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Dive deep into the complex interactions between Polycomb and Trithorax group proteins, including EZH2 and SMARCA4, and understand their roles in transcription and cancer.
Published February 1, 2021
For a quick insight into how Polycomb complexes regulate gene expression, watch our 3-minute video below.
The chromatin environment is crucial for maintaining cell-type-specific gene expression patterns and thereby cell identity. Different chromatin-modifying protein complexes can influence the accessibility of chromatin and local patterns of chromatin modifications. Two such protein families, the Polycomb group (PcG) and Trithorax group (TrxG), have been widely studied for their roles in modulating transcription and influencing cell fate.
PcG and TrxG proteins were originally classified based on their distinct developmental phenotypes in genetic studies in Drosophila. Molecular mechanisms of the encoded proteins have since been studied, and homologs have been identified in many metazoan species. Both classes of proteins associate with several large, multimeric protein complexes, encompassing diverse molecular mechanisms and catalytic activities. PcG proteins are involved in maintaining transcriptional repression, while TrxG proteins modulate chromatin contributing to active transcription (Figure 1)1.
Figure 1. Polycomb and Trithorax group proteins modulate gene expression through the establishment of distinct chromatin states. Transcription initiation leads to the recruitment of TrxG proteins, facilitating an open chromatin environment permissive for active transcription. Termination of transcription leads to PcG recruitment to facilitate a more compact, repressive chromatin environment.
PcG proteins form the Polycomb repressive complexes 1 and 2 (PRC1/PRC2) as well as the Polycomb repressive deubiquitinase complex (PR-DUB) (Figure 2). Genomic binding sites of PRC1 and PRC2 overlap, and both complexes are primarily enriched at CpG-rich promoters of non-transcribed genes, where they contribute to the correct spatiotemporal expression pattern of developmentally regulated genes. Loss of PRC function during development leads to embryonic lethality, and PcG proteins are often found mutated or deregulated in human diseases, including cancer.
Figure 2. Transcriptional regulation by Polycomb repressive complexes. PRCs are recruited to CpG islands (CGIs) of non-transcribed genes, contributing to their maintained repression. PRC2 catalyzes H3K27 methylation, which is recognized by EED of PRC2 and CBX of PRC1. PRC1 catalyzes H2AK119 ubiquitination, which can be bound by JARID2/AEBP2-containing PRC2.2 complexes. H2AK119ub1 can be removed by PR-DUB.
PRC1 complexes contain an E3 ubiquitin ligase (RING1A or RING1B) with activity towards H2AK119, closely associated with a PCGF component (PCGF1-6), which is required for the catalytic activity of the complex to form H2AK119ub1 (Figure 2).
The core PRC2 complex consists of a histone methyltransferase subunit (EZH1 or EZH2), which depends on association with SUZ12 and EED for in vitro activity and the additional association with the histone-binding proteins RBBP4 or RBBP7 for in vivo activity. PRC2 catalyzes mono-, di- and trimethylation of H3K27 (Figure 2).
For more information on the structure and function of PRC1, PRC2, and PR-DUB complexes, please refer to the full guide.
Several molecular mechanisms contribute to the correct genomic targeting of PRCs. Despite the years of studies of transcription factors, non-coding RNAs, and specific DNA features, no clear cause for mammalian PRC recruitment has been identified, although PRCs have been found to bind CpG-rich promoters of non-transcribed genes. Non-core subunits of PRC1 and PRC2 have been shown to guide target site specificity through direct chromatin interactions, strengthened by chromatin-interactions from core subunits. Some studies suggest that non-coding RNAs may be involved in recruiting PRCs to specific loci. Conversely, several studies indicate that RNA transcripts counteract PRC binding by competing for DNA binding surface, thereby providing a ‘sensing mechanism’ to explain selective recruitment of PRCs to non-transcribed genes3.
PRC binding and histone modifications are suggested to confer transcriptional repression via various mechanisms, including prevention RNA polymerase II binding or release, chromatin compaction, or co-transcriptional RNA degradation. Repression depends on the catalytic activities of both PRC1 and PRC2, which may well vary in distinct cell types, developmental stages, and genomic contexts. However, the relative contributions of each complex and modification remain to be understood5.
To learn more about the molecular details of PRC recruitment, download the full guide.
In addition to the developmental defects of PcG mutants in Drosophila, genetic studies in mice have shown that many PcG proteins are essential for normal development, with knockout phenotypes ranging from early embryonic lethality to homeotic transformation or perinatal lethality. Furthermore, mutations in genes encoding PcG proteins are associated with several human congenital disorders, underlining their key role during normal development6,7.
Also, PRC function is often disrupted in many different types of human cancer through deregulation of expression levels or somatic mutations of genes encoding PcG proteins or histone mutants leading to a substitution of the PRC2 substrate lysine (H3K27M)1,8.
The high frequency of cancer-associated mutations along with the promise of ‘epigenetic therapies’ has led to the development of various chemical compounds targeting PcG proteins. These include small-molecule inhibitors blocking the catalytic activity of EZH2 or preventing H3K27me3-binding by EED or CBX proteins or disrupting other protein-protein interfaces. Excitingly, the first EZH2 inhibitor, Tazemetostat, was recently FDA-approved for the treatment of follicular lymphoma and epithelioid sarcoma. Several other compounds are under preclinical and clinical development, including those targeting the catalytic activity of EZH2 or the integrity of PRC2 through degradation or obstruction of binding surfaces10.
