polycomb complexes prc1 and their function in hematopoiesis

Accepted Manuscript Polycomb complexes PRC1 and their function in hematopoiesis Miguel Vidal, Katharzina Starowicz PII: S0301-472X(16)30774-3 DOI: 10.1016/j.exphem.2016.12.006 Reference: EXPHEM 3498 To appear in: Experimental Hematology Received Date: 10 October 2016 Revised Date: 19 December 2016 Accepted Date: 20 December 2016 Please cite this article as: Vidal M, Starowicz K, Polycomb complexes PRC1 and their function in hematopoiesis, Experimental Hematology (2017), doi: 10.1016/j.exphem.2016.12.006 This is a PDF file of an unedited manuscript that has been accepted for publication As a service to our customers we are providing this early version of the manuscript The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain Vidal, Starowicz RI PT ACCEPTED MANUSCRIPT Polycomb complexes PRC1 and their function in M AN U SC hematopoiesis Miguel Vidal*, Katharzina Starowicz EP TE D Department of Cellular and Molecular Biology Centro de Investigaciones Biológicas 28040 Madrid SPAIN email mvidal@cib.csic.es AC C *Corresponding author Phone +34 837 3112, ext 4382 Genomics/Proteomics of Hematopoiesis 8383 words Vidal, Starowicz ACCEPTED MANUSCRIPT Abstract Hematopoiesis, the process by which blood cells are continuously produced, is one of the best studied differentiation pathways Hematological diseases are associated to reiterated mutations in genes encoding important gene RI PT expression regulators, including chromatin regulators Among them, the Polycomb group (PcG) of proteins is an essential system of gene silencing involved in the maintenance of cell identities during differentiation PcG proteins assemble into two major types of Polycomb repressive complexes SC (PRC) endowed with distinct histone tail modifying activities PRC1 complexes are histone H2A E3 ubiquitin ligases and PRC2 trimethylate histone H3 M AN U Established conceptions about their activities, mostly derived from work in embryonic stem cells, are being modified by new findings in differentiated cells Here we focus on PRC1 complexes, reviewing recent evidence on their intricate architecture, the diverse mechanisms of their recruitment to targets and on the different ways in which they engage in transcriptional control We also discuss hematopoietic PRC1 gain-of- and loss-of-function mouse strains, TE D including those that model leukemic and lymphoma diseases, in the belief that these genetic analysis provide the ultimate test bench for molecular AC C EP mechanisms driving normal hematopoiesis and hematological malignancies Vidal, Starowicz ACCEPTED MANUSCRIPT Introduction Blood cells are produced during hematopoiesis, a process where integration of developmental and homeostatic signals guides the generation of appropriate numbers of fully differentiated cells [1, 2] A network of defined RI PT DNA sites, enhancers and promoters, and the transcription factors (TF) that bind to them, results in cell type-specific gene expression patterns [3] All hematopoietic cell types originate in the bone marrow, through hierarchical differentiation from a minute pool of immature hematopoietic stem cells [4, 5] SC TF orchestrate differentiation by establishing and activating a vast collection of enhancers Establishment of new enhancers is initiated in early stages of cell lineage commitment During cell specification, some enhancers are of M AN U transient nature whereas others become cell type-specific after their maintenance in one but not other lineages [6, 7] Accessibility of TF to their cognate sites and interactions with positive and negative transcriptional cofactors is affected by nucleosome density, histone modifications and other molecular alterations corresponding to defined chromatin structures A large TE D collection of chromatin regulators dynamically delineate such structures, from local to high-order, representing the core of epigenetic regulation of gene expression [8-10] Genetic mouse models and the study of mutations in patients underline the crucial impact of chromatin regulation in normal and EP malignant hematopoiesis [11, 