Sequence specificity incompletely defines the genome-wide occupancy of Myc
© Guo et al.; licensee BioMed Central Ltd. 2014
Received: 17 June 2014
Accepted: 22 September 2014
Published: 7 October 2014
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.
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.
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.
c-Myc was initially identified as a proto-oncoprotein and subsequently demonstrated to be a global regulator of transcription -. In vitro, the basic helix-loop-helix leucine zipper (bHLHZip) domain of Myc binds preferentially, albeit very weakly, to double stranded DNA containing the palindrome CACGTG and this is considered the canonical E-box ,. Myc pairs with Max and the heterodimer binds to CACGTG with higher affinity -. A crystal structure of the bHLHZip domains of Myc-Max bound to DNA revealed that the two proteins interact through each protein’s bHLHZip domain and each make specific contacts with four bases . These initial observations and a large number of studies on the effects of Myc on specific genes led to what is now the prevailing model that Myc, in conjunction with Max, binds to E-box sequences and subsequently regulates transcription by Pol II -. However, this model does not explain how specificity for Myc is achieved as there are a number of other bHLHZip protein family members that can bind to the same sequence ,.
Many studies attempting to identify Myc target genes found that the sets of genes regulated by Myc displayed great variation depending on the cell types and conditions used . A significant advancement in understanding Myc function was achieved by two comprehensive studies from the Young and Levens labs ,. Both used inducible systems to show that Myc, when switched on, utilized existing expression programs and globally amplified transcription leading to an increase in the majority of expressed mRNAs. One study concluded that induction of Myc in P493 cells led to increased binding by Myc-Max heterodimers at the E-box containing core promoter sequences of actively transcribed genes . The other study using primary B cells treated with lipopolysaccharide to induce Myc expression found a only a `loose association’ of Myc with E-boxes due to the high frequency of random occurrence of degenerate E-box sequences .
Regulation of gene expression is controlled predominantly through the action of DNA-binding transcription factors that affect both initiation and elongation. A prominent feature of metazoan genomes is the promoter proximal paused Pol II that is found engaged in transcription about 30 to 80 bp downstream of the transcription start site (TSS) on most expressed genes -. The transition of these paused polymerases into productive elongation requires the kinase activity of the Positive Transcription Elongation Factor b, P-TEFb ,. MYC was the first gene shown to be regulated by elongation  and Myc itself associates with P-TEFb - and causes an increase in productive elongation on targeted genes -.
The mechanism of Myc regulation of transcription has been assumed to involve Myc-Max heterodimers binding to E-boxes near TSSs and then influencing the function of the transcription machinery. The discovery that Myc globally regulates essentially all expressed genes , hints that Myc recruitment may be more general. An earlier study showed that Myc occupancy primarily correlated with chromosomal loci with an `open conformation’ ,. These regions are often occupied by the transcription machinery ,. To test the basic assumption that Myc-Max heterodimers predominately occupy high affinity DNA elements in cells, we performed detailed analyses of available human ChIP-Seq datasets for Myc, Max, and Pol II and determined the relationship between sites of occupancy of Myc and Max and locations of high affinity DNA elements. Surprisingly, the global occupancy of Myc and Max strongly correlated with Pol II transcription machinery rather than with sequences that the heterodimer prefers in vitro.
Genome occupancy of Myc and Max correlates with Pol II
These ChIP-Seq datasets were also compared using an algorithm that measures the similarity of peak positions and heights in any two datasets (Figure 2C). A value of 0 means there is no overlap of the signals at any position and 1 indicates the datasets are identical. Myc and Max most closely correlate with each other, as expected. Importantly, the second highest genome-wide correlation for both Myc and Max was Pol II. The correlation of Myc with Pol II would not be expected to be as high as its correlation with Max because of the approximately 100 bp offset of Myc (and Max) from the peaks of promoter proximal paused Pol II. As expected, CTCF was the least well correlated with all datasets because it is bound by its CTC-containing motif mainly in intragenic regions . The correlation analysis was extended to include Fos, Jun, and E2F1 and none of these factors correlated as well with Pol II as Myc and Max (Additional file 1: Figure S2).