You can find more details on the role of PcG proteins in disease in our full guide.
TrxG proteins have been defined as factors suppressing the Polycomb phenotypes in Drosophila, indicating the opposing mechanistic function of the gene products. The TrxG protein complexes have diverse molecular functions with catalytic activities generally associated with active transcription. Many TrxG proteins are essential for preserving transcriptional patterns, and thereby cell fate, and the genes encoding TrxG proteins are often found mutated or deregulated in human cancers12.
The TrxG proteins also segregate into large, multimeric protein complexes. These complexes can be largely sub-divided into two groups: COMPASS/COMPASS-like and SWI/SNF (mammalian homologs called BAF/PBAF).
COMPASS and COMPASS-like complexes are SET-domain containing methyltransferases, catalyzing mono-, di- and trimethylation of H3K4 (Figure 3). The catalytic core of these complexes consists of WDR5, ASH2, RBBP5, and DPY30 (WARD). Each specific complex incorporates a SET-domain-containing methyltransferase (SET1, MLL1/2, or MLL3/4) as well as numerous additional subunits, yielding distinct chromatin binding patterns and additional catalytic activities to the complexes1.
Figure 3. Different COMPASS complexes regulate H3K4 methylation in distinct genomic regions. SET1-COMPASS catalyzes H3K4me3 at the promoters of actively transcribed genes. MLL1/2-COMPASS-like complexes catalyze H3K4me3 at developmentally regulated and bivalent genes also marked by H3K27me3. MLL3/4 COMPASS-like complexes are responsible for the establishment of H3K4me1 at enhancer elements.
You can find more details on different COMPASS and COMPASS-like complexes in the full guide.
The SWI/SNF complexes are ATP-dependent chromatin-remodeling complexes mediating nucleosome sliding or eviction associated with active transcription (Figure 4). In mammalian cells, the two SWI/SNF ATPase homologs, SMARCA4/BRG1 or SMARCA2/BRM each interact with about eight additional proteins to form the core complex, shared by both BAF and PBAF. Additional complex-specific subunits provide functional diversity to the complexes; this includes subunits ARID1A/B and DPF1/2/3 for BAF and PBRM1, BRD7, ARID2, and PHF10 for PBAF.
Figure 4. SWI/SNF protein complexes maintain an open chromatin environment through nucleosome remodeling. SWI/SNF (BAF/PBAF) protein complexes are recruited to CGIs at the promoters of transcribed genes, where they contribute to maintaining an open, permissive chromatin environment through ATPase-dependent chromatin remodeling and exclusion of PRCs.
In Drosophila, both PcG and TrxG proteins are recruited to Polycomb or Trithorax response elements (PREs/TREs). In mammalian cells, unmethylated CpG islands provide docking sites for both types of complexes in response to transcriptional cues. While PcG proteins are recruited to non-transcribed genes, TrxG complexes are recruited to actively transcribed genes, where they contribute to maintaining an open, permissive chromatin environment through nucleosome remodeling and H3K4 methylation (Figure 4). With their opposing functions on chromatin states and mutual inhibition of their respective binding or catalytic function, it appears that the outcome on transcriptional activity depends on the achieved balance of repressive vs activating signals7,8.
See the full guide for more details on molecular mechanisms underlying the recruitment of TrxG proteins.
In knockout mouse studies, developmental phenotypes of TrxG loss-of-function range from early embryonic lethality to later or milder defects, depending on the targeted component. However, both SWI/SNF and COMPASS complexes are required for normal development7.
In line with their importance for maintaining gene expression patterns and cell identity, many TrxG proteins show deregulation in cancer. Check out the full guide for more details.
Due to their frequent misregulation or mutation in human cancers, several compounds targeting TrxG proteins are in preclinical or clinical development. These include small-molecule inhibitors disrupting the WDR5-MLL interaction, thus inhibiting the function of COMPASS-like complexes, and several molecules targeting various SWI/SNF components. Given the described dependencies between SWI/SNF-mutated tumors and PRC2 inhibition, the development of novel drugs targeting SWI/SNF components may provide exciting opportunities for combination treatment in cancers with loss-of-function PRC2 mutations7,8.
Since their discovery in Drosophila, the PcG and TrxG families of chromatin-associated proteins have been extensively studied in organisms ranging from yeast to humans. In recent years, in vitro and in vivo structure-function studies have yielded important information about molecular mechanisms of the PcG and TrxG complexes. This, along with the extensive phenotypic characterization of model organisms and large-scale genomic data from human cancers, has greatly advanced our understanding of how these complexes exert their biochemical and biological roles, both in normal biological contexts and disease.
To summarize, despite the progress in the PcG and TrxG research field, many questions remain open, including molecular details of recruitment of PcG and TrxG complexes, their mechanistic impact on transcription, their biological roles in cancer, and their use as biomarkers and therapeutic targets in anti-cancer therapies.
For more in-depth information on gene regulation by Polycomb and Trithorax proteins, we recommend the following review article: Schuettengruber et al 2017. For a better understanding of what chromatin is and how it functions, you can check out the detailed review by Misteli T. 2020.
If want to learn more about the key techniques used in epigenetics research, refer to our Epigenetics application guide. Also, you can check out our posters on histone modifications and RNA modifications.
If you are studying epigenetic targets in cancer, see our comprehensive Cancer epigenetics guide, which covers histone regulation, DNA and RNA modifications as well as polycomb and chromatin remodeling.