Genomic binding by theDrosophilaMyc, Max, Mad/Mnt transcription factor network

Abstract

The Myc/Max/Mad transcription factor network is critically involved in cell behavior; however, there is relatively little information on its genomic binding sites. We have employed the DamID method 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 as we find that 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. The surprisingly large number of directly bound loci (∼ 15% of coding regions) suggests that the network interacts widely with the genome. Furthermore, we employ microarray expression analysis 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.[Keywords: myc; mad; Drosophila; target genes; transcription] Supplemental material is available at http://parma.fhcrc.org/AOryan.

A role for Myc in facilitating transcription activation by E2F1

“…Such a role would be consistent with the nature of genes apparently regulated by Myc which include not only genes such as the E2Fs that are critical for generating the DNA replication machinery, but also a variety of genes encoding basic metabolic activities essential for growing cells. Indeed, recent large-scale ChIP assays have suggested a very widespread role for Myc in the regulation of transcription (Fernandez et al, 2003;Orian et al, 2003). Our observation of a role for Myc in controlling the activation of E2F genes may provide a mechanism to link the preparation for initiating cell growth with the execution of the G 1 /S transition and the commitment to the cell cycle ( Figure 6).…”

DNA microarray technologies for measuring protein–DNA interactions

“…The methylated sites in the experimental versus control (Dam alone) samples are detected by digestion with a methyl-specific restriction enzyme, amplification, labeling, and hybridization to a microarray. DamID has been used to identify in vivo binding sites in Drosophila [7] and Arabidopsis [44], of sequence-specific TFs [45], DNA methyltransferase [44], chromatin [7] and chromatin-associated proteins using cDNA or PCR amplicon arrays, and more recently, NimbleGen 60-mer tiling arrays [·46].…”

Myc in model organisms: A view from the flyroom

“…This could be due to binding by dMax homodimers, or, dMax could partner other, unidentified factors. Intriguingly, the unique binding of dMax to these genes did not correlate with the presence of E-boxes, perhaps pointing to the latter possibility [17].…”

“…There are some hints, however, that the function of dMax may not be limited to partnering dMyc and dMnt. For example, in experiments designed to map genomic binding by dMyc, dMax, and dMnt, it was found that dMax bound to a large number (365) of genes not bound by dMnt or dMyc [17]. This could be due to binding by dMax homodimers, or, dMax could partner other, unidentified factors.…”