We extended our analyses to eight human cell lines with Myc, Max, and Pol II ChIP-Seq datasets. All eight datasets were combined into a multi-genome metagene analysis and the results clearly indicated that on average, as was found in HeLa cells, Myc and Max were about 100 bp upstream of the promoter proximal paused Pol II and Myc is shifted downstream from Max (Figure 2D). Datasets for the Med1 subunit of Mediator were available for four of these cell lines and the multi-genome analysis displayed a similar distribution for Myc and Med1 including a downstream bulge over the promoter proximal paused Pol II (Figure 2E). These analyses strongly suggest that the Myc might be recruited to these genomic loci by the transcription machinery, with Mediator as a reasonable candidate.
Under stoichiometric conditions with high concentrations of proteins and DNA, Myc-Max heterodimers display relaxed sequence specificity
Dissociation constants of the protein-DNA complexes were determined under the required non-stoichiometric conditions using 0.01 nM radiolabeled probe. MaxL and Myc-MaxL displayed tight binding to CACGTG (Kds of 0.4 nM and 0.1 nM, respectively) (Figure 3D). MaxL did not form a discrete complex with the ATCTAG probe with the concentrations of protein tested (Kd >1 ?M), but instead gave only a smeary band below the position of a tightly bound complex (arrow) (Figure 3E). This is due to initial binding followed by release of the probe during the running of the gel. Myc-MaxL displayed significant affinity for the ATCTAG probe (Kd = 20 nM) (Figure 3F). Competition binding assays under these non-stoichiometric conditions demonstrated that CACGTG containing DNA was able to compete with the binding of MaxL and Myc-MaxL to the CACGTG probe (Figure 3G and H). At 1,000-fold higher concentration, the ATCTAG containing DNA was also able to compete for binding of both Max and Myc-Max to the CACGTG probe (Figure 3G and H). These results indicate that both Max and Myc-Max prefer to bind to the probe containing CACGTG as expected. In the stoichiometric assay described above, 125 nM Myc-Max but not 250 nM Max dimer formed discrete complexes with ATCTAG DNA. In the non-stoichiometric assay, Myc-Max displayed significantly higher affinity for the ATCTAG probe than Max and this difference was seen at 10 and 100 nM protein (Figure 3F). In the competition assay (1 nM protein) the difference between Myc-Max and Max was not seen. The concentration dependent change in the relative binding of Myc-Max and Max to non-specific DNA we observed could be related to the different on and off rates for the two proteins . From all of the in vitro binding studies shown so far, we conclude that Myc-Max demonstrates a sequence preference, but that it also has significant affinity for DNA lacking a canonical E-box.
Determination of the complete sequence preference for Myc-Max and comparison with occupancy in cells
The problem of not knowing the relative affinity of Myc-Max for the previously proposed non-canonical E-boxes was resolved by the PBM assays so we used that information to examine the role intrinsic DNA affinity plays in the occupancy of the heterodimer in cells. A genome browser track comprising the location and relative in vitro occupancy (percent of the top binding site) of each of the top 12 6-mers was generated that graphically displays the range of intrinsic affinities across the genome (Figure 4B). This is an improvement compared to just marking canonical and non-canonical E-boxes without regard to relative affinities of the different sites. Visual comparison of the occupancy of Myc, Max, and Pol II in HeLa cells to the accurate distribution of intrinsic affinities does not provide evidence for a strong correlation between intrinsic affinity and occupancy in cells (Figure 4B). Closer inspection revealed that strong binding sites were not occupied and Myc and Max were found in regions that did not have any of the top 12 6-mer sites (Figure 4C and D).
To further assess whether the intrinsic binding specificity of Myc-Max determines its level of genomic occupancy in the cell, the same Myc sites were rank-ordered by their ChIP-Seq occupancy and compared to the signal of the best 8-mer within a 100 bp window around each peak. The Myc ChIP-Seq signal of the top 30,000 peaks varies about 30-fold (Figure 5B, blue line showing decreasing occupancy from left to right). Using the same x-axis, a second plot was generated that displays the relative affinity of the best 8-mer associated with each of these Myc peaks (Figure 5B, black dots). A slight preference for high affinity 8-mers is visible over the top 5,000 Myc peaks, but the overwhelming conclusion is that 8-mers with a wide range of in vitro occupancies are found around Myc peaks irrespective of the level of in vivo occupancy (Figure 5B). While a statistically significant correlation can be observed between Myc ChIP-Seq occupancy and in vitro 8-mer binding strength, this relationship is weak (Spearman correlation coefficient: ρ = 0.22, P value < 2.2 × 10-16). Had the cellular occupancy correlated well with the affinity for the underlying DNA sequences, there would have been a cloud of black dots clustered around the blue curve in Figure 5B and the Spearman correlation coefficient would have been close to 1. A plot of the same data after ChIP-Seq peaks were grouped into log-scaled bins provides a more detailed view of the high occupancy sites in cells that might be expected to correlate better with intrinsic DNA affinities. However, the huge range of in vitro occupancy scores is clearly found even for the highest occupancy sites (Figure 5C). All these analyses suggest that Myc occupancy is driven only to a small extent by its intrinsic sequence preference, and additional mechanisms are required to recruit Myc to its genomic binding locations in the cell.
