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Mechanical regulation of transcription controls Polycomb-mediated gene silencing during lineage commitment

Abstract

Tissue mechanics drive morphogenesis, but how forces are sensed and transmitted to control stem cell fate and self-organization remains unclear. We show that a mechanosensory complex of emerin (Emd), non-muscle myosin IIA (NMIIA) and actin controls gene silencing and chromatin compaction, thereby regulating lineage commitment. Force-driven enrichment of Emd at the outer nuclear membrane of epidermal stem cells leads to defective heterochromatin anchoring to the nuclear lamina and a switch from H3K9me2,3 to H3K27me3 occupancy at constitutive heterochromatin. Emd enrichment is accompanied by the recruitment of NMIIA to promote local actin polymerization that reduces nuclear actin levels, resulting in attenuation of transcription and subsequent accumulation of H3K27me3 at facultative heterochromatin. Perturbing this mechanosensory pathway by deleting NMIIA in mouse epidermis leads to attenuated H3K27me3-mediated silencing and precocious lineage commitment, abrogating morphogenesis. Our results reveal how mechanics integrate nuclear architecture and chromatin organization to control lineage commitment and tissue morphogenesis.

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Figure 1: Mechanical strain induces transcriptional repression.
Figure 2: Mechanical strain mediates H3K27me3 and RNAPII-S2p occupancy.
Figure 3: Mechanical strain regulates H3K27me3 through localized actin remodelling controlled by Emd and NMIIA.
Figure 4: Strain induces global rearrangement of chromatin.
Figure 5: Nuclear actin mediates the effect of transcription on H3K27me3 occupancy.
Figure 6: Force-mediated adjustment of transcription regulates SC lineage commitment.
Figure 7: NMIIA activity regulates terminal differentiation in vivo.
Figure 8: A model describing mechanical strain-driven regulation of transcription and lineage commitment.

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Acknowledgements

We thank A. Antebi, P. Tessarz and R. Wedlich-Söldner for critical reading of the manuscript, M. Schüler for advice on the bioinformatics analyses, T. Bücher and N. Bremicker for technical assistance, P. Zentis for help with image analyses, and the FACS & Imaging Core Facility of MPI for Biology of Ageing, and CECAD Imaging Facility for support. The Max Planck Genome Centre Cologne is acknowledged for sequencing. This work was supported by the Max Planck Society, the Max Planck Förderstiftung, the Behrens-Weise Foundation (to S.A.W.), and Deutsche Forschungsgemeinschaft through SFB 829 (to C.M.N. and S.A.W.) and through SPP1782 (to C.M.N.). The Myh9 mice were kindly provided by the European Mutant Mouse Archive (EMMA).

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Authors

Contributions

S.A.W. conceived and supervised this study. H.Q.L. designed and performed most of the experiments and analysed data. S.G., C.-Y.C.Y., F.T. and C.G. performed experiments. A.Y., B.H. and C.D. performed the bioinformatics analyses. A.P. and C.M.N. designed experiments, analysed data and provided conceptual advice. S.A.W. designed and performed experiments, analysed data and wrote the paper. All authors commented on and edited the manuscript.

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Correspondence to Sara A. Wickström.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Mechanical strain impacts global transcription and H3K27me3 occupancy.

(a) Estimation of global mRNA expression changes from ERCC spike-ins. Control samples were spiked with mix 1 and strained samples were spiked with mix 2. The observed ERCC ratio (black dots = data points) deviates considerably from the expected ratios (dotted black line). Regression analysis with the ercc-dashboard package (red solid line) indicated a strain-induced drop of polyA + RNA levels to 85% of control cells (n = 3 biological replicates). (b) Western blot analyses of H3K27me3 and RNAPII-S2p show increased levels of H3K27me3 and a decrease in RNAPII-S2p after 12 h of strain. Quantifications show mean + s.e.m. from 4 independent experiments. For full scans of western blots see Supplementary Fig. 8.

Supplementary Figure 2 Kinetics of RNAPII-S2p and H3K27me3 regulation by strain.

