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Originally published In Press as doi:10.1074/jbc.M307733200 on August 8, 2003

J. Biol. Chem., Vol. 278, Issue 43, 42466-42476, October 24, 2003
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E2F6 Negatively Regulates BRCA1 in Human Cancer Cells without Methylation of Histone H3 on Lysine 9*

Matthew J. Oberley, David R. Inman, and Peggy J. Farnham{dagger}

From the McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, Madison, Wisconsin 53706

Received for publication, July 17, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
E2F6 contains a DNA binding domain that is very similar to that of the other members of the E2F family of transcriptional regulators. However, E2F6 cannot bind to all promoters that contain consensus E2F-binding sites. Therefore, we used a combination of chromatin immunoprecipitation and genomic microarrays to identify promoters bound by E2F6 in human cells. Although most of the identified promoters were bound by multiple E2F family members, one promoter was bound only by E2F6. To determine which of the newly identified promoters were regulated by E2F6, we reduced the level of E2F6 by using RNA interference technology. We found that mRNA transcribed from promoters bound by E2F6 was increased after reduction of the amount of E2F6 protein in the cell. Interestingly, many of the E2F6-regulated genes encoded functions involved in tumor suppression and the maintenance of chromatin structure. Specifically, our results suggest that E2F6 represses transcription of the brca1, ctip, art27, hp1{alpha}, and the rbap48 genes. E2F6 has been postulated to mediate transcriptional repression by recruiting a histone H3 methyltransferase to the DNA. However, we found that the E2F6-regulated promoters did not contain histone H3 methylated at lysine 9. To determine the mechanism by which E2F6 regulates transcription, we performed chromatin immunoprecipitation before and after the introduction of small inhibitory ribonucleic acids specific to E2F6. We found that depletion of E2F6 resulted in the recruitment of E2F1 to the target promoters. In summary, we have identified 48 endogenous target genes of E2F6 and have shown that E2F6 can repress target promoters in a manner that does not require histone H3 methylation at lysine 9.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The E2F family consists of six members, E2Fs 1-6, and two obligate heterodimeric partners, DP1 and DP2, which are required for binding to DNA (1). All of the E2Fs contain a conserved DNA binding and dimerization domain, and the different E2F-DP heterodimers can bind to the same consensus sequence. E2Fs 1-5 each contain a C-terminal transactivation domain that can interact with a variety of transcriptional coactivators such as CREB1-binding protein and TFIIH (2, 3). The C-terminal domain also contains sequences required for binding to the pocket protein family of transcriptional repressors (retinoblastoma, p107, and p130). E2Fs 1-3 interact preferentially with retinoblastoma, whereas E2F4 and -5 mainly associate with p107 or p130 (4). Depending upon exactly which proteins associate with the C-terminal domain, E2Fs1-5 can therefore mediate either activation or repression.

E2F6, the most recently identified E2F family member, lacks the C-terminal transactivation domain found in the other E2Fs. Therefore, E2F6 likely cannot serve as a transcriptional activator or bind to the pocket protein family. However, E2F6 has been shown to be a potent transcriptional repressor (5-8). Because E2F6 lacks the pocket protein interaction domain, transcriptional repression may be mediated via interaction with other proteins. Yeast two-hybrid studies have shown that E2F6 can be found in a multiprotein complex with members of the Bmi1-containing Polycomb group complex of transcriptional repressors (9). Other studies have shown that E2F6 is also a component of a complex that contains Polycomb group proteins such as RING1, RING2, MBLR, h-l(3)mbt-like protein, and YAF2 (10). A general function of Polycomb group complexes is to specify developmental patterning, which is accomplished, in part, by the repression of homeobox gene expression (11). E2F6-null mice display homeotic transformations of the axial skeleton (12) and resemble animals lacking the Bmi1 polycomb group protein (13). These studies are consistent with the hypothesis that an E2F6-containing Polycomb group complex is required to silence certain homeotic genes during development. Complexes containing Bmi1 are implicated in the development of cancer through the repression of the p16ink4a and p19arf genes (14), suggesting a specific link between E2F6 and the development of cancer. Also, support of a link between E2F6 and carcinogenesis comes from studies demonstrating that overexpression of E2F6 can alter cell growth parameters. For example, overexpressed E2F6 can inhibit entry into S phase of cells stimulated to exit G0 phase (7) and can also delay the exit from S phase (8).

Although the possible links between E2F6 and cell growth control are intriguing, little is known about the exact genes regulated by E2F6, and it is not yet clear exactly how E2F6 mediates transcriptional regulation. Recently, Ogawa et al. (10) identified a novel euchromatic-specific histone methyltransferase (Eu-HMTase) as part of an E2F6-containing complex from HeLa cells, and this Eu-HMTase was shown to specifically methylate histone H3 at lysine 9 on free histones and mononucleosomes in vitro. This specific histone H3 modification (H3 Me-K9) produced by eukaryotic histone methyltransferases (HMTase), such as SUV39H1, has been shown to recruit HP1 and to create a locally repressive heterochromatic structure (15). The identification of the E2F6-HMTase complex has led to an hypothesis regarding the mechanism of E2F6-mediated transcriptional repression. It has been postulated that the transcriptional repressive activity of E2F6 begins with the recruitment of the Eu-HMTase, which is followed by the methylation of histone H3 at Lys-9, and the subsequent establishment of heterochromatin via recruitment of HP1. However, it has not been rigorously tested whether E2F6 does indeed convert euchromatin into heterochromatin in vivo.

