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The NSL complex maintains nuclear architecture stability via lamin A/C acetylation

Abstract

While nuclear lamina abnormalities are hallmarks of human diseases, their interplay with epigenetic regulators and precise epigenetic landscape remain poorly understood. Here, we show that loss of the lysine acetyltransferase MOF or its associated NSL-complex members KANSL2 or KANSL3 leads to a stochastic accumulation of nuclear abnormalities with genomic instability patterns including chromothripsis. SILAC-based MOF and KANSL2 acetylomes identified lamin A/C as an acetylation target of MOF. HDAC inhibition or acetylation-mimicking lamin A derivatives rescue nuclear abnormalities observed in MOF-deficient cells. Mechanistically, loss of lamin A/C acetylation resulted in its increased solubility, defective phosphorylation dynamics and impaired nuclear mechanostability. We found that nuclear abnormalities include EZH2-dependent histone H3 Lys 27 trimethylation and loss of nascent transcription. We term this altered epigenetic landscape “heterochromatin enrichment in nuclear abnormalities” (HENA). Collectively, the NSL-complex-dependent lamin A/C acetylation provides a mechanism that maintains nuclear architecture and genome integrity.

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Fig. 1: Loss of MOF or its associated complex members KANSL2/KANSL3 leads to nuclear blebbing, MN formation and NERDI.
Fig. 2: Characterization of the transcriptional status of the nuclear abnormalities and DNA damage accumulation.
Fig. 3: MOF and KANSL2 acetylomes reveal the role of lamin A/C acetylation in nuclear lamina architecture.
Fig. 4: Loss of MOF or expression of K311R lamin A leads to genomic instability including chromothripsis.
Fig. 5: Characterization of HENA.
Fig. 6: HENA is dynamic and concomitant with transcriptional silencing of nuclear abnormalities.
Fig. 7: Electron tomography analyses show localized patches of fuzzy nuclear lamina with disrupted submembrane heterochromatin following Mof KO or expression of K311R lamin A.
Fig. 8: Loss of lamin A/C acetylation results in its increased dynamics, increased solubility and weaker nuclear mechanostability.

Data availability

The MS proteomics data for Mof KO have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository (accession number: PXD008539). MALBAC single-cell genomic sequencing data have been deposited to ENA (accession number: PRJEB27801). Source data for all figures in the main paper and its Supplementary Information where statistics were conducted have been provided as Supplementary Table 7. All other data supporting the findings of this study are available from the corresponding author on reasonable request.

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Acknowledgements

We are grateful to B. Sheikh, M. Shvedunova, M. Buck, M. Samata, C. Pessoa Rodrigues, S. Lefkopoulos and G. Semplicio for critical reading of the manuscript. We thank E. Trompouki and R. Sawarkar for fruitful discussions and suggestions. We also thank P. Rawat for help with FRAP and M.-F. Basilicata for help with the generation of the Moffl/-, CreT/+ ESCs. The MPI-IE core facilities (for fluorescence-activated cell sorting, deep sequencing, imaging and mouse care), the EMBL IT facilities (for computing) and the EMBL EMCF (for electron microscopy) have been invaluable for this project. This work was supported by CRC992 (A02) and CRC1381 (B3) awarded to A.A., by an ERC Starting Grant awarded to J.O.K. (336045) and the Swiss National Science Foundation (SNSF 31003A_179418, to O.M.). E.A.R.-Z. is an Emmy Noether Fellow (DFG no. 396913060); P.K. acknowledges the ERC Advanced Grant CardioNect (201203).

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Contributions

A.K. and A.A. conceptualized and designed the experiments and A.K. performed the majority of the experiments with the exception of the following. The in vitro lamin assembly experiments and analysis were performed by R.d.L. and O.M; MS analysis and single-cell genomic DNA bioinformatic analysis were carried out by W.S. and G.M. and by T.R. and J.O.K., respectively; the Kansl2 and Kansl3 conditional KO mice were generated by B.H.; S.G. and H.-R.C. helped with the imaging analysis of the Mof, Kansl2 and Kansl3 KO MEFs; H.K. provided the H3K27me3-mintbody plasmid; J.S. helped with the cell culture and biochemistry experiments; nanoindentation and electron tomography imaging were performed by R.P. and E.A.R.-Z. respectively, together with P.K.; A.K. wrote the manuscript with input from all of the authors; O.M., J.O.K. and A.A. acquired funding; A.A. supervised all aspects of the study. All authors reviewed, edited and approved the paper.

