Fig 2: Zic2 and ES cells pluripotency(A) ChIP-seq track examples of Zic2, Chd4, Mbd3-occupied lineage-specific genes in the ES cell state. Zic2 is found with NuRD at putative enhancers for EphA4, Fgf5 and Otx2. (B) qRT-PCR analyses of the activation kinetics of lineage-specific genes in control (NonT), Zic2, or Mbd3 knockdown embryoid bodies (EBs). Control, Zic2, or Mbd3 knockdown ES cells were induced to form EB for the indicated time points. The expression levels were normalized to Actb. Error bars represent the s.d. of independent measurements of a representative experiment. (C) Model: In the stem cell state (top panel), the zinc finger-containing protein Zic2 functions together with the NuRD repressor complex, setting a poised, yet to be activated, state of developmental genes. Knockdown of Zic2 (bottom panel) and/or NuRD subunits leads to derepression of the same group of developmental genes. See also Figure S5.

Fig 3: Co-occupancy of target genes by NuRD, Wdr5, Sall4, and Suz12. A, Comparison of Wdr5 and NuRD target genes in mESCs. The proportional overlap of both gene data sets was visualized. The NuRD data set was derived from an overlap of Chd4, Hdac1, and Hdac2 target gene sets in mESCs. B, Comparison of NuRD (same as in A), Sall4 and Suz12 target gene sets. C, The shared genes in B were combined into a new data set (105-NuRD/Suz12; 34-NuRD/Suz12/Sall4; 342-NuRD/Sall4). This data set was reduced to genes colocalized by Wdr5. The full Sall4 and Suz12 data sets (same as in C) were compared with the resulting gene data set.

Fig 4: NuRD complex components bind the D4Z4 macrosatellite repeat.(A) Schematic summary of the enChIP procedure. A 3xFLAG-dCas9-HA-2xNLS fusion protein (FLAG-dCas9) consisting of an N-terminal triple FLAG (3xFLAG) epitope tag, catalytically inactive Cas9 endonuclease (dCas9), C-terminal human influenza hemagglutinin (HA) epitope tag and tandem nuclear localization signal (2xNLS) is expressed with one or more guide RNA (gRNA) in an appropriate cell context. Cells are crosslinked, chromatin is fragmented and complexes containing FLAG-dCas9 are immunoprecipitated with an anti-FLAG antibody. After reversing the crosslinks, molecules associated with the targeted genomic region are purified and identified by downstream analyses including mass spectrometry and next-generation sequencing. Adapted from Fujita et al. (2016). (B–E) ChIP-qPCR enrichment of NuRD complex components CHD4 (B), HDAC2 (C), MTA2 (D) and MBD2 (E) along the D4Z4 repeat in MB2401 control myoblasts. The Chr18q12 amplicon contains no CpG dinucleotides and serves as a negative control site, while the TMEM130 promoter is occupied by NuRD complex components in published ENCODE datasets (ENCODE Project Consortium, 2012) and functions as a positive control locus. Error bars denote the standard deviation from the mean of three biological replicates. Statistical significance was calculated by comparing the specific pulldown to the IgG control at each site using a two-tailed, two-sample Mann-Whitney U test. *, p≤0.05; ns, not significant, p>0.05. See also Figure 1—source data 1.10.7554/eLife.31023.005Figure 1—source data 1.Source data for Figure 1.This file contains the source data used to make the graphs presented in Figure 1 and Figure 1—figure supplement 1. GraphPad Prism was utilized to visually represent the quantitative data.

Fig 5: The C terminus of CHD proteins is required for the association with GATAD2 proteins and CDK2AP1. (A) Schematic overview of the cross‐links identified between CHD3 and CHD4 and other NuRD subunits. GATAD2A and GATAD2B mostly cross‐link to the C terminus. CDK2AP1 forms ambiguous cross‐links to the C terminus of both CHD3 and CHD4. (B) X‐ray structure of a dimer of (the C‐terminal parts of) CDK2AP1. One peptide is indicated in gray and the other in blue. Residues that cross‐link to the CHD proteins are indicated in pink. The surface potential of the CDK2AP1 dimer is also shown where blue is positively charged and red is negatively charged. Image is made using ucsf chimera [22]. (C) Sequence alignment of CHD3, CHD4, and CHD5 from human (HS), mouse (MM), zebrafish (DR), and fruit fly (DM) shows the presence of many glutamic acids around the sites of cross‐linking (indicated with red arrows). Used UniProt accession numbers are as follows: CHD3‐HS: Q12873‐1 (1696‐1925), CHD3‐MM: B1AR17‐1 (1622‐1851), CHD3‐DR: X1WBL0‐1 (1525‐1751), CHD5‐HS: Q8TDI0‐1 (1693‐1917), CHD5‐MM: A2A8L1‐1 (1691‐1915), CHD5‐DR: A0A0R4IJ89‐1 (1690‐1919), CHD4‐HS: Q14839‐1 (1685‐1912), CHD4‐MM: Q6PDQ2‐1 (1688‐1915), CHD4b‐DR: F1RBT2‐1 (1728‐1953), and Mi2‐DM: O97159‐1 (1716‐1945). Sequence alignment was generated using clustalx [36]. (D, E) Label‐free purifications of full‐length GFP‐CHD4 and CHD4‐ΔC‐GFP, respectively. Full‐length CHD4 co‐purifies all NuRD subunits in addition to a couple of new interactors (D). C‐terminal truncation of CHD4 leads to a loss of CDK2AP1 and of all other NuRD subunits (E).