions of IFNG and TBX21 in vivo in T_H1-skewed cells (Figure 3D). Significantly, promoter enrichment of NF-κB(p65) is diminished only in WH1- but not non–WH1 mutant–expressing T_H1-skewed cells (Figure 3D and supplemental Figure 5C). CoIP assays similarly show that only WH1- but not non–WH1 mutations disrupt binding of WASp to NF-κB(p65) (Figure 3A). The functional importance of WASp-Notch alliance, uncovered here for the first time in human cells, is critical also in Drosophila, where Wsp mutations result in impaired Notch-mediated cell-fate decisions.³⁶  Together, our studies propose that nuclear-WASp impinges on chromatin-signaling cascade(s) that involves hBRM, Notch, and NF-κB during T_H1-differentiation and identify Thr45 and Arg86 residues of WH1 domain of WASp to be important in this cascade.

We next investigated how the above chromatin-signaling defects affect chromatin remodeling. One mechanism to remodel chromatin is by exchanging histone H2A for H2A.Z, a process catalyzed by SWI2/SNF2-like proteins SRCAP or EP400 (hDomino).^37-39  Because WASp binds EP400 in the nucleus as reported by MS and coIP (Figure 1A-B), and co-enriches with EP400 at the T_H1-gene promoters in vivo as reported by ChIP (Figure 1C), we asked whether WH1 mutants disrupt EP400-dependent, H2A-to-H2A.Z exchange at WASp-target, T_H1-gene promoters. CoIP shows that WASp binding to EP400 is disrupted by WH1- but not non–WH1 mutations (Figure 2D). Sequential ChIP shows impaired coenrichment of endogenous EP400 and transfected WH1 mutants at T_H1-gene promoters (Figure 4A), which we show are also deficient in H3K4me3 modification in cells lacking nuclear-WASp^2,3  or expressing WH1 mutants (Figure 4B). Because EP400 recruitment is positively regulated by H3K4me3,¹⁵  our finding raises the possibility that nuclear-WASp participates in H2A-to-H2A.Z exchange by regulating H3K4me3-dependent binding of EP400 to gene promoters. Indeed, WH1-mutants show a paucity of H2A.Z/H2B-marked heterotypic chromatin in the face of abundant unmodified H2A/H2B-marked chromatin at T_H1-gene promoters, as reported by sequential ChIP (Figure 4A).

Because H2A/H2B-marked hybrid nucleosomes do not reposition as well as H2A.Z/H2B-marked nucleosomes,⁴⁰  we performed qPCR-based DHS assay to determine the degree of promoter compaction. In cells expressing normal or FL-WASp or non–WH1 mutants, T_H1 activation results in increased hypersensitivity of the T_H1-gene promoters reported in fewer amplicons remaining after DNase I treatment. By contrast, in the WH1 mutants, the number of amplicons in T_H0 and T_H1 cells remains essentially unchanged, suggesting suboptimal promoter decompaction of IFNG and TBX21 promoters (Figure 4C). This DHS defect is not observed at the CSF2 locus, a WASp–non-target gene in T_H1 cells. Moreover, we find that decreased enrichment of hBRM at gene promoters in WH1 mutant–expressing T_H1 cells occurs contemporaneously with increased enrichment of EZH2 and the repressive histone mark H3K27me3 (Figure 4B). Because EZH2-like proteins inhibit recruitment and function of SWI/SNF at the promoters,⁴¹  we propose that this defect might further add to the decreased accessibility of nucleosomal DNA at gene promoters in WH1 mutant–expressing cells.

