Volume 56, Issue 4, Pages 580-594 (November 2014) Redundant Mechanisms to Form Silent Chromatin at Pericentromeric Regions Rely on BEND3 and DNA Methylation  Nehmé Saksouk, Teresa K. Barth, Celine Ziegler-Birling, Nelly Olova, Agnieszka Nowak, Elodie Rey, Julio Mateos-Langerak, Serge Urbach, Wolf Reik, Maria-Elena Torres-Padilla, Axel Imhof, Jérome Déjardin  Molecular Cell  Volume 56, Issue 4, Pages 580-594 (November 2014) DOI: 10.1016/j.molcel.2014.10.001 Copyright © 2014 Elsevier Inc. Terms and Conditions

Molecular Cell 2014 56, 580-594DOI: (10.1016/j.molcel.2014.10.001) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 1 Purification of Heterochromatic Loci in Mouse ESCs (A) Map of major satellite and telomere organization. The positions of targeted sequences are shown. (Bottom panel) FISH on metaphase spreads using telomere- (green, left panel) or major satellite-specific (red, right panel) PICh probes. (B) Silver staining of telomere and major satellites PICh from ESCs. Lanes were cut from the same gel. Maj.Sat, major satellite PICh. Tel, telomere PICh. S, Scramble PICh. Purifications were from 2.5 × 107 cells/lane. (C) Histone modification profiles of purified preparations. Enrichment is expressed as compared to the same analysis performed on input histones. (D) Steady-state protein composition of mouse pericentric heterochromatin in ESCs. The 135 enriched proteins are listed and categorized. Bold and italicized factors are known Suvar or Momme factors. Bold factors have been shown to localize to major satellites. Asterisks indicate factors we validated. See also Figure S1 and Table S1. Molecular Cell 2014 56, 580-594DOI: (10.1016/j.molcel.2014.10.001) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 2 Impact of SUV39H Loss on Major Satellite Composition (A) Heatmap of the various histone modifications analyzed on H3 and H4 tails. Results are expressed as the ratios of Inputs Suv39h dn/WT or Maj.Sat Suv39hdn/WT. (B) qPICh plot highlighting major protein changes at mutant major satellites. Factors lost from mutant pericentromeres align along the vertical axis (red oval), while gained factors are found along the horizontal axis (blue oval). See also Figure S2, Table S2, and Table S4. (C) (Left) Combined immunofluorescence and FISH analyses showing AURORA B association to major satellites. (Right) Quantifications of the immuno-FISH data and measurements of AURORA B levels by immunoblots. Error bars represent standard deviation. (D) Immunoblots on purified pericentromere preparations showing the impact of H3K9me3 loss on the maintenance methylation machinery recruitment (UHRF1), while not affecting DNMT3B recruitment. (E) ChIP analyses confirming qPICh findings with the DNA methylation machineries. Error bars represent standard deviation See also Figure S2 and Table S2. Molecular Cell 2014 56, 580-594DOI: (10.1016/j.molcel.2014.10.001) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 3 Effects of DNA Methylation Loss on Pericentromeric Composition (A) Heatmap of the various histone modifications analyzed on H3 and H4 tails. Results are expressed as the ratios of Input DnmtTKO/WT or Maj.Sat DnmtTKO/WT. (B) Immunoblot with purified pericentromeric preparations in the diverse ESCs backgrounds. Suv420h dn is a background in which SUV420H enzymes are abrogated. (C) qPICh plot showing major protein losses (in the red oval), and gains (in the blue oval) at mutant pericentromeres. (D) (Left) Immuno-FISH experiments showing the position of major satellites (green) compared to nuclear lamina (LAMIN B1, red). (Right) Quantification of this phenomenon. Student’s t test was used (see Supplemental Experimental Procedures). (E) LAMIN B1 expression levels in the diverse cell lines used. See also Figure S3, Table S3, and Table S4. Molecular Cell 2014 56, 580-594DOI: (10.1016/j.molcel.2014.10.001) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 4 Epigenetic State of Major Satellites Anticorrelates with DNA Methylation Levels Immunofluorescence staining performed with H3K27me3antibodies (in green) to monitor PRC2 association, and with RNF2 antibodies (in red) to monitor PRC1 recruitment. Extent of colocalization with DAPI signals is shown in white (in %) from more than 100 nuclei for both antibodies (n = 2). H3K9me3 levels and DNA methylation levels at major satellites are indicated below. See also Figure S4. Molecular Cell 2014 56, 580-594DOI: (10.1016/j.molcel.2014.10.001) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 5 BEND3 Binds to Hypomethylated Pericentromeric Regions (A) Immunostaining of endogenous BEND3 in diverse ESCs backgrounds. (Right) Quantifications. Double and triple asterisks represent p values below 0.005 and 0.001, respectively (from a two-sided Student’s t test). (B) Methylation-sensitive DNA affinity assays. (Right) Coomassie gel showing the purity of FLAG-BEND3 preparations, and allowing BEND3 quantifications. (Left) FLAG immunoblots on the fractions: I, 20% of input loaded (0.25 pmol BEND3). U, unbound fraction. B, bound fraction (50% each). A total of 1 pmol of biotinylated major satellites or methyl-major satellites was used. (C) Persistence of BEND3 on metaphase chromosomes from Dnmt TKO cells. See also Figure S5. Molecular Cell 2014 56, 580-594DOI: (10.1016/j.molcel.2014.10.001) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 6 BEND3 Is Involved in PRC2 Recruitment to Major Satellites (A) Colocalization study of BEND3 with H3K27me3 in mouse zygotes. Zygotes microinjected with mRNA for HA-BEND3 were fixed at early (PN2-3), mid (PN4), and late (PN5) pronuclear stages and stained with an HA and H3K27me3 antibodies. The arrow points to HA-BEND3 localization (red), which colocalizes with H3K27me3 (yellow) at the PN5 stage. Embryos are representative of >10 zygotes analyzed from two independent biological replicates. Scale bar, 5 μm. (B) shRNA experiments showing BEND3 is necessary for the recruitment of PRC2 to major satellites in Suv39h dn ESCs. (C) Silver staining of FLAG-BEND3 purification from WT ESCs. Major partners identified by MS are indicated on the right of the gel. (D) shRNA experiment showing BEND3 is necessary for CHD4 recruitment. See also Figure S6. Molecular Cell 2014 56, 580-594DOI: (10.1016/j.molcel.2014.10.001) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 7 Model Highlighting the Mode of Action of Constitutive Heterochromatin Hallmarks Based on Our Data (Top panel) H3K9me3, installed by SUV39H enzymes, controls maintenance DNA methylation, HP1 recruitment, and the CPC complex. DNA methylation controls association to the lamina and prevents PcG “recruiters” from binding. (Middle panel) The loss of H3K9me3 perturbs UHRF1/DNMT1 recruitment. Certain (?) CpG residues become unmethylated, allowing BEND3 to bind and recruit MBD3-NuRD, which in turn allows PRC2 to bind. As substantial DNA methylation is maintained via reduced DNMT1 and unperturbed DNMT3 activities, PRC1 cannot be recruited. Pericentromeric loci remain tightly associated to nuclear lamina. (Bottom panel) The locus is unmethylated, which reduces SUV39H activity via an unknown mechanism. In addition to PRC2 recruitment, PRC1 gets recruited, presumably via the binding of KDM2B (and SCML2?) to unmethylated DNA. Loss of methylated DNA disrupts association to nuclear lamina and leads to a reorganization of the locus. Molecular Cell 2014 56, 580-594DOI: (10.1016/j.molcel.2014.10.001) Copyright © 2014 Elsevier Inc. Terms and Conditions