12] AC C The Polycomb system of chromatin regulators has attracted a great deal of interest and it has been recounted in recent excellent reviews [13-16] Here we summarise advances on Polycomb system activities carried out by Polycomb Repressive Complex(es) of type I (PRC1) and their impact in adult hematopoiesis Much of what is known about the Polycomb system originates from work in other models, mainly mammalian embryonic stem cells (ESCs) and the fly Drosophila melanogaster It is worth noting that while core concepts may be conserved throughout models, Polycomb regulation may hold a great deal of context-specific diversity With this in mind, we first outline PRC1 assemblies, their components and structural motifs in them of potential functionally Then, we consider PRC1 recruiting to targets and ways in which Vidal, Starowicz ACCEPTED MANUSCRIPT they influence transcriptional control Finally, we review lessons from genetic mouse models that illuminate PRC1 function in normal and malignant hematopoiesis RI PT The Polycomb system Polycomb products conform an evolutionary conserved system of chromatin regulators, mainly known for their involvement in developmental processes, from plants to metazoans They are encoded by genes initially identified in SC mutations leading to morphological transformations in embryos and adults of D melanogaster [17] Phenotypically, the alterations are consistent with gains of function of homeotic products, and consequently the notion of their function M AN U as repressors Genes mutated in new fly strains sharing phenotypic features of the original mutant, Polycomb, were collectively branded as the Polycomb group (PcG) of genes [18, 19] The name makes a reference to the additional number of sex combs, a structure present in legs of adult male flies, observed in mutant flies Hence, the names for many of such genes: Posterior sex TE D combs, Sex combs extra, Sex comb on midleg, etc The genetic analysis of fly development revealed another group of mutations, in the so-called Trithorax group (TrxG) of genes, as counteractors of Polycomb phenotypes [20] TrxG genes and their products usually act as activators of gene expression and are EP not considered here (see reviews by [21, 22]) AC C PcG products assemble into diverse multiprotein complexes that fit into either of two major types of biochemical entities, characterised by non-overlapping sets of subunits: type I and type II Polycomb Repressive Complexes (PRC1 and PRC2, respectively) Associated to chromatin through mechanisms not fully understood, they are endowed with histone modifying activities: a protein ligase that monoubiquitylates histone H2A (PRC1), and a lysine methyltransferase specific of histone H3 (PRC2) How PRC1-dependent H2A modification relates to transcriptional control is under active investigation [2325], whereas PcG-modification of histone H3 is known to be central to gene repression [26, 27] The working paradigm, accepted for a long time, sustained that PRC1 activity followed PRC2 Recent research, however, has Vidal, Starowicz ACCEPTED MANUSCRIPT shaped that view into one in which both systems can be interdependent, reinforce each other and oppose gene activation by ATP-dependent nucleosome remodelers and COMPASS histone modifiers found in TrxG complexes A mechanism underlying the antagonistic activities of PcG and TrxG complexes is the transient eviction of PRC from chromatin, at least by RI PT SMARCA4/BRG1, of the SWI/SNF family of remodelers [28] Chromatin sites enriched in PRC1 and PRC2 are scattered throughout euchromatin, and usually, but not always, in overlapping patterns [29], rarely on silenced domains in heterochromatin [30] Characteristically, the Polycomb system SC silences targets important for the maintenance of cell identity during transitions between cell states [31] The extent of Polycomb-modified chromatin varies with cell state, being much larger in differentiated cells than M AN U in cells with developmental potential [32], possibly in relation to restriction of developmental potential through transcriptional repression Of note, while the