Talking to chromatin: post-translational modulation of polycomb group function

Abstract

Polycomb Group proteins are important epigenetic regulators of gene expression. Epigenetic control by polycomb Group proteins involves intrinsic as well as associated enzymatic activities. Polycomb target genes change with cellular context, lineage commitment and differentiation status, revealing dynamic regulation of polycomb function. It is currently unclear how this dynamic modulation is controlled and how signaling affects polycomb-mediated epigenetic processes at the molecular level. Experimental evidence on regulation of polycomb function by post-translational mechanisms is steadily emerging: Polycomb Group proteins are targeted for ubiquitylation, sumoylation and phosphorylation. In addition, specific Polycomb Group proteins modify other (chromatin) associated proteins via similar post-translational modifications. Such modifications affect protein function by affecting protein stability, protein-protein interactions and enzymatic activities. Here, we review current insights in covalent modification of Polycomb Group proteins in the context of protein function and present a tentative view of integrated signaling to chromatin in the context of phosphorylation. Clearly, the available literature reveals just the tip of the iceberg, and exact molecular mechanisms in, and the biological relevance of post-translational regulation of polycomb function await further elucidation. Our understanding of causes and consequences of post-translational modification of polycomb proteins will gain significantly from in vivo validation experiments. Impaired polycomb function has important repercussions for stem cell function, development and disease. Ultimately, increased understanding of signaling to chromatin and the mechanisms involved in epigenetic remodeling will contribute to the development of therapeutic interventions in cell fate decisions in development and disease.

Figure 1

Examples of post-translational modulation of Polycomb group and associated proteins. (a) Simplified model of Polycomb Group (PcG)-mediated repression. The histone methyltransferase EZH2 trimethylates histone H3 at lysine 27, this mark is recognized by chromobox homolog (CBX) proteins via the chromodomain. RNF2/RING1 homologs are E3 ubiquitin ligases for H2A; RYBP binds H2AK119Ub1. Combined, these activities induce/maintain transcriptional repression. Gray boxes depict Polycomb Repressive Complex (PRC)-associated, epigenetically relevant enzymatic activities. (b) RNF2 E3 ligase activity is significantly enhanced in the presence of BMI1 or phosphorylated PCGF2. (c) DNA damage-induced phosphorylation of HIPK2 leads to phosphorylation of CBX4. Phosphorylation of CBX4 at T495 in turn enhances the HIPK2 sumoylation. (d) AKT-induced phosphorylation of EZH2 on S21 impairs its binding to histone H3, thereby inhibiting H3K27 trimethylation. me = methylation, ph = phosphorylation, su = sumoylation; ub = ubiquitylation.

Figure 2

Classification of S/T phosphorylation sites into general kinase recognition sequence categories. At present, not all consensus phosphorylation motifs are known for all kinases. A number of general phosphosite classes have been annotated based on amino acid sequences surrounding S/T phosphorylation sites: proline-directed, acidic, basic and otherwise [97]. (a-c) Sequence logos for Polycomb Group (PcG) phosphorylation motifs of proline-directed ((a), n = 42) acidic ((b), n = 36) and basic ((c), n = 15) categories where the phosphorylated residue (S/T) is centered were generated with Weblogo [98]. Only serine and threonine phosphorylation sites were taken into account when a full 15-mer sequence was available. To avoid sequence bias only non-overlapping human and mouse PcG phosphorylation sites were used. Centered 15-mer sequences were assigned to a motif class sequentially by following a binary decision tree as follows: P at +1 (Pro-directed), 5 or more E/D at +1 to +6 (acidic), R/K at -3 (basic), D/E at +1/+2 or +3 (acidic), 2 or more R/K at -6 to -1 (basic), otherwise. Colors correspond to the chemical properties of the amino acids: hydrophobic (black), basic (blue), acidic (red) and polar (green). (a) Although phosphosite numbers are low and thus no solid deductions are warranted, it is apparent for the proline-directed class (classified by a P in the +1 position), that prolines are not limited to +1, but are abundant in various - and + positions. (b) In the acidic motif, characterized by the presence of the amino acids glutamic acid (E) and aspartic acid (D) in the + residues, E/Ds are present in the - positions. (c) In basic motifs, arginine (R) is predominantly found in -3 positions, in addition to -5.

Figure 3

Hypothetical mechanism for phosphorylation-induced dissociation of Polycomb Repressive Complex (PRC)1 from chromatin. The chromodomain of chromobox homolog (CBX) proteins interacts with H3K27me3. Chromatin dissociation may be the result of ARKS motif methyl-phos switching, by phosphorylation of H3S28 (upper panel). Phosphorylation of conserved residue(s) within the chromodomain of CBX may contribute to chromatin dissociation (lower panel).

Figure 4

Integrated hypothetical model for post-translational modification (PTM)-dependent regulation of Polycomb Repressive Complex (PRC)1-mediated repression. Three independent chromatin states in the context of PRC1 function are recognized: (a) the repressed state, which requires ubiquitylation and sumoylation of PRC1 compounds. Detectable baseline phosphorylation may indicate a prerequisite for PRC function and/or differences in PRC activity at local targets throughout the genome. Signaling to chromatin alters PTM states and chromatin association, and eventually releases PRC silencing (b,c). Observations from our and other groups suggest differential regulation of polyhomeotic homolog (PHC) proteins versus RNF2, BMI1 and chromobox homolog (CBX) proteins; this may involve N-acetylglucosamine (GlcNAc) modification in mammals as well. (c) Whether or not full expression of a Polycomb Group (PcG) target gene requires complete removal or relocation of PHC is currently not known. Likewise, whether ubiquitin-mediated proteolysis of BMI1 and RNF2 is needed to release gene repression is purely speculative. ph = phosphorylation; su = sumoylation; ub = ubiquitylation. Black triangles = H3K27me3; open circles = H3S28ph; unmarked circular/oval structures represent general transcription factors and/or unknown proteins.