Drosophila, graded expression of the maternal transcription factor Bicoid (Bcd) provides positional information to activate target genes at different positions along the anterior-posterior axis. We have measured the genome-wide binding profile of Bcd using ChIP-seq in embryos expressing single, uniform levels of Bcd protein, and grouped Bcd-bound targets into four classes based on occupancy at different concentrations. By measuring the biochemical affinity of target enhancers in these classes in vitro and genome-wide chromatin accessibility by ATAC-seq, we found that the occupancy of target sequences by Bcd is not primarily determined by Bcd binding sites, but by chromatin context. Bcd drives an open chromatin state at a subset of its targets. Our data support a model where Bcd influences chromatin structure to gain access to concentration-sensitive targets at high concentrations, while concentration-insensitive targets are found in more accessible chromatin and are bound at low concentrations. This may be a common property of developmental transcription factors that must gain early access to their target enhancers while the chromatin state of the genome is being remodeled during large-scale transitions in the gene regulatory landscape.In  Drosophila melanogaster embryos, Bicoid (Bcd) protein forms an anterior-to-posterior gradient the embryo , and established a list of robust and reproducible list of 1027 peak Bcd binding regions  to determine the genome-wide binding profile of Bcd to its targets. We performed the ChIP-seq experiments on embryos expressing GFP-tagged Bcd in a ions see . These pions see .bcd mutant embryos, such targets are not expressed. For example, the gap genes buttonhead (btd) and knirps (kni) have anterior expression domains that are not present in bcd mutant embryos , kni expression is reduced to near-background levels (nos hb tsl triple mutants), the kni posterior expression domain has a near wild-type level and anterior boundary  would alter the Bcd ChIP-seq profile. We used the statistical package EdgeR  and αTubulin67C (αTub67C) promoters drive expression levels corresponding to approximately 45% and 25% egg length, respectively. For simplicity, we refer to the uniform lines as low (bcd promoter), medium (mtrm promoter), and high (αTub67C promoter).  is expressed in an anterior stripe in wild-type embryos  as well as interactions between the Bcd target genes themselves. The concentration-dependent activity of uniform Bcd is also apparent in cuticle preparations of embryos expressing the transgenic constructs. The transgenic constructs specify increasingly anterior structures along larval body plan as the concentration of Bcd increases  shows no significant differences between any of the concentrations of uniform Bcd we tested. Concentration-Sensitive III peaks (n = 593) are significantly reduced in binding between the highest and lowest Bcd concentrations, but reductions are not significant between high and medium, or medium and low. Concentration-Sensitive II peaks (n = 138) are significantly reduced in binding at the lowest Bcd level compared to either the medium or the high levels. Finally, Concentration-Sensitive I peaks (n = 152) are significantly reduced in binding at both the medium and the low Bcd levels compared to the highest level . These dAlthough 63 out of 66 previously characterized Bcd-dependent enhancers are identified in our ChIP peaks, the majority of the 1027 peaks identified have not been extensively examined. Within the set of known Bcd targets, there is strong correlation between position of expression and the associated Bcd sensitivity class . To exteThe proportion of peaks overlapping with active enhancers in the Fly Enhancer library varies by sensitivity class. While the Concentration-Sensitive III peaks are the largest class, they have the lowest representation in the Fly Enhancer collection, with 18% of peaks overlapping with a Vienna Tile enhancer reporter. Of these overlapping peaks, 52% overlap with Tiles active at stage 4–6, a smaller fraction than observed for the other peak classes which range from 73% to 77% see . We alsoThe Bcd sensitivity classes are predictive of the expression domains of associated enhancer fragments. Enhancers overlapping with both the Concentration-Sensitive I and II classes drive expression in anterior regions of the embryo, with the Concentration-Sensitive III and Concentration-Insensitive classes driving broad and posterior expression, respectively . This inWe next wanted to determine whether the Bcd-bound regions in each sensitivity class differ at the level of DNA sequence. In vitro, Bcd binds with high affinity to the consensus 5'-TCTAATCCC-3', and that variations on this consensus sequence constitute weak binding sites . If the In further support of this concept, we found little correlation between in vitro binding affinity by electrophoretic mobility shift assay and the in vivo binding properties we observe by ChIP for a selected subset of peaks . At the To measure genome-wide patterns of chromatin accessibility and nucleosome positioning, we performed ATAC-seq  on singlzld mutant embryos. This fraction is higher in Bcd-bound peaks; 402  of the 13,266 open regions in wild-type embryos show significantly reduced accessibility accompanied by increased nucleosome occupancy in those same regions . In contrast, the Concentration-Sensitive III and Concentration-Insensitive classes are both significantly underrepresented   . This subcd mutant embryos . These results indicate that the mechanistic determinants of concentration-dependent Bcd action likely involve a complex interaction between Bcd, DNA, and chromatin structure.These effects of chromatin accessibility impact the availability of sequence motifs for binding Bcd. In wild-type embryos, there is a gradual increase in average motif accessibility from high to low sensitivity, and this difference becomes more pronounced in  embryos , consist085bcd allele, which was originally classified as an ‘intermediate allele’ of bcd (085) forms a gradient from the anterior of the embryo, and is expressed at a similar level as a full-length GFP-Bcd , while binding to the Concentration-Sensitive III and Concentration-Insensitive classes was not . Zld is therefore likely to be one component that