The functions of polycomb products extend beyond their well-known activity as transcriptional regulators to include genome duplication processes. Polycomb activities during DNA replication and DNA damage repair are unclear, particularly without induced replicative stress. We have used a cellular model of conditionally inactive polycomb E3 ligases (RING1A and RING1B), which monoubiquitylate lysine 119 of histone H2A (H2AK119Ub), to examine DNA replication in unperturbed cells. We identify slow elongation and fork stalling during DNA replication that is associated with the accumulation of mid and late S-phase cells. Signs of replicative stress and colocalisation of double-strand breaks with chromocenters, the sites of coalesced pericentromeric heterocromatic (PCH) domains, were enriched in cells at mid S-phase, the stage at which PCH is replicated. Altered replication was rescued by targeted monoubiquitylation of PCH through methylCpG binding domain protein 1. The acute senescence associated with the depletion of RING1 proteins, which is mediated by p21 (also known as CDKN1A) upregulation, could be uncoupled from a response to DNA damage. These findings link cell proliferation and the polycomb proteins RING1A and RING1B to S-phase progression through a specific function in PCH replication.

dPolycomb chromatin modifiers regulate hematopoietic pluripotent stem and progenitor cell self-renewal and expansion. Polycomb complex redundancy and biochemical heterogeneity complicate the unraveling of the functional contributions of distinct components. We have studied the hematopoietic activity of RYBP, a direct interactor and proposed modulator of RING1A/ RING1B-dependent histone H2A monoubiquitylation (H2AUb). Using a mouse model to conditionally inactivate Rybp in adult hematopoiesis, we have found that RYBP deletion results in a reversion of B-1-to-B-2 B-cell progenitor ratios, i.e., of the innate (predominantly fetal) to acquired (mostly adult) immunity precursors. Increased numbers of B-1 progenitors correlated with a loss of pre-proB cells, the B-2 progenitors. RYBP-deficient stem and progenitor cell populations (LKS) and isolated common lymphoid progenitors (CLP) gave rise to increased numbers of B-1 progenitors in vitro. Rybp inactivation, however, did not result in changes of global H2AUb and did not interact genetically with Ring1A or Ring1B deletions. These results show that a sustained regulation of the B-1-to-B-2 switch is needed throughout adult life and that RYBP plays an important role in keeping B-2 dominance, most likely independently of its Polycomb affiliation. RING1 and YY1 binding protein (RYBP) is a component of a subset of type I Polycomb repressive complexes (PRC1), chromatin regulators endowed with histone H2A monoubiquitylating activity (recently reviewed in references 1 and 2). RYBP contacts the C-terminal region of RING1B and its paralog, RING1A (3), heterodimeric RING-type E3 ubiquitin ligases that modify lysine 119 of histone H2A (H2AUb) (4, 5). Canonical PRC1 assemblies are characterized by the presence of both chromobox-containing subunits and oligomerizing SAM motif subunits, PHC proteins (6, 7). Noncanonical PRC1 complexes, on the other hand, contain RYBP or its paralog, YY1-associated factor (YAF1), instead of chromobox proteins (6, 7), as their association with RING1 proteins is mutually exclusive (8). PRC1 association with chromatin not only modifies H2A but also promotes compaction, a structural alteration that usually correlates with transcriptionally repressed states (6, 9-11). PRC1 is recruited to chromatin through PRC2-induced H3K27me3-dependent and -independent mechanisms (6,7,12). In vitro, RYBP-PRC1 is more efficient at histone monoubiquitylation than canonical PRC1 complexes (6), an observation consistent with in vivo evidence showing decreased levels of global H2AUb upon short-hairpin downregulation of RYBP (6, 7, 13). However, the relative contributions to the H2A modification of RYBP-or chromobox-containing PRC1 complexes still are controversial (6,14), possibly because of cell type variations.In the mouse, genetic analysis of PRC1 functions in differentiation often has focused on the hematopoietic compartment (summarized in references 15 and 16) as one of the best known hierarchies of related cell lineages (17). Most defects in PRC1 mutant lines pertain to...