As noted above, all of the E2F family members, including E2F6, can bind in vitro to a consensus element (TTTSSCGC). However, it is not yet clear if the same set of target genes will be regulated by all the E2Fs in vivo. Our group has recently utilized chromatin immunoprecipitation coupled with CpG island microarrays (ChIP-chip) to identify novel in vivo targets of E2F1, E2F4, pRB, and MYC (16-18). CpG islands are tracts of at least 200 bp C + G-rich sequences that are found in the promoters and first exons of about 70% of human genes (19, 20). Because E2F-binding sites are most commonly found within proximal promoter regions and because the consensus binding sequence is GC-rich, the CpG arrays have proved to be very useful for identifying E2F target genes. This present study utilizes an improved version of our previous ChIP-chip protocol to identify genomic targets of E2F6 in human cancer cells. We find that E2F6 regulates genes that are involved in the pathogenesis of neoplasia and in regulating chromatin structure and heredity. We also provide in vivo evidence that E2F6 can repress targets via a mechanism distinct from methylation of histone H3 at lysine 9.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Chromatin Immunoprecipitations (ChIP)—The ChIP procedure was performed as described previously (21) with HeLa and 293 cells, with the following exceptions (see also mcardle.oncology.wisc.edu/farnham). The chromatin was sheared to an average size of 500-2000 bp. After cross-linking reversal and proteinase K digestion, each individual IP was purified with the use of a QIAquick PCR purification kit (Qiagen, Valencia, CA), and samples were eluted with 30 µl of elution buffer. After elution the IPs were examined by gene-specific PCR on the art27 promoter to ensure that the IP successfully enriched the promoter significantly more than did an IgG control. Antibodies used in the ChIP assays include E2F1 (KH20KH95, Upstate Biotechnology, Inc., Lake Placid, NY), E2F4 (sc866 X, Santa Cruz Biotechnology, Santa Cruz, CA), E2F6 (sc8366X, Santa Cruz Biotechnology), Max (sc197, Santa Cruz Biotechnology), RNA polymerase II (sc-899, Santa Cruz Biotechnology), and histone H3 tri-methylated at K9 (Ab8898, Abcam, Cambridge, UK). E2F6 (sc8366P) and TCF4 (sc8631P) peptides were purchased from Santa Cruz Biotechnology.

Amplicon Generation and Labeling—For detailed protocols on these steps see Oberley et al. (21) or mcardle.oncology.wisc.edu/farnham. The generation of amplicons from the individual ChIPs was adapted from Ren et al. (20). Briefly, two unidirectional linkers oligoJW102 (5' gcg gtg acc cgg gag atc tga att c 3') and oligoJW103 (5' gaa ttc aga tc 3') were annealed and blunt-end ligated to the chromatin IPs. Amplicons were created by PCR; each sample consisted of 5 µl of 10x Taq polymerase buffer, 7 µl of 2 mM dNTPs, 3 µl of MgCl2, 6.5 µl of betaine, 2.5 µl of oligoJW102 (20uM), 1 µl of Taq (Promega, M1861), and 25 µl of the blunted and ligated chromatin. PCR was run with one cycle at 55 °C for 2 min, 72 °C for 5 min, and 95 °C for 2 min. Twenty cycles were then run at 95 °C for 0.5 min, 55 °C for 0.5 min, and 72 °C for 1 min. Finally the products were extended at 72 °C for 4 min and then held at 4 °C until purified using the QIAquick PCR purification kit according to the manufacturer's instructions. DNA was quantitated and stored -20 °C until labeling. The amplicons were labeled with amino-allyl dUTP using 3 x 2 µg of the E2F6 IP amplicon (or 3 x 2 µg of the IgG IP amplicon) and 3 x 2 µg of the total input chromatin amplicon, as described previously (22). We coupled the Cy5 dye to the E2F6 IP (or the IgG) amplicon and Cy3 dye to the total reference amplicon, using standard methods (22). Unincorporated dye was then removed with a QIAquick PCR kit, eluting with H2O. The labeled chromatin was then dried with heat in a speed-vac and stored dry at -20 °C until ready for hybridization.

CpG Island Hybridization—The generation and hybridization of the CpG island microarrays have been described elsewhere (17, 22). The hybridized microarrays were analyzed using the Genepix Pro 4.1 (Axon Instruments) software package. This provided a set of raw values for each feature on each array. Features of poor intensity (<500) and those that had obvious blemishes were manually flagged and removed from the putative positive list. To identify clones that are selectively enriched during IP relative to the starting population, the Cy5 and Cy3 channels were normalized across the entire array, by taking the ratio of the medians for all quality features and normalizing them to unity. After normalization, a ratio was generated that was the intensity in the Cy5 channel minus background divided by the intensity in the Cy3 channel minus background. The ratio was generated for all features that met these criteria, and features with ratios above 2 were selected for further analysis. The identified CpG island clones were then sequenced by standard methods using primers corresponding to vector sequences.

PCR Assays—To analyze the identified CpG islands (and other characterized promoter regions), PCRs were performed. Primer sequences for all the ChIP confirmations are available at our website: mcardle.oncology.wisc.edu/farnham. Each PCR mixture contained 2 µl of immunoprecipitated DNA (or 10 ng of each amplicon) and was performed as described previously (16). PCR products were separated by electrophoresis through 1.5% agarose gels and visualized by ethidium bromide intercalation.