influences the accessibility and therefore the apparent in vivo affinity of the peaks that are bound by Bcd but insensitive to its local concentration .All reported experiments were performed on embryos from transgenic and mutant variants of the Fruit Fly bcd mutants refers to embryos derived from E1bcd homozygous mothers. The E1bcd and E1 nosL7 tsl4bcd stocks were from the Wieschaus/Schüpbach stock collection maintained at Princeton University. zld mutants are embryos derived from 294zelda germline clones. Zelda mutant embryos were generated from the 294zld allele (kind gift of Christine Rushlow) as germline clones as described previously  background to facilitate scoring of nuclear density.All ATAC-seq experiments were performed in a E1bcd background. To achieve uniform Bcd expression, the uBcd flies were crossed to a stock expressing a heat shock-inducible flippase in a E1bcd background and the resulting larvae were heat shocked at 37°C. Recombination of the FRT-flanked cassette containing the bcd 3'UTR and 3xP3-RFP was scored by a mosaic loss of RFP expression in the eyes. Mosaic flies were further outcrossed to E1bcd and progeny lacking the bcd 3'UTR were sorted by loss of RFP expression. The resulting flies produced embryos in which the bcd 3'UTR was replaced by the sqh 3'UTR causing a uniform distribution of Bcd along the AP axis. (The uBcd transformants expressed eGFP-tagged Bcd in a graded distribution in the embryo and RFP in the eyes. Transgenic flies containing the uBcd constructs were crossed into a AP axis. bcd cDNA in which the bcd 3'UTR was replaced by the sqh 3'UTR (pBABR GFP-Bcd3'sqh) . This results in a loss of mRNA localization at the anterior pole of the oocyte. A sequence containing the bcd 3'UTR and a 3xP3-RFP reporter flanked by FRT sites was synthesized by GenScript and cloned by Gibson Assembly into the pBABR GFP-Bcd3'sqh plasmid. The FRT-flanked cassette was inserted between the bcd coding sequence and the sqh 3'UTR. The bcd promoter was removed by digesting with AgeI and KpnI and replaced with either the mtrm or the αTub67C promoter to generate the bcd-uBcd, mtrm-uBcd, and αTub67C-uBcd constructs.The uniform Bcd constructs were generated using a pBABR plasmid containing an N-terminal GFP-tagged 085 truncation was generated from eGFP-Bcd  or BestGene (mtrm-uBcd and 085Bcd) and stable transformant lines were established. The mutant cad-GAL4 reporter was injected into a M{vas-int.Dm}ZH-2A, P{CaryP}attP2 stock by Rainbow Transgenic Flies, Inc.The uBcd and Bcdcad enhancer sequences were synthesized by GenScript, amplified using primersThe wild-type cad-GAL4 reporter  was ordered from the Vienna Drosophila Resource Center (VDRC ID 205848/construct ID 210589). The mutated CACCGGCACTCAGTAGAGCA-3’F primer: 5’-R primer: 5’-GCGACCCAAAGACCAGAATA-3’and inserted into the pBPGUw vector  by GatewThe HC_45 and HC_45.4Z sequences were taken from CACCTCGATTTTCTGTTGCCATTTC-3'F primer: 5'-R primer: 5'- CCTGATGGCTGTCCTCTGT-3'and inserted into the pBPGUw vector  by GatewLive embryos were dechorionated in bleach, rinsed in salt solution (NaCl with TritonX-100), and embryos at NC14 were sorted under a light microscope and flash frozen on dry ice. Western blots were performed using a using a rabbit anti-GFP antibody (Millipore Cat # AB3080P) and mouse anti-tubulin antibody (Sigma Cat # T9026) as a loading control. For quantification, the GFP band intensities were normalized to α-tubulin band intensities in each lane. Two biological replicates of 50 embryos were homogenized in 50 μL buffer for each genotype, and 10 μL (=10 embryos) was loaded per lane.et al. used western blots to measure Bcd protein accumulation in the embryo during development, and calculated the total amount of Bcd in the embryo at NC14 to be 1.5 ± 0.2×108 molecules  at NC14 . GFP intensity along the dorsal profile of each embryo was extracted for each frame of the live movies in nuclear cycle 14. The frame with the highest overall intensity in each movie was plotted.Dechorionated embryos of the indicated genotypes were mounted on coverslips overlaid with halocarbon oil and imaged in the mid-sagittal plane on a Leica SP5 laser scanning confocal microscope. Image analysis was performed in MATLAB (A cDNA coding for amino acids 89–154 of the Bcd protein (including the homeodomain) as described in  using pr5’-CAGCCAcatatgCTTTTCGATGAGCGAACG-3’AGCGTAATCTGGAACATCGTATGGGTACTGTTTCATACCCGGCGA-3’5’-GAGCCCggatccctato add a C-terminal HA epitope tag (underlined) and NdeI and BamHI sites (lowercase).E. coli cells using 2 mM IPTG. The protein was purified by affinity chromatography using HisPur Cobalt Resin (Fisher Scientific Cat # 89965) followed by ion exchange chromatography with SP Sepharose Fast Flow resin .The PCR product was cloned into the pET-15b plasmid using NdeI and BamHI, which contains an N-terminal 6xHis tag and T7 promoter, to make plasmid pET-15B-BcdHD. Expression was induced in BL21 (DE3) pLysS d values for each enhancer probe were calculated using the ratio of total shifted probe to free probe.EMSAs were performed using purified Bcd homeodomain and biotin-labeled DNA probes were designed to span ~200 bp in the maximal peak region of Bcd-bound peaks identified by ChIP and corresponding to previously characterized enhancers see . EffectiDrosophila embryos were collected from 0 to 4 hr laying cages, dechorionated in bleach and crosslinked with 180 mL 20% paraformaldehyde in 2 ml PBS + 0.5% Triton X-100 and 6 mL Heptane for 15 min. Crosslinking was quenched with 125 mM Glycine in PBS + 0.5% Triton X-100. Fixed embryos were visually staged and sorted using a dissecting microscope, and all experimental replicates consisted of 200 embryos in nuclear cycle 14. Chromatin immunoprecipitation was performed with an anti-GFP antibody (Millipore) in embryos expressing GFP-tagged Bcd either in a wild-type graded distribution  or uniformly . Sequencing libraries were prepared using the NEBNext ChIP-seq Library Prep master mix kit and sequenced as described in , buffer was removed, and the embryo pellet was flash frozen on dry ice. Frozen pellets were resuspended in Nextera Tagment DNA Buffer +Enzyme and incubated at 37°C for 30 min shaking at 800 rpm. Tagged DNA was purified using a Qiagen Minelute column and eluted in 10 μL. Barcoded sequencing libraries were generated