To determine how H2A.Z-related, chromatin-remodeling defect affects transcription along the DNA translational axis, we investigated transcription initiation and elongation steps at the IFNG locus in T_H1 cells. We mapped ChIP enrichment profiles of RNA Pol II pCTD-Ser5 (transcription-initiation mark), pCTD-Ser2, and CDK9 (both transcription-elongation marks) at 8 different sites within the IFNG locus spanning 5′UTR to 3′UTR (Chr.12: 68 548 000- 68 554 000) (Figure 4D). In T_H1 cells expressing normal WASp, enrichment of pCTD-Ser5 is highest between 5′-TSS and exon-1/intron-1 genomic region, whereas that of pCTD-Ser2 and CDK9 (P-TEFb subunit) are highest toward 3′-end, with the crossover occurring at ∼500 nt downstream of TSS. Such a CTD code identifies most actively transcribed genes.⁴²  By contrast, in WH1 mutant–expressing T_H1 cells, enrichments of pCTD-Ser2 and CDK9 are diminished at the mid–intron-1 region, this despite normal enrichment of pCTD-Ser5 at 5′UTR (Figure 4D). Accordingly, our results endorse the paradigm that H2A.Z-enriched nucleosomes optimize transcription elongation by relieving stalled Pol II within the gene body in a role that is linked to pCTD-Ser2.^43,44  Moreover, because both hBRM and NF-κB(p65) regulate transcription elongation,^45,46  we propose that hBRM and p65 defects also contribute to abnormal T_H1-transcriptional reprograming in a subset of WAS patients carrying hot-spot WH1 mutations.

The clinical phenotypes of genetic disorders are influenced by epigenetic changes that affect gene expression.^47-50  Although epigenetics provide a powerful way to stratify risk genotypes in diseases, how a disease-causing point mutation influences the epigenetic code of the target cell type in which the gene product is expressed is not well understood. Using a disease model of WAS, we provide a SWI/SNF-linked mechanism by which a subset of WAS missense mutations differentially perturb transcription of “core” genes that pattern T_H1-adaptive immunity. We show that a subset of SWI/SNF complexes require functional WASp to execute their locus-specific, chromatin-remodeling functions during T_H1 differentiation in human T cells. Most importantly, the 2 hot-spot WAS missense mutations that clinically manifest XLT-to-WAS disease progression disrupt SWI/SNF activity, whereas mutations causing stable XLT do not. This suggests a direct relationship between gene mutation and epigenetic disease–driving events within the context of T_H1 differentiation for the pathogenic mutations tested. Several lines of evidence propose nuclear-WASp to be an important component in the chromatin-remodeling process during T_H1 development: (1) Nuclear-WASp binds multiple CRCs, in vivo, as determined by MS and coIP; (2) nuclear-WASp co-ChIPs with these CRCs at the same cis-regulatory genomic sites in T_H cells; and (3) WH1 domain, WAS missense mutations disrupt recruitment of CRCs with deleterious consequences on histone H2A-to-H2A.Z exchange at WASp-target gene promoters. Therefore, compared with cytosolic WASp, which participates in cytoskeletal remodeling via ARP2/3-complexes, nuclear-WASp participates in chromatin remodeling via hSWI/SNF-complexes (Figure 5).

Interestingly, not all WAS mutations are damaging to SWI/SNF activity, a finding that correlates with their respective clinical severity grades. Accordingly, WH1 mutations (T45M, R86C) that manifest XLT-to-WAS progression disrupt binding of mutant WASp to SWI/SNF complexes and its recruitment to T_H1-gene promoters, whereas mutations of the basic-domain (A236G) or VCA-domain (R477K) that manifests in XLT do not disrupt these molecular readouts. Inexplicably, whereas 2 of 3 S24F (PH-domain mutation) reported patients manifest XLT and thus conform to the “molecular signatures” identified for other non-WH1 mutants, 1 patient manifests CP effects (XLT-to-WAS progression). Similarly, whereas 3 D485G (VCA mutation) reported patients manifest XLT, 1 patient (D485N) manifests CP effects. Because D485N does not impair ARP2/3-driven actin-nucleation,⁵¹  whether this VCA-domain mutation has a “long-range” effect of disrupting SWI/SNF activity is a possibility that awaits experimental validation.

Notably, the deleterious effect of WH1 mutations on SWI/SNF is limited to certain subunits, being most pronounced on BAF170 and BAF47 but not on BAF53 (ARP4) or β-actin. Because incorporation of BAF170 and BAF47 into nascent-SWI/SNF complex is required for optimal nucleosomal remodeling,²³  our findings suggest that WH1 mutations disrupt neoassembly of higher-order CRCs at WASp-target gene promoters. This BAF defect occurs coordinately with an increased occupancy of EZH2 methylase (polycomb-group family) and the consequent enrichment of histone H3K27me3, a mark of “transcriptionally silent” chromatin. This antagonism between CRCs and EZH2, which has been demonstrated for BAF47 and EZH2 in tumorigenesis,⁵²  might further contribute toward suppressing T_H1-gene activation.