(a) qPCR of RNAPII-S2p-ChIP shows decreased occupancy of RNAPII-S2p at gene bodies of lineage-specific and non-lineage specific PRC2 target genes as well as at constitutively expressed genes already after 3 h of strain (mean + s.e.m., n = 3 independent experiments). (b) qPCR of H3K27me3-ChIP shows no differences in H3K27me3 at promoters upon 3 h of strain (mean + s.e.m., n = 3 independent experiments). (c) Changes in occupancy of RNAPII-S2p become more pronounced after 6 h of strain (mean + s.e.m., n = 3 independent experiments). (d) Only marginal differences in the occupancy of H3K27me3 at promoters upon 6 h of strain (mean + s.e.m., n = 3 independent experiments). (e) Quantification of cell density shows no differences after 12 h of strain (mean + s.d., n = 5 independent experiments, ns = not significant, P = 0.734, Student’s t-test). (f) Quantification of nuclei shows no significant difference in circularity and volume after 12 h of strain (mean ± s.d., n values represent nuclei pooled across 4 independent experiments, ns = not significant, P > 0.6, Mann–Whitney). (g) FACS analysis of proliferating cells (EdU incorporation) shows no differences after 12 h of strain (mean + s.d., n = 3, ns = not significant, P = 0.1, Mann–Whitney). (h) FACS analysis of apoptotic cells (Annexin V) shows no differences after 12 h of strain (mean + s.e.m., n = 6, ns = not significant, P = 0.394, Mann–Whitney).

Supplementary Figure 3 Mechanical strain impacts G/F actin ratio and NMIIA localization without affecting Lamin A/C or Emd protein levels.

(a) Western blot analysis of F- and G-actin fractions shows that 3 h of mechanical strain reduces the G/F actin ratio. The decrease in G/F actin ratio is offset by inhibition of NMII activity by blebbistatin (Bleb). Silver staining of SDS-PAGE gel shows equal amounts of protein loaded (lower panel). (b) Quantification of G/F actin ratio (mean + s.e.m., n = 5, P < 0.05, Kruskal–Wallis/Dunn’s). (c) Immunofluorescence staining of a confocal plane at the level of the nucleus shows increased NMIIA staining around the nucleus and is associated with Emd in cells exposed to 3 h of strain (scale bars 7.5 μm). Right panels show linescans through the nucleus indicating enrichment of NMIIA at the outer nuclear membrane of cells exposed to strain. Dotted blue lines mark the position of the nucleus. A representative of 3 independent experiments is shown. (d) Western blot analysis of LaminA/C and Emd shows no differences in total protein levels upon 6 h of mechanical strain. RNAi-mediated depletion of Emd (siEmd) results in efficient reduction of Emd protein wheras it has no effect on LaminA/C levels (mean + s.e.m., n = 5). For full scans of all western blots see Supplementary Fig. 8.

Supplementary Figure 4 Mechanical strain induces chromatin remodeling.

(a) H3K9me2,3-ChIP shows a decrease in the occupancy of this mark on major satellites (Major sat) and on lamina-associated domains (LAD) upon 12 h of strain. Low levels of this histone mark is detected at promoters of lineage and non-lineage PRC target genes, and this occupancy is further reduced upon strain (mean + s.e.m., n = 3 independent experiments). (b) H3K9me2,3-ChIP shows no apparent change in H3K9me2,3 at major satellites or LADs after 3 h of strain (mean + s.e.m., n = 3 independent experiments). (c) H3K9me2,3-ChIP shows that reduction of H3K9me2,3 at major satellites or LADs starts becoming visible after 6 h of strain (mean + s.e.m., n = 3 independent experiments). (d) H3K27me3-ChIP shows that major satellites and LADs acquire this modification upon 12 h of strain (mean + s.e.m., n = 3 independent experiments). (e) qPCR analysis of a set of genes transcribed from the corresponding LADs in Fig. 3 shows unchanged expression levels after 12 h of strain (mean + s.e.m., n = 3 independent experiments, ns = not significant, Mann–Whitney). (f) Chromosome painting of chromosomes (Chr) 1 and 18 at low magnification shows effect of strain on chromosome territories (scale bar 20 μm). (g) Chromosome painting of Chr1 and 18 shows compact chromosome territories in the nuclear periphery of control cells, whereas in strained or Emd-depleted cells the domains are more diffuse (scale bars 5 μm). Right panels show quantification (mean ± s.d., n values represent cells pooled across 3 independent experiments, P < 0.0237, Kruskal–Wallis/Dunn’s). (h) Chromosome painting of Chr1 and 18 shows condensed chromosomes in telophase compared to interphase. Right panels show quantification (mean ± s.d., n values represent cells).

Supplementary Figure 5 Emd and nuclear actin mediate the effect of strain on transcription and H3K27me3 occupancy.

