Evaluation of Myc E-Box Phylogenetic Footprints in Glycolytic Genes by Chromatin Immunoprecipitation Assays

Defining transcriptional regulatory networks is essential for our understanding of embryonic development, cell growth, and tumorigenesis. Throughout evolution, biologically important genes and their regulatory elements have been selectively conserved (34). Completing sequencing the genomes of a variety of species provides a unique opportunity for the identification of transcriptional regulatory regions through interspecies sequence comparison, also known as phylogenetic footprinting. In particular, noncoding sequences with interspecies sequence identity approaching that of exonic sequences are enriched with putative transcription factor binding sites. A number of approaches that allow the prediction of transcriptional regulatory regions in any genomic region through comparisons of mouse and human sequences have been reported (4, 19, 21, 24). These approaches identify conserved regions that contain putative transcription factor binding sites. However, a purely computational approach tends to be replete with the uncertainty as to whether a predicted cis-regulatory module is biologically functional. Several algorithms have been developed and experimentally evaluated for the discovery of candidate regulatory regions, such as those in the Drosophila genome (2, 26). In mammalian systems, we and other groups have evaluated the functionality of the transcriptional regulation of conserved cis elements. These studies, however, addressed individual genes or a small set of genes rather than a series of functionally related genes, such as those encoding a specific biochemical pathway. In our previous studies, putative direct Myc target genes were randomly selected and subjected to chromatin immunoprecipitation (ChIP) assays to identify Myc binding sites (43). In addition to class I genes in which Myc binding regions are highly conserved among species, we identified another group of genes termed class II genes, in which Myc binding regions do not contain conserved sequences (18). Thus, experimental validation of these computational approaches is particularly important in a well-defined model system involving a set of coordinately regulated genes, such as those encoding components of a metabolic pathway. Although Myc and its target genes have been studied at a broader genome-wide level (6, 7, 14, 16, 17, 23, 25, 27, 29-31, 35, 41), the coupling of comparative interspecies sequence analysis and experimental validation of Myc target genes involved in a single metabolic pathway has not been thoroughly studied. It is particularly intriguing to note that ODC is not only the first identified bona fide Myc target gene, but it also contains a phylogenetically conserved intronic region bearing tandem canonical E boxes (1). MYC overexpression has been suggested to aberrantly enhance tumor glycolysis even in the presence of oxygen, a phenomenon termed the Warburg effect (8, 39). Thus, we chose Myc as a model transcription factor and the glycolytic genes as model target genes to predict Myc binding regulatory regions, which can then be experimentally tested. We have previously found that Myc specifically transactivates LDHA and increases the expression of other glycolytic enzyme genes (32, 37). Numerous studies using global gene expression profiling methods, such as serial analysis of gene expression (SAGE) and DNA microarrays, have found that Myc increases the expression of specific glycolytic enzyme genes, though these increases may be direct or indirect effects of Myc (27-29, 35). To identify the direct target genes of Myc and its binding sites, we and other groups have applied various assays, including an in vitro reporter assay, electrophoresis mobility shift assay (EMSA), and the Myc-estrogen receptor (MYC-ER) system (11). Through the use of the MYC-ER system, several glycolytic genes, such as ENO1, GPI, HK2, LDHA, and PFKM, have been identified as direct targets of Myc (7, 29, 32, 35). However, these experimental approaches did not provide physical evidence that Myc directly activates the transcription of these genes through its association with specific genomic regions. The MYC-ER fusion protein system has been particularly used as a standard for the study of direct Myc target genes, as it allows the identification of MYC-ER-induced targets upon estrogenic ligand stimulation in the presence of cycloheximide, which prevents secondary transcriptional events (11). However, estrogenic ligands and cycloheximide used in these experiments may confound the effects of Myc, and the MYC-ER system is unable to reveal Myc target genes that are in feed-forward loops. In these loops, the expression of terminal target genes are dependent on both Myc and an intermediate transcription factor that is also a direct target of Myc. We reasoned that functionally important genomic regions for Myc binding have been preferentially conserved in the direct target genes. Given that exonic sequence identity in the human and mouse genomes is estimated at about 85% (40), we have performed manual sequence alignments using dot plotting to identify nonexonic regions with at least 65% sequence identity in 30-bp segments. These cutoff criteria are adequately stringent to predict a particular class of Myc target genes, as previously reported (18, 43). We also used the Trafac server to identify potential Myc binding sites in glycolytic genes (19). A number of programs, including RepeatMasker (masking out repeat elements), PipMaker-BLASTZ (sequence alignment algorithm), and MatInspector Professional (transcription factor binding sequence scan), are integrated into the Trafac system to perform phylogenetic footprinting analysis (19). Trafac analysis predicts both canonical and noncanonical E boxes that reside in regions that have at least 50% sequence identity in the human and mouse genomes. Within these conserved segments, we sought to identify canonical Myc binding sites or E boxes with the consensus sequence 5′-CACGTG-3′ and determine whether Myc could bind these regions by ChIP assays (10, 14, 23). By performing a ChIP assay, we can identify immunoprecipitated regions of the genome that are cross-linked to the bound Myc protein by amplifying the Myc-associated DNA fragments by PCR. Our approach using Myc and 14 glycolytic genes as a model provides a unique opportunity not only to evaluate phylogenetic footprinting and determine the architecture of the Myc target glycolytic gene network but also to dissect the molecular basis of Myc-induced altered glucose metabolism. Our results provide evidence that MYC enhances aerobic glycolysis by directly up-regulating the expression of ENO1, GAPD, HK2, LDHA, PFKM, and TPI1 genes, whereas Myc binding to GPI, PGK1, and PKM is diminished or absent in the cases of BPGM, PGAM2 (muscle specific), and PKLR (liver specific). This study indicates that conserved, canonical E boxes are predictive of significant Myc binding to glycolytic target genes, but the absence of canonical E boxes does not exclude the possibility of significant Myc association.