by PCR amplifying the purified DNA using the Nextera DNA Sample Prep kit. Paired-end sequencing was performed on six samples per genotype (n = 5 samples for E1)bcd by the Lewis Sigler Institute for Integrative Genomics Sequencing Core Facility on an Illumina HiSeq 2500.Live embryos expressing a histone (H2Av)-GFP or RFP construct were individually staged on an epifluorescence microscope in halocarbon oil. After the onset of nuclear cycle 14, single embryos were dechorionated in bleach and macerated in cold lysis buffer at t = 12 min into NC14. Samples were pelleted in lysis buffer at 4https://broadinstitute.github.io/picard/). Mapped reads were filtered using samtools  to with digoxygenin-labeled UTP to generate an anti-GAL4 RNA probe. In situ hybridizations were performed according to standard protocols.The GAL4 coding sequence was amplified from a genomic DNA preparation generated from a The Gene Expression Omnibus (GEO) accession number for this study is GSE86966. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been favorably evaluated by James Manley (Senior Editor) and three reviewers, one of whom is a member of our Board of Reviewing Editors.Thank you for submitting your article "Concentration Dependent Chromatin States Induced by the Bicoid Morphogen Gradient" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.The manuscript shows a strong dataset demonstrating that Bicoid influences chromatin accessibility in a concentration-dependent manner, and that Bcd can act as a pioneer factor when at high concentrations. Using fly embryos engineered to uniformly express high, medium, and low levels of Bcd protein, the authors identified four classes of Bcd bound regions, with Concentration-Sensitive I and II classes bound only by high and high/medium levels of Bcd, respectively, driving anterior expression of candidate enhancer fragments, enriched for Bcd motifs, and highly represented in Bcd-dependent chromatin accessibility regions. Concentration-Sensitive III class peaks were bound stronger by high/medium levels of Bcd but lower by low levels of Bcd, driving broad expression of candidate enhancer fragments, not enriched for the transcription factor Zelda (Zld) or Bcd motifs, and not quite dependent on Bcd for chromatin accessibility. Concentration-Insensitive class peaks were bound equally well by all levels of Bcd, driving posterior expression of candidate enhancer fragments, highly enriched for Zld motifs, and not dependent on Bcd for chromatin accessibility. The authors found that the presence and/or the strength of Bcd motifs do not account for in vivo sensitivity to Bcd concentration, as predicted by the binding site affinity threshold model for morphogen function. Rather, the Bcd morphogen gradient is read at the level of chromatin accessibility, established at Bcd concentration-sensitive targets primarily and perhaps even independently by high levels of Bcd, and at concentration-insensitive targets by factors like Zld. The authors further demonstrated using a C-terminus truncated Bcd that the ability of high levels of Bcd to drive chromatin accessibility at concentration-sensitive targets is likely through co-factor recruitment, rather than Bcd cooperative binding. Finally, they turn a Zld dependent target enhancer into a Bcd high level target by changing Zld binding sites into Bcd binding sites thereby increasing the number of Bcd binding sites, and suggest that high levels of Bcd can confer chromatin accessibility.Overall, the paper supports the model put forth, whereby Bcd itself, acting through different classes of genomic binding sites differentially sensitive of Bcd concentration, can create a regulatory logic that delivers spatio-specific regulation of its targets.Although the study was well designed and performed, the reviewers ask that the authors address several criticisms as outlined below.eLife, it might be difficult to read, not only because of the many typos, but the jargon and flow. Examples are: a) What does "strong Bcd gradient" mean? b) Introduction, third paragraph, first sentence: The two phrases seem disconnected. Also, the last sentence of this paragraph could also use some word changes to be more logical; perhaps "Therefore" instead of "However" would flow better. c) A transition is missing between the last two paragraphs of the Introduction. Why is the employed approach better than computational predictions and in vitro measurements? The logic is not obvious. d) Introduction, last paragraph: The use of the word "enhancer" is vague and somewhat misleading. The data reveal distinct classes of Bcd targets not necessarily enhancers. However, further down when referring to the model, it seems appropriate to use "target enhancers."1) The manuscript was difficult to follow at times. For Bcd aficionados it may be easier, but for the more general audience of 2) First section in Results is titled: "Bicoid target gene…other maternal factors, but is physical interaction is not”. Change "maternal" to "patterning" since Bcd binding is changed in zelda mutants .3) With regard to "enhancers whose expression patterns span broadly across the AP axis" – where is the data to support this statement. How many of the 1027 Bcd peaks do these enhancers you speak of comprise – 10 or 100? It is unclear if the following section, are the three specific targets supposed to represent these enhancers? It is not clear because the transition is poor between these paragraphs. Nevertheless, please show the data that led to this conclusion that peaks "overall associate with enhancers[…]"kni posterior enhancer is not expressed" – enhancers are not expressed, genes are expressed; enhancers drive expression. By the way, which enhancer is driving the strong expression seen in the bcd hb nos tsl embryo in 4) The terminology used in the Results (and elsewhere) is incorrect. "the 5) In general, the term "enhancer" is used too loosely in this paper and it is advised that the authors change this. For example, in the last sentence of the second paragraph of the subsection “Bcd binding to genomic targets is concentration dependent” the use of "enhancer" (twice) is premature because it isn't until the following paragraph where a correlation between Bcd peaks and known enhancers is discussed.6) In the third paragraph of the subsection “Bcd binding to genomic targets is concentration dependent”, of the 163 Vienna enhancers that are active in stage 4-6, how many are expressed in a discrete AP domain? I could not find this information and it should be included. Also, why don't all 163 candidate enhancers overlap with a Bcd peak if the 293 overlap with at least one Bcd peak. Also, it is unclear how "77.2%" was derived. 