Locus-Dependent Epigenetic Inheritance of Polycomb-Mediated Gene Silencing

Abstract

During development, it is crucial that gene expression patterns, which define cell phenotypes and “epigenetic states”, are stably inherited. The maintenance of epigenetic states involves changes in repressive histone posttranslational modifications (HPMs), such as H3K27me3 and H2AK119ub1, established and maintained by the Polycomb Group (PcG) of proteins, which assemble into Polycomb Repressive Complexes 1 and 2 (PRC1 and PRC2). One model of Polycomb epigenetic inheritance proposes a positive feedback read-write mechanism, in which the PRC2 histone-methyltransferase complex recognizes the modification on parentally inherited histones and catalyzes the same modification on adjacent newly deposited histones during replication. However, studies in fission yeast, Drosophila and mammalian cells suggest that locus-specific factors such as site-specific DNA-binding proteins also contribute to epigenetic inheritance. To investigate the mechanism of Polycomb-mediated epigenetic inheritance and the possible role of locus-specific factors in human cells, I inserted 5XtetO-CITRINE reporters at two distinct types of loci, Polycomb target genes and housekeeping genes, together with expression and reversible binding of rTetR-CBX7 to establish ectopic Polycomb domains and silencing. I show that upon the release of the rTetR-CBX7 initiator, the Polycomb domain and the silent state are maintained at Polycomb target genes but not at housekeeping genes, suggesting that maintenance of the silent state is locus-dependent. Maintenance of the silenced state at Polycomb target genes is partially dependent on the hydrophobic cage domain of EED, the PRC2 subunit that recognizes H3K27me3, and requires the RING1A/B subunit of PRC1, which catalyzes H2AK119ub. In addition, I show that maintenance is disrupted by point mutations in the DNA-binding domain of MTF2, a PRC2 accessory factor that binds to CpG-rich DNA sequences, supporting the contribution of DNA sequence to epigenetic inheritance. Furthermore, the loss of the silent state at housekeeping genes is partially reversed by DNA deletion that attenuate their transcription, suggesting a potential role for active transcription in counteracting epigenetic maintenance. These findings suggest that the heritability of Polycomb-mediated gene silencing requires DNA sequence-independent histone modification positive feedback but is also dependent on the ability of PRC2 accessory factors to bind to CG rich DNA and is opposed by proximal transcription at some loci.