Genomic sites with higher relative levels of Max
Consideration of the size of the mammalian nucleus, the amount of Myc expressed, and the biochemical parameters of Myc-Max binding to DNA determined here leads to the interesting conclusion that almost all the Myc should be bound to DNA regardless of the influence of specific sequences. Quantification of Myc was recently reported for Myc-inducible P493-6 cells. It was found that before induction there were 13,000 molecules of Myc per cell and 77,000 and 362,000 molecules per cell after 1 or 24 h of induction. Taking into account the estimated volume of a mammalian cell nucleus of 4 × 10-13 liters, this means the concentration of Myc would start at 50 nM before induction and reach 1.5 ?M after 24 h induction. If free DNA was available, Myc-Max should be bound before and after induction since the Kd for even low affinity sites is 20 nM. An analysis of DNase I hypersensitivity, FAIRE, and occupancy of DNA binding transcription factors from ENCODE data in five human cell lines led to the estimation that about 3% of the genome is relatively free of histones and available for binding of factors . This would be about 180,000,000 bp considering there are two genomes per cell. This means that even at the highest concentration of Myc, there are 500 bp of available DNA per Myc molecule and, therefore, all Myc should be bound to DNA. Our in vitro binding data demonstrates that the top 16 8-mers cover the top 65% of the normalized occupancy (see Figure 4). One of these 8-mers should occur on average only every 4,000 bp. Therefore, most of the Myc should be bound to lower affinity sites and this is borne out by our bioinformatic analyses. It has been estimated that there are about 180,000 Pol II molecules engaged in transcription in the average HeLa nucleus and it is likely significant that the level of Myc in a high expressing cell is similar, since Myc correlates highly with the position of engaged Pol II. Sites of occupancy of Pol II and Myc rise concomitantly during induction of Myc in P493-6 cells (Additional file 1: Figure S4), further connecting Myc and promoter proximal paused Pol II .
The specific interaction(s) that bridges Myc with the transcription machinery is not clear, but Mediator is found in a similar position to Myc upstream of the promoter proximal paused Pol II. Like Mediator, Myc and Max peaked upstream of the TSS. Both the Myc and Mediator peaks extended into the downstream region occupied by Pol II. This could be due to crosslinking between the N-terminal transcription activation domain of Myc with Mediator, which has been shown to interact with both Pol II , and Myc , leading to indirect crosslinking through the polymerase to downstream DNA. Perhaps in an analogous manner, the N-terminal domain of KLF3 which interacts with other transcription factors but is not involved in DNA binding has been recently demonstrated to play a major role in genome occupancy . Besides Mediator  and Pol II , Myc has been shown to associate with a number of factors including TRRAP, P-TEFb, chromatin remodeling machinery, DNA repair machinery, and other proteins involved in transcription and other processes ,,,,,.
Our results have clarified the DNA binding preferences of the Myc-Max heterodimer, widening the range of core binding sites bound specifically by Myc-Max and suggesting that non-specific interactions could also be significant. In examining binding to all possible 8-mers it became apparent that sequences flanking the core 6-mers were important. Changing the flanking sequences of the canonical E-box 6-mer from GCACGTGC to TCACGTGA resulted in a four- or five-fold reduction in Myc-Max and Max binding. A similar reduction has been previously noted for Pho4p but not Cbf1p, two yeast bHLH proteins ,. Despite our expanded understanding of the relative interaction potentials of Myc-Max with all possible 8-mers, a strong correlation of Myc occupancy in cells to the underlying sequence could not be made.
