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. 2020 Jan 2;77(1):51-66.e8.
doi: 10.1016/j.molcel.2019.10.018. Epub 2019 Nov 26.

Native Chromatin Proteomics Reveals a Role for Specific Nucleoporins in Heterochromatin Organization and Maintenance

Nahid Iglesias  1 , Joao A Paulo  2 , Antonis Tatarakis  1 , Xiaoyi Wang  1 , Amanda L Edwards  3 , Natarajan V Bhanu  4 , Benjamin A Garcia  4 , Wilhelm Haas  5 , Steven P Gygi  2 , Danesh Moazed  6
Affiliations

Native Chromatin Proteomics Reveals a Role for Specific Nucleoporins in Heterochromatin Organization and Maintenance

Nahid Iglesias et al. Mol Cell. .

Abstract

Spatially and functionally distinct domains of heterochromatin and euchromatin play important roles in the maintenance of chromosome stability and regulation of gene expression, but a comprehensive knowledge of their composition is lacking. Here, we develop a strategy for the isolation of native Schizosaccharomyces pombe heterochromatin and euchromatin fragments and analyze their composition by using quantitative mass spectrometry. The shared and euchromatin-specific proteomes contain proteins involved in DNA and chromatin metabolism and in transcription, respectively. The heterochromatin-specific proteome includes all proteins with known roles in heterochromatin formation and, in addition, is enriched for subsets of nucleoporins and inner nuclear membrane (INM) proteins, which associate with different chromatin domains. While the INM proteins are required for the integrity of the nucleolus, containing ribosomal DNA repeats, the nucleoporins are required for aggregation of heterochromatic foci and epigenetic inheritance. The results provide a comprehensive picture of heterochromatin-associated proteins and suggest a role for specific nucleoporins in heterochromatin function.

Keywords: Bdf1; Clr4; H3K9me; HP1; INM proteins; NPC; SUV39H; Swi6; epigenetic inheritance; nucleoporins.

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Conflict of interest statement

Declaration of Interests

The authors declare no competing interests.