Polycomb system has been associated to transcriptional silencing, increasing evidence, for PRC1 at least, links it also to transcriptional activity (see below) Polycomb proteins participate also in genome stability, through DNA TE D replication and DNA damage repair functions [33-35] but here we will focus on transcriptional control EP PRC1 assemblies: canonical and non-canonical complexes The first Polycomb complex, isolated from Drosophila embryos, was termed AC C PRC1 [36, 37] Mammalian cells contain similar complexes [38], except for the presence of additional subunits corresponding to the multiplicity of homologs Subsequent purifications, however, showed that the term PRC1 encompasses a larger collection of biochemical entities, containing subunits of unexpected relation to PcG products [39-41] All of these complexes share RING finger proteins, while diverging in their content of other subunits In an attempt to organise this heterogeneity, complexes that contain subunits found in the original purifications were termed canonical PRC1 complexes The rest, were grouped into a non-canonical category The division also reflects that, at the time, the little that was known about PRC1 pertained to subunits found in complexes to be named canonical Subsequent attempts to get a more Vidal, Starowicz ACCEPTED MANUSCRIPT granulated classification are based on the content of one of a six member family of the so-called Polycomb group RING finger proteins (PCGFs), because of the reasonable consistency of subunits co-purifying with each o them [42] A minimum set of PRC1 subunits and their organisation in complexes PRC1.1 to PRC1.6, as defined by Reinberg’s lab [42] is listed in RI PT Table 1, and as a diagram in Figure 1A As with all classification attempts, these PRC1 categories use some simplification They are as snapshots determined by the cell type from which SC the complexes are isolated, and although its systematic core may hold, it is already known that PRC1 complexes are very dynamic structures that evolve with progression between cell states [43, 44] An idea of the difficulties in M AN U getting these categories right is the presence of (non-canonical) RYBP in preparations of proteins associated to (canonical) PCGF2/MEL18 [45] The simplest picture is that of a core PRC1 complex, an ubiquitin ligase module, to which a reduced numbers of subunits associate: proteins with oligomerizing and histone reading abilities (PHC, CBX), for canonical forms, and DNA TE D binding proteins, for non-canonical forms Additionally, each of these minimal complexes may contain a heterogeneous collection of subunits, sometimes in a substoichiometrically manner, conforming a pool of PRC1 complexes that EP varies with cell type The core of PRC1 complexes: RING1-PCGF heterodimers that AC C monoubiquitylate histone H2A The RING finger proteins at the centre of each PRC1 complex are structurally related, sharing a N-terminal RING finger, a specialised type of Zn2+ binding motif of the Cys3HisCys4 type [46] and a C-terminal RAWUL (from Ringfinger And WD40 associated Ubiquitin-Like, [47]) domain The paired RING finger motifs make E3 ubiquitin ligases and the RAWUL motifs act as binding platforms for other PRC1 components The PRC1 ubiquitin ligase module (Figure 2) is made of either RING1A or its paralog RING1B, and one of the six PCGF proteins: PCGF1/NSPC1; PCGF2/MEL18; PCGF3; PCGF4/BMI1; PCGF5 and PCGF6/MBLR Each Vidal, Starowicz ACCEPTED MANUSCRIPT RING1 and PCGF component associate through extensive contacts between partially hydrophilic surfaces of their RING fingers [48, 49], thus making heterodimeric RING ubiquitin ligases, a subset of the RING class of ubiquitin E3 ligases (reviewed in [50]) In the multi-step process by which ubiquitin (Ub) is added to proteins, the E3 ligase provides substrate specificity to its transfer RI PT from a specific conjugating enzyme, the E2 element, or Ub carrier [50] PRC1 E3 ligases work with the E2 component UBCH5/UBE2D [48], bound exclusively through contacts in RING1A or RING1B, away from the PCGF moiety [51] Mutations in either of the RING1-PCGF