R2 = 0.19, P = 0.013) as well as in response to challenge  and resting state and challenged levels of cytokine gene transcription with relative abundances of bacteria . These results show that variable environments, even within a small geographic range (<250 km), can drive divergent selection among populations for transcription of genes related to surviving stress.The molecular genetic mechanisms facilitating local adaptation in salmonids continue to be poorly characterized. Gene transcription is a highly regulated step in the expression of a phenotype and it has been shown to respond to selection and thus may be one mechanism that facilitates the development of local adaptation. Advances in molecular genetic tools and an increased understanding of the functional roles of specific genes allow us to test hypotheses concerning the role of variable environments in shaping transcription at known-function candidate loci. To address these hypotheses, wild rainbow trout were collected in their first summer and subjected to metabolic and immune challenges. We assayed gene transcription at candidate loci that play a role in the molecular genetic response to these stresses, and correlated transcription with temperature data from the streams and the abundance and diversity of bacteria as characterized by massively parallel pyrosequencing. Patterns of transcriptional regulation from resting to induced levels varied among populations for both treatments. Co-inertia analysis demonstrated significant associations between resting levels of metabolic gene transcription and thermal regime ( Local adaptation is characterized by local genotype advantage where individuals experience higher fitness in their local environment than any other environment in which they could exist (Kawecki and Ebert QST vs. FST) and correlations of environmental variables or gradients with phenotypic/genetic traits  alleles  to quantify gene transcription combined with novel next-generation pyrosequencing to quantify bacterial community diversity and 1 year of temperature data to provide evidence that local environments drive transcriptional difference, and ultimately, the evolution of local populations. This work provides insight into the mechanisms controlling local adaptation of salmon populations, with implications for how we view adaptation and the management of this species.We sampled six tributaries of Babine Lake  known toOncorhynchus mykiss Walbaum) were collected from each tributary (23–25 August 2010) by dip netting and electroshocking . Fish were placed into heavy plastic bags (60 × 120 cm) containing ambient water from their tributary. The bags were twisted closed to remove any ambient air and oxygen was then bubbled into the water and allowed to accumulate until it filled approximately one-fourth the volume of water in the bag. Bags were sealed and transported (2–6 h) on ice to the Department of Fisheries and Oceans' Fulton River Spawning Channel facility. Fish from each tributary were held in separate cages in a 3 m round tank with water flow-through from Fulton Lake (15 ± 0.5°C). Fish were held for 5 days under starvation to acclimate to hatchery conditions and recover from the capture and transportation stress. Mortality only occurred for several individuals from one population (TA Creek). It is believed that these individuals were chronically stressed prior to sampling due to the presence of many dead fish at the TA Creek sampling location.Approximately 50 young-of-the-year (32–64 mm) rainbow trout  for 1 min. This protocol has been previously shown to elicit an immune response in Chinook salmon fry, Oncorhynchus tshawytscha . We chose genes to represent rate-limiting, and thus regulatory, steps of major metabolic pathways that are important in all tissues. The function of PEPCK and PK were inferred from studies across all levels of life and are widely accepted as rate-limiting steps of gluconeogenesis and glycolysis pathways , and those genes play important roles in determining downstream responses of the immune system. IL-1β and TNFα are involved in activating and modulating responses of the immune system by inducing inflammation and altering expression of other cytokines in fish . All total RNA preparations had purity values of 1.9–2.1 (A260/A280). Based on the concentration calculated using UV spectrophotometry, total RNA was diluted to 100 ng/μL and treated with DNase 1  to remove genomic DNA contamination. Total RNA was converted to complementary DNA (cDNA) using a High Capacity cDNA kit . Reverse transcriptase (RT) reactions contained 1.0 μg of total RNA, 2 μL of random primers (Applied Biosystems), 4 mmol/L each dNTP, 50 U of MultiScribe RT (Applied Biosystems) and 40 U of RNase Inhibitor (Applied Biosystems) in a 1× RT buffer at a final volume of 20 μL. RT reactions were incubated at 25°C for 10 min followed by 37°C for 2 h and were stopped by incubating at 85°C for 5 min. RNA from DNA–RNA hybrids was degraded using 1 U of RNase H  for each RT reaction and incubation at 37°C for 20 min. RT reactions were then diluted to a final volume of 100 μL with ddH2O.Total RNA was extracted from gill tissue using mechanical homogenization of tissue in 0.8 mL of TRIZOL (Invitrogen) following the method of Chomczynski and Sacchi . Total RFour biologically relevant genes for each treatment and two reference genes  were ass0) per technical replicate in LinRegPCR  were combined to create a normalization factor by taking the geometric mean of the N0 values for the reference genes within individuals. Transcription of target genes was then expressed as a ratio of the value for the gene relative to the normalization factor .PCR efficiency for each amplicon was determined using the program LinRegPCR  was amplified with primers corresponding to 786–1063 bp of the E. coli 16S gene , and 50–100 ng of eDNA. Reactions were amplified for 25 cycles of 94°C for 30 sec, 56°C for 30 sec, and 72°C for 40 sec. The PCRs were then split and amplified in triplicate for 20 cycles using adaptor-modified primers (Titanium primers for 454 pyrosequencing) following the same conditions. PCR products were gel-purified, standardized with respect to concentration and pooled. Emulsion PCR was completed by Engencore  using Titanium sequencing chemistry and sequencing was performed on a Titanium PicoTiterPlate  in a GS FLX pyrosequencer (454 Life Sciences). Raw pyrosequencing data were processed, primer sequences trimmed, and low-quality sequences removed using the RDP pyrosequencing pipeline . Despite succession of bacterial communities from season to season, stream microbial communities have been shown to have stable spatial structure from year to year  were deployed in each tributary during initial sampling (August 2010) and recovered from 5 of the 6 tributaries the following spring . Data loggers recorded water temperature every 4 h to an accuracy of 0.5°C. Maximum and minimum water temperatures, average and maximum daily range of water temperature, and the first day with average water temperature above 5°C were calculated.T-tests to test for differences between control and challenged transcription for each gene in each population. To account for multiple tests, we calculated the false discovery rate (FDR) for each challenge and