163/293 = 55.6. Please clarify.Gt and Kr are not in the same expression domain, which is not in align with the statement in the last paragraph of the subsection “Bcd binding to genomic targets is concentration dependent”, and Kr was found enriched in Concentration-Sensitive I peaks that include the Gt anterior enhancer. Though it is brave to conclude from the data presented that Bcd sensitivity class gene expression domains are not defined by repressors, it seems dangerous because so many AP gene domains that have been well studied are defined by repressor interactions. Is it possible that enrichment for specific repressors was not seen because your group of peaks contains peaks that are not real Bcd target enhancers and thus the real ones get diluted out?7) The statement, "we find no evidence that the Bcd sensitivity classes are predominantly defined by repressive interactions." It is worrysome that the authors are making a sweeping statement when there are clearly Concentration-Sensitive I enhancers that bind repressors, in addition to those shown by Chen et al. (2012). For example, anterior cnc and hkb. Also, importantly, it should be shown how many of the peaks in each class are near a TSS (within 500 bp). Do they differ?8) In 9) Text in the first paragraph of the subsection “Sequence composition of ChIP sensitivity classes does not account for in vivo sensitivity to Bcd concentration” is confusing. The authors say their results are not in agreement with those of Ochoa-Espinosa et al. (2005) study, but then in the next paragraph they say their results confirm theirs. Please rewrite for clarity.zld- overlap with Zld binding? How many ATAC-seq peaks overlap with Zld binding? The authors are advised to show Zld binding, Zld motifs, and Bcd motifs in 10) When demonstrating the relationship between Zld and Bcd at Bcd-bound regions, the authors claimed that "Zld-dependent peaks are distributed across each sensitivity class determined by ChIP, with no particular class being significantly more Zld dependent," thus Zld "is unlikely to determine the differential concentration sensitivity of Bcd peaks as a whole." This should be examined more closely. How many of the 855 Bcd peaks that overlap with ATAC open regions overlap with Zld binding? How many of the 402 Bcd peaks with reduced accessibility in 11) Structure function studies (protein deletion mutant here) are often difficult to assess because it is unclear whether the structure, and thus function, of the remaining protein (in this case the homeodomain), not just its presence, is unchanged, which is beyond the scope of this experiment. Therefore, the authors should qualify their conclusion by adding something like "assuming the structure of the homeodomain remains completely intact…"zld- embryos; 2) that accessibility increases when adding Bcd sites (in the absence of Zld sites); and 3) though elaborate, adding Bcd sites to HC-45 instead of Zld sites should render the enhancer active if Bcd can pioneer chromatin.12) The authors showed that a Concentration-Insensitive target caudal (cad), whose chromatin accessibility is Zld-dependent and Bcd-independent, can be converted to a Bcd concentration-sensitive target at anterior region by mutating Zld motifs into Bcd motifs. The authors reasoned that high Bcd levels in anterior regions conferred chromatin accessibility to the transgene. If Bcd-dependent chromatin accessibility at regions with high levels of Bcd is the key to its target expression, the authors should provide additional data to show that Bcd binds stronger to Cad (Zld->Bcd)-GAL4 than to Cad-GAL4 in both wt and 13) Discussion of model in to their target enhancers while the chromatin state of the genome is being remodeled during large scale transitions in the gene regulatory landscape.” to the Abstract.14) The authors should move the last sentence of the paper “This may be a common property of developmental transcription factors that must gain early access eLife. Your revised article has been favorably evaluated by James Manley (Senior Editor), a Reviewing Editor, and 1 reviewer.Thank you for resubmitting your work entitled "Concentration Dependent Chromatin States Induced by the Bicoid Morphogen Gradient" for further consideration at The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:The authors have satisfactorily addressed reviewer concerns. The efforts made to address further experiments  were greatly appreciated.Answers to author questions are answered below:1) Regarding the Kni stainings: We prefer to use the Kni stainings from the first experiment in Reviewer's answer: Yes, please keep it in the supplement.2) Regarding the repressor binding: However, we agree that the data are not entirely clear, and difficult to interpret. Overall we agree that this analysis does not advance the concepts in the manuscript. We did not make it a main figure in the paper for this reason, and one possibility would be to remove the figure and corresponding argument entirely from the last paragraph of the subsection “Bcd binding to genomic targets is concentration dependent”. We would appreciate advice from the reviewers on this point.Reviewer's answer: Please delete the corresponding argument from the last paragraph of the subsection “Bcd binding to genomic targets is concentration dependent”, rather than keeping the new paragraph because it isn't much better with the single repressor focus.3) Regarding the assays on cad and HC45 reporters: The major limitation to measuring chromatin accessibility or performing ChIP on reporter constructs (as suggested in experiments 1 and 2 for our cad reporters) is that the measurements must be able to distinguish between the transgenic (reporter) and endogenous (wild-type) copies of the regulatory element in question.Reviewer's answer: While it is possible to distinguish transgenic from endogenous enhancers by having one primer in the reporter sequence (GAL4), which is likely within ChIPed fragments, and one inside the enhancer , it is not necessary pursuing this venue at this point.With regard to the new HC45 data, the discussion about endogenous HC45 being accessible whether active or not and with or without Zld or Bcd is interesting, though it is formerly possible that local chromatin at the transgene is not behaving the same way as endogenous. Reviewers recommend having this data as a supplemental figure. […] Although the study was well designed and performed, the reviewers ask that the authors address several criticisms as outlined below.1) The manuscript was difficult to follow at times. For Bcd aficionados it may be easier, but for the more general audience of eLife, it might be difficult to read, not only because of the many typos, but the jargon and flow. Examples are: a) What does "strong Bcd gradient" mean? b) Introduction, third paragraph, first sentence: The two phrases seem disconnected. Also, the last sentence of this paragraph could also use some word changes to be more logical; perhaps "Therefore" instead of "However" would flow better. c) A transition is missing between the last two paragraphs of the Introduction. Why is the employed approach better than computational predictions and in vitro measurements? The logic is not obvious. d) Introduction, last paragraph: The use of the word "enhancer" is vague and somewhat misleading. The data reveal distinct classes of Bcd targets not necessarily enhancers. However, further down when referring to the model, it seems appropriate to use "target enhancers."We have made changes throughout the revised manuscript  to make the manuscript more readable and easier to follow.2) First section in Results is titled: "Bicoid target gene…other maternal factors, but is physical interaction is not”. Change "maternal" to "patterning" since Bcd binding is changed in zelda mutants .This wording has been changed to follow the suggestions of the reviewers.3) With regard to "enhancers whose expression patterns span broadly across the AP axis" – where is the data to support this statement. How many of the 1027 Bcd peaks do these enhancers you speak of comprise – 10 or 100? It is unclear if the following section, are the three specific targets supposed to represent these enhancers? It is not clear because the transition is poor between these paragraphs. Nevertheless, please show the data that led to this conclusion that peaks "overall associate with enhancers…"The description of expression patterns that span broadly across the AP axis was meant to refer to the 66 previously identified Bcd-dependent enhancers that overlap with our ChIP-seq peaks. However, upon re-reading this was understandably confusing. We have re-written this section for clarity.4) The terminology used in the Results (and elsewhere) is incorrect. "the kni posterior enhancer is not expressed" – enhancers are not expressed, genes are expressed; enhancers drive expression. By the way, which enhancer is driving the strong expression seen in the bcd hb nos tsl embryo in bcd hb nos tsl embryos stained for Kni from the experiment shown in The embryos of different genotypes in kni expression in this experiment is variable and the levels in some cases were higher than the background levels in the separately stained wild-type controls. This is perhaps due to the small number of germ line clone derived embryos bcd hb nos tsl stained in the tube.We attempted to choose a representative image for this genotype in kni expression, simultaneously staining all genotypes  in the same experiment, mixed in a single tube. In the original manuscript, we presented this data as a supplement to kni expression along the AP axis. These data clearly show that kni expression is reduced to low levels in the absence of Bcd, supporting the possibility that Bcd binding to the posterior kni enhancer is essential for the expression it drives.To address this issue, we therefore repeated this experiment to allow for more quantitative comparisons of We prefer to use the Kni stainings from the first experiment in 5) In general, the term "enhancer" is used too loosely in this paper and it is advised that the authors change this. For example, in the last sentence of the second paragraph of the subsection “Bcd binding to genomic targets is concentration dependent” the use of "enhancer" (twice) is premature because it isn't until the following paragraph where a correlation between Bcd peaks and known enhancers is discussed.We have changed our use of "enhancer" throughout the revised manuscript in instances where it was used prematurely or inappropriately.6) In the third paragraph of the subsection “Bcd binding to genomic targets is concentration dependent”, of the 163 Vienna enhancers that are active in stage 4-6, how many are expressed in a discrete AP domain? I could not find this information and it should be included. Also, why don't all 163 candidate enhancers overlap with a Bcd peak if the 293 overlap with at least one Bcd peak. Also, it is unclear how "77.2%" was derived. 163/293 = 55.6. Please clarify.All 163 candidate enhancers do overlap with Bicoid peaks. But in some cases, more than one Vienna Tile overlaps with a single peak, and in other cases a single Tile overlaps with more than one Bcd peak. The correlation is not one-to-one, so the 163 enhancers only overlap with 151 total Bcd peaks, even though they all overlap with at least one.gt, illustrating the issue of multiple overlaps. Here we see that while VT55790 and VT55792 each overlap a single Bcd peak, that same Bcd peak overlaps with two tiles. The Bcd peak furthest from gt, however, overlaps with only one tile, VT55795. VT55790 and VT55792 were therefore excluded from the plot in The differences in Bcd peak number and Vienna Tile number are shown in The correct number of Bcd peaks that do not correspond to any enhancer candidates in the Fly Enhancer collection is 793 , or 77.2%. The reference to 876 non-overlapping in the text is the sum of the peaks not represented as Tiles [793] + peaks overlapping with Tiles active later in development [42] + the peaks overlapping with inactive Tiles [41]. We thank the reviewers for pointing out the confusion that allowed us to detect and correct this error has been corrected in the manuscript.7) The