Developmental roles of 21 Drosophila transcription factors are determined by quantitative differences in binding to an overlapping set of thousands of genomic regions
- Stewart MacArthur†1, 6,
- Xiao-Yong Li†1, 2,
- Jingyi Li†3,
- James B Brown3,
- Hou Cheng Chu1,
- Lucy Zeng1,
- Brandi P Grondona1,
- Aaron Hechmer1,
- Lisa Simirenko1,
- Soile VE Keränen1,
- David W Knowles4,
- Mark Stapleton1,
- Peter Bickel3,
- Mark D Biggin1Email author and
- Michael B Eisen1, 2, 5Email author
© MacArthur et al.; licensee BioMed Central Ltd. 2009
Received: 26 January 2009
Accepted: 23 July 2009
Published: 23 July 2009
We previously established that six sequence-specific transcription factors that initiate anterior/posterior patterning in Drosophila bind to overlapping sets of thousands of genomic regions in blastoderm embryos. While regions bound at high levels include known and probable functional targets, more poorly bound regions are preferentially associated with housekeeping genes and/or genes not transcribed in the blastoderm, and are frequently found in protein coding sequences or in less conserved non-coding DNA, suggesting that many are likely non-functional.
Here we show that an additional 15 transcription factors that regulate other aspects of embryo patterning show a similar quantitative continuum of function and binding to thousands of genomic regions in vivo. Collectively, the 21 regulators show a surprisingly high overlap in the regions they bind given that they belong to 11 DNA binding domain families, specify distinct developmental fates, and can act via different cis-regulatory modules. We demonstrate, however, that quantitative differences in relative levels of binding to shared targets correlate with the known biological and transcriptional regulatory specificities of these factors.
It is likely that the overlap in binding of biochemically and functionally unrelated transcription factors arises from the high concentrations of these proteins in nuclei, which, coupled with their broad DNA binding specificities, directs them to regions of open chromatin. We suggest that most animal transcription factors will be found to show a similar broad overlapping pattern of binding in vivo, with specificity achieved by modulating the amount, rather than the identity, of bound factor.
Sequence-specific transcription factors regulate spatial and temporal patterns of mRNA expression in animals by binding in different combinations to cis-regulatory modules (CRMs) located generally in the non-protein coding portions of the genome (reviewed in [1–4]). Most of these factors recognize short, degenerate DNA sequences that occur multiple times in every gene locus. Yet only a subset of these recognition sequences are thought to be functional targets [1, 5, 6]. Because we do not sufficiently understand the rules determining DNA binding in vivo or the transcriptional output that results from particular combinations of bound factors, we cannot at present predict the locations of CRMs or patterns of gene expression from genome sequence and in vitro DNA binding specificities alone.
To address this challenge, the Berkeley Drosophila Transcription Network Project (BDTNP) has initiated an interdisciplinary analysis of the network controlling transcription in the Drosophila melanogaster blastoderm embryo [7–12]. Only 40 to 50 sequence-specific regulators provide the spatial and temporal patterning information to the network, making it particularly tractable for system-wide analyses [13–15].
The 21 sequence-specific transcription factors studied
DNA binding domain
A-P early maternal
A-P early maternal
A-P early gap
C2H2 zinc finger
A-P early gap
Receptor zinc finger
A-P early gap
C2H2 zinc finger
A-P early gap
C2H2 zinc finger
A-P early terminal
Receptor zinc finger
A-P early terminal
A-P early gap-like
A-P pair rule
A-P pair rule
A-P pair rule
A-P pair rule
Sloppy paired 1
A-P pair rule
C2H2 zinc finger
C2H2 zinc finger
Tens of functional CRMs have been mapped within the network (for example, [8, 19, 24–26]), which each drive distinct subsets of target gene expression and which have generally been assumed to be each directly controlled by only a limited subset of the blastoderm factors. For example, the four stripe CRMs in the even-skipped (eve) gene are each controlled by various combinations of A-P early regulators, such as BCD and Hunchback (HB), and a separate later activated autoregulatory CRM is controlled by A-P pair rule regulators, including EVE and PRD [24, 27–29].