RNAi Transfections, Westerns, and RT-PCR Assays—293 cells were plated at a density of 6 x 105 on 60-mm plates 24 h before transfection with the siRNAs. siRNAs were purchased from Dharmacon (Louisville, CO) and included siE2F6-01 (AAGGAUUGUGCUCAGCAGCUG-custom order), siE2F6 smart pool (M-003264-00-05), or siGFP (D-001300-01-05). Transfections were performed with OligofectAMINE (Invitrogen) or TransIT-TKO (Mirus, Madison, WI) according to manufacturers' instructions. Western blots were performed as described previously (16). The same antibodies were used for Westerns as described above for the ChIP assays. RT-PCR analysis was performed as described previously (23). Primer sequences for all the RT-PCR assays are available at our website: mcardle.oncology.wisc.edu/farnham.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
The Identification of E2F6 Genomic Binding Sites Using CpG Microarrays—As noted above, very few bona fide target genes for E2F6 have been identified. In fact, we have found that many well characterized E2F target genes are not bound by E2F6. As an example, we have characterized the binding pattern of the E2Fs on the myc promoter, an established E2F target gene (24, 25). By using the ChIP assay, we examined binding of E2Fs 1-4 and 6 in human 293 cells. Primers were used that spanned the consensus E2F-binding site, which is located just upstream of the transcriptional start in the myc promoter. The antibodies to E2F1-4 enriched the myc promoter sequences, indicating that the myc promoter is bound by these proteins in asynchronously growing 293 cells (Fig. 1A). In contrast the E2F6 antibody did not enrich the myc promoter sequences relative to the IgG control sample. Given that the myc promoter was occupied by the other E2Fs, the chromatin structure must be permissible to E2F family member binding. Thus, the absence of E2F6 binding suggests that E2F6 likely requires determinants for binding other than the presence of a consensus E2F-binding site. This indicates that E2F6 may bind to only a subset of promoters bound by other E2F family members.



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  		FIG. 1.
A, the art27 promoter is bound by E2F6 in 293 cells. ChIP analysis was performed with a panel of E2F antibodies using 1 x 107 293 cells per antibody. One sample was precipitated with IgG to control for nonspecific sequence enrichment. The immunoprecipitates, and a sample representing 0.2% of the input, were analyzed by PCR with primers specific for the myc and art27 promoters and 3'-untranslated region of the dhfr gene. Each image was quantitated with ImageQuant software and plotted as a fraction of the input to allow comparisons between fragments. B, E2F6 binding is cell type-specific. ChIP assays were performed and analyzed as in A. The binding profiles of E2Fs 1 through 6 on the art27 promoter were compared in HeLa versus 293 cells.

 

Our laboratory has recently utilized chromatin immunoprecipitation followed by CpG microarray analysis (ChIP-chip) to identify genomic targets of E2F4 (17) and E2F1 (16). Because of the success of these prior experiments, we reasoned that the ChIP-chip technique would be an excellent method to identify a large set of E2F6 target genes. However, prior to beginning the array experiments, it was essential to have a positive control promoter to ensure specific enrichment of E2F6-bound DNA in the immunoprecipitated chromatin. Therefore, we tested a series of additional E2F target promoters for their ability to bind to E2F6. Examination of well characterized E2F target genes (such as dhfr) revealed that E2F6 did not bind to the promoter regions (data not shown). Because promoters that have consensus E2F-binding sites (such as the myc and dhfr promoters) do not bind E2F6, we next tested promoters that bind E2F family members via non-consensus E2F sites (17). Although the promoter for the art27 (Androgen receptor trapped clone 27) gene, which functions as a coactivator of the androgen receptor (26), does not contain a consensus E2F site (as defined as TTTSSCGC), it does have two near consensus E2F sites located at -280 and -109 relative to the transcriptional start site. We have shown previously that the art27 promoter (which was previously called uxt (27)) is bound by several E2F family members in HeLa cells (17). However, binding of E2F6 was not tested in the previous study. We have now demonstrated in vivo binding of E2Fs 1-4 and 6 to the art27 promoter in 293 cells (Fig. 1A). As a negative control, the 3'-untranslated region of the dhfr gene was analyzed. As expected, this region was not enriched by antibodies to any of the E2Fs. Interestingly, E2F6 bound to the art27 promoter in 293 cells but not in HeLa cells (Fig. 1B), despite the fact that E2F6 protein is expressed at significant levels in HeLa cells and that these cells were used to purify the E2F6-containing complex that included the novel Eu-HMTase (10). These data indicate that E2F6 shows target gene selectivity and cell type specificity.

Because E2F6 has been postulated to be a transcriptional repressor, we compared the activity in 293 cells of art27 promoter constructs containing or lacking the sequences that resemble E2F-binding sites (Fig. 2). We transfected each promoter-luciferase construct into 293 cells, along with a cytomegalovirus-Renilla control for transfection efficiency, harvested the cells after 48 h, and measured the promoter activity. Interestingly, we found that deletion of each E2F-like site resulted in increased activity of the art27 promoter, which suggests that E2F6, perhaps in conjunction with other E2Fs (28), acts to repress the transcriptional activity of the art27 promoter. Based on these studies, we have used 293 cells as an experimental system, and the art27 promoter as our positive control, for the study of E2F6-mediated transcriptional regulation of target genes.



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  		FIG. 2.
The regions containing E2F-like sites in the art27 promoter mediate transcriptional repression. 293 cells were transfected as described previously (25) with three art27 promoter deletion constructs driving firefly luciferase expression, along with a Renilla luciferase vector to control for transfection efficiency. The results are plotted as a ratio of the firefly light units to the Renilla light units and are representative of three independent experiments.

