The A3 element was associated with intermediate levels of all three PcG proteins, whereas relatively low PcG protein binding was detected at the A13 region (Fig
The A3 element was associated with intermediate levels of all three PcG proteins, whereas relatively low PcG protein binding was detected at the A13 region (Fig. (Pc), Polyhomeotic (Ph), Posterior sex combs (Psc), and Ring (dRing) ; while PRC2 contains Enhancer of Zeste [E(z)], Extra sex combs, Suppressor of Zeste 12 [Su(z)12], Nurf55, and several other components . In regulatory elements, the PREs, has hindered our understanding of the critical PcG regulation during mammalian development. Results PRC1 and PRC2 proteins have differential binding to the three DNA elements tested In gene , which encodes a sodium-dependent inorganic phosphate co-transporter  and is silent in T cells . Since genes are potentially regulated by PcG proteins, we also selected two regions (A3 and A13) from the gene locus (Table 1). Using ChIP-PCR (chromatin immunoprecipitation followed by PCR using isotope labeled primers) assays, we found that SLC, A3 and A13 displayed differential levels of H3K27me3 binding (Fig. 1A). To test for enrichment of PRC1 and PRC2 components at these loci, we used human resting CD4+ T cells to perform ChIP experiments using antibodies against PRC1 components BMI1 and RING1B, as well as PRC2 component SUZ12. Our data indicated that the three DNA elements SLC, A3 and A13 had differential binding of PcG proteins. The SLC element was highly enriched with all three PcG proteins: SUZ12, BMI1 and RING1B. The A3 element was associated with intermediate levels of all three PcG proteins, whereas relatively low PcG protein binding was detected at the A13 region (Fig. 1B). Similar results were obtained from Upamostat HeLa cells (Fig. 1C) and SW-13 cells (Fig. 1D). Open in a separate window Figure 1 PcG proteins bind to the potential PREs in human Rabbit polyclonal to RAD17 cells.(A) Assessment of H3K27me3 levels at SLC, A3, and A13 regions using PCR. H3K27me3 ChIP DNA samples from resting T cells and their input controls were analyzed using 32P-labeled specific primers. Actin was used as control. Band intensities were quantified using Phospho Imager and indicated below the panel. (B, C, D) PcG proteins SUZ12, BMI1, and RING1B are enriched at the SLC and A3 regions compared to the A13 region in resting T cells (B), HeLa cells (C), and SW-13 cells (D), respectively. ChIP assays were performed using antibodies specific for SUZ12, BMI1, and RING1B with chromatin prepared from CD4+ T cells, HeLa cells and SW-13 cells. The ChIP DNA was analyzed by qPCR using primers specific for the SLC, A3 and A13 regions (primer sequences in Table 1). Table 1 Genomic coordinates of the 3 kb human putative PRE regions and sequences of specific primers used Upamostat in ChIP experiments for each of these regions (sequence information is based on the UCSC hg18 assembly). gene locus, which showed very low level of PcG proteins in both the control and SUZ12 knockdown cells. (C) Knocking down SUZ12 in HeLa cells increased the expression of the endogenous (SLC locus) and (A3 locus) genes but not the (A13 locus) gene. Total RNAs were isolated from HeLa cells transfected with pREP4-Puro-siSUZ12 or a control vector and selected with puromycin. The expression level of the genes was determined by qRT-PCR analysis. To investigate whether these changes at the chromatin level affect expression of endogenous genes near the SLC and A3 loci, we examined the mRNA levels of the genes at the vicinity (Table 1). Interestingly, in HeLa cells, knockdown of SUZ12 resulted in an increased expression level of (near SLC element) and (near A13 element) expression was observed (Fig. 2). Together, these results suggested that normal function of PcG proteins is required for the repressive activities of the putative human PREs. The putative human PREs repress reporter gene expression in by assaying the PRE-mediated silencing effect on the reporter gene expression in adult fly eyes. In this experiment, Upamostat a 3-kb genomic DNA fragment surrounding the SLC, A3 or A13 region was placed next to the gene (within 100-bp) individually in the pCasper3 expression vector (Table 1, Table 2, and Figure S2)..