The different transcriptional regulatory activities of these factors leads them to convey quite distinct developmental fates and morphological behaviors on the cells in which they are expressed. For example, the D-V factors Snail (SNA) and Twist (TWI) specify mesoderm, the pair rule factors EVE and Fushi-Tarazu (FTZ) specify location along the trunk of the A-P axis, and TLL and Huckebein (HKB) specify terminal cell fates.
The blastoderm regulators include members of most major animal transcription factor families (for example, Table 1) and act by mechanisms common to all metazoans . Thus, the principles of transcription factor targeting and activity elucidated by our studies should be generally applicable.
We previously used immunoprecipitation of in vivo crosslinked chromatin followed by microarray analysis (ChIP/chip) to measure binding of the six gap and maternal regulators involved in A-P patterning in developing embryos (Table 1) . These proteins were found to bind to overlapping sets of several thousand genomic regions near a majority of all genes. The levels of factor occupancy vary significantly though, with the few hundred most highly bound regions being known or probable CRMs near developmental control genes or near genes whose expression is strongly patterned in the early embryo. The thousands of poorly bound regions, in contrast, are commonly in and around house keeping genes and/or genes not transcribed in the blastoderm and are either in protein coding regions or in non-coding regions that are evolutionarily less well conserved than highly bound regions. For five factors, their recognition sequences are no more conserved than the immediate flanking DNA, even in known or likely functional targets, making it difficult to identify functional targets from comparative sequence data alone.
Here we extend our analysis to an additional 15 blastoderm regulators belonging to four new regulatory classes: A-P terminal, A-P gap-like, A-P pair rule and D-V (Table 1). We find that these proteins, like the A-P maternal and gap factors, bind to thousands of genomic regions and show similar relationships between binding strength and apparent function. Remarkably, these structurally and functionally distinct factors bind to a highly overlapping set of genomic regions. Our analyses of this uniquely comprehensive dataset suggest that distinct developmental fates are specified not by which genes are bound by a set of factors, but rather by quantitative differences in factor occupancy on a common set of bound regions.
Results and Discussion
We performed ChIP/chip experiments to map the genome-wide binding of 15 transcription factors and analyzed these data along with the six factors whose binding we have previously described. In addition to these 21 factors, we also determined the in vivo binding of the general transcription factor TFIIB, which, together with previous data on the transcriptionally elongating, phosphorylated form of RNA polymerase , provide markers for transcriptionally active genes and proximal promoter regions.
ChIP/chip is a quantitative measure of relative DNA occupancy in vivo
The numbers of genomic regions bound in blastoderm embryos
Number of bound regions
Overlap between antibodies for the same factor
Amino acids recognized
A-P early gap
A-P early terminal
A-P early gap-like
A-P pair rule
Pol II H14*
We carried out an extensive series of controls and analyses to validate the antibodies and array data, and to ensure that our array intensities could be interpreted as a quantitative measure of relative transcription factor occupancy on each genomic region, that is, as a measure of the average numbers of molecules of a particular factor occupying each region (see  for further details).
We used two different methods to estimate FDRs, one based on precipitation with non-specific IgG, and the other based on statistical properties of data from the specific antibody alone. These estimates broadly agree (Additional data file 2). Our previously published quantitative PCR analysis of immunoprecipitated chromatin for regions randomly selected from the rank list of bound regions and also control BAC DNA 'spike in' experiments support the FDR estimates, suggest that the false negative rate is very low for all but the most poorly bound regions, and indicate that the array intensity signals correlate with the relative amounts of genomic DNA brought down in the immunoprecipitation .
Finally, the relative array intensity scores from our formaldehyde crosslinking ChIP/chip experiments broadly agree with the relative density of factor binding detected by earlier Southern blot-based in vivo UV crosslinking [30, 31] (Additional data file 5). For BCD, FTZ and PRD the Pearson correlation coefficients are 0.79, 0.67, and 0.48, respectively, comparing the data from these two assays on the same genomic regions. This agreement is important because it argues that the measured relative signals in both assays are not powerfully influenced by differences in crosslinking efficiency to various DNAs, indirect crosslinking of proteins to DNA via intermediary proteins (which should not be detected by UV crosslinking), or differences in epitope accessibility during immunoprecipitation (which again should be much lower for UV crosslinking). Instead, the correspondence indicates that both these methods provide a reasonable estimate of the relative number of factor molecules in direct contact with different genomic regions in vivo.
