Sequence specificity incompletely defines the genome-wide occupancy of Myc

Abstract

Background: The Myc-Max heterodimer is a transcription factor that regulates expression of a large number of genes. Genome occupancy of Myc-Max is thought to be driven by Enhancer box (E-box) DNA elements, CACGTG or variants, to which the heterodimer binds in vitro.

Results: By analyzing ChIP-Seq datasets, we demonstrate that the positions occupied by Myc-Max across the human genome correlate with the RNA polymerase II, Pol II, transcription machinery significantly better than with E-boxes. Metagene analyses show that in promoter regions, Myc is uniformly positioned about 100 bp upstream of essentially all promoter proximal paused polymerases with Max about 15 bp upstream of Myc. We re-evaluate the DNA binding properties of full length Myc-Max proteins. Electrophoretic mobility shift assay results demonstrate Myc-Max heterodimers display significant sequence preference, but have high affinity for any DNA. Quantification of the relative affinities of Myc-Max for all possible 8-mers using universal protein-binding microarray assays shows that sequences surrounding core 6-mers significantly affect binding. Compared to the in vitro sequence preferences,Myc-Max genomic occupancy measured by ChIP-Seq is largely, although not completely, independent of sequence specificity.

Conclusions: We quantified the affinity of Myc-Max to all possible 8-mers and compared this with the sites of Myc binding across the human genome. Our results indicate that the genomic occupancy of Myc cannot be explained by its intrinsic DNA specificity and suggest that the transcription machinery and associated promoter accessibility play a predominant role in Myc recruitment.

Figure 1

Examples of Pol II, Myc, and Max occupancy. Genome browser tracks show occupancy determined by ChIP-Seq for Pol II, Myc, Max, and CTCF over the indicated gene regions in HeLa cells. The positions of the canonical CACGTG E-boxes are indicated. Regions around (A) chromosome 19 containing 10 genes, (B) PSMB2, and (C) MYC are shown. GRO-Seq data are for IMR90 cells from GSE13518 [34].

Figure 2

Correlation of Myc and Max with Pol II occupancy. (A) Metagene analysis showing the average of 20,886 genes. (B) High resolution heatmaps of the same genes rank-ordered by Pol II occupancy. The region shown is from -2 kb to +2 kb around the TSS. (C) Correlation of the occupancy of the indicated proteins. (D) Metagene analyses of Myc, Max, and Pol II ChIP-Seq datasets from eight different cell lines (HeLa, GM12878, K562, H128, H2171, MM1S, P493, and U87). (E) Metagene analysis of Myc, Max, Med1, and Pol II ChIP-Seq datasets from four different cell lines (H2171, MM1S, P493, and U87). Average occupancies of regions from -1,000 to +1,000 bp around the TSS are shown.

Figure 3

Biochemical analysis of Myc and Max. (A) SDS-PAGE of the indicated recombinant proteins that were expressed in E. coli and purified as described in Methods. (B) EMSA using native polyacrylamide gel electrophoresis with 200 ng of the indicated proteins (250 nM Max dimer and 125 nM Myc-Max) with 0, 0.1, 0.3, 1, or 3-fold molar excess of the indicated dsDNA. The gels were silver stained to show the mobility of the proteins. The arrows indicate protein-DNA complexes. (C) EMSA with simultaneous staining of Max^L and Myc-Max^L. A total of 2.5 pmole of each protein (125 nM) per lane with two levels of the indicated DNA probes. Complexes containing indicated proteins are indicated with arrows. Note that in the Myc-Max prep some dissociation of Max has occurred leading to a low level of Max and Max-DNA species. (D, E, F, G, and H) EMSAs using 0.01 nM of the indicated radiolabeled probe (blue) with the indicated concentration of proteins and competitor DNAs.

Figure 4

Binding of Myc to all possible 8-mers and comparison with genomic occupancy. (A) Fluorescent signal generated by Myc in vitro binding with an array containing all possible 8-mers was normalized. Twelve core 6-mer sequences with the highest in vitro occupancy are shown. The relative affinity of all 8-mers for each 6-mer is shown (10 points if the 6-mer is a palindrome or 16 if it is not). The inset shows the sorted in vitro binding signal for all possible 8-mers. (B) Genome browser view of a region on chromosome 19 comparing Myc, Max, and Pol II occupancy with the distribution of the top 12 6-mers (from A). The height of each 6-mer peak is equal to its relative in vitro occupancy (shown as percent). (C, D) Zoomed in views of two regions shown in (B) that demonstrate the lack of correlation of Myc and Max occupancy with the intrinsic affinity for the underlying DNA determined in vitro.

Figure 5

Comparison of Myc ChIP-Seq occupancy with in vitro binding affinities. (A) The top 30,000 sites occupied by Myc (blue) were rank-ordered and scored by the in vitro occupancy of the best 8-mer in a 100 bp window (y-axis). This was repeated at 30,000 random locations of DNase I-sensitivity (black) and the results were directly compared by ROC analysis (inset). (B) The top 30,000 sites occupied by Myc were rank-ordered by ChIP-Seq signal and scored logarithmically by either normalized ChIP-Seq signal (blue line) or the in vitro occupancy of the best 8-mer in a 100 bp window (black dots). (C) The data in (B) are presented using a default R boxplot (box: 1st to 3rd quartile, line: median, whiskers: 1.5 × interquartile range beyond the box, outliers are stacked) with ChIP-Seq signal in blue and in vitro 8-mers in grey.

Figure 6

Examples of sites with more Max than Myc. Genome browser views of normalized Myc, Max, and ‘Max minus Myc’ occupancy and peaks generated by ChIP-Seq Peak. The distribution of the top 12 6-mers with their relative in vitro occupancies is also displayed. (A) A large region from chromosome 17. (B, C) Close-ups of the two regions with extra Max showing alignment with high scoring 6-mers.

Figure 7

Two models of Myc-Max recruitment. The top panel illustrates the prevailing view of E-box recruitment of Myc-Max followed by interactions of Mediator and other factors that affect transcription. The bottom panel is an alternative view supported by the results presented here in which Myc-Max is recruited by the transcription machinery and bound with a relaxed sequence requirement to promoter DNA. We suggest that the Myc-Max occupancy of the promoter region helps keep the promoter free of nucleosomes and the resulting increased accessibility of the promoter is responsible for the amplification of gene expression caused by Myc.