(a) Immunofluorescence analysis of H3K27me3 and RNAPII-S2p shows that depletion of Emd prevents strain from inducing H3K27me3 accumulation and attenuation of RNAPII-S2p (scale bars 25 μm, mean ± s.d., n values represent nuclei pooled across 3 independent experiments, P < 0.05, P < 0.01, Kruskal–Wallis/Dunn’s). b. Western blot from cells transfected with siRNA targeting Xpo6 (siXPO6) shows efficient depletion of the Xpo6 protein compared to control cells transfected with scrambled siRNA (siScr). A representative of 3 independent experiments is shown. For full scans of western blots see Supplementary Fig. 8. (c) H3K27me3-ChIP qPCR was performed from control or Xpo6-depleted cells that were treated with 5,6-Dichlorobenzimidazole-1-β-D- ribofuranoside (DRB) for 12 h to block RNAPII elongation. DRB treatment prevented the effect of Xpo6 depletion on H3K27me3. A region in the gene body of S26 and IgG (not shown) were used as negative controls (mean + s.e.m., n = 3 independent experiments). (d) qPCR of H3K9me2,3-ChIP shows no change in H3K9me2,3 at lamina-associated domains (LAD) and major satellites (Major sat) upon Xpo6 depletion. A region in the gene body of S26 and IgG were used as negative controls (mean + s.e.m., n = 4 independent experiments).

Supplementary Figure 6 Actin mediates the effects of strain on gene expression.

(a) qPCR analysis of late differentiation genes in Cytochalasin D (CytoD)-treated cells under mechanical strain shows that CytoD treatment prevents strain-mediated repression of differentiation gene expression (mean + s.e.m., n = 5, P < 0.05, P < 0.01, P < 0.001, Kruskal–Wallis/Dunn’s). (b) Depletion of Xpo6 blocks the attenuation of differentiation gene expression in strained cells (mean + s.e.m., n = 4, P < 0.04, P < 0.01, Kruskal–Wallis/Dunn’s). (c) qPCR analyses show that RNAi-mediated depletion of Importin 9 (siIpo9) decreases expression of late differentiation genes (mean + s.e.m., n = 3, P < 0.03, Mann–Whitney).

Supplementary Figure 7 Myh9-deficient mice show defects in epidermal architecture and differentiation.

(a) Immunofluorescence analysis of mouse keratinocytes shows increased H3K27me3 and decreased RNAPII-S2p after 12 h of strain (scale 30 μm, mean ± s.d., n values represent cells pooled across 4 independent experiments, P < 0.05, Mann–Whitney). (b) qPCR analysis of lineage-specific and non-lineage PRC2 target genes in mouse keratinocytes shows that transcription of these genes is surpressed upon 12 h of strain (mean + s.e.m., n = 6, P = 0.027, P < 0.008, Mann–Whitney). (c) Quantification of Ki67-positive proliferating cells from P0 epidermis (mean + s.e.m., n = 4 mice/genotype, ns = not significant, P = 0.0591, Mann–Whitney). (de) Immunofluorescence analysis of Gata-3 (C) and Keratin-6 (K6; D) from P0 hair follicles. Both markers are expressed in Myh9EKO hair follicles. K14 is used to mark progenitors (scale bars 50 μm). A representative image from 3 independent experiments is shown. (f) qPCR analysis of differentiation genes in keratinocytes isolated from Myh9EKO and control mice shows upregulation of differentiation gene expression in Myh9EKO keratinocytes (mean + s.e.m., n = 4 mice/genotype, P < 0.05, Mann–Whitney). (g) Western blot analysis of proteins encoded by late differentiation genes shows increased levels of cornified envelope proteins in Myh9EKO keratinocytes. A representative image from 3 independent experiments is shown. For full scans of western blots see Supplementary Fig. 8. (h) qPCR analysis of a selection of non-PRC2 target genes from Myh9EKO and control E16.5 epidermis shows unaltered expression of these genes (mean + s.e.m., n = 5 mice/genotype, Mann–Whitney). (i) Immunofluorescence analysis of G-actin shows increased G-actin levels in basal keratinocytes of Myh9EKO E16.5 epidermis (scale bars 35 μm). Right panel shows quantification of nuclear actin levels from immunofluorescent images (mean + s.e.m., n = 6 mice/genotype, P = 0.0192, Student’s t-test). (j) Immunofluorescence analysis of Emd and Lamin A/C shows no differences in the protein levels of the two proteins in basal keratinocytes of Myh9EKO E16.5 epidermis (scale bars 25 μm). Right panel shows quantification of Emd and Lamin A/C levels from immunofluorescent images (mean + s.e.m., n = 3 mice/genotype, ns = not significant, P > 0.05, Mann–Whitney).

Supplementary Figure 8 Full scans of all western blots.

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Le, H., Ghatak, S., Yeung, CY. et al. Mechanical regulation of transcription controls Polycomb-mediated gene silencing during lineage commitment. Nat Cell Biol 18, 864–875 (2016). https://doi.org/10.1038/ncb3387

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