Polycomb complexes PRC1 and their function in hematopoiesis Miguel Vidal, Katarzina Starowicz  Experimental Hematology  Volume 48, Pages 12-31 (April 2017) DOI: 10.1016/j.exphem.2016.12.006 Copyright © 2017 ISEH - International Society for Experimental Hematology Terms and Conditions

Figure 1 Schematic representation of the various classes of PRC1 complexes. (A) Except for PCGF components, subunits are represented in a simplified manner that precludes description of paralogs. Protein–protein contacts are not meant to be accurate in all cases because, in many cases, they are 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 association of PRC1 complexes to sites (unmethylated long CpG islands [CGI]) and cooperation between PRC1 and PRC2 to establish chromatin structure at repressed targets. The association of PRC1 with transcriptionally active sites, which is less well characterized, is also indicated. Experimental Hematology 2017 48, 12-31DOI: (10.1016/j.exphem.2016.12.006) Copyright © 2017 ISEH - International Society for Experimental Hematology Terms and Conditions

Figure 2 Schematic representation of the E3 ubiquitin ligase module of PRC1 complexes. The heterodimeric E3 ligase is made of two subunits, one a RING1 protein and another a PCGF protein, associated through their RING fingers. The E2 ligase is not part of PRC1 complexes. It is included, bound to Ub, only to indicate that its recruiting to PRC1 targets is 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 through nucleosome contacts that involve both of the dimeric RING fingers. Experimental Hematology 2017 48, 12-31DOI: (10.1016/j.exphem.2016.12.006) Copyright © 2017 ISEH - International Society for Experimental Hematology Terms and Conditions

Figure 3 Hematopoietic redundancy of RING1A and RING1B paralogs. (A) Lethality associated with 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 bearing a Mx-Cre1 transgene (controls are nontransgenic littermates). The presence of a single allele, whether Ring1A or Ring1B, suffices to provide viability. Data correspond to 6-12 mice of the indicated genotypes. (B) Hematoxylin & eosin-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 indicate 200 μm (top) and 20 μm (bottom). Experimental Hematology 2017 48, 12-31DOI: (10.1016/j.exphem.2016.12.006) Copyright © 2017 ISEH - International Society for Experimental Hematology Terms and Conditions

Figure 4 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 analyzed by Western blot with home-made (RING1A, RING1B, CBX2, and RYBP) or commercially available antibodies (Cell Signaling Technology, US, #5856 [anti-BMI-1]; GeneTex, US, GTX104868 [anti-KDM2B]; Abcam, UK, ab10546 and ab1791 [anti-SKP1 and histone H3, respectively] and Invitrogen, US, PA5-35222 [anti-PCGF6]). Chemiluminiscent signals were quantitated with Image Lab 5.2.1, on images acquired on a Chemidoc (BioRad) device and normalized by histone H3 content. Pooled (four mice per 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, respectively. (B) Representative Western blots. The signal for KDM2B corresponds to the short form, which is far more abundant than the full-length protein. Experimental Hematology 2017 48, 12-31DOI: (10.1016/j.exphem.2016.12.006) Copyright © 2017 ISEH - International Society for Experimental Hematology Terms and Conditions