Corresponding author

Correspondence to Asifa Akhtar.

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Integrated supplementary information

Supplementary Figure 1 Further characterization of the Mof, Kansl2 and Kansl3 KO nuclear abnormalities.

a) Relative Mof mRNA expression (against Rplp0) in control and Mof KO MEFs (4 days). Two-tailed unpaired t-test on n=3 independent biological replicates. Data shown are the mean ± s.e.m. b) Quantification of nuclear abnormalities of Mof fl/-, CreT/- MEFs in the presence or absence of 4OHT; n=2 biological replicates. c) Immunoblotting on whole cell extracts of control and Mof KO MEFs expressing the indicated FLAG-MOF constructs. d) Immunoblotting on whole cell extracts of control and Mof KO MEFs at the indicated time points. e) Immunoblotting on whole cell extracts of control, Kansl2 and Kansl3 KO MEFs. f) Immunoblotting on subcellular fractions of control and Mof KO (2 days) MEFs, where the nucleoplasmic p53 is depicted. Cytopl;Cytoplasmic fraction, Memb;Membrane bound proteins fraction, Nucleopl; Nucleoplasmic fraction, Chrom; Chromatin bound fraction. g) (Same as Fig. 1d) Representative time-lapse imaging snapshots of control, Mof and Kansl2 KO MEFs, transduced with GFP-NLS or GFP-H2B as indicated. 6 independent experiments. The white arrowheads indicate nuclear abnormalities. h) Representative immunostaining of lamin B1 (red) and NUP153 (green) in control, Mof, Kansl2 and Kansl3 KO (4 days) MEFs. dsDNA was counterstained using DAPI. Scale bars, 5μm. i) Representative immunostaining of lamin A (green) and γH2A.X (red) in control and Mof KO (4 days) MEFs. dsDNA was counterstained using DAPI. Scale bars, 5μm. j) Quantification of γH2A.X positive and negative micronuclei on the indicated KO MEFs; Two-tailed unpaired t-test on n=5 (Mof KO) and n=3 (Kansl2 and Kansl3 KO) independent biological replicates. Data shown are the mean ± s.e.m. Uncropped blots of c, d, e, f can be found in Supplementary Fig. 9.

Supplementary Figure 2 Peptide fractionation and SILAC analysis scheme for MEFs and ESCs.

a) Schematic representation of the experimental design used for total proteome and acetylome identification by LC-MS. Cells were labeled with heavy lysine (K8) and arginine (R10) and reciprocal labeling setup (“label swap”) was implemented (Methods). Tryptic peptides were fractionated on a C18 (solid phase extraction) column and underwent two rounds of anti-acetyl (Lys) immunoprecipitation procedures using two different antibody resins. Samples were measured by nano LC-MS/MS (Methods). Additionally, IP input samples were measured. b) Correlation matrix scatter plots with Pearson-r correlation values for acetyl (Lys) sites in replicates from MEFs and ESCs control and Mof KO. Raw ratios were log2 transformed and ratio distributions were adjusted by quartile normalization. c) Histograms representing ratio distributions across replicates in anti-acetyl(Lys) IP samples in MEFs and ESCs. Raw ratio values were log2 transformed (white) and subsequently adjusted by quartile normalization (blue). d) Numerical UpSet diagram representing the overlap between identified acetyl(K)sites within all replicates in MEFs (left panel) and ESCs (right panel). From the total number of 6534 unique acetyl(K)sites in MEFs, 4048 were found in at least 3 replicates. For ESCs, 1964 acetyl(K)sites were found in at least 3 replicates out of 3020 unique acetyl(K)sites. For b, c and d n=6 independent biological replicates.