or RING1-E2 surfaces SC can impair ubiquitin ligase activity Characteristically, canonical PRC1 complexes contain RING1-PCGF2 and RING1-PCGF4 E3 ligases, whereas the remaining E3 ligases are constituents of non-canonical PRC1 complexes M AN U [42] PRC1-dependent modification of histone H2A involves the monoubiquitylation of (predominantly) lysine 119 (K119Ub) or of lysine 120 [52] Histone H2 variant H2A.Z is also monoubiquitylated by PRC1 at lysines 120, 121 and 125 TE D [53-55].The activity of PRC1 E3 ligases is regulated by complex assembly, i.e., their activity follows the establishment of a multiplicity of contacts involving the dimeric RING module, the nucleosome, DNA and the E2 ligase [27] PRC1 recognition of the nucleosome occurs through binding of the E3 EP ligase module that, as other nucleosome-interacting proteins, bind the acidic patch defined by histone pairs H2A and H2B, mostly through contacts AC C involving the RING1 protein These interactions, and the organization of the C-terminal tail of H2A in the nucleosome, position the Ub carrier E2 in the vicinity of the appropriate lysine residue, thus determining the specificity to the modification of the histone substrate [27] (histone H2A can also be ubiquitylated at lysines 127 and 129 by another E3 ligase, [56]) In fact, it appears that E3 ligases in canonical PRC1 complexes may be autoinhibited until engaged with nucleosomal substrates [57] It has been suggested that E3 ligases in canonical, but not in non canonical PRC1, use some stereoselective step previous to their activation, thus explaining their poor performance modifying H2A following forced recruitment to chromatin [58] Most monoubiquitylation of histone H2AUb depends on PRC1 E3 ligases, as Vidal, Starowicz ACCEPTED MANUSCRIPT indicated by the dramatic decrease in levels after compound inactivation of both RING1 paralogs [59] However, additional unrelated E3 ligases (some containing RING1B) can also monoubiquitylate histone H2A under some circumstances, as in tumor cells or in early stages of nucleotide excision RI PT repair of damaged DNA [60-62] The shared C-terminal motif of PRC1 RING finger proteins is an Ub-like folded domain (RAWUL) that participates in interactions with other PRC1 subunits The Ub-like motif in RING1A/RING1B binds through a common SC surface either chromobox (CBX) or RYBP/YAF2 proteins [63] Hence the presence exclusive of one or the other in isolated PRC1 complexes Ub-like motifs of PCGF proteins, instead, associate with specific PRC1 components M AN U For example, PCGF2/MEL18 and PCGF4/BMI1 interact with PHC proteins, whereas PCGF1/NSPC1 binds BCOR [64] It is likely that, as for other multiprotein complexes, PRC1 aggregates form following a cooperativitydriven, hierarchical association of preassembled subunits [65, 66] For example, PCGF1 binds a heterodimer of PRC1 subunits KDM2B/FBXL10- TE D SKP1 only if previously associated to BCOR [67] Subsequently, the complex grows larger after contacts with heterodimers made of RING1A (or RING1B)RYBP/YAF2 or CBX subunits The coexistence of modules may be reflected in the wide range of sizes estimated for PRC1 complexes [42] and in the EP chromatin binding patterns of individual subunits [43] AC C PRC1 subunits preferentially present in canonical complexes Mammalian homologs of Drosophila Polyhomeotic, Polyhomeotic-like 1, and (PHC1, and 3) can participate in polymerizing protein-protein interactions thanks to the their C-terminal SAM (sterile alpha motif) domain [68] This protein motif, present in many other proteins too, has two surfaces for interaction with the same or different SAM-containing proteins and therefore permit the formation of associations of high structural complexity [69] In addition to the SAM domain, PHC paralogs share a so-called homology domain, a FCS-type zinc finger for binding to RING1/PCGF [70] The ability to make large molecular aggregates is evidenced in vivo, both in mammalian and Drosophila cells [70-72] The tendency of PHC proteins to aggregate is Vidal, Starowicz ACCEPTED MANUSCRIPT regulated by addition of O-linked β-N-acetylglucosamine (O-GlcNAc) to serine/threonine strings [73], an activity dependent of the product of the OGlcNAc transferase (Ogt) gene, the homolog of Drosophila super sex combs [74] RI PT A number of proteins of the malignant brain tumor (MBT) family, homologs of the products of Drosophila PcG genes Sex comb on midleg (SCM) and SCM with four MBT (SfMBT), also have a C-terminal SAM domain and appear, substoichiometrically, in preparations of canonical PRC1 [38, 42] It is though SC that the presence of SCML1, SCML2, SCMH1, SFMBT1 and SFMBT2L, in PRC1.4 complexes [75] is due contacts between their SAM domain and the M AN U SAM domain of PHC subunits [76] RYBP/YAF2 and DNA-binding proteins, subunits of non canonical PRC1 complexes RING1 and YY1 binding protein (RYBP) [77] and its paralog, YY1-associated factor (YAF2) [78], are two small, basic proteins with a N-terminal RANBP2- TE D type zinc finger motif They interact with the RAWUL domain of RING1A or RING1B through their C-terminal regions [77] Both RYBP/YAF2 and CBX proteins, despite their dissimilar conformation, contact the same region of RING1B [63] RYBP binds non specifically DNA [79], although the EP physiological relevance of this ability is unknown Instead, RYBP/YAF2 were related initially to a possible recruiting activity, through their partner YY1, the AC C homolog of Drosophila Pleiohomeotic, known to participate in PRC1 recruiting functions in flies [80] In mammalian cells, however, YY1 does not copurify with PRC1 components [42] nor colocalizes with PRC1 or PRC2-bound chromatin [81] A subset of non-canonical PRC1 complexes contain subunits with dedicated DNA-binding domains The best studied is lysine-specific demethylase 2B (KDM2B, or F-box and leucine-rich repeat protein 10, FBXL10), a PRC1.1 component It binds CpG islands (CGI) [82], a singular collection of DNA segments enriched in non-methylated CpG dinucleotides, permissive to transcription that contain embedded over half of all promoters [see 83] Vidal, Starowicz ACCEPTED MANUSCRIPT Legends to Figures Figure Schematic representation of the various classes of PRC1 complexes A, Except for PCGF components, subunits are represented in a simplified RI PT manner that precludes description of paralogs Protein-protein contacts are not meant to be accurate in all cases because in many cases they not yet known Complexes, as depicted, are meant to represent a consensuated view, but cell type-divergent versions are expected Substoichiometric components have been left out B, schematized representation of the SC association of PRC1 complexes to sites (unmethylated long CpG islands, CGI) and cooperation between PRC1 and PRC2 to establish chromatin M AN U structure at repressed targets Association of PRC1 to transcriptionally active sites, less characterized, is also indicated Figure Schematic representation of the E3 ubiquitin ligase module of PRC1 complexes The heterodimeric E3 ligase ins made of two subunits, one a TE D RING1 protein, another a PCGF protein, associated through their RING fingers The E2 ligase is not part of PRC1 complexes It is included, bound to ubiquitin (Ub), only to indicate that its recruiting to PRC1 targets is, EP exclusively, through interaction with the RING1 component of the E3 ligase The divergent orientation of RAWUL domains does not intend to portrait their actual three-dimensional disposition The substrate, histone H2A, is targeted AC C through nucleosome contacts that involve both the dimeric RING fingers (check) Figure Hematopoietic redundancy of RING1A and RING1B paralogs A, lethality associated to the compound inactivation in a mouse model constitutively deficient in RING1A and conditionally deficient in RING1B Double mutation is induced by intraperitoneal administration of poly (I:C) to mice that bear a MxCre1 transgene (controls are non transgenic littermates) The presence of a single allele, whether Ring1A or Ring1B, suffices to provide viability Data correspond to n mice of the indicated genotypes B, Hematoxyline-Eosin 46 Vidal, Starowicz ACCEPTED MANUSCRIPT stained sections of bone marrow [day 12 after p(I:C) treatment] showing