global P-values for each locus using 1000 random permutations of the data. FDRs were calculated for each challenge as the random expectation of the number of significant tests per permutation divided by the number of observed significant tests in the original data. To test for population differences in response to stress, we subtracted mean population resting state transcription from challenged individuals. We then compared population responses in a one-way analysis of variance (ANOVA) for each gene.All analyses were conducted in the statistical software R version 2.14.1  to define the major axes of variation among sites in both transcriptional and environmental datasets. We then correlated the major axes of variation in transcription with the major axes of variation in the environmental data using co-inertia analysis . FDRs were calculated to be very low  indicating that, despite multiple tests, the significance of our results are not obscured by false positives. These results indicate that the challenges we chose do induce transcriptional responses, but that the response is population-specific .Response to challenge: All metabolic and immune genes were differentially regulated following challenge in at least one population. tion TA; . The onlspecific . To suppGene transcription did not correlate with body size (fork length) nor with the order in which sites occur along the axis of the lake for any of the genes or treatments we investigated, suggesting that systematic sampling biases do not influence transcriptional variation. Isolation by distance has been previously demonstrated for the Babine Lake tributary populations using neutral microsatellite markers Koehler ; however16S rRNA libraries were obtained for all six streams sampled in 2011. Sizes of the trimmed and quality filtered libraries ranged from 621 to 3925 sequences (16S rRNA libraries. Flavobacterium spp. accounted for 61% (774 sequences) of all potentially pathogenic organisms detected followed by Acidovorax spp.  and Corynebacterium spp. and Streptococcus spp. . The remaining 16% of sequences were accounted for by 11 genera and their abundances ranged from 1 to 32 individuals detected across all libraries. Overall, the most important potentially pathogenic genus was Flavobacterium spp. which ranged in relative abundance from 0 to 50% of the total bacterial community of sampled tributaries . Loadings for the immune genes were much more diverse, with three genes contributing approximately equally to PC1 for both experimental groups  and one gene loaded onto PC2 (IFNγ).PCA of metabolic gene transcription in control and response to challenge groups identified the first two axis of variation that, respectively, explained 76% and 73% of the overall variation in the data . For the control group, PC1 was loaded primarily by Flavobacterium among tributaries.The first two principal components of the stream temperature dataset  explained 76% of the variation . The first axis was loaded primarily by maximum temperature, maximum daily range, and to a lesser extent average daily range. The second axis was loaded primarily by the first day with average temperature above 5°C. The bacterial community PCA produced one axis that explained 86% of the variation in bacteria communities among sites. This PC was primarily driven by differences in relative abundance of the genus R2 = 0.19, P = 0.013). The strongest associations were PEPCK transcription with average daily temperature range and PK transcription with maximum stream temperatures  and in response challenge . The strongest associations were between Flavobacterium spp. and transcription of the genes IL-1β, CXCL-8, and TNFα  are consistent with results from Fundulus (Whitehead and Crawford PEPCK) indicates that experiencing larger fluctuations in temperature may require tissues to have a greater capacity to produce their own glucose. The negative associations of transcription in response to challenge may reflect an energetic trade-off where populations experiencing extremes more frequently have evolved to have a higher resting state transcription and thus require a reduced transcriptional response compared with those populations less frequently experiencing extremes. Until now, a link between stream temperature and local adaptation in salmonids had not been established  versus metabolic gene transcription  and their relevant environmental parameters also indicates stronger selection imposed by pathogens on juvenile salmonids. Multiple lines of evidence for selection by specific pathogens as well as pathogen diversity on MH and other immune-related loci have been demonstrated for a variety of salmonid species (e.g., Dionne et al. Despite our modest sample size for bacterial community analysis, we discovered a high level of diversity of microbial taxa in this system, consistent with marine studies utilizing massively parallel Flavobacterium spp. to be positively associated with IL-1β, CXCL-8, and TNFα resting state gene transcription among populations of rainbow trout in Babine Lake, indicating that Flavobacterium spp. may be a potent selective agent in this system. One representative of this genus, Flavobacterium psychrophilum, is a cold-water pathogen that is most virulent at low temperatures (Holt et al. Flavobacterium spp. we demonstrated indicates that populations may be trading off the energetic costs of transcribing cytokines with the frequency of infections they experience. Higher levels of resting state transcription in certain populations may reflect the fish's ability or need to respond transcriptionally to infection. A reduced transcriptional response of cytokine genes to a secondary infection has been demonstrated for juvenile rainbow trout that survived a primary infection (Raida and Buchmann We found Flavobacterium psychrophilum, increases in the diversity and virulence of opportunistic pathogens are generally correlated with increasing temperature (Harvell et al. In contrast to In conclusion, we provide evidence for the important role of gene transcription in mediating the process of local adaptation in tributary populations of rainbow trout. By providing a link between local environmental conditions and specific gene transcription profiles, we have strengthened the case that rapid evolution to local environments occurs, and have provided insight into the mechanisms that facilitate local adaptation of natural populations. Specifically, we highlight the role of temperature as a selective force on the transcriptome of salmonids both directly, by affecting the thermal regime fish experience, and indirectly, by influencing coexisting pathogen communities. We also provide the first evidence of local adaptation selection by pathogens on the transcription of immune-related genes. In light of climate change, the strength of selection by these direct and indirect means will undoubtedly change in unpredictable ways, likely leading to complex responses to local environmental variation. Finally, the population-specific response to stress we report reinforces the functional variability among genetically structured populations and emphasizes the need to conserve individual tributary populations to maintain maximal levels of genetic diversity and hence evolutionary potential.


