Characterization of the Contradictory Chromatin Signatures at the 3′ Exons of Zinc Finger Genes (original) (raw)
Figure 2
The 3′ ends of ZNF genes do not display promoter activity.
A) ChIP-seq signal tracks from Ntera2 cells are shown for the ZNF440 gene. H3K4me3 binding is observed at the promoter (top panel), while H3K9me3 localizes to the 3′ end of the ZNF440 gene (bottom panel). The number of sequenced tags is plotted on the y-axis. Shown below the ZNF440 gene schematic are representative constructs for the experiments shown in panels B–D. Promoter regions (from ∼500 bp upstream to ∼100 bp downstream of TSS) and 3′ regions bound by H3K9me3 in vivo were cloned in front of the luciferase cDNA; the 3′ regions were cloned in either the sense (s) or antisense (as) direction (see Table S2 for coordinates of the genomic fragments used for promoter analyses). B–D) Luciferase assays were performed to test for promoter activity at 3′ ends of ZNF genes. The DHFR, ZNF440 and ZNF554 promoters were used as positive controls and an empty vector (EV) was used as a background control. Promoter activity was tested in Ntera2 and DAOY cells B), in U2OS cells (C) and in HEK293 cells (D). In addition, the U2OS and HEK23 cells were also stably transfected with a KAP1 shRNA construct (indicated as KAP1 KD). Fold luciferase was determined based on the empty vector control and is plotted on the y-axis.