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MLL4-associated condensates counterbalance Polycomb-mediated nuclear mechanical stress in Kabuki syndrome

Abstract

The genetic elements required to tune gene expression are partitioned in active and repressive nuclear condensates. Chromatin compartments include transcriptional clusters whose dynamic establishment and functioning depend on multivalent interactions occurring among transcription factors, cofactors and basal transcriptional machinery. However, how chromatin players contribute to the assembly of transcriptional condensates is poorly understood. By interrogating the effect of KMT2D (also known as MLL4) haploinsufficiency in Kabuki syndrome, we found that mixed lineage leukemia 4 (MLL4) contributes to the assembly of transcriptional condensates through liquid–liquid phase separation. MLL4 loss of function impaired Polycomb-dependent chromatin compartmentalization, altering the nuclear architecture. By releasing the nuclear mechanical stress through inhibition of the mechanosensor ATR, we re-established the mechanosignaling of mesenchymal stem cells and their commitment towards chondrocytes both in vitro and in vivo. This study supports the notion that, in Kabuki syndrome, the haploinsufficiency of MLL4 causes an altered functional partitioning of chromatin, which determines the architecture and mechanical properties of the nucleus.

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Fig. 1: KMT2D loss of function destabilizes MLL4–COMPASS complex activity.
Fig. 2: Effects of MLL4 LoF on the clustering of transcriptional biomolecular condensates.
Fig. 3: MLL4 is clustered in transcriptional condensates through its PrLD.
Fig. 4: PcG clustering is affected by MLL4 LoF.
Fig. 5: MLL4 contributes to establishing nuclear mechanical properties.
Fig. 6: Role of PcG in determining the nuclear architecture.
Fig. 7: MLL4 contributes to establishing nuclear mechanical properties.
Fig. 8: MLL4-dependent mechanical properties in cell lineage commitment.

Data availability

The RNA sequencing raw data have been deposited in the Gene Expression Omnibus database under the accession GSE135550. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD021616. Additional data supporting the findings of this study are available from the corresponding authors upon reasonable request. Source data are provided with this paper.

Code availability

The custom-made code for quantifying the cytoplasmic/nuclear ratio of YA/TAZ has been deposited in the public repository GitHub (https://github.com/SZambranoS/RoutinesNucCytoYAP). The custom-made code for quantifying the Brillouin shift has been deposited in the public repository GitHub (https://github.com/ClaudiaBrill/CodeBrillouinFasciani).

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Acknowledgements

We thank the Next-Generation Sequencing Facility at the Department of Cellular, Computational and Integrative Biology (CIBIO) for help with RNA sequencing, the Protein Technology Facility at CIBIO for recombinant protein purification, the Advanced Light and Electron Microscopy Bioimaging Center at the San Raffaele Scientific Institute (Milan, Italy) for STORM imaging acquisition and data analyses, the TIGEM Medaka Core Facility and F. G. Salierno for technical assistance and the CrestOptics–IIT JointLab for Advanced Microscopy for financial and technical support. We thank G. Merla (IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo, Italy). The Genomic and Genetic Disorders Biobank, a member of the Telethon Network of Genetic Biobanks (project number GTB12001), funded by Telethon Italy, and of the EuroBioBank network, provided us with the human primary fibroblasts derived from either healthy donors or donors with Kabuki syndrome. We thank E. Biasini and the members of the Zippo laboratory for helpful discussions and critical reading of the manuscript. We thank D. Allis for sharing the H3.3K27M construct, R. Young for sharing the OptoIDR vectors and Z. Qiuping for the EGFP–Nesprin1/2–KASH vectors. The Mass Spectrometry and Proteomics Core Facility of CIBIO is supported by the European Regional Development Fund 2014–2020. Work in the Zippo group was supported by grants from the Italian Ministry of Health (GR-2011-02351172), Telethon Foundation (GEP13057) and AFM Telethon (AFM 21514). V.P. is the recipient of an AIRC fellowship (21158).

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Authors

Contributions

A.F. and A.Z. conceived of the study, designed the experiments and interpreted the data. S.D. performed the phase separation and immunofluorescence experiments and participated in data analyses. V.P. performed the cellular and molecular biology studies and participated in data analyses. L.F. and S.B. performed the RNA sequencing experiments and computational analyses. F.C. and D.M. performed the molecular biology studies. L.A., F.G., G.O., E.D. and S.Z. performed the computational analyses on imaging and genomic data. R.B. and D.P. performed the mass spectrometry experiments. D.I., C.S. and I.C. performed the in vivo assays in medaka fish. C.T., P.V. and G.R. performed the Brillouin microscopy and computational data analysis. A.Z. supervised the work and wrote the manuscript.