In Escherichia coli the YbeY protein is important for efficient translation at all temperatures and is essential at high temperatures. Deletion mutants of ybeY are defective in protein translation, due to impaired 30 S ribosomal subunits. Here we provide evidence which tie YbeY to the transcription antitermination process. Thus, in ybeY deletion mutants transcription is significantly inhibited when the “nut like” sequences required for transcriptional antitermination are present, while if these sequences are removed transcription is not affected by the mutation.A new group of translation-associated heat shock genes has been recently identified. One of these novel genes is  These elements include three sequences: boxA, boxB and boxC which bind the cellular factors that construct the antitermination complex box elements, RNAP and Rho and secure the transcription antitermination process The formation of the transcription antitermination complex requires an RNA sequences known as “rrn transcript from the precursor state The transcription antitermination process has two roles in addition to its primary function: to assist processing of the mature It is, therefore, clear that the transcriptional antitermination system is critical for several stages of ribosome biogenesis. Here we present evidence suggesting the involvement of a newly-analyzed protein YbeY in the transcription antitermination process.YbeY is a 17 kD heat shock protein that belongs to the UPF0054 family and is highly conserved in bacteria rrn promoter regions fused upstream to a promoterless lacZ gene (nut-like” sequences. The first rrn promoter region was short and contained only the P1 promoter sequence of the rrn promoter (S = short). The second rrn promoter contained the P1 and P2 promoters as well as the nut -like sequences  involved in transcription antitermination (M = medium length). The third and longest promoter region contained the P1 and P2 promoters, nut -like sequences and the tL region of the rrn operon, which is a putative Rho-independent pausing site of transcription (L = long). All the regions also include FIS elements and additional sequences required for transcription initiation. In addition, the lacZ contains four Rho- dependent terminators. Using this experimental system it is possible to examine the affect of YbeY on transcription antitermination.It has been shown that YbeY is essential for correct rRNA maturation, but the molecular basis of this effect has not been elucidated. One factor that could play a role in RNA maturation is the transcription antitermination process which is important for maintaining an optimal elongation rate for correct processing and folding of the RNA. In order to examine the possible effect of YbeY on the transcription antitermination process, we constructed a series of pACYC plasmids (NEW ENGLAND BioLabs) which contain three types of the acZ gene . These pybeY strain, both containing a lacZ deletion (ΔlacZ ). The lacZ mRNA levels were measured by qRT PCR. These lacZ mRNA levels represent the levels of transcription from each promoter. We assumed that if YbeY is involved in transcription antitermination, then the ybeY deletion would not affect transcription from the short promoter region (S) but would alter transcription from the promoter regions which involve the transcription antitermination sequences, i.e., M and L.These plasmids were transformed into the wild type strain and the ΔybeY mutation. In contrast, this mutation results in a significant reduction (100 fold) of transcription from the “M promoter” which contains the transcription antitermination sequences (“nut -like sequences”). The presence of the tL region relieves some of the reduction: transcription from the “L promoter” is reduced only by 50% in the mutant  is unaffected by the absence of YbeY, but the transcription is almost abolished if the promoter region also contains the P2 and nut -like sequences which constitute the antitermination region (M).YbeY is involved in ribosome biogenesis - in its absence ribosomes are defective and translation is reduced equences . TranscrL - the Rho-independent pause site L is a conserved sequence in the leader region of rRNA and various deletions of the tL results in rRNA transcription polarity L may assist a correct transcription antitermination process, and its presence enables the complex to be fully organized and stabilized prior to the transcription of the 16S rRNA. The lack of tL may lead to premature termination during transcription. The effect of tL on transcription can be seen in L remains even in deletion mutants of ybeY where its presence increases the transcription level as well. Yet even in the presence of tL there is still a 50% decrease in transcription of rrn. Such a decrease is probably sufficient to cause the phenotypes seen in the ybeY deletion mutants, as its effect may escalate into the major effect on ribosome biogenesis.The presence of tybeY mutants results from the involvement of YbeY in the transcriptional antitermination process. However, it is also possible that this reduced level results from a lower stability of transcripts in the ybeY mutant. This possibility is ruled out by the experiment presented in ybeY deletion. In contranst, it should be mentioned that the stability of the short transcripts  is affected by the ybeY mutation, but in the opposite directions. Thus, in the absence of YbeY the short transcripts are more stable. This result could be explained by the recent findings of Jacob et al We assume that the reduced level of transcripts overlapping the transcriptional antitermination region in ybeY deletion mutation can be partially complemented by over-expression of the NUS factors involved in transcription antitermination , a wild-type K-12 strain, was