 

Our next step was to perform a high-throughput genomic screen to identify E2F6-binding loci in 293 cells. Our lab has previously screened chromatin immunoprecipitates with CpG island microarrays to identify genomic targets of E2F family members (16, 17). In our previous studies, multiple individual ChIP samples were pooled to obtain enough material to probe a microarray. Here we have adapted several changes to our previously published protocols that have allowed a more rigorous discrimination of robust in vivo targets (Fig. 3A). One critical change was the inclusion of a ligation-mediated PCR step (29) that has allowed a considerable reduction in the quantity of cells required. We performed a ChIP assay on 1 x 107 cells with antibodies specific for E2F6 or an IgG control. After extensive washing, the cross-links were reversed, and the DNA was purified. Next, the E2F6 IP, the IgG IP, and 10 ng of total input DNA were blunt-ended, ligated to a unidirectional linker, and amplified to generate enough DNA to probe the CpG microarrays. Equal amounts of amplicons (6 µg of the E2F6 IP and 6 µg of total input) were labeled with amino-allyl dUTP, and then the E2F6 labeled amplicon was conjugated with Cy5, whereas the reference total input amplicon was conjugated with Cy3. These two labeled pools of DNA were mixed with CoT-1 DNA, which binds the repeat DNA in the samples, and applied to a CpG island microarray under stringent hybridization conditions. The arrays were hybridized overnight at 60 °C, washed, and then scanned on an Axon Instrument 4000B scanner, using GenePix Pro 4.1 software (Axon Instruments) to analyze the array. After manually flagging any obvious blemishes on the resulting image, all features that gave quality signals were included in a data set that was normalized by setting the ratio of all medians to unity.



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  		FIG. 3.
A, experimental design of the ChIP-chip assays. A stepwise description of the ChIP-chip assay is shown; details are provided at mcardle.oncology.wisc.edu/farnham/. B, dot plot of three independent ChIP-chip experiments. The red squares represent a comparison of input amplicons labeled with Cy5 versus Cy3. The yellow squares represent a comparison of the IgG amplicons labeled with Cy5 versus input amplicons labeled with Cy3. The blue squares represent E2F6 amplicons labeled with Cy5 versus input amplicons labeled with Cy3.

 

The results of three array experiments are depicted in Fig. 3B, which is a dot plot on a logarithmic scale where the Cy5 intensity of each feature is plotted on the x axis and the Cy3 intensity of the feature is plotted on the y axis. For the first array hybridization, the total input sample was divided in two; half was labeled with Cy5, and half was labeled with Cy3. These labeled amplicons were then hybridized to an array to give a total versus total comparison (Fig. 3B, red squares). As expected, this plot produced a straight line with a good regression value (R2 = 0.9468) for spotted arrays. Then, as a negative control, an IgG amplicon was labeled with Cy5 and compared with Cy3-labeled total input chromatin (yellow circles); any CpG-islands that were non-specifically enriched by IgG during the ChIP assay were then discarded from further analysis. Finally, the experimental plot is depicted with blue diamonds, which show the results of hybridization of the E2F6 IP amplicon labeled with Cy5 versus the reference total input amplicon that was labeled with Cy3. As is evident from Fig. 3B, there were many features that displayed enriched Cy5 intensities relative to Cy3, and those that were enriched at least 2-fold over total in independent hybridization experiments were chosen for follow up analysis. After repeating each hybridization twice, there were 77 CpG islands that were specifically enriched at least 2-fold higher than starting input chromatin by the E2F6 ChIP; these were not enriched by IgG in either experiment.

The clones from the CpG island library (30) that were spotted onto the arrays have not been sequenced, so all putatively positive clones were sequenced and localized in the human genome using the University of California Santa Cruz Genome Web Browser (genome.ucsc.edu). The majority of the sequenced clones (92.2%) were CpG islands located in the promoters and/or first exons of either characterized genes, mRNAs, ESTs, or predicted genes (Fig. 4). Of the remaining six clones, we were unable to sequence five of them, and the final one did not map to any region of the genome using the April 2003 version of the University of California Santa Cruz Genome Web Browser. Because there is some degree of redundancy of the CpG islands spotted on the arrays, the 77 features that were identified as positive in the two independent hybridizations actually represented 48 independent CpG islands. Of these 48 independent CpG islands, 26 were found in the promoter and 5' region of characterized genes, and 22 were localized to uncharacterized mRNAs, ESTs, or predicted genes. The redundancy on the arrays provided a measure of the robustness of the hybridization procedures. For example, the hp1{alpha} locus was identified as an E2F6 target seven times. Fig. 4 shows the identity of the characterized genes, their function, and whether an E2F consensus binding site exists within 1000 bp upstream of the transcriptional start site. Because Ogawa et al. (10) identified E2F6 in a complex with Mga and Max, E2F6 may also be recruited to promoters via E boxes (CACGTG), due to protein-protein interactions with Max-Mga heterodimers. Therefore, the presence or absence of a consensus E box (CCACGTGG) is also indicated. Interestingly, nearly all of the characterized genes that we have identified through the ChIP-chip screen have either consensus or near-consensus E2F-binding sites and/or E boxes.



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  		FIG. 4.
E2F6 target loci. Shown are the identities and functions of 27 characterized genes identified by ChIP-chip analysis using an antibody to E2F6; also indicated is whether the putative target promoters have E2F or E box-binding sites within 1000 bp upstream of the transcriptional start.

 

We next sought to examine how accurately the relative proportions of DNA sequences present in the amplicons were reflected by the microarray hybridization results. To do so, we performed PCR analysis of the amplicons used to probe the arrays with 14 different primer sets representing sequences that were identified on the array as being at least 2-fold enriched relative to the total input sample (Fig. 5A). As a comparison, amplicons were also made of ChIP assays performed using antibodies to E2F1, E2F4, and RNA polymerase II (RNA pol II). 10 ng of each amplicon was then subjected to equal cycles of PCR with the gene-specific primers and the products electrophoresed on a 1% agarose gel. Semi-quantitative analysis with ImageQuant 5.2 software demonstrated that 13 promoters tested showed at least a 2-fold enrichment in the E2F6 amplicons relative to the starting input chromatin amplicon (Fig. 5A). This demonstrated that under the stringent hybridization conditions that we employed, the microarray data accurately reflected the relative proportions of DNA represented in each amplicon sample that was compared. Interestingly, all of the CpG islands tested were also enriched in the E2F4 and RNA pol II amplicons except for ta-wdrp and htf6. ta-wdrp appears to be an E2F6-specific, RNA pol II-regulated promoter, whereas htf6 was enriched only in the E2F6 amplicons. To date, all of the array-identified CpG islands that we have tested have been enriched in the E2F6 amplicons, suggesting that the hybridization results accurately reflect the DNA proportions present in the amplicons.