How did it become so generally accepted that Myc-Max functioned as a specific DNA binding factor? As shown here and from earlier studies ,-, Myc-Max does have a preference for certain sequences in vitro. Previously, these preferences were determined using EMSAs that routinely included unlabeled carrier DNA. Unfortunately, this leads to an inaccurate determination of the actual range of affinities for different sequences. Many reporter assays have been performed where the presence of a CACGTG E-box sequence enhances the expression of the reporter (for example, ,). In almost all such experiments, only small (2-fold or less) effects are found and sequence-independent effects of Myc are normalized away. The small effects seen are consistent with our model in which having high affinity sites close to the promoter could fine tune the recruitment of Myc. Another issue arises from the misinterpretation of results obtained by programs, such as Multiple Em for Motif Elicitation (MEME) , which discover sequence motifs that are significantly enriched in a population of DNA sequences. However, identification of a motif (with a corresponding low P value) does not mean that sites matching the motif are present in all of the DNA sequences analyzed. In fact, very significant enrichment for a particular motif can be observed even when only a very small fraction of the analyzed sequences contain the motif. A second problem arises because of the length of the sequences analyzed and the resolution of actual binding site. Reasonable quality ChIP-Seq datasets like those analyzed here can determine individual binding sites to within 25 to 50 bps. If the fragments analyzed by a motif discovery algorithm are more than about 100 bp (as they usually are), a particular motif can be present in the sequence analyzed but not be bound (for example, see Figure 1B).
The model we propose for Myc might be applicable to other transcription factors. Significant overlap of occupancy between Myc and other factors such as AP-1 and AP-2 can be observed and two recent ENCODE reports showed co-association of many transcription factors binding to `surprisingly plastic’ DNA sequences ,. Indeed, our results suggest that it may be time to revisit the significance of sequence-specific binding for many transcription factors in higher eukaryotes. The E. coli lac repressor paradigm , in which occupancy of a specific site on a 4 million bp genome is driven by a seven order of magnitude difference between specific and non-specific binding, may not apply to certain human transcription factors. The generally repressive structure of chromatin in eukaryotes can mask most non-specific and specific sites thereby reducing the complexity of DNA that would otherwise be available for binding . In addition, nucleosomes would also obstruct 1-D sliding, one of the important mechanisms used by lac repressor to locate specific sites . The human genome encodes about a thousand DNA binding transcription factors with a wide range of sequence specificity. At one extreme, CTCF is almost always bound to one of several sequence motifs, as determined by differential involvement of its 11 Zinc fingers . TBP on the other hand is found at all promoters regardless of the presence of the TATA sequence it recognizes due to interaction with the transcription machinery . The metazoan genomic landscape may be too complex for all factors to rely solely on sequence specificity for occupancy and function.
We combined an extensive determination of the DNA binding properties of Myc-Max with genome-wide occupancies of Myc, Max, and Pol II and conclude that the affinity of Myc-Max for specific DNA sequences cannot be the main determinant of Myc genomic occupancy in cells. Our results indicate that the range in affinities in vitro for different sequences covers only a little over two orders of magnitude (Kd = 10-8 to 10-10 M). Even taking into account the fact that much of the human genome is not accessible due to chromatin structure, we found that Myc occupancy was not well correlated with affinity for underlying DNA in accessible regions. We found instead a strong correlation of Myc (and Max) occupancy with that of Pol II and on average Myc was located about 100 bp upstream of the promoter proximal paused Pol II. We propose that Myc is a general factor brought to promoters predominately by protein-protein interactions and like TBP, its recruitment to promoters does not strictly require sequence specific binding.
Materials and methods
The alignment files of HeLa, GM12878, and K562 cell lines were downloaded from human ENCODE Project at UCSC . The raw sequence files of H128, H2171, MM1S, P493, and U87 cell lines were obtained from GSE36354. These raw sequences were aligned using ELAND to NCBI Build 36.1 (UCSC hg18) of the human genome. Only sequences that mapped uniquely to the genome with zero or one mismatch were used for further analysis. When multiple sequences mapped to the same genomic position, a maximum of two reads mapping to the same position were used. The sequenced reads were extended 200 bp to account for the size of sequenced fragments and then allocated into 25 base pair bins. The data from each bin were combined to generate the wiggle (WIG) files, which can be uploaded to UCSC genome browser. Max and Myc datasets from HeLa cells were further normalized for the total number of reads and the normalized Myc dataset was subtracted from normalized Max dataset to identify the genomic regions having extra Max compared to Myc.
RefSeq gene list
The complete set of human RefSeq genes was downloaded from the UCSC table browser  on 1 January 2012. A custom annotated set of 20,886 RefSeq genes was generated by merging the TSSs from the same gene within 500 bases of each other, and removing the all TSSs within 1,000 bases of each other. This custom list was used for all analyses.