used in all experiments. The ΔybeY deletion mutation was obtained as described lacZ deletion lacZ or double mutants ΔybeY, ΔlacZ. Plasmid pAC24N encoding Nus factors were transformed from the E. coli ASKA library E. coli MG1655 and its ybeY deletion strain.600 = 0.45. The turbidity readings were performed using the Biotek Synergy EONTM reader, with 1 ml volume in 24 well plates.Unless otherwise stated, cultures were grown exponentially in LB broth (Difco) with aeration at 37°C to ODrrn promoter regions and fused upstream to a promoterless lacZ gene were constructed so that they contained three types of rrn operon promoter regions. The coding sequence of lacZ was PCR amplified using primers containing restriction sites for BamHI and HindIII. The digested PCR product was ligated into pACYC digested with the same restriction enzymes. Next, the promoter region of rrnB was PCR amplified using primers containing restriction sites for XbaI and HindIII. The digested PCR product was ligated into pACYC containing the lacZ product, digested with the same restriction enzymes. The pACYC plasmids encoding the rrnB promoter reign and lacZ were transformed into the lacZ and ybeY lacZ deletion strains. The primers used for plasmids construction are listed in Plasmids (pACYC 184) encoding Total RNA was isolated using the RNA Protect and RNeasy kits (Qiagen) as described by the manufacturer and DNase I (Promega) was used to remove genomic DNA contamination.Reverse transcription was carried out with one microgram of the DNase-treated total RNA using random hexamers (Promega) with ImPromII reverse transcriptase (Promega). Quantitative PCRs were performed using 250 nM of each gene-specific pair of primers in a 20 µl volume with 1 Sybr green PCR master mix (Applied Biosystems). Reactions were run on an ABI 7700 instrument (Applied Biosystems) using the following cycling parameters: 95°C for 10 min, 40 cycles of denaturation at 94°C for 15 second and extension at 60°C for 1 min.lacZ transcript levels the results were normalized to the cat levels in each sample, to account for possible deviations in lacZ levels due to changes in plasmid copy number. The primers used for quantifying the levels of lacZ and cat are listed in For determining For determining the levels of the short transcripts (before the antitermination region) we used the following primers:F: 5-_ TGACACGGAACAACGGCAAACACG_-3 andR: 5-_ TGCATAATACGCCTTCCCGCTACA-_3.


If a gene's promoter were a light switch, you'd probably call an electrician. That's because rather than simply turning on and off in a limited and predictable way, many genes—whose expression is controlled by their promoters—are expressed in bursts, with expression fluctuating randomly over time.What is the molecular basis of this transcriptional bursting? Previous work has suggested, but not directly demonstrated, that gene promoters can assume alternative structural configurations, raising the possibility that the promoter might randomly flip among these alternatives. Transcription requires that the polymerase machinery gain access to the promoter, and that access can be inhibited when DNA is tightly wrapped around histone proteins to form nucleosomes, the basic protein–DNA unit of structure within chromosomes. Researchers have speculated that these alternative promoter configurations might be linked to the number and distribution of nucleosomes within the promoter, but there has been no proof.PHO5, a well-studied yeast gene, does indeed adopt alternative configurations based on nucleosome distribution. And they went further, examining the probability distribution of those nucleosomes and constructing a mechanistic model that accounts for that distribution, and hence the phenomenon of transcriptional bursting.The best way to test that hypothesis is by direct observation, and that is what Christopher Brown, Hinrich Boeger, and colleagues set out to do. Their work confirmed that the promoter of PHO5 genes, and then use the electron microscope to determine the precise configuration of each. The gene's promoter includes three nucleosome binding sites, and they were able to tabulate the relative frequency of each of the eight possible binding states .The key to their study was to chemically “freeze” the promoter regions of multiple Next, they built a mathematical probability model to test various assumptions about the transitions among the binding states. They distinguished three kinds of transitions: assembly of a nucleosome on a binding site, disassembly and removal, or sliding of a nucleosome from one site to another. They found that assembly and disassembly alone accounted for the number of observed nucleosomes, but that sliding was needed to account for their distribution. In particular, it appeared that sliding out of the middle position toward the ends was common. Further, the best fit with the data came when disassembly at the first position occurred only when the second position was unoccupied.PHO5 gene occurs in random bursts, the authors concluded. Further experimentation and modeling allowed them to predict that binding states in which the middle position was unoccupied were the most conducive to transcription. But the transcription rate was not maximal in all conducive states; other, as-yet unidentified factors presumably play a role in determining the degree of transcription for a given conducive state.Since transcription is maximally repressed in the fully occupied state and maximally permitted in the fully unoccupied state, the stochastic transitions among these and the intermediate states suggest that transcription of the The model the authors developed provides a structural basis for transcriptional bursting consistent with a large body of data. While other models might also explain that data, none do so as simply and with as few “working parts” as theirs—generally a sign that a model is pointing in the right direction.Brown CR, Mao C, Falkovskaia E, Jurica MS, Boeger H (2013) Linking Stochastic Fluctuations in Chromatin Structure and Gene Expression. doi:10.1371/journal.pbio.1001621