severe aplasia in double mutant mice, suggesting hematopoietic failure as the likely cause of lethality Scale bars = 200 microns (top) 20 microns (bottom) RI PT Figure Changes in PRC1 protein levels induced by Ring1B inactivation in the indicated hematopoietic cell types A, alterations in primitive (Lin-), lymphoid (Cd19+) and erythroid (Ter119+) progenitors Total cells extracts were SC analyzed by western blot with home made (RING1A, RING1B, CBX2, RYBP) or commercially available antibodies Chemiluminiscent signals, quantitated with and were normalized by histone H3 content Pooled (four mice per M AN U genotype) bone marrow cells were used for immunodepletion of differentiated cells or positive selection of cells expressing Cd19 or Ter119 markers using Miltenyi immunomagnetic purification Values are differences between ratios of average levels in Ring1B KO versus control cells, so that positive or negative figures correspond to upregulated and downregulated levels, TE D respectively B, Representative western blots The signal for KDM2B AC C EP corresponds to the short form, far more abundant than the full length protein 47 Vidal, Starowicz ACCEPTED MANUSCRIPT Table Subunits content in canonical and non-canonical PRC1 complexes Non-canonical (PRC1.1, PRC1.3, PRC1.5, PRC1.6) Common RING1/RING1A, RNF2/RING1B RING1/RING1A, RNF2/RING1B PCGF proteins PCGF2/MEL18 (PRC1.2, PRC1.4) PCGF4/BMI1 (PRC1.2, PRC1.4) PCGF1/NSPC1 (PRC1.1) PCGF3 (PRC1.1) PCGF5 (PRC1.5) PCGF6/MBLR (PRC1.6) Cromodomain proteins CBX2/M33, CBX4/PC4, CBX6, CBX8 (PRC1.1) CBX7, CBX8 (PRC1.2, PRC1.4) CBX3/HP1γ (PRC1.6) Polyhomeotic homologs PHC1, PHC2, PHC3 (PRC1.2, PRC1.4) Sex comb on midleg homologs SCML2, SCMH1, SFMBT1, SFMBT2 (PRC1.4) SC M AN U RYBP* AC C EP Other subunits TE D DNA binding proteins RI PT Canonical (PRC1.2, PRC1.4) KDM2B/FBXL10 (PRC1.1) MGA-MAX, E2F6-TDFP1 (PRC1.6) RYBP, YAF2 (PRC1.1, 3, 5, 6) L3MBTL2, WDR5 (PRC1.6) SKP1, BCOR (PRC1.1) AUTS2, CSNK2A1/CK2a (PRC1.5) *RYBP has been found in some preparations of canonical PRC1 complexes (see text) 48 Vidal, Starowicz ACCEPTED MANUSCRIPT Table Animal and cellular models: loss-of- and gain-of-function of PRC1 genes Gene Model Phenotype Bcor cKO (CreERT ) Impaired ex-vivo expansion of progenitors in liquid and semi-solid cultures [177] In vitro: enhanced proliferation and replating, tendency towards myeloid differentation [177] Hypoplastic thymus, spleen; defective ex vivo expansion of fetal T-cells; impaired B-cell differentiation; transplant ability unaffected Decreases ex vivo proliferation of human progenitors gof, transduced Cbx4 cKO (EaIICre) cKO (LckCre) Ex-vivo, reduced colony formation, differentiation; abolishes reconstitution in competitive transplants TE D KD, shRNA [200] [100, 199] [165] [100, 199] [100, 200] Enhanced ex vivo expansion of HSPCs but not myeloid progenitors; in vivo, T lymphomas develop with variable latency and penetrance in transplanted mice [100, 172] No apparent defects [188] 10 Germinal Centre depletion [103] Little effect on ex vivo proliferation of human + CD34 cord blood cells [200] Impaired long term repopulaltion activity [100] cKO (Cre-ERT ) cKO (Cγ1-Cre) AC C [198, 199] Reduced ex vivo murine HSCs self-renewal; little effect on ex vivo proliferation of human progenitors EP gof, transduced Cbx8 Induced ex vivo differentiation, impaired long term reconstitution in competitive transplants New born lethality if global inactivation; hypoplastic embryonic thymus due to impaired proliferation related to defective thymic stroma (not seen if inactivation restricted to thymocytes); spleen and bone marrow cellularities unaffected gof, transduced Cbx7 SC KD, shRNA M AN U KO RI PT gof (ind CreERT ) Cbx2 References KD, shRNA gof, transduced Kdm2b/ gof, transduced7 Fbxl10 Promotes expansion progenitors ex-vivo, augmented CFC and leukemogenic in vivo [171, 201] gof, Sca1-Tg gof (ind 12 CreERT) In vivo expansion of lymphoid primed progenitors (ind Kdm2b); long term myeloid, lymphoid leukemia (Sca1-Kdm2b) [162, 202] cKO 13 (GATA1Cre) 14 cKO (VavCre) cKO (ind 15 Mx1Cre) Embryonic lethal (GATA1Cre) In adults, reduced