statement, "we find no evidence that the Bcd sensitivity classes are predominantly defined by repressive interactions." It is worrysome that the authors are making a sweeping statement when there are clearly Concentration-Sensitive I enhancers that bind repressors, in addition to those shown by Chen et al. (2012). For example, anterior Gt and Kr are not in the same expression domain, which is not in align with the statement in the last paragraph of the subsection “Bcd binding to genomic targets is concentration dependent”, and Kr was found enriched in Concentration-Sensitive I peaks that include the Gt anterior enhancer. Though it is brave to conclude from the data presented that Bcd sensitivity class gene expression domains are not defined by repressors, it seems dangerous because so many AP gene domains that have been well studied are defined by repressor interactions. Is it possible that enrichment for specific repressors was not seen because your group of peaks contains peaks that are not real Bcd target enhancers and thus the real ones get diluted out?We included this analysis in an attempt to address the Chen 2012 model that repressors define the expression domains of Bcd target genes. The data in 8) In There are several possible reasons why our Bcd ChIP peak list differs from the BDTNP peaks.First, in the manuscript, we compared our ChIP-seq data to BDTNP ChIP-chip data. Second, the BDTNP data were collected from roughly staged embryos (2-3 hour staging), using two replicates per antibody . Our embryos are more carefully staged and limited to early NC14. Third, we used different criteria to select our peaks. After performing the ChIP-seq experiments, we selected the most reproducible peaks across 8 replicates to generate the peak list. We selected these peaks using the Irreproducible Discovery Rate (IDR) approach used by modENCODE to measure reproducibility between genomic datasets, as detailed in the Materials and methods. We are therefore using a different significance threshold to determine which peaks we include in our list, and this approach is different than the FDR ranking used for the BDTNP ChIP data . As such, we expect to generate a list of peaks that differs from theirs. However, as all of the peaks included in our list are highly reproducible across replicates, we are confident that our dataset allows accurate identification of real Bcd peaks at this stage of development. Overall, the Concentration-Sensitive I and II classes had higher ChIP signals than the Concentration-Sensitive III and Insensitive peaks, so these peaks were less likely to be detected in the BDTNP data. Although we acknowledge that some proportion of these types of peaks could be artifacts due to high accessibility, we have filtered our peaks by ATAC-seq accessibility score to mitigate this issue .With respect to the proximity of these peaks to promoters, many early developmental genes have cis-regulatory elements in close proximity to their promoters, for example hunchback P2. It is therefore not clear whether proximity to a promoter rules out a peak as an enhancer, or predicts whether TF binding will be functional. We have added an additional table to the manuscript indicating the number peaks in each class overlapping with Vienna Tile-GAL4 reporters active at stage 4-6 and within 500 bp of a TSS. In both cases there are interesting differences between the sensitivity classes, and these differences are now discussed in the text.hkb and cnc have been previously published and shown to be sensitive to Bcd. In E1bcd mutants, expression of cnc is lost , while anterior hobo expression is confined to a smaller expression domain. . We originally intended to include Hkb immunostaining in Gene expression patterns for the Concentration Sensitive III members 9) Text in the first paragraph of the subsection “Sequence composition of ChIP sensitivity classes does not account for in vivo sensitivity to Bcd concentration” is confusing. The authors say their results are not in agreement with those of Ochoa-Espinosa et al. (2005) study, but then in the next paragraph they say their results confirm theirs. Please rewrite for clarity.number nor strength of Bcd binding sites in an enhancer correlated with the posterior boundary position of gene expression driven by that enhancer. Here we demonstrate that Concentration-Sensitive I and II targets are both enriched for enhancers driving anterior expression and more strongly enriched for Bcd binding sites. Our study therefore disputes the finding that the number of binding sites in enhancers does not correlate with gene expression pattern, but supports the finding that the strength of binding sites does not. This section has been re-written to clarify these two separate points.The Ochoa-Espinosa study concluded that neither the 10) When demonstrating the relationship between Zld and Bcd at Bcd-bound regions, the authors claimed that "Zld-dependent peaks are distributed across each sensitivity class determined by ChIP, with no particular class being significantly more Zld dependent," thus Zld "is unlikely to determine the differential concentration sensitivity of Bcd peaks as a whole." This should be examined more closely. How many of the 855 Bcd peaks that overlap with ATAC open regions overlap with Zld binding? How many of the 402 Bcd peaks with reduced accessibility in zld- overlap with Zld binding? How many ATAC-seq peaks overlap with Zld binding? The authors are advised to show Zld binding, Zld motifs, and Bcd motifs in Using a published list of Zld ChIP-seq peaks , we find that 732 of our Bcd ChIP peaks (71.3%) overlap with Zld peaks. Interestingly, despite the higher representation of Zld motifs in the less sensitive classes, the peaks in the more sensitive classes (I and II) actually overlap with more Zld ChIP peaks:Sensitive I = 125 (82.2%) Zld boundSensitive II = 113 (81.9%) Zld boundSensitive III = 396 (66.8%) Zld boundInsensitive = 97 (67.8%) Zld boundHowever, in terms of Zld dependence, the more relevant feature is whether a peak is open or closed in the absence of Zld. By this metric, none of the peak classes are significantly overrepresented in the Zld dependent group. A barplot showing enrichment of each class in the Zld dependent peaks has been added to Of all ATAC-seq peaks that are open at NC14, 5182 (39.2%) overlap with Zld ChIP peaks.