Using the global and unbiased approach of ChIP-chip assays that interrogate ~24,000 human promoters, we have gained new insight into the function of the oncogene ZNF217. We began by determining that ~half of the in vivo binding sites of ZNF217 are located within proximal promoter regions, and then identified thousands of promoters bound by ZNF217 in three different cancer cell lines. We have established that ZNF217 can function as a transcriptional repressor by demonstrating a) that the majority of ZNF217 target genes are bound by CtBP1/2, b) that most genes bound by ZNF217 show very low expression levels, and c) that reduction in the amount of ZNF217 bound to a promoter can, in some cases, result in increased gene expression. Interestingly, we find that many of the genes bound by ZNF217 in Ntera2 cells function in the neural cell lineage, suggesting that one role of ZNF217 may be to repress specific differentiation pathways.
Although inappropriate expression of ZNF217 has been linked to tumorigenesis (due to amplification at the 20q13 locus in multiple tumor types), the exact mechanisms by which ZNF217 might promote or enhance neoplastic transformation have not been elucidated. It has been proposed that ZNF217 functions as a transcriptional repressor due to its purification in complexes that contain co-repressors such as CtBP2 and co-REST (10
) and histone modifying enzymes such as G9a and LSD1 (12
). However, an understanding of how ZNF217-mediated repression might influence tumorigenesis has not been developed, in large part due to a lack of known ZNF217 target genes. The thousands of ZNF217 target genes that we have now identified provide an excellent data set for testing models of ZNF217-mediated gene regulation. For example, our preliminary analyses of histone modifications of ZNF217 target genes indicates that very low levels of H3me3K9 are found on the target promoters but that about half of the promoters repressed by ZNF217 have high levels of H3me3K27 (see Table S6
). These results suggest that, in some cases, ZNF217 may recruit the PRC2 complex (40
). Further studies examining other histone modifications are in progress.
Our studies support the previous biochemical purification experiments in that we demonstrate that there is a very large overlap between the promoters bound by ZNF217 and the promoters bound by CtBP1/2 in both Ntera2 and MCF7 cancer cells. It is possible that ZNF217 binds to promoters via some or all of its zinc fingers and recruits CtBP1/2 to the promoter regions through the direct protein-protein interactions that have been previously characterized (15
). Alternatively, other DNA binding factors may be the primary contact point between the promoters and the ZNF217/CtBP repressor complex (16
). Our data showing very similar binding patterns of ZNF217 and CtBP2 and our finding that, in general, binding of CtBP2 is not greatly reduced by removal of ZNF217 from a promoter, suggest that ZNF217 and CtBP2 might both be recruited to the chromatin via a different DNA binding protein. This hypothesis is supported by a motif present in many of the ZNF217 binding sites that is distinct from the motif recently identified using in vitro
casting experiments and zinc fingers 6 and 7 of ZNF217 (31
). Although the 8-base motif that we identified does not match any known binding motifs in the TRANSFAC or JASPAR databases, 3 positions of the motif (TCC
or reverse-complement GGA
) do match the conserved GGA that constitutes the core of the DNA binding motif of all ETS family members (41
). Thus, it is possible that an ETS family member may help to recruit the ZNF217/CtBP complex to the DNA. Interestingly, previous studies have shown that CtBP can interact with ELK-3 (also called Net, Sap-2, and Erp), an Ets family member (35
Our finding that thousands of promoters are bound by ZNF217 is consistent with recent ChIP-chip studies of other human transcription factors, such as Men1 (42
), Myc (43
), E2F1 (22
), NFkB (44
), and the estrogen receptor (45
). Further characterization of these target promoters have revealed that changes in binding of the factor does not necessarily lead to changes in gene expression (42
). For example, ~2000 promoters are bound by the transcriptional activator Men1, but only ~5% of these genes showed a change in expression in mice that were nullizygous for the Men1 gene (42
). Similarly, we found that ~5% of the genes identified as ZNF217 targets using ChIP-chip assays showed a change in expression when ZNF217 levels were reduced by siRNA treatment of Ntera2 cells. It is becoming increasingly clear that promoters are regulated by many factors and that loss of a single factor is usually not sufficient to alter the regulation of a promoter. Once the landscape of transcriptional regulation in the human genome has been mapped in more detail, sets of promoters that are commonly regulated by two or more different complexes may be identified. At that point, it may be possible to alter the expression of a greater percentage of genes by removing multiple complexes from the promoter regions.
A major goal of our studies of ZNF217 is to obtain insight into how inappropriate expression of this factor in human cancers can contribute to neoplasia. Interestingly, many of the genes that are bound by ZNF217 encode proteins that are critical in mediating differentiation. We also have shown that ZNF217 levels are down-regulated upon forced differentiation of Ntera2 cells, allowing for the expression of genes involved in differentiation and organogenesis. Thus, we propose that inappropriate expression of ZNF217 may lead to a down-regulation of genes that confer a differentiated phenotype, causing a de-differentiation of the cells and driving them towards a more proliferative and pluripotent phenotype. Future studies will be focused on comparing differentiation potential of cells expressing different levels of ZNF217.