The ZNF217 oncogene is a candidate organizer of repressive histone modifiers

Abstract

The zinc finger protein 217 (ZNF217) is an important oncogene based on the high frequency of amplification and overexpression in many cancer types, but its molecular mode of gene regulation is poorly understood. We purified a complex of nuclear ZNF217-binding proteins by affinity chromatography and identified its components by mass spectrometry as Jarid1b/Plu-1, G9a, LSD1, CoREST and CtBP1. Individual binding of these with ZNF217 was confirmed by co-immunoprecipiation (IP). Known activities of these proteins suggested a role of the ZNF217 complex in histone modification. Using in vitro assays the following activities were demonstrated: Histone H3 lysine 4 trimethyl (H3K4me3) demethylase activity, which co-fractionated with Jarid1b/Plu-1 in anion-exchange chromatography; H3K9 methylation, the known principal activity of G9a; and H3K27 methylation. The latter suggested EZH2 as another ZNF217 binding candidate, which could be confirmed by co-IP. Taken together, these findings suggest that ZNF217 assembles a distinct set of histone modifying proteins at target DNA sites that act synergistically in transcriptional repression.

Figure 1

Nuclear ZNF217 binding proteins identified by affinity chromatography. (A) Fidelity of ZNF217 antisera. Western blotting and immunoprecipitation illustrates the specificity of the anti-ZNF217 antibody used in this study, which stains ZNF217 as a single band. Using [³⁵S]-labeled cellular extract fractions pulse-labeled with [³⁵S]-methionine/cysteine was used to detect ZNF217 by immunopreciptation in nuclear and cytosolic fractions. (B) Affinity chromatographic purification of ZNF217 binding proteins was performed capturing endogenous ZNF217 from nuclear extract with anti-ZNF217 antibodies (bound to the column) under non-denaturing conditions. Shown is an SDS gel that was silver-stained. Individual protein bands were extracted, subjected to tryptic digestion, and analyzed with MALDI-TOF mass spectrometry. The molecular weight of all proteins identified by this method was consistent with their known sizes. (C) RNA interference of ZNF217 by two specific siRNAs against ZNF217 in MCF7 cells was used to determine the presence of ZNF217 following transfections of siRNAs. (D) Immunocytochemical staining of MCF7 cells using ZNF217 antisera followed by secondary labeling with anti rabbit IgG conjugated with Texas Red (Thermo Scientific/Pierce). Fixed cells were counter-stained with DAPI and DAPI labeling was used to merge contrasting images to confirm co-localization.

Figure 2

Co-immunoprecipitation confirming the interaction of ZNF217 with the proteins identified in the affinity purified complex. (A) Anti-ZNF217 antibody mediated immunoprecipitation of Jarid1b/Plu-1 and CtBP1, which were detected by western blotting. Anti-CoREST antibody mediated immunoprecipitation of the same proteins as well as of ZNF217. Anti-Suv39H1 western blotting served as a negative control. (B) Anti-ZNF217 antibody-mediated immunoprecipitation of Flag-tagged G9a and LSD1. (C) Northern hybridization analysis of ZNF217 and BRCA1 following RNA interference of ZNF217 and REST with siRNAs in MCF7 cells. (D) Chromatin immunopreciptation (ChIP) of ZNF217 and Jarid1b/Plu-1 within the human BRCA1 locus following RNA interference of ZNF217 with siRNAs.

Figure 3

H3K4me3 demethylase activity of the ZNF217 complex correlating with Jarid1b/Plu-1. The affinity-purified ZNF217 protein complex was fractionated on a MonoQ column. Individual fractions were tested for demethylase activity by incubation with H3K4me3 substrate. Western blotting with an H3K4 trimethyl-specific antibody demonstrates loss of methylation, i.e., presence of demethylase activity in fractions 6–10. This correlates with the presence of Jarid1/Plu-1 indicated in the corresponding silver stained SDS gel.

Figure 4

H3K9 and H3K27 methylase activity of the ZNF217 complex. (A) Histone H3 tails (1–57) were incubated with S-adenosyl-L- [methyl-¹⁴C]-methionine and aliquots of the affinity purified ZNF217 complex. Autoradiography demonstrated no modification of the GST tag by [¹⁴C] (negative control) and marked incorporation of [¹⁴C] into the H3 (1–57), i.e., into the native H3K9K27 tail sequence (positive control). Mutation to H3K27R or H3K9R decreased [¹⁴C] incorporation while double mutation H3K27R/H3K9R completely abrogated methylation with [¹⁴C]. (B) Anti-ZNF217 antibody mediated immunoprecipitation of EZH2, which was detected by western blot. EZH2 can account for H3K27 methylation, an activity that was not explained by any of the ZNF217 binding partners identified in Figure 1.