The Myc/Max/Mad transcription factor network is critically involved in cell behavior; however, there is relatively little information on its genomic binding sites. The DamID method was used to carry out global genomic mapping of the Drosophila Myc, Max, and Mad/Mnt proteins. Each protein was tethered to Escherichia coli DNA adenine-methyltransferase (Dam) permitting methylation proximal to in vivo binding sites in Kc cells. Microarray analyses of methylated DNA fragments reveals binding to multiple loci on all major Drosophila chromosomes. This approach also reveals dynamic interactions among network members; increased levels of dMax influence the extent of dMyc, but not dMnt, binding. Computer analysis using the REDUCE algorithm demonstrates that binding regions correlate with the presence of E-boxes, CG repeats, and other sequence motifs. Application of the REDUCE algorithm, which correlates binding with the occurrence of DNA sequence motifs, reveals a statistically significant correlation between the E-box CACGTG and the presence of dMnt binding regions. CACGTG enrichment also correlated with dMyc binding in the presence of high dMax levels, for dMax binding in the presence of high dMyc levels, and for genes whose expression is modulated by dMyc. The surprisingly large number of directly bound loci (approximately 15% of coding regions) suggests that the network interacts widely with the genome. Furthermore, microarray expression analysis was employed to demonstrate that hundreds of DamID-binding loci correspond to genes whose expression is directly regulated by dMyc in larvae. These results suggest that a fundamental aspect of Max network function involves widespread binding and regulation of gene expression (Orian, 2003).
A significant gap in understanding of the function of many transcriptional regulatory proteins has been the lack of comprehensive identification of their in vivo binding sites and the genes whose expression they regulate. This problem is especially pertinent for transcription factors such as Myc, Mad/Mnt, Max, and other members of the Max network that function as relatively weak transcriptional regulators, whose consensus binding site is ubiquitous, and whose expression elicits profound effects on cell growth and proliferation. Standard methods of target gene evaluation do not reliably differentiate between genes bound and directly regulated by Myc and Mad from genes whose expression is altered as a secondary or later consequence of Myc or Mad induction. In principle, it is important to know about both sets of genes, but it is also crucial to distinguish between them. The DamID method employed in this paper permits determination of transcription factor binding site regions in live cells and is not dependent on chemical cross-linkers or PCR primers. Because it involves 'marking' of DNA in chromatin by a methyltransferase linked to a transcription factor, even transient or low affinity interactions with DNA, as well as proximity to regions distal to the binding site (through looping or higher-order folding), might be detected. Because a cDNA array was used to detect targeted methylation regions, only binding sites within a few kb of transcription units are detected. Therefore, enumeration of dMax network binding sites is likely to be an underestimate. The mapping resolution also does not permit precise pinpointing of the binding site within each probed locus, although the REDUCE analysis strongly suggests that E-box motifs within target loci mediate the protein recruitment (e.g., as for Mnt target bicaudal) (Orian, 2003).
The validity of the approach is strongly supported by several lines of evidence. (1) The degree of overlap between dMyc, dMax, and dMnt binding regions is consistent with the relationship between E-box binding and heterodimerazation with Max established previously for the vertebrate proteins as well as for their orthologs in Drosophila. Importantly, the GAGA factor, a ubiquitous transcription factor unrelated to the dMax network, displays only minimal overlap with dMnt binding sites, suggesting the results are specific for binding by dMax network transcription factors. Furthermore, studies in mammalian cells have shown both overlapping and nonoverlapping functions and target genes for Myc and Mad proteins in agreement with DamID findings. (2) Using a ChIP assay, the direct binding of dMyc and dMnt to a DamID-defined target gene, bic (bicaudal), was demonstrated. In addition, the mammalian orthologs of at least 18 genes identified as binding targets for dMyc, dMax, and dMnt in this study have been demonstrated to be direct targets for vertebrate Myc using ChIP. (3) Application of the REDUCE algorithm, which correlates binding with the occurrence of DNA sequence motifs reveals a statistically significant correlation between the E-box CACGTG and the presence of dMnt binding regions. CACGTG enrichment also correlated with dMyc binding in the presence of high dMax levels, for dMax binding in the presence of high dMyc levels, and for genes whose expression is modulated by dMyc. (4) A substantial set of target genes identified in the Drosophila gene expression microarray analysis, employing larvae overexpressing dMyc, correspond to target genes defined by DamID. In addition, target genes identified in this study are in accord with genes regulated by Myc and Mad as described in several recently published gene-expression studies in vertebrate systems (Orian, 2003).