Otx2 ablation causes photoreceptor degeneration through a non-cell-autonomous mechanism involving Otx2 function in the supporting RPE. Surprisingly, photoreceptor survival does not require Otx2 expression in the neural retina, where the related Crx homeobox gene, a major regulator of photoreceptor development, is also expressed. To get a deeper view of mouse Otx2 activities in the neural retina, we performed chromatin-immunoprecipitation followed by massively parallel sequencing (ChIP-seq) on Otx2. Using two independent ChIP-seq assays, we identified consistent sets of Otx2-bound cis-regulatory elements. Comparison with our previous RPE-specific Otx2 ChIP-seq data shows that Otx2 occupies different functional domains of the genome in RPE cells and in neural retina cells and regulates mostly different sets of genes. To assess the potential redundancy of Otx2 and Crx, we compared our data with Crx ChIP-seq data. While Crx genome occupancy markedly differs from Otx2 genome occupancy in the RPE, it largely overlaps that of Otx2 in the neural retina. Thus, in accordance with its essential role in the RPE and its non-essential role in the neural retina, Otx2 regulates different gene sets in the RPE and the neural retina, and shares an important part of its repertoire with Crx in the neural retina. Overall, this study provides a better understanding of gene-regulatory networks controlling photoreceptor homeostasis and disease.During mouse retinal development and into adulthood, the transcription factor Otx2 is expressed in pigment epithelium, photoreceptors and bipolar cells. In the mature retina,  A handful of signalling pathways and transcription factors families are reused for multiple purposes in plant and animal development OTX2 and CRX are associated with severe ocular and retinal diseases such as microphthalmia, retinitis pigmentosa, cone-rod distrophy and Leber’s congenital amaurosis Otx1 and Otx2 is required to specify the retinal pigment epithelium (RPE) All three members of the Otx family of homeodomain transcription factors, Otx1, Otx2 and Crx play critical roles in development and function of the mammalian retina. Mutations in human Otx2 has been addressed in the adult eye. At this stage, RPE cells express Otx2 alone while photoreceptor and bipolar cells co-express Otx2 and Crx. Photoreceptor cells express Crx at a higher level than Otx2 while bipolar cells, express Otx2 at a higher level than CrxOtx2 causes exclusive, progressive and complete degeneration of photoreceptors Otx2 deletion is restricted to RPE cells, indicating a non-cell-autonomous, RPE-based mechanism for photoreceptor degeneration. Furthermore, RPE-specific expression of Otx2 in conditionally ablated retinas rescues photoreceptor disease, showing that Otx2 expression in photoreceptor and bipolar cells is not required cell-autonomously for survival Otx2 knockout in the whole retina has identified mostly RPE-specific genes as Otx2 direct target genes. Despite the recognized role of Otx2 in photoreceptor cell specification and development and the maintenance of its expression in photoreceptor and bipolar cells, the function of Otx2 in the adult neural retina remains unknown.The function of Here, genome-scale identification of Otx2 binding sites in the neural retina and comparison with Otx2 binding sites in the RPE was done as a first step toward the characterization of Otx2 target genes in both compartments. Otx2 genome occupancy was analysed using two independent Otx2 chromatin immuno-precipitation strategies followed by massively parallel sequencing (ChIP-seq), providing a robust map of Otx2 binding sites in both compartments of the adult retina. We show that cis-regulatory regions bound by Otx2 in the RPE and in the neural retina are mostly non-overlapping and have very different structural features, indicating tissue specific activities. Finally, comparison to Crx genome occupancy clearly points to redundant Otx2 and Crx functions in the neural retina.Otx2−GFP/+Otx2 knock-in mice expressing an Otx2-GFP fusion protein 6 sequence reads were mapped to the genome for each condition. MACS algorithm with a <1% FDR threshold was used to identify peaks, henceforth referred to as Otx2-bound regions (OBRs). NR-WT and NR-GFP assays respectively yielded 15448 and 5997 OBRs, while RPE-WT and RPE-GFP assays respectively yielded 2941 and 3766 OBRs , we set out to compare the genomic targets of Otx2 in each compartment. We dissected eyes from 4–5 weeks old mice and carried out Otx2-ChIP-seq separately on RPE and NR nuclei. In order to enhance the reliability for identified Otx2-bound sites, we performed two parallel experiments using two different antibodies: first, 766 OBRs . ControlFor each experiment, peak distribution across functional domains of the genome was analysed using Cis-regulatory Element Annotation System (CEAS) . For all−15, Fisher Exact P-value) sensory perception of light stimulus (p = 1.6×10−14), vision (p = 2.6×10−11), detection of light stimulus (p = 1.1×10−6), response to light stimulus (p = 8.2×10−6). By contrast, such enrichment was absent in the 5903 closest genes corresponding to the non-core set of peaks. This indicates that the intersection of GFP and WT datasets in the NR corresponds to a core set of high confidence Otx2 bound regions, with strong relevance to NR function. Similarly, in the RPE, 55.6% of the OBRs detected in the WT assay were common with the OBRs detected in the GFP assay. A total core set of 1638 