Binding to thousands of genomic regions over a relatively narrow range of occupancies
Percentage of genes whose transcription start site is within 5 kb of ChIP/chip peaks
% genes close to 1% FDR peaks
% genes close to 25% FDR peaks
A-P pair rule
A quantitative continuum of binding and function
Our earlier analyses of the six maternal and gap A-P factors showed that although these proteins bind to large number of regions, the most highly bound regions clearly differ in many regards from the more poorly bound, many of which may not be functional targets. Parallel analyses of the other 15 factors demonstrate the same trends.
First, for those factors for which a significant number of target CRMs are known, the few hundred most highly bound regions are enriched for these targets. Transgenic promoter, genetic, in vitro DNA binding and other data have identified a set of 44 CRMs as direct targets of subsets of the A-P early factors and 16 CRMs as direct targets of particular combinations of D-V regulators [8, 25, 32]. Figure 4 and Additional data file 8 show that the 500-bp ChIP/chip peaks that overlap CRMs known to be targets of at least some members of a given regulatory class are bound by all members of that class, on average, at higher levels than the majority of genomic regions at which these proteins are detected.
Taking all of these five analyses into account, the few hundred most highly bound regions have characteristics of likely functional targets of the early embryo network. Although some poorly bound regions are also likely to be functional targets at this time, including ones weakly modulating transcription of housekeeping genes (for example, ), many do not appear to be classical CRMs that drive transcription in the blastoderm. A minority do become more highly bound in the later embryo and may be active then (our unpublished data), but the binding to many others we feel is likely to be non-functional, including that to most of those in protein coding regions.
Our analysis contrasts with the predominant qualitative interpretation of in vivo crosslinking data by other groups studying animal regulators [32–46]. Many of these groups have also shown that factors bind to a large number of genomic regions. They have not, however, noted the many differences between highly bound and poorly bound regions shown in Figures 4 to 8. In addition, with only a few exceptions [43, 44, 46], they have not seriously considered the possibility that some portion of the binding detected is non-functional. We suspect that similar correlations between levels of factor occupancy and likely function of bound regions will be found for other factors once quantitative differences amongst bound regions are considered.
Factors bind to highly overlapping regions
To calculate the probability that this extensive co-binding occurs by chance, we used the Genome Structure Correction (GSC) statistic , which is a conservative measure that takes into account the complex and often tightly clustered organization of bound regions across the genome. For the great majority of the pair-wise co-binding shown in Figures 9a, b, these probabilities have Bonferroni corrected P-values < 0.05 (all instances with z scores ≥4 in Figure 9c, d) and, thus, the overlap is highly unlikely to have occurred by chance. With such extensive co-binding, it is not surprising that some regions are bound by many factors. Averaged over all regulators, 88% of their top 300 peak windows are bound by 8 or more factors and 40% are bound by 15 or more factors (Additional data file 13).
Several recent in vivo crosslinking studies have also noted significant overlap in binding between some sequence-specific factors in animals [32, 34, 37, 44, 46]. In these other cases, however, the overlapping factors are known to have related functions and, thus, the co-binding is less surprising. Work using the DamID method showed a high overlap in binding when transcription factors with different functions and specificities were ectopically expressed in tissue culture cells , and it was suggested that these binding 'hotspots' were non-functional storage sites. In contrast to these other studies, we have found overlapping binding for a larger number of regulators, many of which are well characterized as having distinct biological and transcriptional regulatory specificities. The binding we have measured is for endogenous factors, and the greatest overlap in binding is at known and probable functional targets. Thus, it does not seem that overlapping patterns of binding reflect either shared functions or a lack of function. Instead, we must ask how the undoubtedly distinct specificities of the blastoderm factors arise despite the overlap.
Quantitative differences in binding correlate with biological and transcriptional regulatory specificity
Other work, however, cautions against assuming that all of the lower level interactions shown in Figure 10 result in transcriptional regulation. In the case of the binding of A-P gap factors to the eve autoregulatory element, transgenic promoter analysis indicates that this binding is not sufficient to detectably activate this CRM in early stage 5 embryos . A similar argument can be made for binding of A-P pair rule factors to the eve stripe CRMs [24, 27, 28, 49]. In these cases, either this lower level binding is non-functional or it plays an augmentary role only in the context of multiple promoter elements. It is not sufficient for regulation on its own.
