Cavalli Lab - The Polycomb and Trithorax Group proteins page

Polycomb group (PcG) and trithorax group (trxG) proteins regulate expression patterns of many developmental genes. Their function is best understood in the regulation of homeotic genes, where these proteins are able to maintain, respectively, silenced or active states throughout development. These proteins raised considerable interest in recent years, both because the basic regulatory mechanisms that involve these factors are fascinating, and because they play key roles in a variety of normal cellular processes and in disease.

A brief introduction to Polycomb and Trithorax:

Polycomb group (PcG) proteins are highly conserved regulatory factors that were initially discovered in Drosophila. PcG genes are best known for their role in maintaining silent expression states of Hox genes during development, while trithorax group (trxG) proteins maintain Hox gene expression patterns in the appropriate spatial domains. PcG and trxG proteins are also involved in the regulation of normal cell proliferation, and their mutation has been linked to defects in stem cell fates and to cancer. They act by regulating chromatin structure and chromosome architecture at their target loci.

PcG proteins form multimeric complexes that exert their functions by modifying chromatin structure and by regulating the deposition and recognition of multiple post-translational histone modifications. Three major PcG protein complexes have been described. The first, named PhoRC, contains the DNA-binding protein Pho (this is the Drosophila name, the homolog in mammals is YY1). The second complex, named the E(Z)/ESC complex or Polycomb Repressive Complex 2 (PRC2), contains four core proteins: the histone methyltransferase Enhancer of Zeste (E(Z)), Extra sex combs (ESC), Suppressor of zeste-12 (SU(Z)12), and nucleosome-remodeling factor 55 (NURF-55). E(Z) trimethylates lysine 27 of histone H3 (H3K27me3), and, to a lesser extent, lysine 9 of histone H3 (H3K9me3). A third complex, named PRC1, recognizes these methylation marks via the chromodomain of the Polycomb (PC) protein. PC is a stoichiometric component of PRC1, together with Polyhomeotic (PH), Posterior Sex Combs (PSC), and dRING. In mammals, the duplication of many PcG genes allows variations in complex composition, which differ with cell type and developmental stage.

TrxG proteins are a somewhat heterogeneous group, but they are characterized by complementary mechanistic properties to the PcG. Within trxG members, some bind specific sequences of DNA. A second class class of trxG members is composed by SET domain factors like Drosophila Trx and Ash1 and vertebrate MLL, as well as their associated proteins. A third class of trxG factors comprises protein components of ATP-dependent chromatin remodeling complexes like the SWI/SNF or the NURF complexes, and includes proteins (such as one component of the NURF complex) specifically capable to "read" the histone methylation marks laid down by the SET domain proteins.

In Drosophila, PcG proteins repress their target genes by binding to specific DNA elements called Polycomb Response Elements (PREs). Analysis of known PREs has revealed the presence of binding sites (usually in multiple copies) for several DNA-binding proteins, such as Pleihomeotic (PHO) and Pleihomeotic-like (PHOL), GAGA factor (GAF)/Pipsqueak (PSQ), Zeste and DSP. Other studies have suggested possible additional roles for other proteins, such as the corepressor CtBP and the DNA binding factors Grainyhead (GRH) as well as members of the Sp1/KLF family. Therefore, a large number of proteins might contribute to PcG recruitment at PREs. Each PRE has a different number and topological organization of binding sites for these factors, possibly providing the basis for the specificity of PRE function.

PREs have only been characterized in Drosophila so far. In general, PREs might be simply defined as DNA elements necessary and sufficient for recruitment of PcG complexes and for PcG-dependent silencing of flanking promoters. Many of the PcG binding sites identified by chromatin immunoprecipitation in vertebrates might correspond to this criterion. Their DNA sequences are likely to be fairly different from fly PREs, since three of the DNA-binding factors involved in PcG recruitment, GAF, Pipsqueak and Zeste, are not conserved in vertebrates.

