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bioRxiv · 10.64898/2026.06.03.729812

Heterochromatin organization and liquid-liquid phase separation: it is not about if but about when

Romero, H.·Arroyo, M.·Zhadan, A.·Muzzopappa, F.·Zhang, H.·Qin, W.·Mahmoud, M.·Leonhardt, H.·Erdel, F.·Cardoso, M. C.
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Abstract

Heterochromatin is a membraneless compartment within the cell nucleus. In recent years, a controversy arose on whether heterochromatin organization is driven by liquid-liquid phase separation or not. While many heterochromatin proteins were shown to undergo liquid-liquid phase separation in vitro, other studies reported that this does not happen in cells. Here, we tested the ability of heterochromatin proteins to generate heterochromatin barrier compartments in cells. We found that, while several proteins (H1.0, H1.4, HP1alpha, HP1beta, Mbd1, Mbd2 and MeCP2) form barrier compartments in mouse and/or human cells this differs between cell types. In addition, not all compartments in the same cell form barriers. We established and experimentally validated a model that predicted the ability to form barrier compartments is dependent on the protein accumulation in heterochromatin followed by the competition between compartments for the nucleoplasm pool of the protein and resulted in larger size for the barrier compartments. These findings resolve the existing controversy and rationalize how in cells heterochromatin compartments form and compete to establish dynamic barriers to the entry and exit of its components. HighlightsHeterochromatin barrier formation differs between proteins, cell lines and heterochromatin compartments within the cell. Barrier formation depends on heterochromatin anchors, including ligands and other scaffolds. Barrier compartments are defined by their larger size and higher protein enrichment. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/729812v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@a63021org.highwire.dtl.DTLVardef@a20362org.highwire.dtl.DTLVardef@8c2390org.highwire.dtl.DTLVardef@72dde3_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Romero, H., Arroyo, M., Zhadan, A., Muzzopappa, F., Zhang, H., Qin, W., Mahmoud, M., Leonhardt, H., Erdel, F., Cardoso, M. C.. 2026-06-07. Heterochromatin organization and liquid-liquid phase separation: it is not about if but about when. https://doi.org/10.64898/2026.06.03.729812

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