To our knowledge, this is the first demonstration that the chromatin immunoprecipitation assay can be used to clone promoters which are direct in vivo targets of a mammalian site-specific DNA binding protein. This provides a powerful new approach to examine direct transcription factor targets in an unbiased manner which does not rely on previous characterization of a consensus sequence or a prior knowledge of gene expression patterns. Although others have used similar approaches to isolate genomic fragments (6
), those studies did not use subsequent experiments to confirm in vivo binding of the factor of interest to the isolated DNA. Due to this lack of in vivo confirmation, it is difficult to assess the validity of the previous protocols. Also, sequence analysis of the clones isolated in the previous studies indicated that the cloned fragments corresponded to nonpromoter regions, such as introns (5
). One study did find that 3 out of 43 clones isolated after in vitro incubation of genomic DNA with purified Ets-1 protein were promoters; however, the authors did not confirm in vivo binding of ETS1 to these 3 clones or to the other 40 isolated clones. Therefore, it is difficult to be sure if any of the clones in that study were bona fide in vivo targets of ETS1.
Utilizing the chromatin immunoprecipitation assay to clone fragments bound by E2F family members, we found that 64% (9 of 14) of the clones characterized were bona fide in vivo E2F binding sites. Characterization of the three highest-affinity clones revealed that they correspond to promoter regions (Fig. ), providing validation that novel E2F-regulated promoters can be isolated by using this protocol. Future studies will be performed to determine whether any of the remaining clones are promoters. A recent study using high-density microarray analysis (31
) found that about 7% of the mRNAs represented on the microarray were responsive to overexpression of E2Fs. These results suggest that a high percentage of mammalian genes might be regulated by direct binding of E2F to the promoter region. If this estimate of the number of E2F target genes is correct, then it is not surprising that we did not isolate one of the several dozen well-characterized E2F target promoters in the set of nine positive clones that we analyzed. However, one of our ChET clones (ChET 9, which corresponds to the promoter region of the KIAA0160 gene) was shown to be upregulated by E2F overexpression in the microarray analysis (31
). The fact that this gene was isolated by two independent screening methods for E2F target genes provides strong evidence that this promoter is indeed a direct target of E2F family members and that the chromatin immunoprecipitation cloning technique can identify E2F-regulated promoters.
Summary of information obtained relating to the ChET promoter clones.
One of the E2F4 clones, ChET 4, displayed high-affinity E2F binding in vivo and corresponded to the promoter region for the beclin 1
gene. Beclin 1 was isolated through its ability to interact with bcl-2 and has been postulated to possess tumor suppressor activity in breast cancer (1
). It is interesting that the gene for a potential tumor suppressor protein was isolated as an E2F target gene because E2F regulation is thought to play a significant role in tumorigenisis. Further experiments examining the nature of the role E2F plays in Beclin 1 regulation may provide further insight into the role of E2F in tumor development.
We found that two of the high-affinity E2F binding clones did not contain E2F sites which closely matched the consensus sequence. It is important to note that others have previously shown that site-specific DNA binding proteins can regulate transcription through sequence elements that diverge from the consensus. For example, CREB, Ets-1, and AML1 can regulate expression of the human T-cell receptor beta chain promoter through nonconsensus binding sites (12
) and a nonconsensus site mediates regulation of the atrial natriuretic factor by serum response factor (13
). Computer inspection suggests that the ChET clones may contain multiple low-affinity E2F sites, each of which diverges from the known consensus. Perhaps a combination of weak binding sites allows for cooperative recruitment of the E2F complex in vivo. It is also possible that the promoter context may greatly influence E2F binding efficiency within the cellular environment. We have previously shown that some (e.g., CCAAT and YY1) but not all (e.g., Oct1, Ap2, and NF1) transcription factor sites can synergize with E2F sites to activate transcription (47
). It is possible that this synergy was mediated by cooperative DNA binding. Also, others have shown that Sp1 can physically interact with E2F family members and that binding of Sp1 can influence the occupancy of a nearby E2F site (20
). Each of the three characterized ChET clones contains at least one consensus Sp1 binding site (Fig. ). Finally, others have shown that E2Fs can interact with other sequence-specific DNA binding proteins, such as C/EBPα (17
). Interestingly, we have recently shown that E2F1 can be recruited to promoters which contain C/EBPα binding sites but lack E2F consensus sites (Graveel and Farnham, unpublished). It remains to be determined if C/EBPα and/or other protein-protein interactions are mediating the recruitment of E2F to the promoters we have cloned. However, recruitment of E2F through the recognition sequence of another DNA binding protein could explain why some of the cloned fragments failed to show robust competition of a consensus E2F site in vitro.
