Genomic binding by the Drosophila Myc, Max, Mad/Mnt transcription factor network - PubMed (original) (raw)

. 2003 May 1;17(9):1101-14.

doi: 10.1101/gad.1066903. Epub 2003 Apr 14.

Bas van Steensel, Jeffrey Delrow, Harmen J Bussemaker, Ling Li, Tomoyuki Sawado, Eleanor Williams, Lenora W M Loo, Shaun M Cowley, Cynthia Yost, Sarah Pierce, Bruce A Edgar, Susan M Parkhurst, Robert N Eisenman

Affiliations

Genomic binding by the Drosophila Myc, Max, Mad/Mnt transcription factor network

Amir Orian et al. Genes Dev. 2003.

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 ( approximately 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.

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Figures

Figure 1

Figure 1

Comparison of chromatin profiles for the Drosophila Max network. Scatter plot comparisons of chromatin binding profile data sets of the dMax network proteins derived from the entire cDNA array. The average binding is presented as Cy5:Cy3 log ratios between dMax–Dam:Dam vs. dMnt–Dam:Dam, r = 0.49 (A); dMax–Dam:Dam vs. dMyc–Dam:Dam with dMax expressed at low levels, r = −0.43 (B); dMax–Dam:Dam vs. dMyc–Dam:Dam with dMax expressed at high levels, r = 0.17 (C); dMnt–Dam:Dam vs. dMyc–Dam:Dam where dMyc–Dam is coexpressed with low levels of dMax, r = 0.26 (D); dMnt–Dam:Dam vs. dMyc–Dam:Dam where dMyc–Dam is coexpressed with high dMax levels, r = 0.71 (E); dMnt–Dam:Dam vs. dGAF–Dam, r = 0.11 (F). Ratios were calculated using a full set of experiments as described in Materials and Methods. Pearson correlation coefficients (r) were calculated for each set. Statistically significant binding targets are indicated by colored solid dots. Blue, dMax–Dam; green, dMyc–Dam; red, dMnt–Dam; brown, dGAF–Dam; purple, overlapping targets. The bicaudal (bic) gene is marked by open black circle and is printed twice on the array.

Figure 2

Figure 2

Venn diagrams depicting the number of shared genes within the dMax network. Criteria for calculating statistically significant targets are described in Materials and Methods. (A) Comparison of dMnt and dMax targets. (B,D) dMyc data sets generated in the presence of low dMax levels. (C,E,F) dMyc data sets generated in the presence of high dMax levels.

Figure 3

Figure 3

Map of dMax network binding across Drosophila chromosomes and repetitive elements. Annotation of binding targets was preformed using the Drosophila gene collection subset (Rubin et al. 2000). Individual genes are depicted as vertical gray bars. dMyc, dMnt, and dMax binding sites are denoted by green, red, and blue solid circles, respectively. Color intensity is directly linked to binding P values as depicted at the bottom. (A) dMax network binding to the four Drosophila chromosomes. A partial list of genes found to be orthologs of direct targets of c-Myc (Fernandez et al. 2003) are indicated by black triangles and gene names. dMyc-regulated genes in the larval expression study that were detected in the the DamID analysis are denoted by purple (activated) and pink (repressed) arrows. (B) dMax network binding to repetitive elements.

Figure 4

Figure 4

Occupancy of the bic promoter by dMyc and dMnt proteins correlates with changes in histone acetylation. (A) Diagram of the bic promoter, the CACGTG E-box is denoted as an open box. The PCR primer set (−50 left primer, +75 right primer) used is denoted by upper black arrows. (B) Promoter occupancy was determined using monoclonal antibodies to dMyc (lanes 5,6), dMnt (lanes 7,8), acetylated histone H3 (lanes 11,12), and acetylated histone H4 (lanes 13,14). ChIP using genomic extracts derived from dMyc-expressing cells (odd-numbered lanes) or dMnt-expressing cells (even-numbered lanes) is described in Materials and Methods. Input, 2 ng non-IP genomic DNA; bic, the bic E-box specific primers; kis, control primers for the kis gene promoter serving as a loading control; αIgGp and αIgGm, Anti-mouse polyclonal and monoclonal IgG antibodies. Enrichment is represented as the ratio between the bic and the kis PCR products as described in Materials and Methods. (C) Ecdysone treatment leads to endogenous dMnt recruitment to the bic promoter and correlates with deacetylation of histone H3 and H4. Ecdysone treatment and ChIP were as described in Materials and Methods. Amplification linearity was tested in the presence of ecdysone (lanes 2–4). All other abbreviations are as depicted in Figure 4B.

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