Volume 53, Issue 4, Pages 631-644 (February 2014) Mislocalization of the Centromeric Histone Variant CenH3/CENP-A in Human Cells Depends on the Chaperone DAXX  Nicolas Lacoste, Adam Woolfe, Hiroaki Tachiwana, Ana Villar Garea, Teresa Barth, Sylvain Cantaloube, Hitoshi Kurumizaka, Axel Imhof, Geneviève Almouzni  Molecular Cell  Volume 53, Issue 4, Pages 631-644 (February 2014) DOI: 10.1016/j.molcel.2014.01.018 Copyright © 2014 Elsevier Inc. Terms and Conditions

Molecular Cell 2014 53, 631-644DOI: (10.1016/j.molcel.2014.01.018) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 1 Effects of CenH3 Overexpression in Breast Cancer and Model Cell Lines (A) Western blotting of total extracts from MCF10-2A and BT20 and BT474. Proteins showing at least 2-fold differences in their expression levels are highlighted in red. Antibodies are indicated on the right of each western blot. A 2-fold dilution series is represented with the gradient bar. The asterisk possibly represents the unphosphorylated form of HJURP. (B) Western blotting of total extracts from the control (HeLa-WT) and CenH3 overexpressing (HeLa-eCenH3) cell lines. Antibodies are indicated on the right of each western blot. A 2-fold dilution series is represented with the gradient bar. (C) Fluorescent microscopy visualization of CenH3 and CENP-B shows spreading of eCenH3 to chromosome arms. The scale bar represents 5 μm. (D) Single mitotic chromosome showing eCenH3 spreading to chromosome arms. The scale bar represents 2 μm. Molecular Cell 2014 53, 631-644DOI: (10.1016/j.molcel.2014.01.018) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 2 MNase-ChIP-Seq of wtCenH3 and eCenH3 Mononucleosomes (A) Scheme outlining strategy for purifying and sequencing DNA associated with CenH3 mononucleosomes from either WT or CenH3-overexpressing cells. (B) SYPRO-Ruby staining of CenH3 mononucleosome (IP) compared to their respective inputs. A 2-fold dilution series is represented with the gradient bar. (C) Southern blotting of DNA purified from CenH3 mononucleosome compared to their respective inputs (labeling is same as Figure 2B). Probes are indicated on the left. Southern blots have been inverted for presentation purposes. (D) Pie charts representing proportion of full-length reads from HeLa-WT input (all mononucleosomes) and wtCenH3 and eCenH3 IP mapping to one of three genomic sequence categories. (E) Example of peak distribution and read density of input, wtCenH3, and eCenH3 mapping to a nonrepetitive sequence on chromosome 18. Annotation of the centromere derives from the University of California, Santa Cruz Genome Browser gap track. Molecular Cell 2014 53, 631-644DOI: (10.1016/j.molcel.2014.01.018) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 3 eCenH3 Outside the Centromere Is Enriched at Active Regions and Forms a Heterotypic Tetramer with H3.3 (A and B) Fold enrichment of intersection between peaks of wtCenH3, eCenH3, and eH3.3 and peaks of various histone marks and histone variants (A) and genes, promoters, active enhancers, and regions bound by transcription factors (B) compared to randomized peaks. A blue line indicates what would be expected by chance. (C) Nucleosome positioning of histone variants at bound CTCF sites. H2A.Z extracted at both low and high-salt (HS) conditions. (D) Nucleosome positioning of histone variants at active enhancers (centered on the peak of p300 binding). (E) CenH3 ChIP-qPCR of active regions at CTCF sites, active enhancers, and the MYC gene in BT474 or BT20, compared to MCF10-2A. A thick blue line represents 2-fold enrichment. Error bars indicate the SD from triplicate experiments. (F) Distribution of DNA read lengths from MNase-ChIP-seq experiments for input and eCenH3 IP at alpha satellites and nonrepetitive sequences. (G) Boxplot of the proportion of G+C for input reads (146–149 bp), eCenH3 reads (130–136 bp [short]), and eCenH3 reads (146−149 bp [long]). The black line in each box plot represents the median value. A thick blue line represents the mean G+C value of input reads mapping to α satellites. Molecular Cell 2014 53, 631-644DOI: (10.1016/j.molcel.2014.01.018) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 4 Homo- and Heterotypic CenH3 Particles Exhibit Distinct DNA Protection Length and Genomic Localization at Functional Elements (A) Western blotting of CenH3 mononucleosome (IP) isolated from HeLa cell lines compared to their respective inputs. A 2-fold dilution series is represented with the gradient bar. Specific antibodies are indicated on the left. (B) Ratio between replicative H3 and H3.3 