–zld embryos). We see the same pattern with the Bcd ChIP peaks: although 732 (71.3%) of Bcd ChIP peaks overlap with Zld ChIP peaks, only 402 (39.1%) are dependent on Zld for accessibility.However, only 2675 (20.2%) are dependent upon Zld for accessibility  overlap with Zld binding. Therefore, reduced accessibility in the absence of Zld appears to be a strong predictor of Zld binding, while Zld binding itself is not necessarily as predictive of Zld dependence, in agreement with .Of the 855 Bcd peaks that overlap with ATAC open peaks, 654 (76.5%) overlap with Zld binding. Of the 402 with reduced accessibility in Zld and Bcd motif representation in each class was included in the original manuscript in 11) Structure function studies (protein deletion mutant here) are often difficult to assess because it is unclear whether the structure, and thus function, of the remaining protein (in this case the homeodomain), not just its presence, is unchanged, which is beyond the scope of this experiment. Therefore, the authors should qualify their conclusion by adding something like "assuming the structure of the homeodomain remains completely intact…"We have now qualified this conclusion in the manuscript.12) The authors showed that a Concentration-Insensitive target caudal (cad), whose chromatin accessibility is Zld-dependent and Bcd-independent, can be converted to a Bcd concentration-sensitive target at anterior region by mutating Zld motifs into Bcd motifs. The authors reasoned that high Bcd levels in anterior regions conferred chromatin accessibility to the transgene. If Bcd-dependent chromatin accessibility at regions with high levels of Bcd is the key to its target expression, the authors should provide additional data to show that Bcd binds stronger to Cad (Zld->Bcd)-GAL4 than to Cad-GAL4 in both wt and zld- embryos; 2) that accessibility increases when adding Bcd sites (in the absence of Zld sites); and 3) though elaborate, adding Bcd sites to HC-45 instead of Zld sites should render the enhancer active if Bcd can pioneer chromatin.We agree that being able to perform experiments 1 and 2 would be valuable. However, these experiments present significant technical issues, as described in detail below. Experiment 3 was performed, and confirms the hypothesis that addition of Bcd sites to HC45 renders the enhancer “active”. We will discuss experiment 3 first, followed by our attempt at experiments 1 and 2.To perform experiment 3, we generated three HC45 transgenic reporter lines . We used the HC45 wild-type and +4 Zld site sequences reported in Xu et al., 2014, and introduced 4 Bcd sites at the same positions used to make the +4 Zld site reporter to make the new +4 Bcd site reporter. We replicated the previously reported result, that HC45 is inactive in blastoderm embryos, and that adding 4 Zld sites confers activity to the reporter. Similar to addition of Zld sites, addition of Bcd sites also confers activity to the reporter see .We performed this experiment at the suggestion of the reviewers, and are encouraged by the positive result, but we have some reservations about whether this provides stronger evidence of “pioneering” activity than our prior results with the cad enhancer. HC45, while inactive in blastoderm embryos, has open chromatin at this stage according to our ATAC data. The wildtype sequence has one Zld binding site, and seven Bcd binding sites. HC45 requires neither Zld (as cad does) nor Bcd for chromatin accessibility, in the sense that it retains chromatin accessibility in embryos maternally mutant for either factor. As such, it is unclear what drives the phenomenon of HC45 “acquiring activity” upon addition of Zld or Bcd sites. By analogy to the hsp70 system, where GAGA-factor pioneers the acquisition of open chromatin, thus allowing HSF to interact with binding sites and activate transcription, it would appear that neither Zld nor Bcd are operating like a pioneer (GAGA-factor), and are instead possibly functioning like transcriptional activators (HSF). On the other hand, we cannot rule out that addition of Zld or Bcd binding sites further enhances chromatin accessibility at HC45, or drives remodeling of chromatin to favor an ‘active’ versus an ‘inactive’ state. To address such questions, we would need to be able to measure differences in binding state and chromatin accessibility at these reporters (as suggested in experiments 1 and 2 above), and as described below, this is technically difficult. For these reasons, we have chosen to incorporate this new result with HC45 only as a supplement to zld germline clones were produced by crossing 294zld/FM7 females to C is that the measurements must be able to distinguish between the transgenic (reporter) and endogenous (wild-type) copies of the regulatory element in question. This effectively limits our analysis to fragments of DNA that we can distinguish by SNPs. To pursue this, we sequenced the cad enhancer in genomic DNA preps from single adult flies, from every possible genetic background that would be present in the flies used in this experiment. The reporter constructs were generated by injecting plasmid DNA into flies with an attp2 landing site and then crossed to white females to isolate transformants. Primer set 1 amplifies a product using the "wild-type" or non-transgenic SNP, primer set 2 amplifies a product using the transgenic SNP, and primer set 3 amplifies a product in the cad enhancer that is identical between the wild-type and transgenic sequences. These primers were successful at distinguishing between the wild-type and transgenic SNP sequences in genomic DNA, as primer 2 only gives a product in DNA from a fly with the cad-GAL4 construct. Similar results were obtained by qPCR on intact genomic DNA.A second technical limitation is how to effectively measure differences in chromatin accessibility at a single locus by a qPCR based assay that can leverage the presence of the SNP to distinguish between the transgenic and endogenous enhancer copies. ATAC-seq sample preparations are themselves not suitable for PCR-based quantification approaches due to the insertion of adapter sequences at the time of chromatin fragmentation at accessible sites. On the other hand, the feasibility of Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE)-qPCR