We found the EMSA activity in the flow through of the scrambled HS4C9 oligo-affinity column

We found the EMSA activity in the flow through of the scrambled HS4C9 oligo-affinity column. HS4C9 oligonucleotide. ChIP-qPCR analysis using antibodies against ILF2, MCM5, RAD50 and p300 confirmed the association of the DARRT complex with the HS4 region (Figure 3; Supplementary Figure 2B). In addition to HS4, our ChIP qPCR results showed small but significant enrichment of ILF2, RAD50 and MCM5 Rabbit Polyclonal to DDX3Y at the -globin promoter and at the -globin replication origin -Rep-1 in K562 cells (Figure 3A). The p300 ChIP qPCR indicated strong binding to the HS4 region, and weaker but significant binding to other LCR and promoter sites, suggesting that it bound to multiple sites at the globin (Figure 3A). To test whether DARRT is also associated with normal erythroid cells, we carried out ChIP analysis using antibodies against ILF2, ILF3, MCM5 and RAD50 in human CD34+ derived primary erythroid cells (Figure 3B). All antibodies showed a significant enrichment at the HS4 site. In addition, in the normal erythroid cells, ILF2, ILF3 and MCM5 showed substantial enrichment at the -Rep-1 region of the globin IR. This suggests redundancy in the binding sites for DARRT in the normal -globin complex and this redundancy may account for some of the variation in effects of local deletions on globin replication (Forrester et al, 1990; Kitsberg et al, 1993; Cimbora et al, 2000; Mechali, 2001). Open in a separate window Figure 3 ChIP qPCR of the DARRT protein components. ChIP-qPCR assays showing the recruitment of DARRT components at the -globin locus in (A) K562 and (B) human primary LY310762 erythroid cells differentiated from human CD34+ cells. Large text letters in each of the eight graphs indicate which antibody was used for that set of chromatin immunoprecipitations. The ChIP, methodology, antibodies and sequences of the primers are presented in the Supplementary data. ChIP-PCR gels showing the relative enrichments are LY310762 provided as Supplementary Figure 2B. Fold enrichments were calculated relative to an IgG control. Each figure represents the average of three biological replicates and is expressed as means.e.m. The components of DARRT are all most strongly enriched over HS4 in K562 cells. In the primary erythroid cells, the components are also enriched strongly over the previously described -globin origins of replication (-Rep-1). Co-immunoprecipitation (co-IP) and immunodepletion experiments were carried out to test whether DARRT exists as a single homogeneous complex. Immunoprecipitating antibodies against ILF2, ILF3, MCM5 and RAD50 showed that these proteins can mutually co-immunoprecipitate each other from K562 nuclear extract, thereby suggesting that DARRT exists as a complex in the nuclear extract before fractionation (Figure 4A). Furthermore, treatment of protein extracts with DNase and RNaseA before immunoprecipitation did not have any effect on the co-IP results, suggesting that these proteins are not bound together by any RNA or DNA intermediates. Immunodepletion experiments with purified DARRT complex showed that when sufficient antibodies against the MCM5 and RAD50 proteins were added to clear them from the supernatant, there was co-clearance of each other and of ILF3, MCM3, ILF2 and DNA-PK from the IP supernatant, showing that these proteins are entirely present in a complex with MCM5 and RAD50 (Figure 4B). In addition, we show that ILF2 and RAD50 can be co-immunoprecipitated with an antibody against MCM5 in erythroid cells prepared from freshly isolated human lineage-negative LY310762 CD34+ cells (Mahajan et al, 2009), indicating that DARRT also exists in primary erythroid cells (Figure 4C). Open in a separate window Figure 4 DARRT exists as a single homogeneous multiprotein complex containing the DNA-binding component ILF2. (A) Co-immunoprecipitations (co-IP) of K562 nuclear extracts using antibodies against ILF2, ILF3, MCM5 and RAD50 show that all the tested components of DARRT co-immunoprecipitate from nuclear extracts. Nuclear extracts (150 g) were immunoprecipitated with 10 g of antibodies or isotype-specific IgG and LY310762 western blotting was performed. (B) Co-Immunodepletions of some of the DARRT components showing that components of DARRT are all associated in a single complex in purified DARRT preparations. For each immunodepletion, 3 g of the purified DARRT complex and 15 g of each of the antibody and control normal IgG were used. The supernatant and immunoprecipitated material was analysed by western blot using antibodies against various proteins as shown in the figure. (C) The co-IP in primary human erythroid cells showing that DARRT components co-immunoprecipitate in nuclear extracts from normal.