First, we used the previously described GSC statistic for the likelihood that two factors bind the same regions more frequently than expected by chance, but this time focusing only on the overlap between highly bound regions. All 441 pair-wise comparisons of overlap were computed between the 300 regions bound most highly by each factor (Figure 13a) and separately between the 300 next most highly bound regions (Figure 13b). For both cohorts, not surprisingly given our earlier analysis, co binding between most pair-wise combinations of factors occurs far more frequently than expected by chance, even where the proteins belong to different regulatory classes (z scores ≥4 in Figure 13a, b). However, for the top 300 bound regions, there is an obvious further preferential overlap among A-P early regulators as well as a moderate preference among the A-P pair rule factors and the D-V factors (Bonferroni corrected Mann Whitney tests suggest that, taken collectively, the preferential co-binding among A-P early regulators is highly significant (P < 9 × 10-15), while that among the A-P pair rule factors and the D-V factors is moderately significant (P < 2 × 10-3) for both; Additional data file 13). The next most highly bound cohort shows reduced preferential co-binding within regulatory classes, with only that among A-P early regulators being significant (P = 7 × 10-9; Figure 13b; Additional data file 13).
Thus, across a broad array of mostly uncharacterized genomic regions the levels of binding of transcription factors correlate with the expectation that factors with more similar functions show more similar binding specificity. Consistent with our previous observation that highly bound regions appear more functionally significant, the distinctions in binding preferences between regulatory classes is larger on the most highly bound regions. Just as on the known CRMs, however, the distinctions between the different classes are relatively modest, suggesting that the regulatory specificity of transcription factors in general may be fuzzier than widely realized and perhaps also suggesting a role for post-DNA-binding events to increase the distinctions between factors.
All of the preceding analyses consider binding to short genomic regions. The target genes of blastoderm factors, however, are often found associated with several such regions (for example, Figure 10). Thus, while the above analyses establish that regulators show quantitative preferences for binding to individual genomic regions, they do not establish if they exhibit preferences for different genes.
The GO terms associated with the 301 to 600 ranked peaks show much less difference between each factor and regulatory class, consistent again with these less highly bound regions playing a lesser role in determining biological specificity and function.
Some of the types of gene preferentially bound by the regulatory classes readily fit expectations; for example, the strong association of genes involved in A-P axis specification, patterning by pair rule genes, and trunk and head segmentation with A-P early and A-P pair rule factors. Others are unexpected, such as the preference of D-V regulators for a series of GO terms related to eye development. Most likely the differences between factors revealed in these heat maps reflect differences due to target genes that are strongly patterned along the A-P axis versus those strongly patterned along the D-V axis. Because important effectors of blastoderm regulators' functions are patterned along both body axes, because the early factors both activate or repress target genes, and because GO terms imperfectly capture and categorize the biological function of each gene, this analysis does not provide a complete description of the different specificities of each factor at the target gene level.
A general model for animal transcription factor binding and function
What mechanism, though, drives the extraordinarily extensive, overlapping pattern of binding? We speculate that the pattern is a natural consequence of these factors' intrinsic DNA binding specificities (as measured in vitro), the relatively high concentrations at which they are expressed in nuclei in which they are active, chromatin structure, and the law of mass action.
Most animal transcription factors recognize short degenerate DNA sequences that occur frequently throughout the length of most genes [1, 5, 6]. It has long been proposed on thermodynamic grounds that the majority of transcription factor molecules would be bound to DNA in the nucleus, rather than be free in solution [50–52]. In eukaryotes, only a subset of the genome is fully accessible to sequence-specific DNA binding factors because of the presence of nucleosomes [53–60]. Any several hundred base-pair segment of such accessible DNA will likely contain moderate to high affinity recognition sequences for a large proportion of transcription factors. Since many of the blastoderm factors are present at concentrations of many tens of thousand of molecules per cell [30, 61], they may well be able to significantly occupy these sites, generating a highly overlapping pattern of binding focused at open chromatin regions.
