Charge segregation in the intrinsically disordered region governs VRN1 and DNA liquid-like phase separation robustness.

VERNALIZATION1 (VRN1) is a transcriptional repressor involved in plant vernalization that undergoes liquid-liquid phase separation (LLPS) with DNA. The naturally occurring VRN1-like proteins contain two B3 DNA binding domains connected by an intrinsically disordered region (IDR). The IDR length in VRN1-like proteins has a broad distribution, while the charge segregation pattern is largely conserved. We studied the effect of IDR length and charge segregation on DNA-induced VRN1 phase separation. When only neutral residues (Pro-Ser repeats) were used, the phase separation behavior is sensitive to IDR length, changing from gel-like aggregates (L=40) to liquid-like droplets (L=100-120) and clear solution (L=160). When a pair of continuous patches of positive and negative residues were added to the IDRs, all the VRN1 variants formed robust and durable droplets with DNA independent of the IDR length. To test how robust the system is, we introduced folded green fluorescent protein or the enzyme GPX4 into VRN1 variants with charge segregation in IDR, the resulting proteins form LLPS with DNA as well. Our study implies that VRN1-like proteins use conserved charge segregation pattern to retain functional LLPS during evolution, and demonstrates the possibility of using this system to design novel biosensors or bio-factories by introducing various functional modules.