Corresponding authors

Correspondence to Alessandra Fasciani or Alessio Zippo.

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The authors declare no competing interests.

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Extended data

Extended Data Fig. 1 KMT2D haploinsufficiency affects MLL4 activity.

a, Schematic representation of KMT2D gene and the corresponding MLL4 protein. The position of the inserted mutations in exon 39 and the relative changes in the coding sequences are highlighted. b, qRT-PCR of KMT2D in WT and MLL4Q4092X MSCs, normalized on GAPDH level. Data are means + SEM (n=3 independent experiments); unpaired two-tailed Student’s t-test was applied for statistical analysis. c, Western Blot analysis of MLL4 protein in WT and MLL4Q4092X MSCs by using a specific antibody recognizing a central portion of the protein; Lamin B1 was used as loading control. d, qRT-PCR of KDM6A in WT and MLL4Q4092X MSCs, normalized on GAPDH level. Data are means + SEM (n=3 independent experiments); unpaired two-tailed Student’s t-test was applied for statistical analysis. e, Representative images and quantifications of immunostaining for MLL4 in WT and MLL4Q4092X MSCs grown on the same coverslips. WT MSCs were pre-labelled with CellTrace Violet.Scale bar, 20 µm. Box plots indicate the median (middle line), the first and third quartiles (box), and the 10th and 90th percentile (error bars) of the fluorescence intensity. The number of analyzed nuclei is reported in figure as n; Student’s two-tailed unpaired t-test was applied for statistical analysis. f, Representative images and quantifications of immunostaining for PA1 in WT and MLL4Q4092X MSCs. Scale bar, 20 µm. Box plots indicate the median (middle line), the first and third quartiles (box), and the 10th and 90th percentile (error bars) of the fluorescence intensity. The number of analyzed nuclei is reported in figure as n; unpaired two-tailed Student’s t-test was applied for statistical analysis. g, Western Blot analysis of MLL4 and UTX in WT and MLL4P4093X MSCs; Lamin B1 was used as loading control (n=1). h, Quantifications of immunostaining for MLL4, PA1, UTX, H3K4me1 and H3K27ac in WT and MLL4P4093X MSCs. Box plots indicate the median (middle line), the first and third quartiles (box), and the 10th and 90th percentile (error bars) of the fluorescence intensity. The number of analyzed nuclei is reported in figure as n; Student’s two-tailed unpaired t-test was applied. for statistical analysis (****P<0.0001).

Source data

Extended Data Fig. 2 KMT2D truncating mutations cause MLL4 LoF.

ad, Representative images and quantifications of immunostaining for MLL4 (a), H3K4me1 (b), H3K27ac (c) and UTX (d) in primary fibroblasts from healthy donor or Kabuki patients. Scale bar, 20 µm. Box plots indicate the median (middle line), the first and third quartiles (box), and the 10th and 90th percentile (error bars) of the fluorescence intensity. The number of analyzed nuclei is reported in figure as n; unpaired two-tailed Student’s t-test was applied for statistical analysis (****P<0.0001). e, Quantifications of immunostaining for BRD4 and MED1 in WT and MLL4Q4092X MSCs grown on the same coverslips. WT MSCs were pre-labelled with CellTrace Violet Scale bar, 20 µm. Box plots indicate the median (middle line), the first and third quartiles (box), and the 10th and 90th percentile (error bars) of the fluorescence intensity. The number of analyzed nuclei is reported in figure as n; unpaired two-tailed Student’s t-test was applied for statistical analysis. f, Western Blot analysis of BRD4 and MED1 in WT and MLL4Q4092X MSCs; histone H3 was used as loading control. Signal quantifications are reported as bar plots. Data are means + SEM of 4 independent experiments for BRD4 and 5 independent experiments for MED1; one-tailed Student’s t-test was applied for statistical analysis. gi, Representative images and quantifications of cluster intensity for BRD4 and MED1 immunostaining in WT and MLL4P4093X MSCs (g) on in primary fibroblasts from healthy donor or Kabuki patients (h, i). Box plots indicate the median (middle line), the first and third quartiles (box), and the 10th and 90th percentile (error bars) of the fluorescence intensity. The number of analyzed nuclei is reported in figure as n; unpaired two-tailed Student’s t-test was applied for statistical analysis (****P<0.0001).