Supplementary Figure 3 Characterization of the MOF dependent proteome and acetylome deregulation on MEFs and ESCs.

a) Adjusted liquid chromatography profiles for particular IP fractions that exhibit different hydrophobic properties (Methods). b) Graphic representation of histone multiplicity (Supplementary Tables 3, 4 and 6). c) Correlation between different multiplicities of acetylated sites identified in MEFs and ESCs Mof KO. Log2 fold changes of a two-sided t-test performed on n=6 SILAC ratios. Histone acetyl (Lys) sites are highlighted in red and H4K16ac in green. The red line and gray field correspond to a linear smooth fit regression with a 0.95 confidence interval respectively. d) Immunoblotting on whole cell extracts of control and Mof KO ESCs. Uncropped blots in Supplementary Fig. 9. e) Overlap of the MEFs and ESCs Mof KO proteome and acetylome datasets. f) Heatmap illustration of the quantitative proteome changes of Mof KO MEFs and ESCs. Dendrogram illustrates Euclidean distance clustering with 4 identified clusters. Color range indicates the log2 fold changes. g) Enriched gene sets of Supplementary Fig. 3f. (Supplementary Table 5). h) Same as Supplementary Fig. 3f for the commonly detected acetyl (Lys) sites. i) Enriched gene sets of Supplementary Fig. 3h. (Supplementary Table 5). j) Enriched gene sets of the MEFs and ESCs-only acetylome. (Supplementary Table 5). k) Heatmap illustration of all quantified histone acetyl (Lys) sites, in MEFs and ESCs Mof KO. Color range indicates the log2 fold changes. Different multiplicity values were averaged (Supplementary Fig. 3b, c and Supplementary Table 6). Not identified sites are marked as a crossed box. l) Enriched gene sets of the statistically significantly hypoacetylated targets in MEFs and ESCs Mof KO. Color scale depicts the p-value of the statistically enriched terms (Supplementary Table 5). m) Circos plot representation of the GO analysis overlap between significantly hypoacetylated Mof KO targets in MEFs and ESCs. Yellow lines represent similar GO terms (Supplementary Table 5). For panels g, I, j, l, m the n=number of dataset genes within each GO term is defined in Supplementary Table 5.

Supplementary Figure 4 Conservation analysis of the lamin A/C acetyl(Lys)sites.

a) Left; Representative immunostaining of lamin B1 (green) and OCT4 (red) in control and Mof KO (4 days) ESCs. dsDNA was counterstained using DAPI. Scale bars, 5μm. Right; Quantification of nuclear abnormalities in control and Mof KO ESCs. n=2 biological replicates. b) Phylogenetic tree of lamin A/C in the indicated organisms based on complete protein sequences. The maximum-likelihood tree was reconstructed using PhyML. (Uniprot annotations; P02545 – Homo sapiens, P48678 - Mus musculus, P13648 - Gallus gallus, B3DKC5 - Danio rerio, Q60HF0 - Macaca fascicularis, Q21443 - Caenorhabditis elegans, F1MYG5 - Bos Taurus, Q03427 - Drosophila melanogaster). c) Vertebrate lamin A/C protein sequence alignment. The lysine (K) residues are highlighted in red. The coiled-coil domain is framed and the detected acetyl(K)sites from the MEFs SILAC are marked with asterisks. Alignment was performed using the SeaView software employing the clustal omega algorithm.

Supplementary Figure 5 Characterization of lamin A/C acetylation in MEFs.