has to our knowledge not been reported in the literature, but it is likely that samples prepared by the FAIRE-seq technique would remain compatible with qPCR analysis. We therefore pursued this possibility in order to address this question. We fixed and sorted embryos by stage, homogenized, and sonicated genomic DNA as described for ChIP-seq in the Materials and methods. Following sonication, we isolated soluble chromatin by phenol-chloroform extraction as described in . We tested the effectiveness of this approach using wild-type (Oregon-R) embryos, and qPCR primers that would give predictable results based on chromatin accessibility measured by ATAC-seq in this study. We used three optimized primer sets that amplify short regions around the hunchback coding sequence. These regions amplified by these primers should be highly accessible (hb ChIP2), accessible (hb ChIP2), and inaccessible (hb P2 +4) at nuclear cycle 14. A plot showing the enrichment of the qPCR products over an input chromatin sample is shown in zld->bcd), we performed FAIRE on wild-type (white) embryos and embryos expressing either the cad-GAL4 or cad(zld->bcd)-GAL4 in either a wild-type or –zld background. Unfortunately, when we performed the FAIRE experiment, the primers that effectively distinguish between the transgenic and endogenous cad enhancer in intact genomic DNA do not distinguish between the wild-type and transgenic alleles on a sheared DNA sample. Both the wild-type and transgenic primer sets gave a low signal compared to the highly accessible hb ChIP2 region, and are indistinguishable between embryos with and without the transgenic reporter:This result was encouraging. To perform the requested experiment to measure accessibility at cad (WT) and cad(Since we cannot even detect the transgene (or the endogenous copy) in a sheared DNA prep, and since we are limited to this single priming site to distinguish the alleles of this enhancer, we feel that we have exhausted our options for measuring the accessibility of the transgenic reporters directly, and are technically unable to address this point sufficiently.Similarly, to measure differential binding of Bicoid to these reporters by ChIP (suggested experiment 1), we would be constrained by the same technical limitations as described for measuring chromatin accessibility via SNP based PCR. We emphasize that we recognize the potential power of such an experimental approach, but regrettably for this particular set of reporter constructs, despite our hardest efforts, we cannot address these questions within the limited time available for revision of this manuscript.zld mutant embryos, neither is the enhancer accessible, nor is the enhancer active. Suggested experiment 1 is designed to confirm an expected result, namely that adding Bcd sites results in greater Bcd binding in both wild-type and zld mutant embryos. This is likely to be the case. Suggested experiment 2 is designed to show the presumed positive correlation between chromatin accessibility and the activity state of the reporter construct. We are not aware of any documented example of an enhancer that shows activity without an accessible chromatin signature. Similar to the wild-type enhancer in zld mutant embryos, mutating the Zld motifs to a non-functional DNA sequence eliminates the activity of this enhancer in wild-type embryos . When we instead convert the Zld motifs to Bcd motifs (Z->B), we recover enhancer activity, albeit in a different spatial pattern  Discussion of model in This has been added to the Discussion.14) The authors should move the last sentence of the paper “This may be a common property of developmental transcription factors that must gain early access to their target enhancers while the chromatin state of the genome is being remodeled during large scale transitions in the gene regulatory landscape.” to the Abstract.We have made this change in the manuscript.The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:The authors have satisfactorily addressed reviewer concerns. The efforts made to address further experiments  were greatly appreciated.Answers to author questions are answered below:1) Regarding the Kni stainings: We prefer to use the Kni stainings from the first experiment in Reviewer's answer: Yes, please keep it in the supplement.We have kept this figure in the supplement.2) Regarding the repressor binding: However, we agree that the data are not entirely clear, and difficult to interpret. Overall we agree that this analysis does not advance the concepts in the manuscript. We did not make it a main figure in the paper for this reason, and one possibility would be to remove the figure and corresponding argument entirely from the last paragraph of the subsection “Bcd binding to genomic targets is concentration dependent”. We would appreciate advice from the reviewers on this point.Reviewer's answer: Please delete the corresponding argument from the last paragraph of the subsection “Bcd binding to genomic targets is concentration dependent”, rather than keeping the new paragraph because it isn't much better with the single repressor focus.We agree that the discussion of the BDTNP repressor binding is not a valuable addition to the manuscript. We have deleted the text, and panel A from 3) Regarding the assays on cad and HC45 reporters: The major limitation to measuring chromatin accessibility or performing ChIP on reporter constructs (as suggested in experiments 1 and 2 for our cad reporters) is that the measurements must be able to distinguish between the transgenic (reporter) and endogenous (wild-type) copies of the regulatory element in question.Reviewer's answer: While it is possible to distinguish transgenic from endogenous enhancers by having one primer in the reporter sequence (GAL4), which is likely within ChIPed fragments, and one inside the enhancer , it is not necessary pursuing this venue at this point.With regard to the new HC45 data, the discussion about endogenous HC45 being accessible whether active or not and with or without Zld or Bcd is interesting, though it is formerly possible that local chromatin at the transgene is not behaving the same way as endogenous. Reviewers recommend having this data as a supplemental figure.We agree with this point. We will keep the HC45 data in