Figure_9.tif (1.19 MB)

Recruitment of Pax2 to the cwp1-3 and myb2 promoters.

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posted on 15.02.2012, 00:55 by Shen-Fung Chuang, Li-Hsin Su, Chao-Cheng Cho, Yu-Jiao Pan, Chin-Hung Sun

(A) Microarray analysis. Microarray data were obtained from the 5′▵5N-Pac and pPPax1 (or pPPax2) cell lines during vegetative growth. Fold changes are shown as the ratio of transcript levels in the pPPax1 (or pPPax2) cell line relative to the 5′▵5N-Pac cell line. Results are expressed as the means ± standard error of at least three separate experiments. (B) Pax2 and Pax1 overexpression generated similar gene expression patterns. The Venn diagrams illustrate the overlap of altered gene expression between the Pax2 and Pax1 overexpressing cells. Thirty eight and 185 genes were up-regulated (i.e. increased levels of gene expression relative to the control) in the Pax1 and Pax2 overexpressing cells, respectively. Among them, nineteen genes overlap. Fifty four and 172 genes were down-regulated in the Pax1 and Pax2 overexpressing cells, respectively. Among them, thirty genes overlap. (C) ChIP assays. The non-transfected WB cells were cultured in growth medium for 24 h and then subjected to ChIP assays. Anti-Pax2 antibody was used to assess binding of Pax2 to endogenous gene promoters. Preimmune serum was used as a negative control. Immunoprecipitated chromatin was analyzed by PCR using primers that amplify the 5′-flanking region of specific genes. At least three independent experiments were performed. Representative results are shown. Immunoprecipitated products of Pax2 yielded more PCR products of pax2, cwp1, cwp2, cwp3, myb2, and ran promoters, indicating that Pax2 was bound to these promoters. The 18S ribosomal RNA gene promoter was used as a negative control for our ChIP analysis.