Source data

Extended Data Fig. 3 Effects of MLL4 LoF on the clustering of MED1 and BRD4.

a, b, Representative images and 3D reconstruction of the distribution of BRD4 (a) and MED1 (b) clusters in WT and MLL4Q4092X MSCs; Scale bar, 20 µm. Quantification of the number of BRD4 and MED1 clusters in WT and MLL4Q4092X MSCs. Box plots indicate the median (middle line), the first and third quartiles (box), and the 10th and 90th percentile (error bars). The number of analyzed nuclei is reported in figure as n; unpaired two-tailed Student’s t-test was applied for statistical analysis. c, Graphical representation of the experimental design adopted to measure the clustering of the MED1-IDR tagged with mCherry and fused to the Cry2 module. A single blue light stimulation (488nm, 50% light intensity, 2 seconds) was applied (green bars) followed by acquisition of mCherry signal (red bars). d, Quantification of the fluorescence intensity of MED1-IDR, measured in WT (n=39) and MLL4Q4092X (n=47) MSCs at the indicated time points. Box plots indicate the median (middle line), the first and third quartiles (box), and the 10th and 90th percentile (error bars) of the fluorescence intensity. The number of analyzed nuclei is reported in figure as n; unpaired two-tailed Student’s t-test was applied for statistical analysis. e, Representative images of light-stimulated MSCs expressing the mCherry-Cry2 construct, at different time points; scale bar, 10 µm. f, Representative images of light-induced clustering of MED1-IDR in MSCs, after a single pulse of stimulation (2 seconds, 488mn wavelength), stained for MLL4 and BRD4; scale bar, 10 µm. Pearson coefficient between MED-IDR and MLL4 or BRD4 was determined. Box plots indicate the median (middle line), the first and third quartiles (box), and the 10th and 90th percentile (error bars) of the fluorescence intensity. The number of analyzed nuclei is reported in figure as n.

Extended Data Fig. 4 Characterization of the MLL4-specific PrLD.

a, Representative images of dual immunostaining for MLL4 and BRD4 or MED1 in WT MSCs; scale bar, 10 µm. Asterisks indicate Golgi aspecific signal. Pearson coefficient between MLL4 and BRD4 or MED1 was determined. Box plots indicate the median (middle line), the first and third quartiles (box), and the 10th and 90th percentile (error bars). The number of analyzed nuclei is reported in figure as n. b, Representative images of immunostaining for MLL4 in WT MSCs, and in cells carrying KMT2D truncating mutation on one (MLL4Q4092X/WT) or both alleles (MLL4Q4092X/ Q4092X). c, Graphical representation of the Prion-like amino acid composition of MLL4 retrieved by PLAAC analysis. d, Analyses on the KMT2D gene showing the constrained coding regions (CCR), the known clinical variants (clinVar), the protein changing variants, and the conservation pattern (GERP). The square indicates the genomic regions codifying for the MLL4-PrLD. e, SDS-PAGE and Coomassie staining of purified MLL4-PrLD and MLL4-PrLDΔQ recombinant proteins. f, Phase separation of MLL4-PrLD recombinant protein visualized and quantified by fluorescence microscopy, in presence of increasing concentration of NaCl or 1,6-hexanediol; scale bar 5 µm. g, Phase separation of MLL4-PrLDΔQ at different protein concentrations; scale bar 5 µM. h, Measurements of the relative amount of condensed MLL4-PrLDΔQ versus protein concentration. Red line represents the regression line. i, Measurements of the number of formed MLL4-PrLD clusters versus the expression level in NIH3T3 cells. The quantification is the result of three independent experiments. Red line represents the regression line. j, Measurement of the mean fluorescent intensity in NIH3T3 cells expressing the OptoMLL4-PrLD or the OptoMLL4-PrLDΔQ, respectively. Box plots indicate the median (middle line), the first and third quartiles (box), and the 10th and 90th percentile (error bars) of the fluorescence intensity. The number of analyzed nuclei is reported in figure as n. Unpaired two-tailed Student’s t-test was applied for statistical analysis.