To date, the majority of well-characterized E2F target promoters have been shown to be cell cycle regulated and activated by E2F overexpression. In contrast, our three novel E2F target promoters are constitutively expressed in growing versus differentiated U937 cells. It is perhaps not surprising that E2F target promoters isolated using an unbiased approach show expression profiles different from those of the well-characterized E2F target promoters. According to microarray analyses, hundreds of genes are regulated by E2F family members (16
). It is highly unlikely that this large number of mRNAs, which encode proteins having highly diverse biological functions, will all show exactly the same expression pattern in all cell types.
It is interesting that the mRNA produced by each of the three novel promoters displayed unique expression profiles when normal versus tumor human primary samples were examined; one mRNA was constitutively expressed, one mRNA was downregulated in the tumor sample, and one mRNA was highly upregulated in tumor RNA. Interestingly, one of the promoters that we cloned which displayed high-affinity binding in vivo was shown to be repressed, not activated, by E2F1. Although most E2F target genes studied to date are activated in response to overexpression of E2F1, it has been shown that the cyclin D1 promoter is also repressed by E2F1 (48
). In addition, the recent microarray analysis by Muller et al. provided evidence that E2Fs can both activate and repress cellular genes, although their data did suggest that most E2F-mediated repression was indirect (31
). Additional evidence supporting E2F-mediated repression of the ChET 8 promoter can be extrapolated from a recent study examining the cell cycle fluctuations of thousands of human mRNAs (4
). We have extracted the expression profiles of E2F1 and KIAA0254, the mRNA driven by the ChET 8 promoter, from the published microarray data. Interestingly, ChET 8 mRNA levels are inversely related to E2F1 mRNA levels (data not shown). Collectively, these findings support a role for E2F1 in repression of the ChET 8 promoter. Further experiments need to be performed to characterize similarities and differences between the promoters which are directly activated and those which are directly repressed upon overexpression of E2F1. However, these observations suggest that the nature and context of the E2F binding site may influence the role that E2F plays in regulation of a promoter.
In summary, the data presented in this paper establish the basis for cloning novel promoters regulated by specific transcription factors through chromatin immunoprecipitation techniques. Our initial data suggest that the E2F consensus binding sequence may not account for all potential in vivo E2F targets, possibly due to the roles of interacting proteins within the cellular environment. Importantly, the possibility that E2F family members can regulate promoters that lack consensus binding sites may aid in the understanding of microarray studies which show that hundreds of mRNAs can respond to overexpression of E2Fs (16
). Also, we find it most interesting that the expression profiles of the genes identified by using this unbiased approach are quite different from the expression profiles of the previously characterized E2F target genes. Finally, of particular interest are ChET 9 and ChET 4. ChET 9 contains a consensus E2F binding site and shows high-affinity binding in vivo and in vitro. Interestingly, the KIAA0160 mRNA which is transcribed by ChET 9 is upregulated in two different tumor types. The protein encoded by the KIAA0160 mRNA has high homology to a Drosophila
protein called Su(z)12. This protein was isolated as a suppressor of a mutation of the gene for zeste, a site-specific DNA binding transcription factor. Although no characterizations of Su(z)12 have been performed; another suppressor of zeste, Su(z)2, is known to be a locus-specific chromosome binding protein. Therefore, it is possible that KIAA0160 will be involved in transcriptional regulation. ChET 4, which shows high-affinity E2F in vivo binding but does not contain a consensus E2F site, is the promoter region for the beclin 1
gene, a putative tumor suppressor gene. The Beclin 1 protein is thought to effect the degradation of cellular proteins and has been shown to be significantly downregulated in human breast carcinomas (26
). Our future studies will be focused on understanding the role of Beclin 1 and KIAA1060 in neoplastic transformation.