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  		FIG. 5.
Confirmation of E2F binding to identified target promoters. A, ChIP analysis was performed using 293 cells with antibodies specific to E2F1, -4, -6, pol II, or with IgG as a control. Amplicons were generated for each antibody, the IgG IP, and the total starting input chromatin. To examine whether the promoters were enriched relative to total in the amplicons, 10 ng of each sample was subjected to 28 cycles of PCR using promoter-specific primers. B, a standard ChIP assay was performed using 293 cells, and promoters were examined using gene-specific primers.

 

Although the experiments described above confirmed that sequences corresponding to the identified CpG islands were indeed enriched in the amplicons used to probe the array, they did not address the reproducibility of the ChIP assay itself. It was possible that some of the identified targets might not be reproducibly precipitated in subsequent experiments or that the apparent enrichment in the amplicons might have been an artifact due to the amplification procedure. Therefore, we next examined binding of E2F1, E2F4, E2F6, and Max to a subset (n = 11) of the identified CpG islands in two independent experiments using the standard chromatin immunoprecipitation analysis (Fig. 5B). Due to space constraints, we have chosen to show a selected number of promoters that are either genes that have been shown, or are suspected, to play a role in cancer pathogenesis or genes that had interesting binding patterns in the amplicons. Fig. 5B shows that the promoters for hp1{alpha} and rbap48, two genes important for chromatin organization and maintenance (31, 32) as well as the promoter for ctip, a BRCA1-interacting protein (31), bind E2Fs 1, -4, -6, and Max. ta-wdrp bound to E2F6 and RNA polymerase II but not to E2F1 or E2F4. htf6 was not enriched in any of the immunoprecipitated samples, suggesting that it was a false-positive. To date, 8 of 11 of the tested array-identified loci have been confirmed to be robustly bound by E2F6 in several independent E2F6 ChIP assays, giving a confirmation rate of 73%. We previously performed a CpG microarray comparison of an E2F6 ChIP to a no antibody ChIP using our previous methods (17) which did not employ ligation-mediated PCR, and we found that the empirically determined false-positive rate for that protocol was significantly higher (data not shown), suggesting that our experimental design adapted from the yeast studies of Ren et al. (29) represents a significant improvement over our previously published studies (16, 17). However, despite ever increasingly sophisticated statistical analysis of the genome-wide ChIP-chip data currently being performed in yeast (for example, see Ref. 33), independent and stringent empirical examination of types I and II error rates will continue to be required to validate the global conclusions presented of genome-wide DNA-binding factor targets due to experimental error. These issues will become more pronounced as genome-wide analysis moves increasingly toward human studies where there are three orders of magnitude more DNA than in yeast. Therefore, in this study, any target we call positive for E2F6 binding has been confirmed in multiple independent analyses (see below).

The Promoters Identified via ChIP-chip Analysis Are Bona Fide E2F6 Target Genes—To demonstrate that the enrichments of the DNA sequences examined are the result of specific E2F6 antibody-epitope interactions, we performed two different experiments. First, we demonstrated the specificity of our antibody using Western blot analysis (Fig. 6A). For these experiments, we compared the signals detected on the Western blot with the E2F6 antibody before and after introduction into the cells of an E2F6-specific small interfering (si) RNA (E2F6-01). RNA interference represents a highly specific method for the knockdown of practically any desired protein and is accomplished by the design of a short, double-stranded RNA (siRNA) that is precisely complementary to the mRNA of the protein of interest (34). OligofectAMINE-E2F6 siRNA complexes were formed, and 2 x 105 293 cells were incubated with either 100 nM E2F6-01, 100 nM of an siRNA specific for the green fluorescent protein (GFP), or the OligofectAMINE reagent alone for 48 h. Whole cell extracts were made of each treatment and electrophoresed on an SDS-PAGE gel, and the proteins were transferred to a nitrocellulose membrane. The membrane was then probed with antibodies specific for E2F6 or E2F4. The E2F6 protein is ~35 kDa (6), and our antibody recognized a single protein on the membrane that migrated at ~35 kDa (Fig. 6A). In contrast to the mock- or 100 nM GFP siRNA-treated cells, treatment with 100 nM of the E2F6-01 siRNA resulted in a significant reduction in E2F6 protein expression. Equal protein loading was shown by re-probing the same blot with E2F4 antibody (Fig. 6B). These results demonstrate that the E2F6 antibody used in these experiments is specific for the E2F6 protein.



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  		FIG. 6.
The E2F6 antibody specifically recognizes the E2F6 protein. A, 293 cells were transfected with siRNAs specific for E2F6, GFP, or a mock control, and whole cell extracts were made after 48 h. Western analysis was performed using an antibody to E2F6. B, the same membrane as in A was stripped and then probed with an E2F4 antibody; the membrane was also probed with actin as a loading control (data not shown). C, prior to use in the ChIP assay, the E2F6 antibody was pre-incubated with a 5- or 10-fold excess by weight of an epitope-blocking peptide for the E2F6 antibody or with an irrelevant peptide for an antibody to TCF4. ChIP analysis was then performed and promoters examined by gene-specific PCR.

