DNA replication licensing occurs on chromatin but how the chromatin template is regulated for replication remains mostly unclear. DNA re-replication and chromosomal polyploidy. Reduction of WDR5/RBBP5 also prevented the activation of H2AX Kit checkpoint caused by re-replication but not by ultraviolet or X-ray irradiation; and the components of MLL complexes co-localized with the origin recognition complex (ORC) and MCM2-7 replicative helicase complexes at replication origins to control the levels of methylated H3K4. Downregulation of WDR5 or RBBP5 reduced the methylated H3K4 and suppressed the recruitment of MCM2-7 complexes onto replication origins. Our studies show the MLL complexes and H3K4 methylation are required for DNA replication but not for DNA damage restoration. gene (Miotto and Struhl 2008 2010 Earlier studies have shown the actively transcribed chromatin areas appear to replicate DNA early in S-phase (Karnani et al. 2010 Rampakakis et al. 2009 In eukaryotes the transcriptionally active chromatin regions are usually enriched with trimethylated lysine 4 (K4) in histone H3 (H3K4) (Greer and Shi 2012 Martin and Zhang 2005 The MLL histone methyltransferase complexes each composed of a member of the MLL SET-domain protein family and other essential parts including ASH2L DPY30 and WD40 proteins WDR5 Ginsenoside Rg2 and RBBP5 catalyze the mono- and tri-methylations of H3K4 (Greer and Shi 2012 Higa et al. 2006 Jiang et al. 2011 Martin and Zhang 2005 Wysocka et al. 2005 With this statement we show the MLL-WDR5-RBBP5 methyltransferase complexes and H3K4 methylation are required for DNA replication in human being cells. RESULTS Reduction of WDR5 suppresses DNA re-replication in Geminin-deficient cells Since DNA re-replication in one eukaryotic cell cycle would lead to chromosome polyploidy and genome instability (Blow and Dutta 2005 we investigated the potential involvement of histone changes in DNA replication by analyzing this specific type of DNA replication. In metazoans both Geminin and CRL4CDT2 negatively and individually regulate the replication licensing activity of CDT1 for DNA replication (Higa et al. 2006 Jin et al. 2006 Mihaylov et al. 2002 We examined whether DNA re-replication induced by irregular activation of CDT1 is definitely controlled by histone changes in human being colorectal malignancy HCT116 cells that contain a pseudo-diploid genome (Ballabeni et al. 2004 Reduced manifestation of Geminin by specific siRNAs activates CDT1 and consequently induces Ginsenoside Rg2 chromosomal DNA Ginsenoside Rg2 re-replication (Ballabeni et al. 2004 Mihaylov et al. 2002 Zhu et al. 2004 advertising the formation of enlarged nuclei that contain more than 4N DNA content (Fig.?1A-D). Downregulation of Geminin also caused prominent nuclear staining of H2AX (Fig.?1G) indicating the activation of the replication/DNA damage checkpoints by the presence of elongation forks during DNA re-replication (Ballabeni et al. 2004 Jin et al. 2006 We found that siRNA-mediated reduction of WDR5 a key component of the MLL-WDR5-RBBP5 methyltransferase complexes that mono- and trimethylate H3K4 (Wysocka et al. 2005 led to the Ginsenoside Rg2 marked reduction on the formation of enlarged nuclei caused by Geminin Ginsenoside Rg2 deficiency (Fig.?1A-F). Reduction of WDR5 also dramatically decreased the number of cells that were positive for H2AX staining in Geminin-deficient cells (Fig.?1G). Circulation cytometry (FACS) analyses also exposed that depletion of WDR5 eliminated the percentage of cells that contain >4N DNA content material induced by Geminin deficiency (Fig.?1C D). These studies indicate that reduced manifestation of WDR5 is sufficient to suppress DNA re-replication in Geminin-deficient cells. Fig. 1. Reduced manifestation of WDR5 suppresses re-replication induced by Geminin deficiency. (A) HCT116 cells were transfected with 50?nM siRNAs of Ginsenoside Rg2 luciferase (control) Geminin Geminin+WDR5 and WDR5 for 48?h. The cells were fixed with paraformaldehyde … The MLL-WDR5-RBBP5 methyltransferase complexes are required for re-replication Because WDR5 functions as an essential component of the MLL methyltransferase complexes (Wysocka et al. 2005 we also monitored the effects of WDR5 downregulation on H3K4.