Regulation of Polyhomeotic Condensates by Intrinsically Disordered Sequences That Affect Chromatin Binding

Area of Science:

• Cell Biology
• Molecular Biology
• Genetics

Background:

• Polycomb group (PcG) complex PRC1 forms nuclear condensates called Polycomb bodies.
• The PRC1 subunit Polyhomeotic (Ph) has a sterile alpha motif (SAM) domain involved in condensate formation and chromatin organization.
• Ph's SAM domain alone (mini-Ph) can form phase-separated condensates in vitro, suggesting SAM-driven phase separation in Polycomb body formation.

Purpose of the Study:

• To investigate the role of intrinsically disordered regions (IDRs) in Ph in regulating Polycomb condensate formation and chromatin binding.
• To determine how specific low-complexity regions within Ph influence condensate size, number, and chromatin association.

Main Methods:

• Systematic deletion analysis of Ph's intrinsically disordered regions (IDRs).
• Live imaging of transfected Drosophila S2 cells to observe condensate dynamics.
• Chromatin fractionation experiments to assess chromatin binding of Ph variants.

Main Results:

• Each of Ph's three identified IDRs uniquely impacts condensate size, number, and morphology.
• Removal of the central, glutamine-rich IDR resulted in large, chromatin-excluding condensates, similar to mini-Ph.
• Deletion of the glutamine-rich IDR decreased chromatin binding, while deletion of other IDRs increased it.

Conclusions:

• All three IDRs, through their interactions, regulate Ph condensate size and number.
• Tight chromatin binding by Ph IDRs antagonizes Ph SAM-driven phase separation.
• These findings reveal the intricate regulation of biological condensates within a single protein.