Another interesting finding is that the 2 hot-spot WH1 mutations selectively impair promoter recruitment of hBRM but not BRG1, the 2 homologous central ATPases of mammalian SWI/SNF complexes that direct distinct cellular processes. BRM targets Notch-regulated gene promoters, whereas BRG1 targets gene promoters regulated by zinc-finger proteins.²⁶  Consequently, we show that promoters lacking hBRM contemporaneously lack recruitment of the core components of Notch signaling in vivo, resulting in impaired chromatin-activity of NF-κB, a downstream Notch1 target.³²  This finding is immediately relevant to the immune dysregulation in WAS, because NF-κB is critical for T_H1 development.³⁵  Because removal of H2A/H2B-containing nucleosomes is required for the specific binding of NF-κB,⁵³  we propose that WASp through its effects on H2A-to-H2A.Z exchange facilitates NF-κB binding to gene promoters in T_H1 cells. Therefore, BRG1, whose enrichment at T_H1-gene promoters is unaffected, cannot substitute for the lost hBRM functions during T_H1 transcriptional-reprogramming. This suggests that the functional effects of BRG1 and BRM are influenced by the lineage-specific transcription factors they coassociate with, a paradigm that supports the “Activator Model” for SWI/SNF recruitment to promoters.⁵⁴  However, we cannot rule out the possibility that the promoter-enriched BRG1 maybe “nonfunctional,” and, if so, BRG1 defect could also contribute to impaired chromatin signaling.

In addition to its effects on hBRM/BAF promoter activity, WH1 mutations perturb other reaction steps of the multistep chromatin-remodeling process.^55,56  WH1 mutants interfere with the enrichment of H2A/H2A.Z-marked heterotypic chromatin at T_H1-gene promoters via its effects on EP400, hSWR1/hDomino-homolog that catalyzes H2A.Z deposition in vivo.^20,39  Because H2A.Z imparts increased mobility to the peri-TSS nucleosomes,⁵⁷  the H2A.Z deficiency observed with WH1-domain mutants affects chromatin decompaction at T_H1-gene promoters reported in a DHS profile that is nonconducive to recruiting transcription factors.⁵⁸  Because H2A.Z is also important in DNA repair, determining whether and how WASp effects on H2A-to-H2A.Z exchange at other genomic loci affects this process will begin to clarify why certain WAS mutations associate with genomic instability in lymphocytes.⁵⁹  Given the importance of chromatin remodeling in biology, including in cancer development,^48,49,60-63  we predict that the WASp:SWI/SNF alliance may affect multiple other-cell biological processes including tumor suppression, the disruption of which may further influence the phenotypic variations in WAS. Notwithstanding, the implications of our studies should be limited to the T_H1 development, because we have not studied megakaryocytes, the major cell type responsible for the early findings of thrombocytopenia (XLT) in WAS. Although thrombocytopenia was shown to be VCA-domain–independent,⁶⁴  clarification of whether WAS mutations differ in their proclivity to perturb other aspects of megakaryocyte biology is essential. Irrespective of the multiple potential pathways that will eventually be determined to contribute to the complete disease-causing spectrum of WAS, our study provides the beginnings of a novel epigenetic basis to predict symptom severity in WAS, at least as contributed by T_H cell dysregulation.

The authors thank Jerome Parness (University of Pittsburgh) for insightful discussions and critical reading of the manuscript, M. Balasubramani of the University of Pittsburgh Proteomic Core Facility for analyzing the MS data, and Fabio Candotti for providing WAS patient T-cell line. We thank the University of Iowa Dance Marathon for providing laboratory space.

This research was supported by National Institutes of Health National Institute of Allergy and Infectious Diseases grants R01 AI073561 and R01 AI084957.

Contribution: K.S. performed the majority of the experiments; S.S. generated all patient mutants and assisted in ChIP and PCR assays; S.-S.H. performed the EMSA/Gel-shift assays; and Y.M.V. conceived the study, designed the experiments, analyzed the data, and wrote the paper.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Yatin M. Vyas, Division of Pediatric Hematology-Oncology, University of Iowa Children’s Hospital, Iowa City, IA 52242; e-mail: yatin-vyas@uiowa.edu.