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. 2024 Mar 7;15:1781. doi: 10.1038/s41467-024-45773-3

Fig. 6. Kdm1a condensates exhibit LLPS properties.

Fig. 6

A The PrDOs algorithm identified intrinsically disordered regions at the N-terminus of mouse Kdm1a. B Kdm1a scheme illustrating the separation of the IDR and catalytic domain. C Time-lapse images of the nuclei of HEK293 cells expressing GFP-Kdm1a. Fusion and fission events of Kdm1a puncta were captured in a 2 second interval. Scale bar: 5 µM. D GFP-Kdm1a expression in HEK293 cells produces green-fluorescent puncta with rapid fluorescence recovery after photobleaching (FRAP) (n = 4 independent experiments) Scale bar: 2.5 µM. E GFP-Kdm1a droplets disappeared few minutes after treatment with 1,6-hexanediol. Scale bars: 5 µM. F Quantification of GFP-Kdm1a foci > 0.3 µm per cell before and after 1,6-hexanediol treatment. (n = 17 cells in 3 independent experiments; Mann-Whitney test, two-tailed: ***pval = 0.0004). Scatter plot represents mean ± SEMs. G Scheme of experiments in hippocampal PNCs. E17 Kdm1a f/f embryos infected with a lentiviral vector co-expressing CRE recombinase and GFP (LV-CRE-GFP) or a control lentiviral vector that only expresses GFP (LV-GFP). The same elements (created by B.dB.) in a different order were used in80 (Lipinski et al., KAT3-dependent acetylation of cell type-specific genes maintains neuronal identity in the adult mouse brain. Nat Comm 2020 May 22;11(1):2588. 10.1038/s41467-020-16246-0). H RT-qPCR analysis demonstrates that de-repression of upDEGs also occurs in Kdm1a-deficient PNCs. (control, n = 3; CRE, n = 3; two-way ANOVA: ****p-val < 0.0001). Boxplots indicate median value, interquartile range, minimum and maximum value, and individual data points. I Schematic representation of the human Kdm1a variants used in rescue experiments. J RT-qPCR analysis demonstrates that hKDM1A protein restores non-neuronal genes repression in Kdm1a-deficient PNCs. However, a truncated KDM1A protein lacking the N-terminal IDR is unable to restore the transcriptional phenotype found in Kdm1a-deficient PNCs. (control, n = 4; CRE, n = 6; Flag-hKDM1A, n = 5 and Flag-(w/oIDR)hKDM1A, n = 4; two-way ANOVA: **p-val2 <  0.001; NS pval >0.01). Boxplots as indicated in panel H. K Schematic model of Kdm1a function at the boundary of H3K27me3 and H3K27ac domains. The loss of Kdm1a causes changes in H3K4 methylation levels, alters the balance between H3K27me3/H3K27ac and disrupts chromatin compartmentalization leading to the de-repression of PCR2-repressed nonneuronal genes that interact with active loci. Kdm1a’s IDR is essential to maintain non-neuronal repression, suggesting that liquid-liquid phase separation is involved in the transcriptional and epigenetic alterations described in Kdm1a-ifKOs. Source data are provided as a Source data file.