Source data

Extended Data Fig. 5 KMT2D truncating mutations affect PcG clustering.

a, Western Blot analysis of EED, EZH2 and SUZ12 in WT and MLL4Q4092X MSCs; histone H3 was used as loading control. b, qRT-PCR of BMI and RING1B in WT and MLL4Q4092X MSCs, normalized on GAPDH level. Data are means + SEM (n=3 independent experiments); unpaired two-tailed Student’s t-test was applied for statistical analysis. c, Quantifications of immunostaining for H3K27me3, BMI, and RING1B in WT and MLL4P4093X MSCs. Box plots indicate the median (middle line), the first and third quartiles (box), and the 10th and 90th percentile (error bars) of the fluorescence intensity. The number of analyzed nuclei is reported in figure as n; unpaired two-tailed Student’s t-test was applied for statistical analysis (****P<0.0001). df, Representative images and quantifications of immunostaining for H3K27me3 (d), BMI (e), and RING1B (f) in primary fibroblasts from healthy donor or Kabuki patients; scale bar, 20 µm. Box plots indicate the median (middle line), the first and third quartiles (box), and the 10th and 90th percentile (error bars) of the fluorescence intensity. The number of analyzed nuclei is reported in figure as n; unpaired two-tailed Student’s t-test was applied for statistical analysis (****P<0.0001). g, qRT-PCR of KMT2D in WT and MLL4Q4092X MSCs, expressing CRISPRa with or without crRNA targeting KMT2D promoter. Retrieved data were normalized on GAPDH level. Data are means + SEM (n=4 independent experiments); unpaired two-tailed Student’s t-test was applied for statistical analysis. h, i, Quantifications of immunostaining for MLL4, H3K4me1, BMI, RING1B and H3K27me3 in WT and MLL4Q4092X MSCs expressing CRISPRa with or without crRNA targeting KMT2D promoter. Box plots indicate the median (middle line), the first and third quartiles (box), and the 10th and 90th percentile (error bars) of the normalized fluorescence intensity. The number of analyzed nuclei is reported in figure as n; Student’s two-tailed unpaired t-test was applied for statistical analysis (****P<0.0001).

Source data

Extended Data Fig. 6 MLL4 abundance modulates chromatin compaction and nuclear architecture.

a, b Reconstructed 3D images of nuclei retrieved from images of WT and MLL4P4093X MSCs (a) or primary fibroblasts from healthy donor and Kabuki patients (b). Scale bar, 5 µm. The nuclear area, volume and flattening were determined and represented as box plots indicating the median (middle line), the first and third quartiles (box), and the 10th and 90th percentile (error bars). The number of analyzed nuclei is reported in figure as n; unpaired two-tailed Student’s t-test was applied for statistical analysis (****P<0.0001). c Measurements of nuclear shape in WT and MLL4Q4092X MSCs expressing CRISPRa with or without crRNA targeting KMT2D promoter. The nuclear area, volume and flattening were represented as box plots indicating the median (middle line), the first and third quartiles (box), and the 10th and 90th percentile (error bars). The number of analyzed nuclei is reported in figure as n; unpaired two-tailed Student’s t-test was applied for statistical analysis (****P<0.0001). d qRT-PCR of LMNA in WT and MLL4Q4092X MSCs, normalized on GAPDH level. Data are means + SEM (n=3 independent experiments); unpaired two-tailed Student’s t-test was applied for statistical analysis. e Quantifications of immunostaining for LMNA and phosphorylated LMNA/C (pLMNA) in WT and MLL4P4093X MSCs. Box plots indicate the median (middle line), the first and third quartiles (box), and the 10th and 90th percentile (error bars) of the fluorescence intensity. The number of analyzed nuclei is reported in figure as n; unpaired two-tailed Student’s t-test was applied for statistical analysis (****P<0.0001). f, g Representative images and quantifications of cellular area detected by Phalloidin staining in WT and MLL4Q4092X MSCs (f) or in the same cells expressing either EGFP or EGFP-Nesprin-KASH (g). Scale bar, 20 µm. Box plots indicate the median (middle line), the first and third quartiles (box), and the 10th and 90th percentile (error bars) of the cellular area. The number of analyzed cells is reported in figure as n; unpaired two-tailed Student’s t-test was applied for statistical analysis (****P<0.0001). h, i Representative images and quantifications of immunostaining for H4K16ac in WT and MLL4P4093X MSCs (h) or in WT primary fibroblasts from healthy donor or Kabuki patients (i). Box plots indicate the median (middle line), the first and third quartiles (box), and the 10th and 90th percentile (error bars) of the normalized fluorescence intensity. The number of analyzed nuclei is reported in figure as n; unpaired two-tailed Student’s t-test was applied for statistical analysis (****P<0.0001).