The number of reads within 10,000 bases of the TSS of each RefSeq gene was tabulated without binning. The average value of the lowest 2,000 of 20,000 data points was subtracted from each position, and then data were normalized so that the area under each curve was equal. For Figure 2D, the data generated from the previous step were summed at each position using eight cell types from ENCODE (HeLa, GM12878, and K562) and GSE36354 (H128, H2171, MM1S, P493, and U87). Figure 2E was generated from GSE36354 (H2171, MM1S, P493, and U87).
Generation of heatmaps
Heat maps were generated using the program R . Genes were rank-ordered based on the sequence density for Pol II from -2 kb to +2 kb from the TSS. Using this order, base pair resolution sequence density for Pol II, Myc, Max, and CTCF for 20,886 genes was displayed without binning. The raw images were 21,000 × 4,000 pixels each and were adjusted identically using the gamma adjustment in Corel PhotoPaint (version X3) to allow visualization of the wide range of data (Figure 2B).
Correlation of datasets
For Figure 2C, a total of 4 WIG tracks were generated using ENCODE data including Myc, Max, Pol II, and CTCF. The reads at each position were sorted in ascending order, and the cumulative percentage was calculated using R. The lowest 95% of the data points were subtracted as background since they represent mainly isolated single reads and the remaining reads were normalized to reads per million. The difference between two tracks was quantified as the sum of the absolute values of two tracks at each point. The correlation between two transcription factors was calculated within the range of 0 to 1, with a correlation of 1 indicating that the two tracks have a perfect match and 0 denoting total independence.
A peak finding algorithm (ChIP-Seq Peak)  was applied to determine precise position and height of each significant peak of Myc, Max, and Pol II. The height is equal to the number of reads contributing to the peak not the highest value at the summit of the peak. The peak positions with heights at least 30 (about 10% of highest peak height) were used to retrieve the sequences within 50 bases of peak locations. To generate Max minus Myc peaks, the Myc and Max datasets were first normalized so that the total reads were 1 million and then subtracted from each other before the ChIP-Seq Peak algorithm was applied. The peaks identified were further annotated with the RefSeq genes and RNA expression levels (GSE23316) across the genome. A total of 351 genes that have a peak of extra Max within 250 bp of their TSSs were classified into three Gene Ontology (GO) categories . The normalized HeLa Myc and Max ChIP-Seq datasets, the Max minus Myc dataset, and its associated ChIP-Seq Peak dataset are available on GEO (GSE43227).
Expression and purification of proteins
Coding sequences of Myc (gi:29839758), MaxL (gi:21704261) and MaxS (gi:21704263) were amplified from HeLa cDNA and individually cloned into pET21a (C-terminal His-tag). All proteins were expressed in BL21 star E. coli after overnight induction of MaxL and MaxS at 18°C or 4 h induction of Myc at 37°C. Soluble Max proteins were purified over Ni-NTA and Mono Q for MaxS or Mono S for MaxL as described for Gdown1 . Yields were approximately 20 mg per liter. Myc was purified over Ni-NTA and Mono Q in the presence of 7 M urea yielding 7 mg of Myc per liter. Both Myc-Max heterodimers were prepared by mixing equal moles of Myc and Max in 6 M urea followed by step dialysis against 4 M, 2 M, 1 M, and 0.5 M urea with 500 mM HGKEDP (25 mM HEPES, pH 7.6, 15% glycerol, indicated KCl, 0.1 mM EDTA, 1 mM DTT, and 0.1% of a solution of saturated PMSF in isopropanol) for 1.5 h each, then against 100 mM HGKEDP overnight. The renatured Myc-Max proteins were finally purified over Mono Q in HGKEDP. All protein samples were aliquotted and kept at -80°C. In the absence of urea, Myc was prone to aggregation, but Max and Myc-Max chromatographed cleanly and behaved well. Myc-Max heterodimers partially dissociated over time generating a small amount (<10%) of Max homodimers.