a) Left; immunoblotting on subcellular fractions of WT/K417R OST-lamin A MEFs. Right; Nuclear abnormalities quantification; Two-tailed unpaired t-test on n=3 independent biological replicates. b) Relative mRNA expression (against Rplp0) of the OST-tagged WT, K97R, K108R, K311R and K486R derivatives (n=3 independent biological replicates) of Supplementary Fig. 5c. c) Nuclear abnormalities quantification in MEFs expressing the indicated lamin A derivatives. Statistical comparisons against WT lamin A using ordinary one-way ANOVA on n=3 independent biological replicates. d) Relative mRNA expression (against Rplp0) of the OST-tagged WT, K311R and K311Q lamin A derivatives (n=3 independent biological replicates) of Fig. 3c. e) Immunoblotting on the indicated subcellular fractions of MEFs expressing WT, K311R or K311Q lamin A. f) Left; Immunoblotting on whole cell extracts of immortalized Lmna KO MEFs expressing WT, K311R or K311Q OST-lamin A. Right; Nuclear abnormalities quantification. Two-tailed unpaired t-test on n=3 independent biological replicates. g) Representative immunostaining for OST-tag (green) and lamin B1 (red) on control and Mof KO K319R/Q MEFs. h) Nuclear abnormalities quantification of Supplementary Fig. 5g. Ordinary one-way ANOVA on n=3 independent biological replicates. i) Representative immunostaining for WT/K59R GFP-lamin B2 (green) and lamin B2 (red). j) Nuclear abnormalities quantification in WT/K59R lamin B2 MEFs. Two-tailed unpaired t-test on n=3 independent biological replicates. k) TEM analysis of paracrystalline arrays. K311R and WT lamin C show similar striped patterns and structural organization of the paracrystalline arrays. Scale bar, 20 nm. l) Quantification of total RNA Pol II and RNA Pol II-S2P. Nonparametric one-way ANOVA followed by a Kruskal-Wallis test. n numbers are stated in the graph and represent nuclear structures counted. m) left:Representative immunostaining of NUP153 in WT and K311R OST-lamin A MEFs. Right: Quantification of NUP153 in main nuclei and nuclear abnormalities of K311R MEFs. Two-tailed nonparametric Mann-Whitney test. n numbers are stated in the graph and represent nuclear structures counted. n) H4K16ac intensities on MEFs expressing WT, K311Ror K311Q OST-lamin A. Means were compared by a Kruskal-Wallis test on n=3 independent biological replicates. The lower and upper hinges correspond to the first and third quartiles. The whiskers extends from the hinge to the largest or smallest values respectively, no further than 1.5 * IQR (inter-quartile range) from the hinge. The middle line represents the median (the middle value of the dataset). Data beyond the end of the whiskers are plotted individually. Data shown are the mean ± s.e.m for a, b, c, d, f, h, j, l, m. For g, i, m dsDNA was counterstained using DAPI and scale bars represent 5μm. Uncropped blots of a, e, f can be found in Supplementary Fig. 9.

Supplementary Figure 6 Sensitivity of lamin A/C acetylation to HDAC inhibition and representative genomic DNA read-depth plots for control, Mof KO and K311R lamin A MEFs.

a) Immunoblotting on whole cell extracts of control and Mof KO upon panobinostat (200nM) and bufexamac (5μM) treatment. b) Quantification of nuclear abnormalities in control and Mof KO MEFs treated with the indicated panobinostat and bufexamac concentrations; Ordinary one-way ANOVA on n=3 independent biological replicates. c–e) Normalized read-depth along the genome for typical WT control primary MEFs. Three different cells are shown. f) Normalized read-depth along the genome for a Mof KO MEF exhibiting chromothripsis, shown in Fig. 4i. g) Normalized read-depth along the genome for a K311R lamin A MEF exhibiting chromothripsis, shown in Fig. 4j. h) Normalized read-depth plots and CBS segmentation results for selected Mof KO cells. CBS segments were further computationally processed (Methods) to generate high confidence segments used in Fig. 4e, f. i) Immunoblotting on subcellular fractions of MEFs expressing the indicated OST-lamin A derivatives. j) Normalized read-depth plots and CBS segmentation results for selected K311R lamin A MEFs. CBS segments were further computationally processed (Methods), to generate high confidence segments used in Fig. 4g, h. Uncropped blots of a, i can be found in Supplementary Fig. 9.