In addition to modifications at the chromatin level, regulation at the level of nuclear architecture influences the regulation of PcG target genes. In mice, it has been reported that nuclear re-organization is coupled to Hox gene activation in early development. In Drosophila, homologous chromosomes pair in interphase nuclei, and transgenic PREs typically silence more strongly when they are present in two copies on homologous chromosomes. This notion of pairing is reinforced by the finding that PRE-containing sequences can also pair with homologous sequences located on different chromosomes, and that these long distance nuclear interactions reinforce PcG-mediated silencing.

Therefore, multiple mechanisms cooperate to drive regulation of gene expression by PcG and trxG proteins. This is likely very important in light of the fact that these proteins regulate a large number of genes, sometimes maintaining the memory of transcriptional states, while in other cases their regulation is more flexible. These multiple mechanisms may be important to ensure the necessary regulatory plasticity, while providing sufficient robustness to the regulated state.

Recent reviews for further readings

1:	Lanzuolo C, Orlando V.
	Memories from the polycomb group proteins Annu Rev Genet. 2012;46:561-89. doi: 10.1146/annurev-genet-110711-155603. Epub 2012 Sep 17. PMID: 22994356 [PubMed - in process]

2:	Pirrotta V, Li HB.
	A view of nuclear Polycomb bodies. Curr Opin Genet Dev. 2012 Apr;22(2):101-9. doi: 10.1016/j.gde.2011.11.004. Epub 2011 Dec 16. Review. PMID: 22178420 [PubMed - indexed for MEDLINE]

3:	Holec S, Berger F
	Polycomb group complexes mediate developmental transitions in plants. Plant Physiol. 2012 Jan;158(1):35-43. doi: 10.1104/pp.111.186445. Epub 2011 Nov 15. Review. No abstract available. PMID: 22086420 [PubMed - indexed for MEDLINE]

4:	Bantignies F, Cavalli G
	Polycomb group proteins: repression in 3D. Trends Genet. 2011 Nov;27(11):454-64. doi: 10.1016/j.tig.2011.06.008. Epub 2011 Jul 25. Review. PMID: 21794944 [PubMed - indexed for MEDLINE]

5:	Fisher CL, Fisher AG.
	Chromatin states in pluripotent, differentiated, and reprogrammed cells. Curr Opin Genet Dev. 2011 Apr;21(2):140-6. doi: 10.1016/j.gde.2011.01.015. Review. PMID: 21316216 [PubMed - indexed for MEDLINE]

6:	Schuettengruber B, Martinez AM, Iovino N, Cavalli G.
	Trithorax group proteins: switching genes on and keeping them active. Nat Rev Mol Cell Biol. 2011 Nov 23;12(12):799-814. doi: 10.1038/nrm3230. Review. PMID: 22108599 [PubMed - indexed for MEDLINE]

7:	Margueron R, Reinberg D.
	The Polycomb complex PRC2 and its mark in life. Nature. 2011 Jan 20;469(7330):343-9. doi: 10.1038/nature09784. Review. PMID: 21248841 [PubMed - indexed for MEDLINE]

8:	Beisel C, Paro R.
	Silencing chromatin: comparing modes and mechanisms. Nat Rev Genet. 2011 Feb;12(2):123-35. doi: 10.1038/nrg2932. Epub 2011 Jan 11. Review. PMID: 21221116 [PubMed - indexed for MEDLINE]

9:	Sawarkar R, Paro R.
	Interpretation of developmental signaling at chromatin: the Polycomb perspective. Dev Cell. 2010 Nov 16;19(5):651-61. doi: 10.1016/j.devcel.2010.10.012. Review. PMID: 21074716 [PubMed - indexed for MEDLINE]

10:	Mills AA.
	Throwing the cancer switch: reciprocal roles of polycomb and trithorax proteins. Nat Rev Cancer. 2010 Oct;10(10):669-82. doi: 10.1038/nrc2931. Review. PMID: 20865010 [PubMed - indexed for MEDLINE]

11:	Sauvageau M, Sauvageau G.
	Polycomb group proteins: multi-faceted regulators of somatic stem cells and cancer. Cell Stem Cell. 2010 Sep 3;7(3):299-313. doi: 10.1016/j.stem.2010.08.002. Review. PMID: 20804967 [PubMed - indexed for MEDLINE]