Binding reactions were 25 mM HEPES (pH 7.6), 100 mM KCl, 1 mM DTT, 0.1 mM EDTA, and 0.01% Triton. For reactions analyzed on silver stained gels, 200 ng of protein and 0, 0.1, 0.3, 1, or 3-fold molar excess of unlabeled dsDNA were used in each reaction. For the reactions using radiolabeled probes, each strand of the indicated probe was end-labeled with ?-32P-ATP and then annealed to generate dsDNA. Each reaction contained 0.01 nM labeled dsDNA and 0.01 pM, 0.1 pM, 1 pM, 0.01 nM, 0.1 nM, 1 nM, 10 nM, or 100 nM protein. For competition assays 0.01 nM labeled CACGTG probe, 1 nM of the indicated protein and the indicated amounts of non-labeled competitor DNA were used. After 30 min at room temperature, Ficoll (2% final) was added to each reaction and the samples were run on a 6% polyacrylamide gel in 0.5X Tris/glycine buffer at 10 mA for 2 h. The gels were then analyzed by silver staining or phosphorimaging.
Protein-binding microarray (PBM) experiments were performed as described previously . Briefly, 4 x 44 K arrays (Agilent Technologies; AmadID 015681) containing the `all 10-mer’ universal PBM design were used. Arrays were incubated with a PBS buffer based protein mixture of 10 nM His-tagged Myc/Max heterodimer or Max homodimer, 2% milk, 200 ng/?L BSA, 50 ng/?L Salmon Testes DNA, and 0.02% TX-100. Bound protein was tagged with 10 ng/?L anti-His antibody conjugated to Alexa 488 (Qiagen; 35310) in PBS with 2% milk. The microarrays were scanned using a high-resolution GenePix 4400A scanner (Molecular Devices). Data were analyzed to obtain fluorescence intensities for all 8-mers, as described previously . The raw median intensities and normalized in vitro occupancies for Myc-Max and Max-Max across all 8-mers are provided in Additional file 3: Table S2. Genome tracks were created by identifying all 8-mers in the human genome (NCBI build 36, hg18) that contain a core 6-mer that appeared in the top 200 8-mers (these 6-mers were CACGTG, CGCGTG, CACATG, CACGAG, CATGCG, AACGTG, CATGAG, CATATG, CATGCA, CATGCT, GTGCAC, and CTCGAG). Although only the central 6-mer was shown in the genome track, the in vitro occupancy scores were obtained by normalizing the observed intensity for each 8-mer to the intensity of the highest ranked 8-mer (in this case, CCACGTGG). The raw PBM data as well as the 8-mer intensities have been deposited in GEO (GSE58570) and the genome track for the top 12 6-mers has been deposited in GEO (GSE43227).
Statistical analyses were performed in R. Fisher’s exact test (dhyper) was used to compare the frequency of CACGTG sites in Myc-occupied regions and in all accessible DNA (DNase I hypersensitive regions ). A similar analysis was performed using Pol II ChIP-Seq peaks instead of CACGTG sequences. In Figure 5A, in vitro Myc 8-mer occupancies were compared using the Wilcoxon rank-sum test (wilcox.test with the paired parameter set to false). A receiver operating characteristic (ROC) curve was used to assess the enrichment of sites with high in vitro 8-mer occupancies in the Myc ChIP-Seq peaks. A ROC curve is a plot of false positive rate (1-specificity) versus true positive rate (sensitivity) computed for different 8-mer occupancy cutoffs. Each point on the ROC curve corresponds to one cutoff. Sequences with 8-mer occupancy above or below the cutoff are predicted positives (that is, predicted to be bound by Myc in vivo) or predicted negatives (that is, predicted not bound by Myc in vivo), respectively. In Figure 5B, the correlation between Myc ChIP-Seq and in vitro occupancies was tested using Spearman’s rank correlation coefficient (cor.test with the method parameter set to Spearman).
JG and JJC cloned and expressed the Myc and Max proteins used. JG and FKF performed EMSAs. JS and RG performed the PBM assays and analyzed results with suggestions from DHP. TL, KAN, RG, and JS performed bioinformatic analyses with contributions from JG and DHP. DHP made the original observations that led to the study. JG, KAN, and DHP did most of the writing of the manuscript in consultation with all others. All authors approved the final manuscript.
We wish to thank Dan Weeks and Miles Pufall for thoughtful, spirited discussions. This research was supported by National Institutes of Health grant GM35500 to DHP, a PhRMA Foundation Research Starter Grant to RG, an American Heart Association postdoctoral fellowship 12POST12040106 to JG, a Duke Translational Medicine Quality Framework postdoctoral fellowship to JS, a University of Iowa Presidential Graduate Fellowship to KAN, and in part through computational resources provided by the University of Iowa.
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