The Drosophila Gene Ontology Database was used to derive an unbiased classification of genes associated with dMax network binding regions. Many of the dMax network targets identified are genes that fit well with the established biological functions of Myc and Mad. In addition, a significant number of targets point to new pathways likely to be regulated by the network. The data demonstrate both binding to, and regulation of, genes encoding proteins broadly involved in biosynthetic processes, in accord with genetic and biochemical analyses, demonstrating that Myc is involved in cell growth in Drosophila and vertebrates, and from earlier global gene expression studies. The DamID binding loci also include genes involved in cell cycle and DNA replication. The list of putative dMax network targets also reveals potential novel pathways such as mitochondrial biogenesis and function, as well as vesicular transport. Other pathways known to be linked to Myc such as apoptosis, proteolysis, and the immune response are also reflected in the list of dMax network target genes as are a number of transcription factors (Orian, 2003).
The findings demonstrate a surprisingly large number (968) of binding sites for proteins of the dMax network. Considering that the array represents a random sampling of ~50% of Drosophila coding regions, a conservative estimate is that dMax network proteins interact with ~2000 genes, and this is likely to be an underestimate. It is important to note however that dMax network proteins do not bind profligately to DNA, as evidenced by the low degree of overlap with GAGA factor, the general correlation of E-box sequences with binding, and the lack of association with repeat elements linked to HP1 binding previously. HP1 is predominantly localized to pericentric heterochromatin, and its binding is associated with silenced chromatin structure. The lack of association of dMyc, dMax, or dMad with such elements may indicate that the network proteins are primarily associated with genes that are subject to ongoing transcriptional modulation. These findings are in accord with extensive ChIP assays in human cells. That study suggested that 8%-10% of cellular genes associate with Myc and in general display enhanced histone H3 and H4 acetylation (Orian, 2003).
The large number of binding sites and regulated target genes identified in this study contrasts with earlier ideas of Myc function that posited a small number of critical targets. However, not all binding sites necessarily result in direct transcriptional regulation by dMax network factors. This is evident from the dMyc-dependent gene expression data carried out in growing third instar larvae. At this developmental stage, 31% (89/287) of the Myc binding loci (as determined in Kc cells) displayed altered mRNA epression in larvae. Of genes that were detected as overlapping targets of all three proteins or of only dMyc and dMnt, 48.6% and 60.5% respectively, displayed concomitant changes in mRNA levels upon Myc induction. Interestingly, Myc binding and histone acetylation at mammalian genes has been described, whose expression does not appear to change in response to induction of Myc. One possible explanation is that Myc binding to a subset of genes, although not immediately affecting gene expression, confers a permissive state on chromatin allowing binding by other cis-acting factors at later times (Orian, 2003).
The many dMax targets detected that are shared with dMyc and dMnt most likely represent binding by dMyc-dMax and dMnt-dMax heterodimers. However, the extent of nonoverlap between binding sites for these proteins is more extensive than expected. For example, it was found that dMax expressed at low levels binds to 365 genes that do not overlap with either dMnt or dMyc targets. However, 15% of these binding loci are regulated by dMyc in the larval expression analysis. Thus, the degree of overlap is probably influenced by the temporal pattern and levels of dMyc expression. This has implications for tumorigenesis where vertebrate Myc proteins are often dramatically overexpressed. This work provides evidence that such overexpression may shift the spectrum of target genes relative to those expressed in normal cells (Orian, 2003).
Max homodimers bind E-boxes with relatively low affinity and in mammalian cells are inhibited by phosphorylation from binding DNA. Although it is not know known whether dMax homodimers are similarly blocked from binding to DNA in vivo, the idea is favored that the large number (365) of unique dMax binding sites and the lack of correlation with E-boxes reflects dimerization and DNA binding by dMax with as-yet-unidentified interacting proteins. Interestingly, in mammalian cells Max has been found, in association with the bHLHZ protein Mga, in E2F6 repression complexes. Similarly, unique sites found for dMnt and dMyc may represent non-E-box DNA binding through formation of higher-order complexes. For mammalian Myc, interaction of Myc-Max heterodimers with the Miz-1 protein has been shown to direct Myc to non-E-box sites. It is likely that associations with other partners may redirect dMyc and dMnt to unique binding sites. If so, the findings indicate that such interactions may be extensive and are an important part of dMax network function (Orian, 2003).
The canonical E-box sequence alone is unlikely to be sufficient to determine specific binding by dMax network proteins and, indeed, many E-box-containing promoters are not associated with Max network proteins. One possibility is that other sequences in the vicinity of an E-box may play a role in target gene specificity. For example, the DRE, which correlated with binding of all three dMax network proteins is located within <1 kb of many of these E-box sequences. Therefore, it is tempting to hypothesize that the DRE operates in cis with adjacent E-boxes to recruit protein complexes that will either promote activation or repression. Alternatively, the proximity of DRE and E-box sites may reflect coordinate regulation of the same genes through distinct signaling pathways (Orian, 2003).