12:	Schuettengruber B, Chourrout D, Vervoort M, Leblanc B, Cavalli G.
	Genome regulation by polycomb and trithorax proteins. Cell. 2007 Feb 23;128(4):735-45. PMID: 17320510 [PubMed - in process]

Top PART 2. Polycomb and Trithorax group proteins

This table lists PcG and trxG proteins in humans and flies, as well as proteins that may be involved at recruiting them to their target genes. The left column indicates whether the proteins are found in well-characterized biochemical complexes. A brief note describes the main function of each protein. In the Pubmed links column, main papers describing the proteins are hyperlinked. When too many papers have been published on a given protein, I have put a link to a review discussing it in more detail. * indicates that the protein exist but its function in the PcG or trxG pathway is still not clear Note: This table is constantly under revision. should you see mistakes or have updates, please send me an email
	Drosophila melanogaster	Homo sapiens	Notes
PcG/trxG recruiters
	Dsp1	HMGB2	Dsp1 is an HMG box protein. It assists Pho in PcG recruitment at Drosophila PREs. HMGB2 is involved in YY1 in silencing of D4Z4 repeats
	Grh	GRHL1	Fly Grh helps PcG recruitment at one PRE
	Gaga factor / Trl	?	Fly Trl is involved for PcG recruitment at some PREs although is classified as a trxG protein. It is a Zn-finger sequence specific protein that binds the GAGAG motif. It also contains a BTP/POZ domain that is generally involved in protein-protein interactions
	Lolal	?	Lola-like binds Trl and acts as a PcG protein
	Psq	?	Psq co-purifies with components of the PRC1 complex and binds the same sequences as Trl
	Zeste	?	Zeste found within PRC1 but also linked to trxG-mediated activation
Fly PhoRC complex Identified in 2006	Pho	YY1 *	Fly PhoRC binds PREs and is involved in recruitment of PcG proteins to PREs. Pho also forms a second complex named INO80, likely to be involved in chromatin remodelling. Pho can recruit the histone methyltransferase E(z) to the Ubx PRE. In vitro, it can also recruit PRC1 components to DNA independent on the action of E(z). Whether PhoRC exist in human cells is unknown, but its homolog YY1 ca, recruit PcG proteins to target genes
	Phol	YY2 *
	dSfmbt (CG16975)	L3MBTL2 *
	?	E2F6	E2F6 forms two different multimeric complexes containing PcG proteins, one with RING1A, RING1B and MBLR, and the other one with EZH2, E(PC) and Sin3A
	?	BCL6	BCL6 is a BTB containing protein (similar to Drosophila Krüppel, but it is not known whether it is a true homolog) that was suggested to recruit PcG proteins to its target genes via the corepressor BCOR complex
	Rbf	RB1 RBL1	Human Retinoblastoma protein represses genes in a PcG-dependent manner to block cell proliferation. This pathway was not yet identified in other organisms
	?	PLZF	PLZF has been shown to bind to the HoxD complex and to bind Polycomb proteins on chromatin. This sequence specific DNA binding protein contains a Zn-Finger domain and a BPB/POZ domain that is generally involved in protein-protein interactions. Plzf mutants strongly derepress the HoxD locus in the embryonic hindlimb bud, PLZF binds to Bmi-1 and recruits it to HoxD
PcG complexes
PRC2 complex H3K27 trimethylation activity characterized in 2002	E(z)	EZH2 EZH1	SET domain histone methyltransferase, specific for H3K27.
		EZH2 EZH1
	Esc	EED	EED is required for the histone methyltransferase activity of EZH2 within the PRC2 complex
	Escl
	Su(z)12	SUZ12	Like EED, SUZ12 is a critical minimal component required for enzymatic activity of the PRC2 complexes. In Drosophila, Su(z)12 drives nucleosome binding by PRC2.
	Nurf-55 (Caf1)	RBBP4/RbpAp48 RBBP7/RbAp46	Drosophila Nurf55 and Su(z)12 form the minimal nucleosome binding module of PRC2 in vitro
		RBBP4/RbpAp48 RBBP7/RbAp46
PRC2-associated factors	Pcl	PCL1/PHF1 PCL2/MTF2 PCL3/PHF19/MTF2L1	Pcl was found in a PRC2 complex-subtype in flies, extracted from late developmental stages. This complex has histone deacetylation activity dependent on Rpd3. Pcl is perfectly colocalized with PC in polytene chromosomes, suggesting that it links PRC2 to PRC1.
	Rpd3	HDAC2
	Sir2	SIRT1	SIRT1 was found in a subtype of PRC2 complex in human and flies, linking polycomb silencing to histone deacetylation and cancer.
PRC1 complex Identified in 1999. PC identified as a reader of the H3K27me3 mark in 2002. H2A ubiquitylation activity found in 2004.		RING1/RING1A/RNF1	RING domain protein that is essential for the H2A ubiquitylation function of RING1B within the hPRC1L complex (see below)
	Sce/dRing	RNF2/RING1B/RING2	RING1B is the catalytic subunit responsible for the H2A ubiquitylation within the hPRC1L complex. Sce/dRing is the corresponding protein in Drosophila
	Pc	CBX2/HPC1/M33 CBX4/HPC2 CBX6 CBX7 CBX8/HPC3/PC3	Chromo domain protein, reader of the H3K27me3 mark. Required for silencing in flies. In mammals, multiple homologs exert different functions by forming alternate PRC1 complexes (Refs a b c)
	Ph-p	PHC1/EDR1/HPH1 PHC2/EDR2/HPH2 PHC3/EDR3/HPH3	Polyhomeotic proteins are SAM and Zn-finger proteins that are stoichiometric components of PRC1 and required for silencing. In flies, Polyhomeotic has additional phenotypes when compared to Polycomb, suggesting that it may have additional functions. Flag-tagged Ph and Psc were the starting components leading to the identification of the PRC1 complex in 1999.
	Ph-d

