Fig 2: Zic2 colocalizes with Mbd3-NuRD Complex in ES Cells and functions in regulating gene expression(A) Binding profiles in mouse ES cells demonstrating the presence of the indicated histone modifications at Zic2, Chd4 and Mbd3 co-bound regions (2,047 regions, highlighted in pink), Chd4-Mbd3 only regions (1,726 regions, highlighted in brown) and Zic2 only regions (5,945 regions, highlighted in purple) within 50 kb around the Zic2 or Chd4 peak. H3K4me1 and p300 data are from (Creyghton et al., 2010), and H3K4me3 and H3K27me3 data are from (Mikkelsen et al., 2007). (B–C) Scatter plots demonstrating differentially expressed genes, as assessed by RNA-seq in Zic2 knockdown versus Mbd3 (B) and Mta2 (C) knockdowns. Genes significantly misregulated are shown in blue and green, respectively. Correlation coefficients are shown. RNA-seq after Zic2 knockdown was performed and analyzed in four replicates. RNA-seq after Mbd3 or Mta2 knockdown was performed and analyzed in duplicates. (D–F) MA plots demonstrating differentially expressed genes after (D) Mbd3, (E) Zic2 and (F) Zic3 knockdown in ES cells. Significantly changed genes after Mbd3 knockdown are shown in blue. Genes up-regulated in both Mbd3 knockdown and knockout datasets are highlighted with red circles, and genes down-regulated in both Mbd3 knockdown and knockout datasets are highlighted with green circles. Genes regulated by Mbd3 were changed in the same direction after Zic2, but not Zic3, knockdown. See also Figure S3 and Table S1.

Fig 3: BCL11B p.R3S substitution disrupts interaction with NuRD and PRC2 complexes. (A) Immunocytochemistry of HEK293T cells expressing wild-type BCL11B (top) and BCL11B p.R3S (bottom). Cells were stained with an anti-BCL11B monoclonal antibody and co-stained with DAPI to visualize nuclei. (B) Co-immunoprecipitation studies. BCL11B and BCL11B p.R3S were immunoprecipitated with the anti-FLAG antibody from transiently transfected HEK293T cells, and the corresponding blot was probed with antibodies to BCL11B (red), MTA2 (green), HDAC2 (green), EZH2 (green) and SIRT1 (green). Comparison of lanes 4 (wild-type protein) and 6 (p.R3S) demonstrates that the patient-derived BCL11B mutant does not interact with the NuRD or PRC2 complexes, however, the p.R3S substitution did not alter interaction of the protein with the class III histone deacetylase SIRT1 (33). (C) Schematic outlining BCL11B interaction with the NuRD and PRC2 complexes.

Fig 4: Zic2 interacts with the Mbd3-containing NuRD complex in embryonic stem cells(A) A clonal ES cell line expressing Flag-tagged Zic2 was generated, and the resulting protein complexes were affinity purified and analyzed by SDS-PAGE, silver staining and mass spectrometry. (B) MudPIT analysis of the relative abundance of Zic2 and Mbd3-NuRD subunits are shown. dNSAF values represent the distributed normalized spectral abundance factor. All the core components of Mbd3-containing NuRD (Chd4, Mbd3, Mta1/2, Gatad2a/b, Hdac1/2, Rbbp4/7 (Allen et al., 2013) as well as known NuRD-associated proteins Cdk2ap1 and Sall4 (Kloet et al., 2014; Lu et al., 2009; Spruijt et al., 2010) were reproducibly identified in the Flag-Zic2 purifications. (C–D) Endogenous immuneprecipitations (IP) followed by Western blotting confirm the interaction of Zic2 with subunits of the Mbd3-NuRD complex. (C) NuRD subunit IP followed by Zic2 Western blotting. Aff4, which interacts with Cdk9 within the Super Elongation Complex, served as a negative control for the IP experiments. (D) Zic2 IP followed by Mbd3-NuRD subunit Western blotting. (E) Size exclusion chromatography of mouse ES cell nuclear extracts demonstrated that the elution pattern of Zic2 overlaps to a larger extent with Mbd3 and Mta2 than with Mbd2. Elution profile corresponding to 1.5 MDa and 670 KDa are indicated with arrowheads. See also Figure S2.