obtained by mass spectrometry from three independent eCenH3 purifications. Error bars indicate the SD from triplicate experiments. (C) Western blotting against H3 from eCenH3 mononucleosomes separated on a triton acid urea (TAU) gel compared to eH3.1. (D) Scheme of sequential immunoprecipitation used to purify homotypic and heterotypic mononucleosomes from eCenH3 cells. (E) Western blot of sequential immunoprecipitation. Input represents all mononucleosomes. IP1 represents particles that have only undergone IP for eCenH3. FT corresponds to flowthrough of the IP2 against H3 or control IgG. IP2 corresponds to the bound fraction of the IP against H3 or control IgG. The percentage of loaded material is indicated above each lane. The asterisk corresponds to IgG light chain. Western blot antibodies are indicated on the left. (F) Southern blotting of sequential immunoprecipitation. Labeling is the same as in Figure 4E. Each lane was loaded with 30 ng of DNA. Probes are indicated on the left and molecular weight (in bp) on the right. (G) Proportion of reads from ChIP-seq of homo- and heterotypic particles mapping to three types of genomic sequence. (H) Read length distributions for homo- and heterotypic particles. (I and J) Nucleosome occupancy of homo- and heterotypic particles at bound CTCF sites (I) and active p300 enhancer peaks (J). Molecular Cell 2014 53, 631-644DOI: (10.1016/j.molcel.2014.01.018) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 5 DAXX Is Involved in eCenH3 Localization at Chromosome Arms (A) Fluorescent microscopy visualization of CenH3 and CENP-B after treatment with the indicated siRNAs. The scale bar represents 5 μm. (B) Histogram representing the fluorescence of CenH3 present in chromosome arms of metaphase spreads from the two cell lines treated with the indicated siRNAs. Error bars are the SEM calculated from the indicated numbers of analyzed chromosomes summarized in Figure S5C. p values represent pairwise comparison of siLuc (control), and all other siRNAs are treated cells from the same cell line with a Mann-Whitney U test. (C) Western blot of cellular proteins fractionated into “soluble” (containing cytoplasmic and some nuclear proteins), “salt-extractable” (principally nuclear proteins), and “insoluble” pellet fractions in order to assess the distribution of protein in each fraction. Aliquots of each extract corresponding to an equal number of cells and a dilution series were loaded onto the gel in order to obtain directly comparable signals. (D) Western blot analysis of anti-DAXX or rabbit IgG immunoprecipitates from nuclear extract of wtCenH3 or eCenH3 cell lines. Antibodies are indicated on the left. (E) Recombinant H3.1-H4, H3.3-H4, or CenH3-H4 complexes were subjected to stringent (binding at 0 and 5 M NaCl and washes at 1 M NaCl) or less-stringent (binding at 0 and 25 M NaCl and washed at 0 and 5 M NaCl) GST pulldown assay with either GST or GST-DAXX HBD (179–389). Molecular Cell 2014 53, 631-644DOI: (10.1016/j.molcel.2014.01.018) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 6 Presence of the CenH3 Heterotypic Particle Causes Occlusion of CTCF (A) ChIP-qPCR of CenH3 in HeLa cell lines using mock (siLuc)- or DAXX (siDAXX)-depleted cells at each of five different CTCF and enhancer loci. Error bars indicate the SD from triplicate experiments. (B) ChIP-qPCR of CTCF in WT and eCenH3 cells lines (left) and MCF10-2A, BT-20, and BT474 cell lines (right) with siLuc or DAXX-depleted cells at the same 5 CTCF loci. Error bars represent SD. Significant p values are presented in Table S4. Error bars indicate the SD from triplicate experiments. Molecular Cell 2014 53, 631-644DOI: (10.1016/j.molcel.2014.01.018) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 7 Overexpressing CenH3 Provides a Survival Advantage after DNA Damage (A) Western blot of total extracts from siRNA-treated cells to verify efficiency of the indicated siRNAs as well as DNA damage indicators in absence (0 nM CPT) or presence (200 nM CPT) of CPT. The time point analyzed is after 3 days of siRNA treatment followed by 4 days of CPT treatment. (B) Mean growth curve of wtCenH3 or eCenH3 mock- or DAXX-depleted in the absence (0 nM) or 200 nM of CPT carried out in triplicate. Error bars indicate the SD from triplicate experiments. (C) Colony formation assay after ionizing radiation with increasing γ ray dose. Error bars represent SD. Error bars indicate the SD from triplicate experiments. Molecular Cell 2014 53, 631-644DOI: (10.1016/j.molcel.2014.01.018) Copyright © 2014 Elsevier Inc. Terms and Conditions