pools of HSPCs and CLPs, differentiation skewed towards myeloid lineages, variable loss of lymphoid cells; loss of repopulation ability [162] 11 49 Vidal, Starowicz ACCEPTED MANUSCRIPT Table Animal and cellular models: loss-of- and gain-of-function of PRC1 genes Gene Model Phenotype References L3mbtl3 KO Embryonic lethal due to anemia; defficient maturation to granulocytes, erythrocytes Pcgf1/N KD, shRNA6 spc1 Mouse progenitors: mild enhancement of exvivo expansion (larger if Runx1-deficient cells); long term reconstitution unaffected Impaired + ex-vivo expansion of human CD34 cord blood cells) [179, 200] Pcgf2/ Mel18 Early postnatal lethality; hypocellular bone marrow, thymus and spleen; severe decrease of circulating lymphoid cells; enhanced proliferation of a reduced HSC pool, diminished B-cell compartment; enhanced repopulating activity on competitive transplants [179, 200] Early postnatal lethality; hypocellular bone marrow, thymus, spleen and reduced number of circulating cells; very limited repopulating ability; decreased pool of adult HSCs Increased oxidative stress-induced DNA damage 160, 165, 169, 199, 204] RI PT cKO (VavCre) 14 Pcgf5 cKO (Cre-ERT ) Pcgf6 KD, shRNA Phc1 KO Rnf2/Ri ng1B cKO (ind 15 Mx1Cre) cKO (Cre-ERT ) AC C Rybp [164] No apparent defects [205] No effect on ex-vivo expansion of human + CD34 cord blood cells [200] Early postnatal lethality; no differences in circulating cells, hypoplastic spleen in neonates; transient expansion of embryonic progenitors; fetal liver cells without repopulation activity, arrested B-cell development [161, 206208] Hypocellular bone marrow, enlarged pool of hyperproliferative myeloid progenitors [170] EP Hypocellular bone marrow, thymus and spleen; exhaustion of HSC pool, impaired lymphoid differentiation TE D M AN U SC KO Pcgf4/B KO mi1 double Ring1A Ring1B [203] + KD, shRNA Impaired ex-vivo expansion of human CD34 cord blood cells KO + cKO (Cre-ERT ) Impaired ex-vivo expansion of progenitors lethal aplasia KO + cKO (LckCre) conversion of T-lineage progenitors to the Bcell fate [175] cKO (ind 15 Mx1Cre) cKO (Cre-ERT ) B cell development altered with B1 progenitors expansion at an expense of B2 progenitors [176] cKO, conditional inactivation 4’-hydroxytamoxifen induced Cre recombinase 50 [200] [33], this paper Vidal, Starowicz ACCEPTED MANUSCRIPT gain of function conditional gain of function, inducible Cre integrated at the ubiquituously expressed Rosa26 locus KO, constitutive inactivation KD, knocked down expression after retro or lentiviral transduction of hematopoietic progenitors gain of function after retro or lentiviral transduction of hematopoietic progenitors Ubiquitous recombination driven by a EIIaCre transgene Thymus-specific recombination driven by a LckCre transgene 10 Germinal Center (GC)-specific recombination driven by a Cγ1Cre transgene 11 Sca1-Tg, drives expression embryonic and adult hematopoetic tissues, predominantly T cells 12 Doxycicline-inducible inactivation within a Vav-Cre expression domain 13 Early embryo hematopoietic deletion driven by Gata1Cre transgene 14 Ubiquitous hematopoietic recombination driven by a VavCre transgene upon Cre-induced 15 Hematopoietic (predominant) recombination driven by an inducible Mx1Cre transgene AC C EP TE D M AN U SC RI PT 51 Vidal, Starowicz ACCEPTED MANUSCRIPT Table Protein levels of PRC1 subunits in hematopoietic cells Progenitors Bone marrow* - + LKS/LKS ratio CD19 + 1,16 0,52 0,02 1,08 0,41 2,4 1,6 Thymus* 0,27 0,14 0,04 0,03 0,03 0,19 0,09 1,14 0,45 0,26 0,76 0,63 0,46 0,06 0,27 0,24 0,12 CBX7 1,05 0,01 0,32 0,52 0,22 CBX8 1,34 0,05 0,54 0,5 0,28 0,61 0,16 0,47 1,42 0,88 0,3 0,12 1,01 0,8 0,39 0,29 0,11 0,27 0,07 0,04 0,54 0,31 0,46 0,09 0,09 0,26 bd 0,41 0,54 0,33 0,08 bd bd 0,33 0,5 0,5 0,4 1,19 0,45 0,21 1,38 1,07 1,11 0,81 1,16 RYBP 0,54 0,23 0,53 0,38 0,31 YAF2 0,27 0,38 0,86 0,77 0,68 M33 PHC2 1,29 CBX4 0,99 KDM2B 2,77 SKP1 1,11 PCGF1 PCGF6 L3MBT L2 MGA MAX/MI N CK2a † 0,82 1,09 AC C # 0,76 EP PCGF5 0,81 TE D BCOR M AN U Non-canonical RI PT Spleen* BMI1 Neutrophils Ter119 SC PRC1 subunits RING1 B RING1 A Canonical # mass-spectrometry data from [159] *quantitative western blot (our data), relative to values in Lin cells; cells isolated as indicated in legend to Figure † below detection 52 Vidal, Starowicz ACCEPTED MANUSCRIPT Table Models that investigate functions of PRC1 products in malignant hematopoiesis Gene Model Phenotype References Activity Bcor Mll-Af9 transformed CD34 human progenitors + KD shRNA + [173] Impaired ex-vivo maintenance of transformed phenotype Cbx7-transduced Eu-Myc progenitors in a transplantation model Accelerated onset of lymphomas [172] ON Mll-Af9-transduced KO progenitors in a transplantation model [188] SC Cbx8 RI PT Cbx7 ON M AN U Required for initiation and maintenance of leukemic transformation Mll-Enl-ER knock in progenitors + KD shRNA ON [189] ON Reduced ex-vivo expansion Diffuse large B cell lymphoma cells + KD shRNA [103] Kdm2b/ Fbxl10 TE D Cell differentiation G12D VavCre-KRas + cKO ON [162] EP Accelerated in vivo myeloid transformation B, T lymphoid leukemias + KD shRNA TS [162] Defective growth AC C Hoxa9-Meis1 + shRNA KD cotransduced progenitors, transplanted ON [171] Impaired in vivo leukemogenesis Mll-Af9 + shRNA KD cotransduced progenitors, xenotransplanted + Mll-Af9 transformed CD34 human progenitors + shRNA KD ON [173] Impaired in vitro growht/leukemogenesis Pcgf4/B mi1 Hoxa9 + Meis1 transformed KO progenitors, transplantated Unable to repopulate in secondary transplant 53 ON [190] ON Vidal, Starowicz ACCEPTED MANUSCRIPT Table Models that investigate functions of PRC1 products in malignant hematopoiesis Gene Model Phenotype References Activity Runx1 D171N + Bmi1 cotransduced progenitors, transplanted [190] TS RI PT Shortened latency in appearance of MDS/AML [209] Bcr-Abl progenitors + KD shRNA Impaired ex-vivo expansion ON [191] Plzf-Rara-transduced KO progenitors SC Defective ex-vivo transformation, differentiation +/- Eµ-Myc transgenic, Bmi1 mice ON [169] M AN U Reduced Eµ-myc in vivo lymphomagenesis +/- Dnmt3a R882H-transduced Bmi1 transplanted progenitors, ON [192] Decreased HSC pool expansion -/- Ink4a + KO progenitors, transplanted ON [197] Rnf2/Ri ng1B -/- Ink4a + KO TE D Increased repopulation capacity, myelofibrosis TS [170] Accelerated onset of lymphomas [173] AC C EP Mll-Af9 + shRNA KD cotransduced progenitors, xenotransplanted + Mll-Af9 transformed CD34 human progenitors + KD shRNA TS Impaired leukemogenesis/ex vivo-maintenance of transformed phenotype Mll-Af9 leukaemic cells + KD shRNA ON [188] No effect - double Mll-Af9 + shRNA KD cotransduced progenitors, Ring1A/ xenotransplanted + Ring1B Mll-Af9 transformed CD34 human progenitors + shRNA KD [173] Impaired (partially) leukemogenesis/ex vivomaintenance of transformed phenotype Mll-Af9-transduced double KO progenitors 54 ON [193] Vidal, Starowicz ACCEPTED MANUSCRIPT Table Models that investigate functions of PRC1 products in malignant hematopoiesis Gene Model Phenotype References Activity ON AC C EP TE D M AN U SC RI PT Impaired ex-vivo transformation and transplant 55 AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT EP TE D M AN U SC RI PT Polycomb PRC1 complexes are chromatin modifiers with histone H2A E3 ligase activity Canonical PRC1 compact chromatin, non-canonical PRC1 contain DNA binding subunits PRC1 targets in differentiated cells are transcriptionally repressed and active Both hematopoietic differentiation and homeostasis are regulated by PRC1 PRC1 subunits promote/restrict hematopoietic transformation in a cell-context manner AC C • • • • • ... content in canonical and non-canonical PRC1 complexes Non-canonical (PRC1. 1, PRC1. 3, PRC1. 5, PRC1. 6) Common RING1/RING1A, RNF2/RING1B RING1/RING1A, RNF2/RING1B PCGF proteins PCGF2/MEL18 (PRC1. 2, PRC1. 4)... domain and the M AN U SAM domain of PHC subunits [76] RYBP/YAF2 and DNA-binding proteins, subunits of non canonical PRC1 complexes RING1 and YY1 binding protein (RYBP) [77] and its paralog, YY1-associated... shared C-terminal motif of PRC1 RING finger proteins is an Ub-like folded domain (RAWUL) that participates in interactions with other PRC1 subunits The Ub-like motif in RING1A/RING1B binds through