In addition, REDUCE analysis has revealed a number of unexpected correlations. For example, association was found between dMyc and AT-rich sequences when dMax levels are limiting. In several loci examined, these AT-rich regions occur in the vicinity of genes lacking E-boxes, perhaps reflecting dMyc association with as-yet-undefined binding proteins when dMax levels are limiting. REDUCE analysis of dMax binding regions failed to detect a binding correlation with CACGTG. However, when high levels of dMyc were expressed together with dMax-Dam, REDUCE analysis of dMax binding regions found the E-box significantly correlated with binding. This is in accord with data that dMax homodimers bind only weakly to E-boxes, and that Max binding is largely directed by its heterodimeric partners. Perhaps the AT- (and CG-) rich sequences influence architecture of the binding site or serve as binding motifs for factors that enhance dMax network protein association with DNA (Orian, 2003).
Taken together, these data suggest a rather more complex picture of the functioning of Max network transcription factors than has been considered previously. The results suggest extensive yet specific interaction with chromatin probably encompassing thousands of binding sites and directly affecting expression of hundreds of genes. In addition, the DamID results indicate the possibility of several different modes of Myc, Max, and Mad/Mnt interactions. These include binding to partner proteins yet to be identified as well as potential cooperation with other transcription factors. Earlier experiments have shown that Myc and Mad expression is under tight control by the cell. Such control is likely to be important in balancing the multiple protein-protein and DNA binding interactions inferred from the data (Orian, 2003).
Vertebrate Myc, Max, and Mad/Mnt are all members of the bHLHZ class of transcription factors in which the bHLHZ domains mediate highly specific heterodimerization of Myc and Mad with Max as well as sequence-specific DNA binding. To determine whether dMnt heterodimerizes with dMax, in vitro binding assays were carried out. Chimeric proteins in which GST was fused to the dMnt bHLHZ domain or the dMnt DeltaZIP bHLH domain were bacterially expressed and purified. GST alone or GST fusion proteins were incubated with in vitro-transcribed and in vitro-translated [35S]methionine-labeled dMax, dMyc, or each of the three dMnt splice forms. The GST-dMnt bHLHZ fusion protein interacted specifically with dMax but did not interact with dMyc or any of the dMnt splice forms. The GST-dMnt DeltaZIP bHLH fusion protein failed to interact with dMax, dMyc, or any of the dMnt splice forms. These results demonstrate that dMnt is able to heterodimerize with dMax through the bHLHZ domain but is unable to homodimerize or interact with dMyc. In addition, the leucine zipper domain is required for dMnt heterodimerization with dMax (Loo, 2005).
Mammalian Mad family members and Mnt contain an amino-terminal SID that is required for transcriptional repression. The SID domains of Mad proteins have been previously demonstrated to interact with the second paired amphipathic helix (PAH2) domain of the Sin3 corepressor. Because dMnt possesses a region similar to mammalian SIDs, GST pull-down experiments were performed to determine whether dMnt associates with the PAH2 domain of dSin3. GST or GST-dSin3 (PAH2) fusion protein was incubated with labeled, in vitro-transcribed, and in vitro-translated dMnt splice forms. GST-dSin3(PAH2) interacted specifically with the two dMnt splice forms containing SID but did not bind the dMnt DeltaSID form. No interaction of the dMnt splice forms with GST alone was observed (Loo, 2005).
Vertebrate Myc-Max and Mad-Max heterodimers have been demonstrated to bind specifically to the consensus E-box sequence CACGTG, and Drosophila Myc-Max dimers bind the same E-box sequence. In vitro-translated dMnt, dMnt DeltaZIP, or dMnt DeltaSID were tested together with dMax in electrophoretic mobility shift assays and it was determined that dMnt-dMax and dMnt DeltaSID-dMax heterodimers bind specifically to a labeled CACGTG oligonucleotide. As expected, given that dMnt DeltaZIP is unable to heterodimerize with dMax, no specific binding to the labeled oligonucleotide was observed in the sample containing dMnt DeltaZIP and dMax (Loo, 2005).
The DNA binding activity of these proteins is related to their transcriptional function. Synthetic reporter genes, as well as cellular target genes, are transcriptionally activated by both vertebrate and Drosophila Myc-Max heterodimers and are transcriptionally repressed by mammalian Mad-Max and Mnt-Max. Activation and repression by these groups of heterodimers are dependent on the presence of the E-box sequence. It was found that expression of dMnt and dMax repress twofold the transcription of a reporter construct containing an E-box (CACGTG), as compared to empty vector control. In contrast, the dMnt DeltaSID and dMnt DeltaZIP splice forms fail to influence transcriptional activity relative to empty vector. Taken together, these results indicate that dMnt, like vertebrate Mad and Mnt proteins, dimerizes with dMax, binds E-box sequences, and represses transcription of an E-box-containing promoter in a SID-dependent manner (Loo, 2005).
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