 

We next performed a ChIP assay in which the E2F6 antibody was pre-incubated with E2F6 blocking peptides at 5- and 10-fold excess by weight. As a control, a blocking peptide for the TCF-4 antibody was also pre-incubated with the E2F6 antibody. As shown in Fig. 6C, the art27, brca1, and ctip promoters were robustly enriched by the E2F6 antibody, whereas the myc promoter was not, as demonstrated previously. A 5-fold excess of E2F6 blocking peptide was enough to completely abrogate the ability of the E2F6 antibody to enrich the art27, brca1, and ctip loci, whereas pre-incubation of the E2F6 antibody with a 5- or 10-fold excess of TCF-4 peptide did not prevent these loci from being enriched. This demonstrated that the loci we chose for further examination of the mechanism(s) of E2F6-mediated regulation of transcription are immunoprecipitated by the ChIP procedure as a result of specific antibody-epitope interactions and are not non-specifically enriched as a result of the procedure itself.

As described above, we have performed an in vivo screen to identify novel genomic locations to which E2F6 is bound and have confirmed specific binding of E2F6 to these promoters in multiple, independent ChIP experiments. However, a key question is whether the observed binding of E2F6 to promoters has functional consequences on gene transcription. Our approach to this issue was to examine the effects of a temporary knockdown of E2F6 protein expression on the mRNA production from the putative E2F6 target promoters. During the course of these experiments we found that a mixture of four double-stranded siRNAs specific to the E2F6 mRNA reduced the E2F6 mRNA to a greater extent than did the E2F6-01 siRNA described above (data not shown). Therefore, we transfected 293 cells with the E2F6 siRNA mixture or the GFP siRNA. The GFP siRNA was used to control for non-specific effects that could simply be caused by the transfection of exogenous siRNA into cells. The cells were harvested after 96 h, and levels of mRNA of specific transcripts were examined using RT-PCR with primers than spanned introns to ensure that we were monitoring only mature, spliced mRNA. Fig. 7A shows that by 96 h the levels of e2f6 mRNA were greatly depleted, whereas levels of gapdh mRNA remained unchanged. The levels of art27 mRNA were increased by reduction of the amount of E2F6 protein. This was expected due to the fact that the art27 promoter binds E2F6, and deletion of the E2F-like sites in the promoter leads to increased transcriptional activity (Fig. 2). Interestingly, RNAi-mediated knockdown of E2F6 resulted in the up-regulation of brca1 and its binding partner ctip, both of which play important roles in neoplasia (35, 36), and of hp1{alpha} and rbap48, genes that control heterochromatin formation (31), and chromatin assembly and heritability (32), respectively. As expected, because the myc promoter does not bind E2F6, the levels of myc mRNA were unaffected by E2F6 siRNA transfection. Therefore, not all E2F target genes are affected by reduction of the levels of E2F6 protein in this system. These results indicate that many of the promoters identified as E2F6 targets in the ChIP-chip analysis are indeed transcriptionally regulated by E2F6. Interestingly all E2F6-binding promoters that we have examined appear to either be up-regulated or unchanged (data not shown) upon depletion of E2F6 from 293 cells; we have not found any mRNA, other than the E2F6 mRNA itself, to be down-regulated by the siE2F6 treatment. Our data support the hypothesis that E2F6 appears to act exclusively as a negative regulator of transcription of the promoters to which it is bound, in accordance with previous findings from other laboratories (5-8).



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  		FIG. 7.
E2F6 represses transcription from target promoters via occlusion of activating E2Fs. A, RT-PCR analysis was performed using gene-specific primers spanning introns of the indicated mRNAs following treatment of 293 cells with siRNAs specific to E2F6 or GFP. B, 96 h after introduction of siRNAs, the cells were cross-linked with formaldehyde and subjected to ChIP analysis, using an antibody to E2F6. 0.2% of the total input of each sample was also subjected to PCR to show that equal amounts of starting input chromatin were used. C, RNAi-ChIP analysis was performed as in B with antibodies specific to E2F1, pol II, histone H3 trimethyl-K9, and IgG. Individual promoters were examined via gene-specific PCR.

 

E2F6 Can Repress Transcription by Competing with Other E2Fs for Promoter Occupancy—We next sought to test the hypothesis that E2F6 binds to DNA as part of a Polycomb group complex containing the Eu-HMTase specific for methylation of lysine 9 of histone H3 (10). If this model is correct for all E2F6-regulated promoters, one would predict that target promoters would be bound by histone H3 methylated at lysine 9 in the presence of E2F6 but that reduction of E2F6 protein would cause a loss of this specifically modified histone. To test this hypothesis, we have coupled the siE2F6 knockdown strategy with a direct examination of promoters using ChIP (RNAi-ChIPs). 293 cells were treated with the siE2F6 mixture or with siGFP for 96 h. First, we directly examined the effect of E2F6 siRNA treatment on E2F6 binding to the art27, brca1, ctip, rbap48, and hp1{alpha} promoters, relative to siGFP control (Fig. 7B). As a reference for the starting amount of chromatin, a 1:500 dilution of the starting input chromatin was amplified to allow comparison between the siGFP and siE2F6 treatments. We found that siE2F6 treatment resulted in greatly reduced enrichment of these loci following ChIP. This indicates siE2F6 treatment not only lowers E2F6 mRNA levels in 293 cells (Fig. 7A) but also results in the loss of E2F6 protein from target promoters (Fig. 7B). Next, ChIP analysis was performed with antibodies specific to E2F1, RNA pol II, histone H3 tri-methylated at lysine 9, or IgG alone (to control for nonspecific chromatin immunoprecipitation), using 2 x 106 cells per antibody. The immunoprecipitates were analyzed by PCR using primers specific for the E2F6 target promoters (Fig. 7C). Interestingly, we did not observe histone H3 to be methylated at lysine 9 on any of these E2F6 targets, indicating that at least a subset of E2F6-regulated promoters are not repressed by recruitment of the Eu-HMTase and subsequent transient heterochromatin formation. To demonstrate that the antibody specific for lysine 9 methylation of histone H3 functions in the ChIP assay, we examined the htf6 promoter. We showed the htf6 promoter does not bind to RNA pol II (Fig. 5A) and that the htf6 mRNA is transcribed at very low levels in 293 cells (data not shown), indicating that this locus likely has a heterochromatic structure in these cells. We found robust histone H3 methyl K9 signals that were immunoprecipitated equally after either siRNA treatment. Therefore, we are confident that the antibody is able to function in a ChIP assay. Most interestingly, we found that levels of E2F1 bound to the E2F6 target promoters increased upon the removal of E2F6 from the examined promoters, indicating that one mechanism of E2F6 repression of targets may be the occlusion of other activating E2F family members.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The E2F family of transcription factors has been shown to play a critical role in controlling normal and tumor cell proliferation. However, very little is known about the most recently discovered family member, E2F6. Therefore, we used an unbiased approach to identify genes regulated by this factor, beginning with a ChIP-chip screen of about 8000 human CpG islands, followed by RNAi experiments to demonstrate that the promoters bound by E2F6 are in fact regulated by this factor. Finally, we performed RNAi followed by ChIP analysis to investigate the mechanism by which E2F6 mediates transcriptional repression of the identified target genes.

