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Atypical Mowat-Wilson patient confirms the importance of the novel association between ZFHX1B/SIP1 and NuRD corepressor complex.
Verstappen G, van Grunsven LA, Michiels C, Van de Putte T, Souopgui J, Van Damme J, Bellefroid E, Vandekerckhove J, Huylebroeck D.
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Mutations in ZFHX1B cause Mowat-Wilson syndrome (MWS) but the precise mechanisms underlying the aberrant functions of mutant ZFHX1B proteins (also named Smad-interacting protein-1, SIP1) in patients are unknown. Using mass spectrometry analysis, we identified subunits of the NuRD corepressor complex in affinity-purified Zfhx1b complexes. We find that Zfhx1b associates with NuRD through its N-terminal domain, which contains a previously postulated NuRD interacting motif. Interestingly, this motif is substituted by an unrelated sequence in a recently described MWS patient. We show here that such aberrant ZFHX1B protein is unable to recruit NuRD subunits and displays reduced transcriptional repression activity on the XBMP4 gene promoter, a target of Zfhx1b. We further demonstrate that the NuRD component Mi-2beta is involved in repression of the Zfhx1b target gene E-cadherin as well as in Zfhx1b-induced neural induction in animal caps from Xenopus embryos. Thus, NuRD and Zfhx1b functionally interact, and defective NuRD recruitment by mutant human ZFHX1B can be a MWS-causing mechanism. This is the first study providing mechanistic insight into the aberrant function of a single domain of the multi-domain protein ZFHX1B/SIP1 in human disease.
Tandem affinity purified Zfhx1b complexes contain multiple subunits of NuRD. (A) Graphic presentation of the mouse Zfhx1b protein and the N-terminally 3XFS-tagged variant. The N-terminal and C-terminal zinc finger domains (NZF and CZF), Smad-binding domain (SBD), homeodomain-like domain (HD), the C3H type zinc finger (light grey box) and the CtBP interacting sites (CBS) are shown. (B) Schematic representation of the variant tandem affinity purification procedure. The first purification involves an in-batch Flag-IP of extracts of HEK293T cells, containing transiently synthesized 3XFS-Zfhx1b proteins. Upon elution with 3XFLAG peptides, the eluted proteins were applied to a streptactin-agarose column for a second purification. Precipitation was done using acetone. (C) Brilliant blue G-colloidal staining of the purified proteins from HEK293T cells transiently producing 3XFS-Zfhx1b or 3XFS-tag only. Co-purified proteins were identified by MS.
Figure 2. Zfhx1b associates with several subunits of the NuRD complex. (A) Extracts of HEK293T cells containing transiently synthesized 3XFS-Zfhx1b or 3XFS tag as negative control were precipitated in a Flag-dependent manner and eluted using 3XFLAG peptides. Western-blot analysis was performed using antibodies directed against the NuRD subunits. (B) IP of HEK293T cell extracts with Zfhx1b-specific or control anti-Flag antibodies, followed by Western-blot analysis using NuRD subunit-specific antibodies.
A dominant-negative variant of Mi-2β interferes with Zfhx1b-mediated repression of specific target promoters in reporter constructs. HEK293T cells were transiently transfected with constructs coding for Zfhx1b, wild-type or mutant Mi-2βK750C polypeptides, and the E-cadherin (A), or 3TPlux reporter constructs (B). Cells were harvested 48 h after transfection and were analyzed for luciferase activity. β-galactosidase activity corrected luciferase values are given.
Figure 4. Zfhx1b associates with NuRD via its N-terminal domain. (A) Graphic presentation of wild-type (WT) full-length (FL) and mutant Zfhx1b proteins used in this study. (B) Western-blot detection showing co-immunoprecipitation of Flag-MTA2 and RbAp48, and HA-HDAC2 with the respective Zfhx1b proteins (triangle) tested.
Figure 5. Defective NuRD recruitment by a mutant ZFHX1B protein that causes atypical MWS. (A) Alignment of Sall1, Zfhx1a, mouse, Xenopus laevis and human wild-type Zfhx1b, and mutant ZFHX1B amino-terminal domains. The conserved 12 amino acid motif (italics) in Sall1 that is sufficient for NuRD recruitment and the critical residues (*) that abolished binding to NuRD and repression by Sall1 when mutated are indicated. Note that mouse Zfhx1a and mouse, human and Xenopus laevis Zfhx1B but not the Atypical Zweier mutant human Zfhx1b contain the conserved motif (bold) within their N-terminus. (B) Schematic presentation of the ZFHX1BWT and ZFHX1BAZmut proteins used in this study. (C) Western-blot detection showing co-immunoprecipitation of endogenous NuRD subunits with ZFHX1BWT but not ZFHX1BAZmut. Note that CtBP binding by ZFHX1BAZmut proteins is unaffected.
Figure 6.XZfhx1b depends on intact XMi-2β activity in early neural tissue formation. (A) Whole-mount in situ hybridization with a cRNA probe for XZfhx1b and XMi-2β at the indicated developmental stages; ba, branchial arches; ev, eye vesicle; nc, neural crest; nt, neural tube; s, somites. (B) The indicated plasmids were transfected into HEK293T cells and cells were analyzed 48 h later for the XBMP4 promoter-driven luciferase activity. β-Galactosidase-corrected luciferase values are given. Note that ZFHX1BWT represses the activity of the reporterconstruct in a dose-dependent manner, while repression by ZFHX1BAZmut proteins is significantly reduced. (C) QRT–PCR expression analysis of the indicated genes on total RNA isolated from animal caps derived from embryos injected with XZfhx1b RNA alone (200 pg/blastomere), co-injected with MOs against XMi-2β, co-injected with MOs against XMi-2β and hMi-2β RNA, co-injected with control MOs, or non-injected. Values were corrected for H4. Note that overexpression of XZfhx1b represses XBMP4 and the BMP-dependent gene XVent-1 and induces the neural gene N-CAM, while knock-down of XMi-2β alleviates XZfhx1b-dependent repression and induction of the respective genes.
cdh4 (cadherin 4) gene expression in X. laevis embryo, NF stage 11, assayed via in situ hybridization.
cdh4 (cadherin 4) gene expression in X. laevis embryo, NF stage 18, assayed via in situ hybridization.
cdh4 (cadherin 4) gene expression in X. laevis embryo, NF stage 28, assayed via in situ hybridization, anteriorleft, dorsal up.