	Psc	PCGF1 (NSPc1) PCGF2/RNF110/ZFP144 (MEL18) PCGF3 PCGF4 (BMI1) PCGF5 PCGF6 (MBLR)	RING domain protein that is essential for the H2A ubiquitylation function of RING1B. BMI1 is a central component of the hPRC1L complex that contains RING1A, RING1B, HPH2 and PC3. In Drosophila, there is a second gene highly similar to Psc, named Su(z)2, which binds to similar sites in polytene chromosomes (Ref) In mammals, multiple homologs exert different functions by forming alternate PRC1 complexes (Refs a b c)

		ZNF134
	Scm	SCMH1
	TAFs
trxG complexes
SWI/SNF complex Characterized in 1994 trxG function for Brm discovered in 1992	Brm	SMARCA2/BRM	The Brm complex is a chromatin remodelling complex that uses the energy of ATP to remodel nucleosome structure and/or position, opening up chromatin structure. The Brm protein is the ATP-dependent engine of the complex. Moreover, this protein has a Bromodomain which drives binding to acetylated histones. In human, there are two subcomplexes, containing either BRM or BRG1. Brm complexes are involved in a large number of gene activation processes, probably beyond their function as antagonists of PcG-mediated silencing.
		SMARCA4/BRG1
	Osa	ARID1A/BAF250
	mor	SMARCC2/BAF170
	Snr1	SMARCB1/hSNF5/BAF47
NURF complex Identified in 1995 Reader of the H3K4me3 mark identified in 2006	Iswi	SMARCA1/SNF2L/ISWI	The NURF complex, like the Brm complex, is a chromatin remodelling complex that uses the energy of ATP to remodel nucleosome structure and/or position, opening up chromatin structure. The ATP-dependent engine is Iswi/SNF2L in this case. In addition, this complex "reads" the histone H3K4me3 mark via the PHD finger of its largest subunit: Nurf-301/BPTF.
	Nurf-38
	Nurf-55 (Caf1)	RBBP4/RbpAp48 RBBP7/RbAp46
	E(bx)/Nurf-301	BPTF
TAC1 complex Identified in 2001 Trx function in H3K4 methylation found in 2004.	Trx	??? Human homologs exist (see below for Trx homologs MLL1-3), but the homolog complex has not been found. Instead, MLL complexes have been isolated, which are not known in flies.	The TAC1 complex seems to activate transcription of its target genes by stimulating transcription initiation as well as elongation. This occurs at Hox genes, but also at genes that were previously unknown to be regulated by trxG proteins like heat-shock genes. No TAC1-homologous complex has been isolated in human.
	nej/dCBP
	Sbf1
Ash1 complex	Ash1	??? Homologs exist in human, but it is unknown whether they form a complex	In Drosophila, Ash1 was shown to directly interact with dCBP and activate gene expression.
Ash1 complex	nej/dCBP
MLL complexes Identified in 2002	??? Again, fly homologs of the MLL subunits exist, but no MLL complexes have been isolated in flies	MLL/ALL-1/HRX/TRX1 MLL2 MLL3	These are the human Trx complexes. There are most likely multiple MLL complexes, each containing a different MLL subunit, which can function as a H3K4 methyltransferase. It is unclear whether the different MLLs have different functions. The WDR5 subunit binds to methyl H3K4 and stimulates further methylation by the SET domain of MLL. The ASH2L also regulates catalysis of trimethylation by MLL. In yeast, a similar complex exist, called COMPASS.