In addition to the independent interactions of transcription factors with their target DNA sequences in open chromatin, some of the overlap in binding may likely arises from protein-protein interactions in which a factor associates with an accessible region as a result of direct interactions with protein molecules bound to the region. Such indirect binding - whether between transcription factors, or mediated by the large numbers of co-factors associated with CRMs - could explain the frequent absence of high affinity DNA recognition sequences for proteins bound to a given region. Like protein-DNA interactions, protein-protein interactions are enhanced when protein concentrations are high and, thus, in vivo could also mediate low level binding at non-functional sites.
How is it possible to have so much binding that has no or little effect on transcription? Natural selection clearly acts on CRMs to preserve the proper number, arrangement and affinity of recognition sequences for whichever factors are needed for its activity. There is also evidence that selection acts against sites that might interfere with activity . Purifying selection will remove any 'spurious' binding that interferes with the proper expression of a gene. But weak binding that has only a small or no affect on transcription could well be tolerated in many cases. Just as there is a quantitative continuum of binding, there may also be a continuum of effects on transcription, and ultimately on phenotype.
We have mapped genome-wide in vivo DNA binding for the largest group to date of animal transcription factors acting in a given tissue at the same time. The work supports and extends our previous studies indicating that animal sequence-specific transcription factors bind in vivo across a quantitative continuum to highly overlapping regions close to a large percentage of genes [11, 31]. Highly bound genes include strongly regulated known and likely targets, moderately bound genes include unexpected targets whose transcription is regulated weakly, and poorly bound genes include thousands of non-transcribed genes and likely non-functional targets [9–11, 22, 31]. Factors with distinct biological specificities have highly overlapping patterns of binding. However, quantitative differences in binding to common targets generally correlate with each factor's known specificity, though these specificities appear to be more fuzzy and less distinct than commonly assumed, with a high proportion of shared targets. We propose that the broad DNA recognition properties of animal transcription factors and the relatively high concentrations at which they are expressed in cells focuses them to bind to highly overlapping sets of open chromatin regions. Our work illustrates that the qualitative analyses of in vivo DNA binding data that have widely been employed fail to reveal some of the most significant features of how transcriptional regulators behave in cells, and highlights the importance of a detailed quantitative interpretation of DNA binding patterns.
Materials and methods
In vivoformaldehyde crosslinking of embryos and chromatin purification
Embryos were collected in population cages for 1 hour, and then allowed to develop to the required stage before being harvested and fixed with formaldehyde . Embryo aging times were determined based on the transcription factor analyzed: for A-P maternal, gap, and terminal factors, as well as D-V maternal and a subset of D-V zygotic factors, including SNA and TWI, the embryos used were 2 to 3 hours old (mainly between late stage 4 and early stage 5), while for A-P pair rule, and the D-V zygotic factors, MED, MAD, and Schnurri (SHN), the embryos were 2.5 to 3.5 hours old (mainly at mid- to late stage 5). The chromatin used for immunoprecipation was isolated from the fixed embryos by CsCl gradient ultracentrifugation and then fragmented to an average size of about 700 bp.
Affinity purified antibody production
All of the antibodies used were immunoaffinity purified from rabbit antiserum. The two anti-PRD antibodies, PRD 1 and PRD 2, were available from a previous study . The MAD and MED antisera were a generous gift from L Raftery , the RUN antiserum from E Wieschaus, and the TFIIB antibody from R Tjian. For other factors, antibodies were produced in rabbits immunized with recombinant His-tagged fusion proteins expressed and purified in Escherichia coli using the Invitrogen Gateway system. Rabitts were immunized with either the full length protein (Dichaete (D), HRY, SLP1, Daughterless (DA), DL, SNA, and TWI) or portions of the protein (TLL amino acids 110 to 259, SHN amino acids 1,617 to 1,750, and SHN amino acids 2115 to 2,279). Immunoaffinity purifications were performed using E. coli-expressed purified recombinant His-tagged proteins. The amino acid sequences used (listed in Table 2) were chosen to exclude regions with any significant homology to other Drosophila proteins, as previously described . Additional results demonstrating the specificity of the antibodies are provided in Additional data file 1.
Chromatin immunoprecipitation and DNA hybridization to high density microarrays
Chromatin was immunoprecipitated and the resulting DNA was amplified and hybridized to Affymetrix Drosophila Genomic Tiling Arrays as previously described . For each antibody, duplicate immunoprecipitations were performed along with duplicate control IgG immunoprecipitations. These were each hybridized to separate arrays as were duplicate input DNA samples. All raw microarray data (CEL files) have been deposited at Array Express [E-TABM-736] . In addition, these and more processed forms of the data are available from the BDTNP's public web site, together with more detailed information about antibodies used and so on .