peaks was deduced. DAVID analysis indicated that the 1374 closest genes were enriched in specific ontology terms (−5), cell junction (p = 1.0×10−5), metal-ion binding (p = 3.5×10−4), eye development (p = 2.2×10−3), melanocyte differentiation (p = 2.4×10−3) whereas these enrichments were absent or strongly reduced (p>8.8×10−3) in the 2151 non-core specific closest genes. Therefore, the intersection of GFP and WT datasets in the RPE also represents a core set of high confidence OBRs with relevance to specialized function of the RPE.We next examined to which extent the double antibody approach enhanced the reliability of OBR identification, by comparing the pairs of datasets obtained in the NR and the RPE . In the gy terms : cell adNNT/C. The distribution of Otx2 binding motifs in 1 kb of genomic sequence centred on all core OBRs was evaluated. In each tissue, we found a prominent peak of TAATCC sites at the centre of the OBRs , MEME-ChIP and Motiflab  and S2. the OBRs , indicatGene promoters and regulatory regions often lie close to CpG islands, which have a higher GC percentage than genome average. To test whether Otx2 bound regions display specific base content, we compared the percentage of GC in 1000 bases around the centre of OBRs to 1000 random genome sequences of the same length . The GC To evaluate the relevance of core vs non-core datasets, we examined whether the closest corresponding genes were enriched in known Otx2 target genes. In the RPE, Otx2 target genes identified in previous microarray studies 2 tests supported the statistical significance of these results. Hence, filtering the most conserved OBRs also distinguishes relevant genes in the NR.Among the thousands of genome sites bound by transcription factors, no clear indication has emerged so far that could help recognizing those that have prominent regulatory role. We used our knowledge of Otx2 direct target genes to evaluate whether additional features such as chromatin marks or sequence properties could help predicting the relevance of OBRs. The rationale was the following: if a given criteria is relevant to cis-active function of an OBR, then target-gene OBRs should have a lower rank according to this criteria . We usedAlthough Otx2 is expressed in photoreceptor and bipolar cells of the adult mouse retina, its role in the NR, especially in photoreceptors, remains unclear. Previous studies have shown that RPE-specific rescue of Otx2 expression following whole knockout is sufficient to maintain healthy photoreceptors, suggesting that Otx2 is dispensable in these cells. This raises three hypotheses: i) either Otx2 transcription factor has no function in the NR, or ii) these functions are not essential and do not lead to any visible phenotype, or iii) compensatory mechanisms in the NR mask the effects of Otx2 deletion. All these hypotheses imply that Otx2 would have different functions in NR and RPE. To test this idea, the two confident NR and RPE core OBR sets were compared to determine whether Otx2 binds to the same locations in both tissues . Out of The observation of a different OBR location for 384 genes in the NR and in the RPE suggested potential difference of Otx2-bound cis-regulatory elements organization in both tissues. For instance, several PR specific genes have been shown to bear important cell-specific regulatory elements very close to their transcription start site (TSS) −32), supporting the significance of this result. Other enhancer sites for GFY and NFY were enriched to a lesser degree  performs all Otx functions in eye and retina development. Mammalian Otx proteins can rescue some defects of otd mutant flies although to various extents Otx genes have acquired specialized sub-functions through evolution. Indeed, each member of the family has specialized activities, some of which being unique. For instance, Otx1 can rescue gastrulation defects in Otx2 knockout mice but fails to develop anterior head Otx1 knockout mice but fails to recover the lateral semicircular canal of the inner ear in vivo binding and in vitro transactivation properties argue in favour of their redundancy in the neural retina and hence bring a simple explanation for the absence of photoreceptor specific Otx2 knockout phenotype.Otx1, Otx2 and Crx have a common evolutionary origin. In All experiments were conducted under UE guidelines approved by local and state ethical committees. TL received the authorization to experiment #06-261 from the DDPP of the Préfecture des Alpes Martimes, France, and protocols used in this study were approved by the local ethical committee CIEPAL-Azur (permit #Nce/2011-25).http://farnham.genomecenter.ucdavis.edu/pdf/FarnhamLabChIP%20Protocol.pdf).For RPE ChIP, 40 mouse eyes were dissected to remove cornea, lens and neural retina. RPE/choroid eye cups were directly cross-linked in 1% formaldehyde in DMEM at room temperature for 10 min then quenched by adding glycine at a final concentration 125 mM and incubated at room temperature for 5 min. Eye cups were washed twice in cell wash buffer . RPE/choroid nuclei (around 5 million) were then isolated with a dounce (pestle B) in cell lysis buffer . Eyecups were removed and RPE/choroid nuclei suspension was obtained. The rest of ChIP procedures were performed as described on Farnham Lab web site  and the antibody raised against GFP were applied to wild type chromatin and used as controls for WT and Otx2-GFP assays, respectively. The final DNA precipitates were dissolved in 20µl of TE .An antibody raised against OTX2  and an antibody raised against GFP  were used to precipitate chromatin–Otx2 complexes from wild type mice in the WT assay, and chromatin-Otx2-GFP complexes from Otx2ChIP-seq experiments were performed according to standard protocols as previously described