The experiments described here represent a major technical advance over our previous ChIP-chip experiments. Our detailed protocol, beginning with formaldehyde cross-linking and ending with array analysis, is provided on our web site (mcardle.oncology.wisc.edu/farnham/) and in Oberley et al. (21). A critical new step in our protocol is the inclusion of a ligation-mediated PCR (29) that allows a great reduction in the number of cells needed to probe an array. In our recent analysis of E2F1 target genes (16), we pooled 50 individual E2F1 IP samples to obtain enough material to probe the array; for the analyses described in this work, a single E2F6 IP was sufficient. This reduction in cell number will be critical for future studies on the identification of target genes of transcription factors in human tumor samples.

Many transcription factors, including E2Fs, regulate promoters via binding sites that range from -400 to +200 relative to the transcriptional start site (37, 38). Accordingly, most of the identified E2F6 target genes contain consensus or near-consensus E2F sites in the promoter region. However, several of the E2F6 target genes do not have a consensus E2F-binding site between -1000 and +150 relative to the transcriptional start (e.g. coaster and recql), and yet we detect robust signals in the E2F6 immunoprecipitates. It is possible that, in addition to binding to consensus sites, E2F6 can also bind to a sequence that is unrelated to the consensus site. It is also possible that E2F6 binds to a site located at a great distance from the transcription start site, and we are detecting the signal due to a small population of very long chromatin fragments. However, this is unlikely because amplicon generation preferentially creates DNA fragments that are between 200 and 500 bp in length (data not shown), and thus large fragments are not abundant in the sample used to probe the array. However, it remains possible that E2F6 binds at a great distance upstream of the start site and is brought into the basal promoter region through protein-protein interactions. E2F family members have been demonstrated to interact with other site-specific DNA-binding proteins. Examples include the interactions of E2F1 with Sp1 (39, 40), E2F2 with YY1 (41), and E2F3 with TFE3 (42). Also, a Max/Mga heterodimer was purified in the E2F6 complex from HeLa cells (10), which suggests that E2F6 could be recruited to a promoter via an E box, the Max/Mga-binding site. Both the coaster and recql promoters have near consensus E boxes (CACCTG) that could presumably bind Max/Mga heterodimers. In support of the hypothesis that near-consensus E boxes can bind Max family members, Moriuchi et al. (43) found that mutation of an E box with precisely the sequence (CACCTG) found in the coaster and recql promoters abrogated E box-binding protein-mediated regulation of CXCR4 transcription in vivo. Also, we have shown that Max is bound to the E2F6 target promoter ta-wdrp, which lacks an E box. These observations support the hypothesis that E2F6 may exist in an in vivo complex with Max/Mga heterodimers.

To begin to address the mechanism by which E2F6 regulates target genes, we utilized RNAi to transiently knock down E2F6. We first demonstrated that the mRNA levels for the identified E2F6 target genes were sensitive to the reduction of E2F6 protein. The mRNA levels were all increased upon reduction of E2F6, allowing us to firmly conclude that brca1, ctip, hp1{alpha}, and art27 are bona fide E2F6 target genes. These results are in accordance with previous studies suggesting that E2F6 is a transcriptional repressor. As a next step in understanding how E2F6 can mediate transcriptional repression, we then performed a ChIP assay after introduction of E2F6-specific siRNAs. The results of this experiment provide several interesting insights into the mechanisms of E2F6-mediated transcriptional regulation. First we found that the amount of RNA pol II bound to the E2F6 target genes was unchanged after the depletion of E2F6, despite a dramatic up-regulation of transcription. This indicates that the E2F6-bound promoters are occupied by the same levels of RNA polymerase whether they are firing at a low or high rate, i.e. the regulation of transcription is at a step after the recruitment of RNA polymerase II. Studies are in progress to examine whether RNA pol II becomes phosphorylated at serine 5 following E2F6 depletion, which is a marker of RNA pol II elongation (44-46). Our results also indicate that the E2F6-bound promoters are in an open chromatin confirmation; if the binding of E2F6 had caused heterochromatin formation, then the promoters would not be accessible for binding by RNA polymerase II. Second, we were unable to find evidence that the Eu-HMTase was recruited to the E2F6 target promoters, as the E2F6 target promoters showed no binding of histone H3 dimethylated (data not shown) or trimethylated at lysine 9. Ogawa et al. (10) postulated that, due to HP1{gamma} recruitment and heterochromatin formation, E2F6 would be part of a stably bound transcriptional repression complex. At odds with this model, we found that E2F6 could be removed from target promoters, resulting in transcriptional activation. Third, we have found significantly increased binding of E2F1 to the brca1, ctip, rbap48, and hp1{alpha} promoters after siE2F6 treatment. Therefore, our findings suggest that E2F6 can regulate transcription in an Eu-HMTase independent manner by blocking access of activating E2F family members to an E2F-binding site.

