Polycomb-like 3 promotes polycomb repressive complex 2 binding to CpG islands and embryonic stem cell self-renewal

Area of Science:

• Epigenetics and Gene Regulation
• Stem Cell Biology
• Molecular Oncology

Background:

• Polycomb repressive complex 2 (PRC2) plays a vital role in gene silencing through histone H3 lysine 27 trimethylation (H3K27me3).
• PRC2 is implicated in developmental transitions, embryonic stem cell (ESC) differentiation, and cancer progression.
• Understanding the regulatory mechanisms of PRC2 is key to deciphering its roles in these processes.

Purpose of the Study:

• To investigate the function of Polycomb-like 3 (Pcl3) within the PRC2 complex.
• To determine Pcl3's role in embryonic stem cell (ESC) self-renewal and differentiation.
• To elucidate how Pcl3 influences PRC2 activity, including H3K27me3 modification and target gene recruitment.

Main Methods:

• Mass spectrometry to identify Pcl3 interacting partners.
• Co-immunoprecipitation to confirm interactions with PRC2 core components.
• Pcl3 knockdown and overexpression studies in ESCs.
• Teratoma and embryoid body formation assays.
• Western blotting to assess H3K27me3 levels.
• Chromatin immunoprecipitation and sequencing (ChIP-seq) to map PRC2 binding sites.
• Site-directed mutagenesis of the Pcl3 Tudor domain.

Main Results:

• Pcl3 was identified as a novel component of the PRC2 complex, interacting with Suz12 and other core PRC2 subunits.
• Pcl3 knockdown in ESCs led to increased spontaneous differentiation, specifically impacting self-renewal.
• Pcl3 levels directly correlated with H3K27me3 levels, with Pcl3 depletion reducing and overexpression increasing H3K27me3.
• ChIP-seq analysis showed Pcl3 co-localization with Suz12, and Pcl3 depletion reduced Suz12 binding at over 60% of PRC2 target loci.
• Mutation of the Pcl3 Tudor domain impaired H3K27me3 formation, highlighting its functional importance.
• Pcl3 and Pcl2, though in distinct PRC2 complexes, target overlapping genomic regions, particularly CpG islands.

Conclusions:

• Pcl3 is a critical component of PRC2 essential for maintaining ESC self-renewal.
• Pcl3 promotes PRC2 catalytic activity, evidenced by its regulation of H3K27me3 levels.
• Pcl3 plays a significant role in recruiting PRC2 to a subset of its genomic targets, influencing gene expression programs.
• The Tudor domain of Pcl3 is vital for its function in H3K27me3 modification.