Figure 6.

Identification of additional direct Suz12 target genes. (A) Scatter plot showing the intensities of the spots on the CpG-island microarrays. Two independent experiments were performed to identify Suz12-bound loci in SW480 cells. In red are the spot intensities from a representative array that was hybridized with Suz12 and input amplicons. Shown in green are the spot intensities of a representative array that was hybridized with IgG and input amplicons. Red spots that enriched at least threefold were selected for further analysis. The spots shown in blue were enriched at least threefold in both the Suz12 and IgG control immunoprecipitations and represent DNA fragments that precipitate nonspecifically in the ChIP procedure. (B) Confirmation and mapping of Suz12 binding to a 5-kb region of each promoter identified in A was performed using custom oligonucleotide promoter arrays. The arrays were prepared and hybridized as in Figure 5. Shown are the fold enrichments from the Suz12 and input comparisons of six promoters: four true positive targets and two (TLK2 and PVALB) false positives. (C) Confirmation of the results shown in B using PCR analysis. Independent ChIP experiments were performed in SW480 cells using antibodies to RNA Polymerase II (lane 1), Suz12 (lane 2), and a control IgG (lane 3). Three dilutions of the input chromatin are also shown (lanes 4–6). The precipitated chromatin was analyzed with PCR using primers specific to the promoters of the indicated genes.