Unformatted text preview: ters over the IgG control in A2058 cells, and this was confirmed using a different anti‐PAX3 antibody (Invitrogen), albeit with less efficiency (Fig. 4.2A). After validating the PAX3 ChIP assay on A2058 cells, we assessed binding of our selected genes (Table S1) in HEM1455 and A2058 cells. Enrichment in PAX3‐IP relative to control IgG‐IP was observed for most of the genomic sites analysed (Fig. 4.2B), which is consistent with these genes being direct downstream targets of PAX3 in these cell types. However, there was no enrichment in NFKβ2 genomic site in either HEM1455 or A2058, indicating it is not a target of PAX3 in either cell. 84 Chapter IV – Medic, Rizos and Ziman, 2011 Figure 4.2. PAX3 binding to target genes in melanocytes and melanoma cells. (A) PAX3 binding to MITF and DCT promoters in A2058 cells was assessed by end‐point PCR. ChIP assay was performed with two different PAX3 antibodies (from DSHB, lane 3; or Invitrogen, lane 5) and matching control IgG antibodies (lanes 4 and 7). Non‐specific GAPDH promoter was included as a negative control for PAX3‐IP; and IP with AcH3 (acetylated Histone H3, lane 6) serves as positive IP control. Blank (lane 1) is no‐template control for PCR reaction. (B) Graph shows PAX3 binding to potential target genes in HEM1455 and A2058 cells, quantified by qPCR. For each target site, enrichment in PAX3‐
IP was normalised to the input DNA and calculated as a fold increase over normalised IgG‐
IP. Only enrichment above the enrichment value for the non‐specific site (GAPDH) was considered true enrichment. Therefore, for each IP, specific promoter enrichment was subtracted from non‐specific promoter enrichment (which was 5.58‐fold for both HEM1455 IP1 and 2 and 2.6 and 5.135‐fold for A2058 IP1 and 2, respectively). Asterisk (*) indicates statistically significant fold difference, p<0.05. Interestingly the overall fold enrichment at all sites is higher in A2058 compared to HEM1455, indicating a stronger binding of PAX3 to its targets in melanoma cells. In melanocytes four genomic sites, NES, TPD52, BCL2L1 and PTEN, do not show enrichment in PAX3‐IP, suggesting that PAX3 regulates different pathways in melanocytes and melanoma cells. The fact that these genes are involved in differentiation (NES), proliferation (TPD52), and cell survival (BCL2L1 and PTEN) signifies PAX3 involv...
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This document was uploaded on 03/06/2014 for the course CHEMISTRY 12 at National University of Singapore.
- Spring '14