G-quadruplex folding in Xist RNA antagonizes PRC2 activity for stepwise regulation of X chromosome inactivation

Area of Science:

• Epigenetics
• Molecular Biology
• Genetics

Background:

• The regulation of Polycomb repressive complex 2 (PRC2) by RNA is not well understood.
• PRC2 can bind RNA G-quadruplexes (rG4s), but their in vivo folding and binding preferences are unknown.

Purpose of the Study:

• To investigate the role of folded rG4s in Xist RNA in regulating PRC2 activity and gene silencing.
• To determine if PRC2 preferentially binds folded or unfolded rG4s.

Main Methods:

• Utilized the X-inactivation model in mouse embryonic stem cells.
• Identified folded rG4s in Xist RNA.
• Assessed PRC2 binding affinity to folded and unfolded rG4s.
• Measured PRC2 histone methyltransferase activity and H3K27me3 enrichment.
• Mutagenized rG4s to assess their impact on PRC2 recruitment, release, and catalytic activity.

Main Results:

• Multiple folded rG4s were identified in Xist RNA.
• PRC2 preferentially binds folded rG4s.
• High-affinity rG4 binding inhibits PRC2's histone methyltransferase activity.
• Stabilizing rG4s antagonizes H3K27me3 enrichment on the inactive X chromosome.
• Mutagenizing rG4s did not affect PRC2 recruitment but promoted its release and activation, leading to misplaced H3K27me3 marks and compromised gene silencing.
• Xist-PRC2 complexes were entrapped in the S1 compartment, preventing translocation to S2.

Conclusions:

• Xist RNA G-quadruplex folding is a critical regulator of PRC2 activity.
• rG4 folding controls H3K27me3 enrichment and stepwise gene silencing on the inactive X chromosome.
• This mechanism ensures proper chromosome-wide gene silencing by regulating PRC2 localization and activity.