		WDR5
		ASH2L
		RBBP5/SWD1
		C10orf9/CFP1
PcG/trxG related proteins
H3K4 demethylase	Lid	JARID1C/XLMR/SMCX JARID1B/PLU-1	Lid was classified as a trxG protein, but it has been later shown to possess H3K4 demethylase activity, which would fit better with a PcG activity. This same enzymatic activity is also found in the human proteins RBP2 (Refs a, b), and PCGF6/MBLR/RING6A (Ref).
H3K27 demethylases	dUTX	UTX JMJD3	These proteins were found to be able to demethylated tri- and dimethylated histone H3 K27. Biochemically, the mammalian components associate with MLL (TrxG) complexes. In flies, dUTX colocalizes with the elongating form of RNA pol II. Whether their genetic function is to be classified in the trxG remains to be determined.
	Asx	ASXL1 ASXL2	Drosophila Asx gene mutations have both PcG and trxG phenotypes.
TIP60 complex	dom	EP400/P400	The Domino protein has a DNA-dependent ATPase domain and belongs to the SWI/SNF family. It is a member of the Tip60 complex, and is involved in repression of Hox genes in flies. More recently, it has also been involved in histone replacement following DNA-damage.
	E(Pc)	EPC1	Chromosomal protein sharing some sites with PC in polytene chromosomes in flies. Like E(z), it behaves as a suppressor of heterochromatin position effect variegation, suggesting that both proteins play a role in heterochromatin in addition to PcG-mediated silencing.
NURD complex	Mi-2	CHD4/Mi-2	Mi-2 is the ATP-dependent engine of the NURD chromatin remodelling complex, which couples nucleosome remodelling to histone deacetylation. Drosophila Mi-2 was proposed to make a link between transcriptional repression in early embryogenesis and PcG-mediated silencing in late embryogenesis
	kis	CHD7	Kismet is a chromodomain containing ATP-dependent helicase. Originally identified in flies as a suppressor of mutant Polycomb phenotypes, it was later shown to be involved in RNA pol II transcriptional elongation. Little is known about the function of the human counterpart CHD7.
	tna	ZMIZ1	Tonally is a fly Zn-finger protein that, like kis, suppresses Pc mutant phenotypes and is required for expression of Hox genes. Not much is known about its putative human homologue ZMIZ1.
	kto	MED12L/TRAP230/TRALP	Drosophila Kohtalo and Skuld encode the homologs of the two largest subunits of "Mediator", a transcriptional coactivator complex. They interact directly and their mutants have the same phenotypes. Their trxG function has not been further characterized sofar.
	skd	THRAP2/TRAP240	Skuld/TRAP240. See comment to Kohtalo

For obvious reasons, this list does not include the work in our lab. For this, please go to the lab publications page. Moreover, this list is certainly not perfect. If you have important additions or updates that you wish to be included, please write me an email.