Primary array analysis
The data from the complete set of six arrays from each ChIP/chip experiment were processed using TiMAT  as described previously  to derive peak window locations, bound regions, 1% and 25% FDR thresholds for both the IgG and Symmetric null tests, and so on. Bound regions were associated with the gene (from release 4.3 of the D. melanogaster genome) whose 5' end was closest to the array intensity peak in the bound region. To identify the closest transcribed gene, the subset of release 4.3 annotations that completely overlap regions bound by RNA polymerase II in our ChIP-chip experiments was used.
Correlation between ChIP/chip and UV crosslinking results
Relative percentages of UV crosslinking to defined restriction fragments  and the corresponding mean oligo ChIP/chip scores of the same genomic regions are plotted as scatter plots in Additional data file 5. Pearson correlation coefficients were calculated for each plot.
Analysis of enrichment down the rank lists of recognition sequences, GO terms, distance to transcribed genes, genomic locations, and PhastCons scores
Enrichment of recognition sequences, GO terms, distance to transcribed genes, genomic locations and phastcons scores were determined essentially as described in . A statistical analysis of the significance of these plots is presented in Additional data file 4.
Distribution of ChIP/chip peak scores
In Figure 4, peaks were distributed by the mean ChIP/chip peak scores in the 500-bp peak window. For A-P early factors, a peak was associated with A-P early CRMs if the peak single nucleotide position was contained within one of the CRMs extended by 250-bp flanking regions. For D-V factors, a peak was associated with D-V CRMs in the same way.
Overlap of bound regions between transcription factors
Overlap of bound regions between two transcription factors in Figure 9 was measured by the percentage of single nucleotide peak locations of one factor contained in 1% FDR bound regions of the other factor. The top 300 peaks (1-300) and separately peaks 301 to 600 of each factor were used in the analysis. Overlap of one factor by multiple factors was measured by the percentage of peaks of that factor contained in 1% FDR bound regions of a defined number of other factors (Additional data file 13).
To calculate the liklihood z score that overlap occurs by chance (Figures 9c, d and 13a, b), z-scores were computed using the GSC statistics . A null distribution of feature-feature overlap was computed by selecting pair-wise block samples from the genome, and in each block in the pair the annotations of one of the two features of interest were swapped to yield artificial overlaps. The resulting null distribution is more realistic than that derived from other methods in that the complex and often tightly clustered organization of each feature across the genome is preserved, resulting in a much larger (conservative) estimate of standard deviation than derived via other methods. Like most methods, including feature start-site randomization, this null is Gaussian, and hence after centering, the only quantity that needs to be estimated is precisely the standard deviation.
Heat map analysis of binding of transcription factors to CRMs
In Figure 12a a CRM is defined as being bound by a transcription factor if it was overlapped by at least 300 bp by one of the factor's 1% FDR bound regions, or for CRMs less than 300 bp long, if the CRM was completely overlapped by a 1% FDR region. In Figure 12b, the binding intensity of a transcription factor to a CRM is defined by the highest 675-bp smoothed window score in the CRM for that factor, without regard to FDR threshold. The window scores for each factor were placed on the same scale by setting the highest 675-bp window contained on the whole array data to 10.
Heat map analysis of correlation of scores of bound regions between transcription factors
In Figure 13c, d, for each transcription factor, the score associated with each 500-bp peak window was derived from the mean score of oligos in the window. The scores for the equivalent 500-bp windows for each of the other 20 factors were then derived from the mean oligo scores from those datasets, without regard to any FDR threshold. Scores for each pair-wise comparison of factors were used to calculate the Pearson correlation between the top 300 bound regions (1-300) and separately for regions from 301 to 600 on the ChIP/chip rank list. Because the original data for the PRD 1 antibody was derived from a different array scanner than that used for the other factors and because we found that a subtle scaling difference between the two scanners affected the correlation coefficients, the PRD 1 data used in all of Figure 13 were from a replica set (PRD 1*) that used the same Affymetrix G7 scanner used to derive data for the other factors.