Extended Data Fig. 7 Rescuing chromatin compartmentalization re-establishes nuclear mechanical properties.

a, b Representative images and quantifications of immunostaining for H3K27me3 (a) and BMI (b) in WT and MLL4Q4092X MSCs untreated or treated with TSA (1.5 µM for 8h). Scale bar, 20 µm. Box plots indicate the median (middle line), the first and third quartiles (box), and the 10th and 90th percentile (error bars) of the normalized fluorescence intensity. The number of analyzed nuclei is reported in figure as n; unpaired two-tailed Student’s t-test was applied for statistical analysis (****P<0.0001). c Representative images and quantification of immunostaining for H3K27me3 in WT, MLL4Q4092X or MLL4Q4092X MSCs expressing either H3.3WT or H3.3K27M. Scale bar, 20 µm. Box plots indicate the median (middle line), the first and third quartiles (box), and the 10th and 90th percentile (error bars) of the normalized fluorescence intensity. The number of analyzed nuclei is reported in figure as n; unpaired two-tailed Student’s t-test was applied for statistical analysis (****P<0.0001). d Representative maps of stiffness distribution in WT, MLL4Q4092XMSCs, MLL4Q4092X MSCs expressing H3.3K27M or MLL4Q4092X MSCs treated with TSA for 24h; Scale bars, 10 µm. Bar plot representing nuclear Brillouin shift in the different conditions. Data are means + SEM (n=4 independent experiments); unpaired two-tailed Student’s t-test was applied for statistical analysis (****P<0.0001).

Extended Data Fig. 8 Clustering dynamics of PcG-associated condensates.

a Quantification of the number and the area of the light-induced droplets of BMI-Cry2 at different time points. First panel shows the distribution changes in the time window of 300 seconds post-stimulation. Second panel shows the formation and disassembly of BMI-Cry2 clusters within a time window of 35 minutes. b Quantification of the relative frequency of small (<0.1μm2) and large (>0.1μm2) droplets of BMI-Cry2 at different time points. c Representative images of dual immunostaining for BMI1 and RING1B (upper panels) or H3K27me3 (middle panels) or BRD4 (lower panels) in WT MSCs; scale bar, 10 µm. Pearson coefficient was represented as box plots, indicating the median (middle line), the first and third quartiles (box), and the 10th and 90th percentile (error bars). The number of analyzed nuclei is reported in figure as n. d Representative images of immunostaining for H3K27me3 and RING1B in NIH3T3 expressing BMI-Cry2, after light-induced clustering, Scale bar, 10 μm. Pearson Coefficient between BMI-Cry2 and H3K27me3 or RING1B was represented as box plots indicating the median (middle line), the first and third quartiles (box), and the 10th and 90th percentile (error bars). The number of analyzed nuclei is reported in figure as n.

Extended Data Fig. 9 Targeting ATR rescues the YAP/TAZ activity.

a Quantifications of immunostaining for YAP/TAZ in WT and MLL4P4093X MSCs. Box plots indicate the median (middle line), the first and third quartiles (box), and the 10th and 90th percentile (error bars). The number of analyzed nuclei is reported in figure as n; unpaired two-tailed Student’s t-test was applied for statistical analysis (****P<0.0001). b Western Blot analysis of YAP, TAZ and phosphorylated YAP (Ser127) in WT and MLL4Q4092X MSCs; GAPDH was used as loading control. c Representative images of the distribution of ATR in WT and MLL4Q4092X MSCs. Scale bar, 20 µm. d Representative images and quantifications of immunostaining for YAP/TAZ in WT and MLL4Q4092X MSCs untreated or treated with different concentrations of the ATR inhibitor VE-822. Scale bar, 20 µm. Data are represented as box plots, indicating the median (middle line), the first and third quartiles (box), and the 10th and 90th percentile (error bars). The number of analyzed nuclei is reported in figure as n; unpaired two-tailed Student’s t-test was applied for statistical analysis (****P<0.0001). e Representative images and quantification of phH2A.X in WT and MLL4Q4092X MSCs. Scale bar, 20 µm. Box plots indicate the median (middle line), the first and third quartiles (box), and the 10th and 90th percentile (error bars) of the phH2A.X distribution per cell. The number of analyzed nuclei is reported in figure as n; unpaired two-tailed Student’s t-test was applied for statistical analysis. f Volcano plot of differentially expressed genes between WT and MLL4Q4092X MSCs. Vertical blue lines indicate the chosen cutoff (−2>fold change>2), horizontal red line indicates Wald test-derived p-value cutoff of 0.05. g Heat map of k-means clustering analysis of expressed genes in WT MSCs untreated or treated with ATR inhibitor at the indicated time points. h Venn diagram showing the overlap between the ATR responsive genes identified in WT and MLL4Q4092X MSCs. i qRT-PCR of the indicated genes in WT, MLL4Q4092X (i) and MLL4P4093X MSCs (k) untreated or treated with ATR inhibitor at the different time points. The expression level was normalized on GAPDH level. Data are means + SEM (n=3 independent experiments); unpaired two-tailed Student’s t-test was applied for statistical analysis (***P<0.001).