You will find here basic material on the subject, with particular attention to the Drosophila field. Some main protein components of the PcG and the trxG are presented, and their target DNA elements, the Polycomb and trithorax response elements (PREs and TREs), are presented and discussed. Examples of approaches to study PREs and TREs are given. Please note, this is not intended to present a balanced overview of the field and thus most of the work done by our colleaugues is not directly shown or referred to. I strongly recommend to read one the recent excellent reviews published on the subject in order to deepen the understanding of the issues raised in this introductory slide show.

This example shows a typical approach that can be used to study molecular requirements for the action of PRE and TRE elements in Drosophila. This approach consists in analyzing specific sequences of PRE/TRE and/or the role of specific PcG or trxG components in transgenic constructs. These particular examples are taken from research done in our lab.

3) An example of the study of nuclear compartmentalization and Polycomb proteins

This example illustrates that PcG proteins form nuclear compartments, and that a Drosophila PRE/TRE is able to induce long-distance interactions in the three dimensional space of the cell nucleus. These interactions can have functional consequences such as increased, PcG dependent, gene silencing and can be heritable through cell division and meiosis. Moreover, the RNAi machinery affects them, suggesting that other cellular components besides PcG proteins are important for the regulation of the nuclear organization of PcG target genes.

	Index
Part 1	Polycomb history and introduction
Part 2	Polycomb and trithorax group proteins
Part 3	Landmark Polycomb and trithorax discoveries
Part 4	In-house developed Polycomb tutorials
Part 5	External Polycomb Links
Part 6	Other teaching material
Part 7	Montpellier teaching

Year	Brief description of the main findings	Pubmed link
1978	Ed Lewis's founding Polycomb paper identifying a role for the Pc gene in the regulation of homeotic genes	go!
1985	Characterization of the trithorax gene as a regulator of homeotic gene expression Role of PcG proteins in the maintenance of homeotic gene expression, i.e. in the process of "cellular memory"	go! go!
1988	Antagonism between Polycomb and trithorax genes	go!
1989	Polytene chromosome binding pattern of Pc	go!
1991	Identification of Bmi-1, the first mammalian PcG gene Role of Bmi-1 in Cancer	go! go: a! b!
1992	Involvement of Trithorax in leukemia	go!
1993	Characterization of PREs in Drosophila Chromatin IP of Polycomb	go: a! b! c! go!
1994	Bmi-1 action as a bona fide mammalian PcG protein	go!
1997	Analysis of PcG proteins in plants PcG proteins and epigenetic regulation of gene expression by "cosuppression"	go! go!
1999	Purification of the PRC1 complex Role of PcG in cell proliferation	go! go!
2000	trxG proteins and histone acetylation	go: a! b!
2001	Link between PcG proteins and the basal transcriptional machinery PcG proteins and genomic imprinting in mammals	go: a! b! go!
2002	Characterization of the E(z)-Esc / PRC2 complex - Histone methyltransferase activity trxG proteins and histone methylation	go: a! b! c! d! go: a! b!
2003	Binding of the PC chromo domain to histone H3 methylated at Lysine 27 PcG proteins and X-inactivation Polycomb as a Sumo E3 protein	go: a! b! go: a! b! go!
2004	PRC1 proteins mediate histone ubiquitination Identification of a PRC3 complex related to PRC2 and identification of histone H1 methylation activity	go! go!
2005	Identification of a link between PcG proteins and DNA methylation Role for PcG proteins in the phenomenon of transdetermination in Drosophila	go: a! b! go: a! b!
2006	Genome-wide mapping of the downstream target sites for PcG proteins	Drosophila: a! b! c! Human Mouse
2007	Discovery of H3K27me3 demethylases	a! b! c! d!
2009	1- Crystal structure of EED reveals a mechamism for maintenance of H3K27me3 through the cell cycle (partially supports an earlier work by the Helin lab) 2- Identification and initial characterization of the first mammalian PREs	1a! 1b! 2a! 2b!
2010	Various links between PcG proteins and noncoding RNAs (earlier work had pointed to a link between PcG proteins and a ncRNA in X inactivation, but in 2010 the data were broadly generalized and, in particular, SUZ12 was shown to be an RNA-binding protein.	a! b! c! d!
2012	Identification of alternate mammalian PRC1 complexes, suggesting that each of them may have specific functions	a! b! c!
2013	...will it be it your turn ?