Mann-Whitney tests were applied to the binding intensity data of transcription factors to CRMs (Figure 12b), overlap GSC Z scores between factors (Figure 13a, b), and Pearson correlation of intensity scores of peak windows between factors (Figure 13c, d) and are reported in Additional data file 13. Each data set was divided into two categories by factor regulatory classes. The Mann-Whitney test was one-sided, with the null hypothesis that the two categories of data followed the same distribution. Bonferonni corrected values are provided where stated.
Heat map analyses of the association of bound regions with GO terms
In Figure 15, each bound region is associated with the 'biological process' GO term for the gene whose transcription start site was closest to the array intensity peak in the bound region. The non-redundant set of the 7 most enriched GO terms associated with the top 300 bound regions of each factor were used in the analysis. Negative logged probabilities from a hypergeometric distribution were used to measure the association of the top 1 to 300 and 301 to 600 bound regions of each factor with a GO term. The scores of different factors were put on the same scale by setting the most enriched value to 10.
Additional data files
The following additional data are available with the online version of this paper: further evidence that the antibodies used specifically recognize the transcription factors they were raised against in the embryo (Additional data file 1); a table that shows for each factor the numbers of genomic regions bound in blastoderm embryos determined by the symmetric null FDR test and the IgG control FDR test (Additional data file 2); figures plotting down the ChIP/chip rank list in 200-peak cohorts the enrichment of factor recognition sequences using the conventions shown in Figure 2 (Additional data file 3); a table that shows statistical evidence that the top 200 ChIP/chip peaks are significantly enriched over all peaks in the 1% FDR set for the values plotted in Figures 2, 5, 6 and 8 (Additional data file 4); scatter plots comparing relative levels of mean UV crosslinking and mean ChIP/chip scores across a series of highly and poorly bound genomic regions (Additional data file 5); tables listing the genomic coordinates of regions bound by each factor for the 1% FDR data set, and information on the locations and scores of peak windows, and on the closest gene and closest transcribed gene for each peak (Additional data file 6); tables listing the genomic coordinates of regions bound by each factor for the 25% FDR data set, and information on the locations and scores of peak windows, and on the closest gene and closest transcribed gene for each peak (Additional data file 7); figures showing the fraction of bound regions in different cohorts distinguished by ChIP/chip score and, for some factors, the fraction of those bound regions that overlap known CRMs, using the conventions shown in Figure 4 (Additional data file 8); figures plotting down the ChIP/chip rank list in 200-peak cohorts the five most highly enriched GO terms of the closest gene using the conventions shown in Figure 5 (Additional data file 9); figures plotting down the ChIP/chip rank list in 200-peak cohorts the median distance to the closest gene and the distances to closest genes transcribed or patterned in blastoderm embryos using the conventions shown in Figure 6 (Additional data file 10); figures plotting down the ChIP/chip rank list in 200-peak cohorts the percent of peaks found in intergenic, intronic and protein coding regions using the conventions shown in Figure 7 (Additional data file 11); figures plotting down the ChIP/chip rank list in 200-peak cohorts the PhastCons scores of 500-bp peak windows using the conventions shown in Figure 8 (Additional data file 12); tables listing the values plotted in the heat maps in Figures 9, 12 and 13, percentages of the top 300 1% FDR peaks bound by 1, 8 or more, 15 or more or 21 factors, and the results of Mann-Whitney tests applied to the data in Figures 12 and 13 (Additional data file 13); a figure showing the pattern of ChIP/chip scores on the eve gene for both factor and negative control immunoprecipitations for all antibodies shown in Table 2 (Additional data file 14).
Berkeley Drosophila Transcription Network Project
chromatin immunoprecipitation followed by microarray analysis
false discovery rate
Genome Structure Correction.
This work is part of a broader collaboration by the BDTNP. We are grateful for the frequent advice, support, criticisms, and enthusiasm of its members. We thank Laurel Raftery, Eric Wieschaus, and Robert Tjian for their generous gifts of antisera. The in vivo binding data and computational analyses were funded by the US National Institutes of Health (NIH) under grants GM704403 (to MDB and MBE). Additional computational and evolutionary analyses were funded by NIH grant HG002779 (to MBE). Work at Lawrence Berkeley National Laboratory was conducted under Department of Energy contract DE-AC02-05CH11231.
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