Supplementary Figure 7 In depth characterization of the nuclear abnormalities epigenetic landscape.

a) Quantification of H3K27me3 and H3K27ac normalized to DAPI in the main nuclei, nuclear blebs and micronuclei of Mof KO (4 days); Nonparametric one-way ANOVA followed by a Kruskal-Wallis test. b) Representative immunostaining of H4K8ac (green) in Mof and Kansl2 KO (4 days) MEFs. c) Quantification of H4K8ac/DAPI in the main nuclei, nuclear blebs and micronuclei of Mof and Kansl2 KO (4 days); Nonparametric one-way ANOVA followed by a Kruskal-Wallis test. d) Representative immunostaining of K311R OST-lamin A MEFs for ATAC-see (white) and H3K27me3 (red). Arrows indicate nuclear abnormalities enriched for H3K27me3. e) Quantification of DAPI/dsDNA in the main nuclei and micronuclei of K311R lamin A MEFs; Two-tailed nonparametric Mann–Whitney test. f) Quantification of histone H3/DAPI in the indicated nuclear structures of Mof KO MEFs; Nonparametric one-way ANOVA followed by a Kruskal-Wallis test. g) Quantification of DAPI in the indicated nuclear structures of K311R lamin A MEFs; Nonparametric one-way ANOVA followed by a Kruskal-Wallis test. h) Representative immunostainings upon incubation with blocking peptides indicating specificity of the detected signal. Arrows indicate nuclear abnormalities. i) Quantification of bulk ATAC-see in MEFs expressing the indicated lamin A derivatives. Dots represent mean signal intensity. Nonparametric one-way ANOVA followed by a Kruskal-Wallis test. j) Immunoblotting on whole cell extracts of WT and K311R lamin A MEFs. Uncropped blots in Supplementary Fig. 9. k) Quantification of nuclear abnormalities on control and Mof KO (2 days) MEFs in the presence of the indicated GSK343 concentrations; Ordinary one-way ANOVA on n=3 independent biological replicates. l) Quantification of H3K27me3/DAPI in the main nuclei and nuclear abnormalities of control and Mof KO (2 days) MEFs in the presence of the indicated GSK343 concentrations; Nonparametric one-way ANOVA followed by a Kruskal-Wallis test. m) Quantification of Pol II-S2P in the main nuclei and nuclear abnormalities of control and Mof KO (2 days) MEFs in the presence of the indicated GSK343 concentrations; Nonparametric one-way ANOVA followed by a Kruskal-Wallis test. The data shown are the mean ± s.e.m. for a, c, e, f, g, k, l, m. dsDNA was counterstained using DAPI and scale bars of 5μm where applied to b, d, h. For a, c, e, f, g, l, m, n numbers are stated in the graphs and represent nuclear structures counted.

Supplementary Figure 8 Defective nuclear lamina architecture and impaired nuclear mechanics upon loss of MOF and lamin A/C acetylation.

a) Representative electron tomographic slices showing preserved nuclear structure in Mof control and WT lamin A MEFs. N; nucleus. b) Quantification of electron tomographic micronuclei heterochromatin density in Mof KO and K311R lamin A MEFs. Data shown are the mean ± s.e.m. n=15 nuclear structures counted. c) Quantification of electron tomographic submembrane (120 nm) heterochromatin density in the 1-μm-vicinity of fuzzy nuclear lamina sites; Two-tailed nonparametric Mann–Whitney test. The data shown are the mean ± s.e.m. n numbers are stated in the panel and represent single cells counted. d) Quantification of electron tomographic global nuclear heterochromatin density measured; Two-tailed nonparametric Mann–Whitney test. Data shown are the mean ± s.e.m. n numbers are stated in the panel and represent single cells counted. e) Relative Emerald-lamin A FRAP over time in Lmna KO MEFs overexpressing WT or K311R lamin A. Two-tailed nonparametric Mann–Whitney test for n=45 WT and n=50 K311R cells. f) Immunoblotting on whole cell extracts of MEFs upon Mof or Kansl2 KO (4 days). Uncropped blots in Supplementary Fig. 9. g) Nuclear and cyto-indentations are performed in triplicate for each cell (left image) and for each isolated nucleus (right image). Pink crosses indicate sites of nanoindentation. Scale bar; 20μm. h) Schematics of the nanoindentation experiment performed and representative force distance curve used to calculate the effective Young’s Modulus. Fit of the indentation curve used to obtain the Young’s Modulus shown in black. i) Cytoindentation quantification on intact control and Mof KO (4 days) MEFs; Two-tailed nonparametric Mann–Whitney test. Data shown are the mean ± s.e.m. n numbers are stated in the panel and represent single cells counted. j) Nanoindentation nuclei stiffness quantification in intact control and Mof KO (4 days) MEFs; Two-tailed nonparametric Mann–Whitney test. Data shown are the mean ± s.e.m. n numbers are stated in the panel and represent single cells counted. k) Nanoindentation stiffness quantification on freshly isolated control and Mof KO (4 days) MEFs nuclei; Two-tailed nonparametric Mann–Whitney test. Data shown are the mean ± s.e.m. n numbers are stated in the panel and represent single nuclei counted.