http://liulab.dfci.harvard.edu/MACS/) http://www.ebi.ac.uk/bertone/software.html), with respect to the coordinates of the beginning and end of RefSeq transcripts. Due to possible overlap of one peak from a dataset with two peaks from another, total peak number may slightly vary.Clustering for WT and Otx2-GFP assays was performed by first generating density (.wig format files) counting the number of tags in a 25pb sliding window for each ChIP-seq data set. Peak detection was performed using the MACS software  supplemented with 10% fetal calf serum , 50 units/ml penicillin, 50 µg/ml streptomycin and 2 mM L-glutamine (Invitrogen).IRBP promoter cloned into the pSEAP2-basic vector . The pSEAP2-Basic (promoter-less) and pSEAP2-Control (SV40 promoter) vectors were used as negative and positive controls respectively. In standard assays, 105 HeLa cells per well were seeded in 24 wells plates and transfected by the CaPO4 method with unless indicated, 1 µg IRBP-SEAP, 0.25 µg GFP or GFP-Otx2 or GFP-Crx expression vector and 0.1 µg beta-galactosidase expression vector. After 40 hours incubation, secreted alkaline phosphatase (SEAP) and beta-galactosidase activities were measured. SEAP activity was normalized with beta-galactosidase activity. Normalized SEAP background in the absence of Otx2 expression vector was taken as 1 fold activation. Three independent experiments were performed in duplicate to generate each data.Transcriptional activity of the Otx2 and Crx proteins was assayed using the region –66 to +68 of the Table S1HOMER enriched motifs in the NR.(XLSX)Click here for additional data file.Table S2HOMER enriched motifs in the RPE.(XLSX)Click here for additional data file.Table S3Closest genes lists.(XLSX)Click here for additional data file.


Many bacteria possess multiple bEBPs, which are activated by diverse environmental stimuli. In this work, we assess the ability of a promiscuous, constitutively-active bEBP—the AAA+ ATPase domain of DctD from Sinorhizobium meliloti—to activate transcription from all σ54-dependent promoters for the characterization of the σ54 regulon of Salmonella Typhimurium LT2.Sigma54, or RpoN, is an alternative σ factor found widely in eubacteria. A significant complication in analysis of the global σ54-dependent promoters in Salmonella under a single condition. These promoters are controlled by a variety of native activators and, under the condition tested, are not transcribed in the absence of the DctD AAA+ ATPase domain. We also identified a novel σ54-dependent promoter upstream of STM2939, a homolog of the cas1 component of a CRISPR system. ChIP-chip analysis revealed at least 70 σ54 binding sites in the chromosome, of which 58% are located within coding sequences. Promoter-lacZ fusions with selected intragenic σ54 binding sites suggest that many of these sites are capable of functioning as σ54-dependent promoters.The AAA+ ATPase domain of DctD was able to drive transcription from nearly all previously characterized or predicted σ54-dependent promoters of the S. Typhimurium LT2 σ54 regulon under a single growth condition, this approach is likely to be valuable for examining σ54 regulons in other bacterial species. The S. Typhimurium σ54 regulon included a high number of intragenic σ54 binding sites/promoters, suggesting that σ54 may have multiple regulatory roles beyond the initiation of transcription at the start of an operon.Since the DctD AAA+ ATPase domain proved effective in activating transcription from the diverse σ The constant subunits constitute the RNA polymerase core (RNAP), which has the polymerization activity; the σ subunit determines promoter recognition and functions in the Eσ-promoter transition from closed complex to open complex (isomerization). The primary σ factor in a bacterium, such as σ70 in Escherichia coli, controls transcription of most housekeeping genes in the cell; alternative sigma factors have specialized regulons that function in the response to environmental stressors or morphological changes, or in developmental systems  has unusually diverse regulons, with genes that function in a variety of cellular processes, including flagellar biogenesis, response to nitrogen starvation, transport and metabolism of carbon substrates, and tolerance to heavy metals gT  . An isoglutamine . S. Typhal media , or nitral media . Media sSinorhizbium meliloti DctD AAA+ ATPase domain  with an N-terminal 6x-His tag. This plasmid was constructed by digestion of pHX182 . Linkers consisting of complementary oligonucleotides  described in  [rpoN cells were compared and analyzed using a 2-tailed Student’s T-test. Data shown for each promoter construct represents ≥3 biological replicates.The DctD250 expression plasmid pTG4 was introduced into eviously  with thee (ml))] . Ratios The DNA microarray and ChIP-chip data were deposited in NCBI GEO under accession number GSE25849.Amp: Ampicillin; bEBP: Bacterial enhancer-binding protein; ChIP: Chromatin immunoprecipitation; Eσ: RNA polymerase holoenzyme; LB: Luria-bertani media; LM-PCR: Ligation-mediated PCR; NB: Nutrient broth; RNAP: RNA polymerase; Spc: Spectinomycin; Str: Streptomycin; UAS: Upstream activation sequence.The authors declare that they have no competing interests.DS harvested RNA, carried out promoter-reporter assays, and drafted and revised and prepared the manuscript. JF prepared cDNA and preformed DNA microarrays assays and applied DNA from the ChIP to the microarrays. SP and MM produced the microarrays and protocols and performed the statistical analysis of the microarray therein. JM performed the bioinformatic analyses. TH conceived, designed, and coordinated the study, harvested RNA, and performed ChIP pulldowns. AK conceived, designed, and coordinated the study, harvested RNA, and drafted and revised the manuscript. All authors read and approved the final manuscript.Complete list of microarray results. The file contains data for all genes within operons that showed up-regulation by σ54.Click here for fileSequences used to generate Position-Specific Scoring Matrix.Click here for fileOligonucleotides used.Click here for file