Figures

Figure 1.
Figure 1.. A strategy for isolation of native chromatin domains.
A, Outline of the strategy. B, Representative coomassie- (left) and silver-stained (right) SDS polyacrylamide gels showing proteins recovered by purification of 3×Flag-Swi6. Bottom, western blot showing that histones containing the heterochromatin mark, H3K9me2, were recovered using Flag-Swi6 nChIP. Molecular weight markers in kilodalton are shown on the left. C, Flag (blue) and H3K9me3 (red) ChIP-seq showing that Flag-Swi6 efficiently pulled-down every heterochromatin domain during native purifications. Native purification ChIP-seq (top four rows) and conventional ChIP-seq (Xlinked, crosslinked, bottom six rows) are shown. Reads mapped to different heterochromatin regions in the indicated genotypes are presented as reads per million (number in bracket in the first row of each ChIP-seq data). Top, chromosome coordinates. D, Volcano plot displaying the TMT-based quantitative MS result of Flag-Swi6 nChIPs. The plot shows log2 ratios of averaged peptide MS intensities between Flag-Swi6 and reference (no tag) eluate samples (x axis) plotted against the negative log10 P values (y axis) calculated across the triplicate datasets (one-tailed Student’s t test, n = 3 biological replicates). Note maximum upper values were set for the x and y axis to accommodate all detected proteins in the plot. A dashed horizontal black line marks the P value = 0.05. Major Flag-Swi6 pulled-down protein groups relevant for this study are color coded as indicated and selected protein names are denoted. The full dataset of specific co-precipitating proteins is given in Table S1. E, Silver-stained SDS polyacrylamide gels showing proteins recovered by purification of Flag-Swi6 in the indicated mutant backgrounds. F, Volcano plot displaying the label-free MS result of Flag-Swi6 nChIPs plotted as in (D) against the reference (Flag-Swi6 nChIP in a H3K9R mutant background). Dashed black lines mark the P value = 0.05 (horizontal) and an enrichment of 4-fold compared to the reference (vertical). Major Flag-Swi6 pulled-down protein groups relevant for this study are color coded as indicated, and selected protein names are denoted. Note maximum upper values were set of the x and y axis to accommodate all detected proteins in the plot. n = 3 biological replicates. The full data set of specific co-precipitating proteins is given in Table S2. G, same as (F) but showing the nuclear periphery proteins pulled-down in Flag-Swi6 nChIPs. See also Figure S1.
Figure 2.
Figure 2.. Identification of proteins specifically associated with different types of heterochromatin.
A, Representative silver-stained SDS polyacrylamide gels showing proteins recovered by purification of Flag-Swi6 in wild-type (wt) and dcr1Δ cells. B-F, label-free MS (B-E) and TMT-based quantitative MS (F) experiments plotted by relative protein abundance (total peptide intensity divided by molecular weight [MW] multiplied by a 1000 (B-E) or 10 (F)) on the x axis and log2 ratio (intensity of peptides originating from the Flag-Swi6 versus Flag-Swi6 mutant background IP) on the y axis. Note maximum upper values were set on the x and y axis to accommodate all detected proteins in the plot. A dashed vertical line marks a fold enrichment = 4 (B-E) or 2 (F) compared to the reference. Venn diagrams (top right) denote the number of proteins common (blue) or enriched (white) in the Flag-Swi6 wt versus mutant background IP, left or right of the dashed vertical line, respectively. Major Flag-Swi6 pulled-down protein groups relevant for this study are color coded as indicated, and selected protein names are denoted. n = 1 (C-E) or 3 (B, F) biological replicates. The full data set of specific co-precipitating proteins is given in Table S2. See also Figure S2.
Figure 3.
Figure 3.. Heterochromatin versus euchromatin proteomes.
A, Schematic of nucleosome-associated proteins used for isolation of native euchromatin (left) and heterochromatin (right) domains. B, Western blotting analysis showing that endogenous Flag-Swi6, Bdf1-Flag and Bdf2-Flag are expressed at similar levels. (C, D) Representative silver-stained SDS polyacrylamide gel (C) and western blot (D) showing that while Swi6 and Bdf1/2 pull-downs contained histones with heterochromatin- and euchromatin-associated modifications, respectively. E, Volcano plot displaying the MS analyses of the histone modification content in the immunoprecipitated chromatin fractions of Flag-Swi6 and Bdf2-Flag plotted as in (Figure 1D). n = 3 biological replicates. The full data set of specific histone modifications is given in Figure S3C. F, Volcano plot displaying the TMT-based quantitative MS result of Flag-Swi6 nChIPs plotted as in (Figure 1D) against Bdf1-Flag nChIPs. Dashed vertical lines mark a fold enrichment = 2 compared to the reference. The names of known heterochromatin proteins (top graph) and nuclear periphery proteins (bottom graph) are denoted. n = 3 biological replicates. The full dataset of specific co-precipitating proteins is presented in Table S3. G, same as (F) but showing the label-free MS analysis of protein content in the immunoprecipitated chromatin fractions of Flag-Swi6 versus Bdf2-Flag. n = 3 biological replicates. The full dataset of specific co-precipitating proteins is presented in Table S3. H, Diagram of known nuclear periphery proteins. Proteins in bold were present in the immunoprecipitated chromatin fraction by Flag-Swi6. I, Venn diagrams of full dataset from (F) and (G) showing the number of shared and specific proteins in the indicated immunoprecipitated chromatin fractions. J, Summary of the most abundant shared factors (left column) and complexes (right column) identified by MS in Flag-Swi6 IP versus Bdf1-Flag and Bdf2-Flag IPs. See also Figure S3.