One E2F6 target gene identified in our studies is brca1, a tumor suppressor that is often mutated or inactivated in familial breast cancer (35) or silenced via promoter hypermethylation in non-familial cases of breast cancer (47). That E2F6-mediated repression of brca1 transcription might be another mechanism that contributes to BRCA1 down-regulation in cancer is a tantalizing hypothesis. Our results indicating that the loss of E2F6 from promoters allows for activating E2F transcription factors to bind are especially interesting when considering previous studies of the brca1 promoter. Wang et al. (48) have previously shown that the brca1 promoter contains E2F-binding sites that can mediate transcriptional activation by exogenously expressed E2F1. Therefore, we suggest that the transcriptional activity of the brca1 promoter is controlled by the level of occupancy of the E2F site by either the activator E2F1 or the repressor E2F6. Targeted therapeutic intervention of E2F6 may allow an increased amount of BRCA1 to be produced (which may reduce tumorigenicity), whereas removal of E2F1 would be predicted to reduce the amount of BRCA1 in the cell (Fig. 8). We recognize that although certain promoters, such as brca1, may be conversely regulated by E2F1 versus E2F6, not all promoters will be similarly affected. For example, Croxton et al. (28) has shown that E2F1 can repress certain promoters. Also, we have shown that deletion of the E2F sites in the art27 promoter results in a dramatic up-regulation of promoter activity, indicating that the main effect of occupancy of the art27 promoter by E2Fs is transcriptional repression. Perhaps certain of the other E2Fs that bind to the art27 promoter, in addition to E2F6, cause transcriptional repression. Clearly, the function of individual E2Fs will vary, depending upon the specific target promoter analyzed.



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  		FIG. 8.
Inactivation of different E2F family members will have different effects on E2F target genes. A schematic is shown representing the competition of E2F6 and E2F1 on the brca1 promoter. Targeted inactivation of E2F6 would result in increased levels of BRCA1 protein and the restoration of tumor suppression function. In contrast, inactivation of E2F1 would allow occupancy of the promoter by E2F6, resulting in down-regulation of BRCA1 protein and loss of tumor suppression function.

 

Interestingly, in addition to brca1, several other of the identified E2F6 target genes play a significant role in breast cancer. One such gene, ctip, encodes a protein that directly binds to BRCA1, modulating its response to DNA damage (36). The direct suppression of both of these genes by E2F6 might have additive effects in conferring growth advantages to tumorigenic cells. We also demonstrated that hp1{alpha} was transcriptionally repressed by E2F6 binding to the promoter. It has been shown previously (49) that down-regulation of hp1{alpha}, but not hp1{beta} or -{gamma}, has been correlated with highly invasive and metastatic breast cancer cells. Significant decreases in the amount of HP1{alpha} protein were also found in metastatic tumors, relative to the primary tumors, in seven of nine patients with breast cancer. The authors concluded that HP1{alpha} may play a significant role in the silencing of genes involved in invasion and metastasis. We suggest that occupancy of the E2F site in the hp1{alpha} promoter by E2F6 would down-regulate HP1{alpha} protein levels, contributing to neoplastic transformation but that removal of E2F6 from the cell would result in increased HP1{alpha} and the regain of silencing of critical promoter regions. In light of the association between breast cancer and E2F6 target genes, our future studies will be to examine whether E2F6 is up-regulated in breast cancer cells lines and/or metastatic breast cancer samples.

In summary, our results indicate that coupling ChIP to CpG island microarray analysis is a powerful tool to identify targets of specific transcription factors that have been implicated in the development of cancer. By using this unbiased approach, we identified E2F6 target genes and revealed a possible link between E2F6 and breast cancer. Finally, via RNAi-based depletion of E2F6, we showed that E2F6 functions as a repressive transcription factor in a histone methyltransferase-independent manner on target promoters in human cancer cells.


   FOOTNOTES
 
* This work was supported in part by United States Public Health Service Grants CA45240, CA09135, and CA14520 and the NIEHS Training Grant ES07015-24 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{dagger} To whom correspondence should be addressed: McArdle Laboratory, University of Wisconsin Medical School, 1400 University Ave., Madison, WI 53706. Tel.: 608-262-2071; Fax: 608-262-2824; E-mail: farnham{at}oncology.wisc.edu.

1 The abbreviations used are: CREB, cAMP-response element-binding protein; Eu-HMTase, euchromatin-specific histone methyltransferase; ChIP-chip, chromatin immunoprecipitation-microarray analysis; ChIP, chromatin immunoprecipitation; siRNA, small inhibitory ribonucleic acid; RT, reverse transcriptase; myc, myelocytomatosis oncogene; art27, androgen receptor trapped clone 27; brca1, breast cancer 1; ctip, CTBP-interacting protein; rbap48, retinoblastoma-associated p48; hp1{alpha}, heterochromatin protein 1; htf6, human teratocarcinoma finger; gapdh, glyceraldehyde-3-phosphate dehydrogenase; RNA pol II, RNA polymerase II; GFP, green fluorescence protein. Back


   ACKNOWLEDGMENTS
 
We thank Tim Huang for supplying the CpG island microarrays used in this study, Antonis Kirmizis for technical advice and helpful discussions, and the University of Wisconsin Biotechnology Center for sequencing analysis and primer synthesis.



   REFERENCES
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 DISCUSSION
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