Supplementary Figure 9 Unprocessed scans of immunoblots.

Red boxes show cropped regions. Molecular weights (kDa) are indicated.

Supplementary information

Supplementary Information

Supplementary Figures 1–9 and Supplementary Video and Table titles/legends.

Reporting Summary

Supplementary Table 1

MEF_input—Proteome deregulation following Mof KO. Two-tailed t-test applied.

Supplementary Table 2

ESC_input—Proteome deregulation following Mof KO. Two-tailed t-test applied.

Supplementary Table 3

MEF_Acetylome deregulation following Mof KO. Two-tailed t-test applied.

Supplementary Table 4

ESC_Acetylome deregulation following Mof KO. Two-tailed t-test applied.

Supplementary Table 5

Gene Ontology analysis.

Supplementary Table 6

Histone acetyl (lysine) sites multiplicity comparison for Mof KO.

Supplementary Table 7

Statistics source data.

Supplementary Video 1

Live-cell imaging of a control MEF cell transduced with GFP–NLS (green). Time intervals of 15 min are shown.

Supplementary Video 2

Live-cell imaging of a Mof KO MEF transduced with GFP–NLS (green). Time intervals of 24 min are shown.

Supplementary Video 3

Live-cell imaging of a Kansl2 KO MEF transduced with GFP–NLS (green). Time intervals of 15 min are shown.

Supplementary Video 4

Live-cell imaging of a Mof KO MEF transduced with GFP–H2B (green). Time intervals of 15 min are shown.

Supplementary Video 5

Live-cell imaging of a Mof KO MEF transduced with GFP–NLS (green). Time intervals of 2 min are shown. Rupture of the main nucleus is observed.

Supplementary Video 6

Live-cell imaging of a Mof KO MEF transduced with GFP–NLS (green). Time intervals of 2 min are shown. Rupture of the main nucleus is observed.

Supplementary Video 7

Live-cell imaging of a Mof KO MEF transduced with GFP–NLS (green). Time intervals of 2 min are shown. Rupture of the nuclear abnormality followed by main nuclei rupture is observed.

Supplementary Video 8

Live-cell imaging of a Mof KO MEF transduced with GFP–NLS (green). Time intervals of 2 min are shown. Continuous rupture of the nuclear abnormality is observed.

Supplementary Video 9

Live-cell imaging of a WT lamin A MEF transduced with GFP–NLS (green). Time intervals of 15 min are shown.

Supplementary Video 10

Live-cell imaging of a K311R lamin A K311R MEF transduced with GFP–NLS (green). Time intervals of 15 min are shown.

Supplementary Video 11

Live-cell imaging of a Mof KO MEF transduced with GFP-H3K27me3-mintbody (green). Time intervals of 15 min are shown.

Supplementary Video 12

3D reconstruction of a Mof KO MEF (4 d) where the main nuclei (dark blue) with NERDI as well as the MN (light blue) are visible.

Supplementary Note

Mof KO SILAC proteome and acetylome data analysis.

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Karoutas, A., Szymanski, W., Rausch, T. et al. The NSL complex maintains nuclear architecture stability via lamin A/C acetylation. Nat Cell Biol 21, 1248–1260 (2019). https://doi.org/10.1038/s41556-019-0397-z

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