Figure 4.
Figure 4.. Genome-wide localization of NPC and INM proteins and role for the Lem2 and Nur1 proteins in perinuclear anchoring.
A, ChIP-seq tracks showing the localization of the indicated proteins to representative heterochromatin domains. Reads are presented as reads per million (number in bracket). Top, chromosome coordinates. B, Same as in (A), but showing an expanded view of Lem2-TAP and Nur1-TAP ChIP-seq at rDNA regions. C, Representative images of live cells with Lem2-mKO2 and Gar1-GFP are shown. Dotted line indicates the nuclear periphery. Scale bar, 5 μm. D, Left, Representative 3D reconstructions of the nucleolus (green) and the nucleus (light gray). The nucleolus and nucleus were reconstructed based on the signals of the Gar1-GFP nucleolar marker and the Cut11-mKO2 nuclear membrane marker, respectively. Signal boundaries are shown with clipping planes for Cut11-mKO2. Right, top, the volume (μm3/y-axis) of the Gar1-GFP nucleolar domain plotted for the strains indicated in the x-axis. The average volume of n = 40–70 cells was calculated for each strain. Right, bottom, the ratio of the volume of the nucleolus to the volume of the nucleus plotted for the strains indicated in the x-axis. The average ratio of n = 40–70 cells was calculated for each cell separately and the averages from all cells were plotted for each strain. Error bars indicated SEM. P values of pairwise unpaired t-tests of each strain compared with the #1 wt strain are indicated with asterisks. n.s = not significant, *p<0.05, ***p<0.001. E, Images of live cells with Gar1-GFP nucleolar marker and Cut11-mKO2 nuclear membrane marker showing the fragmentation of the Gar1-GFP-stained nucleolus and its localization outside the nucleus in lem2Δ, nur1Δ, and double mutant cells (white arrows). White arrowhead highlights deformation of the nuclear envelop in lem2Δ cells. Percentage (%) of cells showing fragmentation is indicated (bottom right), calculated as the average ± s.d. from the two clones of each mutant strain. Scale bar, 2 μm. See also Figure S4.
Figure 5.
Figure 5.. Npp106 is required for clustering of heterochromatin foci and epigenetic inheritance.
A, Representative images of live cells with GFP-Swi6 and Taz1-mKO2. Dotted lines indicate the nuclear periphery. Scale bar, 5μm. B, Number of GFP-Swi6 foci is plotted as the percentage of cells with 1–4 foci (blue bar) or with more than 4 foci (yellow bars) for each strain (n = 200–300 cells). C, Average number of GFP-Swi6 foci (n = 2 biological replicates) for the indicated strains. D, Average number of Taz1-mKO2 foci (n = 2 biological replicates) for the indicated strains. E, Images of live cells with Cnp1-GFP centromeric marker in npp106+ and npp106Δ cells. Scale bar, 5μm. F, Map of 10XtetO-ade6+ reporter gene inserted in place of the ura4+ gene between pmp20+ and mug135+ genes. Thin arrows indicate primer locations for ChIP-qPCR analyses in H and I panels. G, Silencing assays of 10XtetO-ade6+ on low-adenine medium lacking tetracycline (Low Ade - TET) or containing tetracycline (Low Ade + TET) to assess heterochromatin establishment and maintenance, respectively, in TetR-Clr4DCD, clr4+ and epe1+ or epe1D cells containing the indicated NPC mutations. H, I, H3K9me3 (H) and Swi6 (I) ChIP-qPCR analyses of 10XtetO-ade6+ reporter gene and surrounding regions in cells with the above genotypes grown for 24 hr in the absence (-TET, left) or presence (+TET, right) of tetracycline. fbp1+, used as control, is a gene located in euchromatin. Values are shown as % input. Error bars, s.d.; n = 3 biological replicates. J, Silencing assays of 10XtetO-ade6+ showing that heterochromatin inheritance is enhanced in alm1Δ cells. 3 different alm1Δ isolates are presented. See also Figure S5.
Figure 6.
Figure 6.. Summary of heterochromatin and euchromatin proteomes.
A, Summary of heterochromatin, euchromatin, and common chromatin complexes (in bold) identified by nChIP. Proteins in red in chromatin/shared are proteins that have been shown previously to be involved in silencing. B, Summary of histone modifications associated with euchromatin and heterochromatin. C, Model for nuclear pore complex (NPC)-mediated clustering of heterochromatic loci via interactions between Swi6 and the inner ring subcomplex. Centromeres are further associated with the periphery via interactions with the Nur1-Lem2 inner nuclear membrane complex. D, Diagram of the nucleus showing representative heterochromatin- and euchromatin-associated proteins and localization of specific chromosome regions to the nuclear periphery via interactions with Nur1-Lem2 or NPCs. The NPCs associated with heterochromatin are shown in red.
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