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Extended Data Fig. 10 Targeting ATR restores chondrogenesis and osteogenesis.

a qRT-PCR of chromatin architecture genes in WT and MLL4Q4092X MSCs expressing CRISPRa with or without crRNA targeting KMT2D promoter. Retrieved data were normalized on GAPDH level. Data are means + SEM (n=3 independent experiments); unpaired two-tailed Student’s t-test was applied for statistical analysis. b, c Quantifications of immunostaining for YAP/TAZ in WT and MLL4Q4092X MSCs expressing CRISPRa with or without crRNA targeting KMT2D promoter (b) or in WT and MLL4Q4092X MSCs expressing either H3.3WT or H3.3K27M.(c). Box plots indicate the median (middle line), the first and third quartiles (box), and the 10th and 90th percentile (error bars). The number of analyzed nuclei is reported in figure as n; unpaired two-tailed Student’s t-test was applied for statistical analysis (****P<0.0001). d Differentiation of WT and MLL4Q4092X MSCs into adipocytes, osteocytes or chondrocytes were detected was detected by Oil red, Alizarin red or by Alcian blue staining, respectively. Scale bar, 100 µm. e qRT-PCR of the indicated genes during differentiation of WT and MLL4Q4092X MSCs towards chondrocytes. The expression level was normalized on GAPDH level. Data are means + SEM (n=3 independent experiments); unpaired two-tailed Student’s t-test was applied for statistical analysis. f Chondrogenic differentiation of WT and MLL4Q4092X MSCs or MLL4Q4092X MSCs expressing exogenous MLL4 was detected by Alcian blue staining. Scale bar, 500 µm. g Bar plot of the relative quantification of Ethmoid plate (EP), Palatoquadrate (PQ), Ceratohyal (CH) and Ceretobranchials 1 to 5 (CBs) cartilage length in Ctrl, KMT2D 5’UTR-morpholino and KMT2D 5’UTR-morpholino-treated with ATR inhibitor. Data are means + SEM (n=10 animals; n=12 animals for CH quantification in the KMT2D morphants); two-tailed Student’s t-test was applied for statistical analysis (****P<0.0001). h Bar plot of the relative quantification of Palatoquadrate (PQ), Ceratohyal (CH), Operculum (OP), Cleithrum (CL) and fifth ceratobranchial (CB) cartilage length in KMT2D 5’UTR-morpholino and ATRi-treated KMT2D 5’UTR-morpholino medaka fish. ata are means + SEM (n=10 animals); two-tailed Student’s t-test was applied for statistical analysis (****P<0.0001). k Bar plot of the relative quantification of Palatoquadrate (PQ), Ceratohyal (CH), Operculum (OP), Cleithrum (CL) and fifth ceratobranchial (CB) mineralization in KMT2D 5’UTR-morpholino and ATRi-treated KMT2D 5’UTR-morpholino medaka fish. Data are means + SEM (n=10 animals); two-tailed Student’s t-test was applied for statistical analysis (****P<0.0001).

Supplementary information

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Supplementary Tables 1–4

Supplementary Video 1

Time-lapse live imaging showing MED1–IDR clustering, induced by blue light stimulation, in WT MSCs.

Supplementary Video 2

Time-lapse live imaging showing MED1–IDR clustering, induced by blue light stimulation, in MLL4Q4092X MSCs.

Supplementary Video 3

Time-lapse live imaging showing optoMLL4 (MLL4PrLD) clustering, induced by blue light stimulation, in NIH3T3 cells.

Supplementary Video 4

Time-lapse live imaging showing optoBMI (BMI–Cry2) clustering, induced by blue light stimulation, in NIH3T3 cells.

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Unprocessed western blots and gels.

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Unprocessed western blots and gels.

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Fasciani, A., D’Annunzio, S., Poli, V. et al. MLL4-associated condensates counterbalance Polycomb-mediated nuclear mechanical stress in Kabuki syndrome. Nat Genet 52, 1397–1411 (2020). https://doi.org/10.1038/s41588-020-00724-8

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