An obvious implication is that the presence of PRC1 at a locus does not necessarily equate with repression, although formal proof would require analyses at the single cell level. Open in a separate Mycophenolic acid window Figure 6 Correlation of ChIP-sequencing and RNA-sequencing in BF and Hs68 cells. RING1 and RING2. (D) Lysate from cells expressing Flag-tagged CBX4 (left) or CBX6 (right) were immunoprecipitated with antibodies against CBX4, 6, 7 and 8 as indicated. Following SDS-PAGE, the Flag-tagged proteins were identified by immunoblotting. Ig refers to the position of the immunoglobulin heavy chain. gb-2014-15-2-r23-S1.ppt (626K) GUID:?ADA11372-DC59-4294-96EB-D551031A15E9 Additional file 2: Figure S2 ChIP-PCR analyses of multiple PRC1 proteins at representative loci. Each dataset includes a screenshot of the CBX7 binding profile across the locus (top), with a diagram showing the position of the PCR primer sets relative to the organization of the suspected target gene. The primer sequences are described in Additional file 6: Table S3. The panels show the enrichment observed with the indicated antibody at each primer set as a percentage of input. Grey bars show values for a control IgG antibody. (A) GATA6 in BF cells, (B) CCND2 in BF cells, (C) MEIS1 in BF cells and (D) NRN1 in Hs68 cells. gb-2014-15-2-r23-S2.pptx (176K) GUID:?0BAFABBB-0D17-4923-B26B-6182BAABC0D0 Additional file 3: Table S1 List of candidate PRC1 target loci in the BF and Hs68 strains of HF. Alphabetic list of loci associated with PRC1 ChIP-seq peaks in HFs, showing the number of PRC1 proteins identified at the locus for the BF and Hs68 strains. TRUE and FALSE indicate whether LEPR the locus is usually transcriptionally active in BF and Hs68, as judged by RNA-sequencing. gb-2014-15-2-r23-S3.xlsx (51K) GUID:?974C5913-0382-4637-B269-746A63F58784 Additional file 4: Physique S3 ChIP-PCR showing differential binding of PRC1 proteins in BF and Hs68 cells. Each dataset includes a screenshot of the CBX7 binding profile across the locus (top), with a diagram showing the position of the PCR primer sets relative to the organization of the suspected target gene. The primer sequences are described in Additional file 6: Table S3. The panels show the enrichment observed with the indicated antibody at each primer set as a percentage of input. Grey bars show values for a control IgG antibody. (A) TBX2, (B) TBX4 and (C) RUNX3. gb-2014-15-2-r23-S4.pptx (121K) GUID:?62D22DB0-7CA1-446B-8329-07ABBC36AAE0 Additional file 5: Figure S4 ChIP-seq and RNA-sequencing profiles of the HOX clusters in BF and Hs68 cells. Upper tracks in each physique show the profiles of DNA sequence tag densities following ChIP-seq with antibodies against CBX6, CBX7, CBX8, H3K27me3 and H3K4me3 in the BF and Hs68 strains of HDF as indicated. The lower tracks show duplicate RNA-sequencing data for the corresponding genomic regions in either BF or Hs68 cells. The tag densities were normalized to the same maximum (numbers on left). gb-2014-15-2-r23-S5.pptx (315K) GUID:?A0B0E18B-2F7F-4872-8A26-9C40AB2AF971 Additional file 6: Table S2 List of antibodies used for ChIP. Table S3. List of oligonucleotide primers for PCR analysis of immunoprecipitated chromatin. Table S4. List of oligonucleotide primers used to assess RNA levels by reverse transcription and quantitative PCR. gb-2014-15-2-r23-S6.docx (25K) GUID:?21B8A916-832E-49B9-ABE9-4D9C21933A91 Abstract Background Polycomb group proteins form multicomponent complexes that are important for establishing lineage-specific patterns of gene expression. Mammalian cells encode multiple permutations of the prototypic Polycomb repressive complex 1 Mycophenolic acid (PRC1) with little evidence for functional specialization. An aim of this study is usually to determine whether the multiple orthologs that are co-expressed in human fibroblasts act on different target genes and whether their genomic location changes during cellular senescence. Results Deep sequencing of chromatin immunoprecipitated with antibodies against CBX6, CBX7, Mycophenolic acid CBX8, RING1 and RING2 reveals that this orthologs co-localize at multiple sites. PCR-based validation at representative loci suggests that a further six PRC1 proteins have comparable binding patterns. Importantly, sequential chromatin immunoprecipitation with antibodies against different orthologs implies that multiple variants of PRC1 associate with the same DNA. At many loci, the binding profiles have a distinctive architecture that is preserved in two different types of fibroblast. Conversely, there are several hundred loci at which PRC1 binding is usually cell type-specific and, contrary to expectations, the presence of PRC1 does not necessarily equate with transcriptional silencing. Interestingly, the PRC1 binding profiles are preserved in senescent cells despite changes in gene expression. Conclusions The multiple permutations of PRC1 in human fibroblasts congregate at Mycophenolic acid common rather than specific sites in the genome and with overlapping but unique binding profiles in different fibroblasts. The Mycophenolic acid data imply that the effects of PRC1 complexes on gene expression are more subtle than simply repressing the loci at which they bind. Background Polycomb-group (PcG).