Drosophila and yeast. We find evidence of a role for Chd1 in H3 dynamics in both organisms. Using genome-wide ChIP-on-chip analysis, we find that Chd1 influences histone turnover at the 5′ and 3′ ends of genes, accelerating H3 replacement at the 5′ ends of genes while protecting the 3′ ends of genes from excessive H3 turnover. Although consistent with a direct role for Chd1 in exchange, these results may indicate that Chd1 stabilizes nucleosomes perturbed by transcription. Curiously, we observe a strong effect of gene length on Chd1's effects on H3 turnover. Finally, we show that Chd1 also affects histone modification patterns over genes, likely as a consequence of its effects on histone replacement. Taken together, our results emphasize a role for Chd1 in histone replacement in both budding yeast and Drosophila melanogaster, and surprisingly they show that the major effects of Chd1 on turnover occur at the 3′ ends of genes.Chd proteins are ATP–dependent chromatin remodeling enzymes implicated in biological functions from transcriptional elongation to control of pluripotency. Previous studies of the Chd1 subclass of these proteins have implicated them in diverse roles in gene expression including functions during initiation, elongation, and termination. Furthermore, some evidence has suggested a role for Chd1 in replication-independent histone exchange or assembly. Here, we examine roles of Chd1 in replication-independent dynamics of histone H3 in both  Drosophila and that, surprisingly, while it promotes exchange of histones at the beginning of genes, it prevents exchange at the ends of genes. Finally, we show that Chd1 helps determine the characteristic pattern of chemical modifications of histone H3 found over actively transcribed gene sequences.Nucleosomes prevent transcription by interfering with transcription factor binding at the beginning of genes and blocking elongating RNA polymerase II across the bodies of genes. To overcome this repression, regulatory proteins move, remove, or structurally alter nucleosomes, allowing the transcription machinery access to gene sequences. Over the body of a gene, it is important that nucleosome structure be restored after a polymerase has passed by; failure to do so may lead to activation of transcription from internal gene sequences. Interestingly, although nucleosomes constantly move on and off of promoters, they are relatively stable over the bodies of genes. Thus, the same nucleosomes that are removed to allow a polymerase to pass by must be reassembled in its wake. Here, we examine the role of an ATP–dependent chromatin remodeling protein, Chd1, in regulating nucleosome dynamics. We find that Chd1 is important for exchange of the histone H3 in both yeast and  Eukaryotic genomes are packaged as chromatin, whose fundamental repeating subunit, the nucleosome, is composed of 147 bp of DNA wrapped 1.7 times around an octameric histone core. Nucleosomes may interact with each other to form higher-order levels of chromatin packaging necessary to compact an entire genome within a nucleus. This genome packaging strategy leads to a dominant theme in eukaryotic gene regulation: nucleosomes tend to repress gene expression, and a large array of gene regulatory mechanisms in eukaryotes operate by strengthening or weakening the repressive effects of nucleosomes on gene expression Genome-wide nucleosome mapping studies indicate that although the majority of a eukaryotic genome is typically covered with regularly spaced nucleosomes, nucleosome depleted or nucleosome free regions are frequently found over promoters and at the 3′ ends of genes  in vitroAlthough nucleosomes over transcribed genes appear to be relatively stable One factor implicated in the regulation of transcribed chromatin is the ATP-dependent chromatin remodeling enzyme Chd1. Chd1 is the founding member of a family of highly conserved chromatin remodeling enzymes found throughout eukaryotes chromodomains, a central Snf2/Swi2 type helicase domain and a C-terminal domain that mediates DNA or nucleosome binding Drosophila melanogaster, the chromodomains do not play an important role in its localization to chromatin In vitro assays show that Chd1 has the ability to assemble, remodel, slide and promote regular spacing of nucleosomes Drosophila polytene chromosomes show that Chd1 associates with both promoters and transcribed regions of active genes CHD proteins typically have a pair of N-terminal in vitroSchizosaccharomyces pombe, Chd1 (Hrp1) acts at a subset of promoters to disassemble nucleosomes close to the transcription initiation site Drosophila, following fertilization of an egg, sperm chromatin is decondensed, protamines are removed and replaced with nucleosomes whose only form of histone H3 is the replication-independent variant H3.3 chd1 mutants, H3.3 levels in decondensing sperm chromatin are greatly reduced and unevenly distributed, suggesting a role for Chd1 in the replication-independent assembly or distribution of H3.3 nucleosomes Several studies suggest mechanisms for how Chd1's biochemical activity may relate to these biological functions. Chd1 can promote transcription and catalyze activator dependent, promoter specific nucleosome remodeling in vivo role for Chd1's nucleosome remodeling activity. Nucleosomes are typically regularly positioned over genes in wild type yeast cells chd1Δ mutant, this positioning is largely lost over gene bodies chd1Δ yeast cells. Curiously, micrococcal nuclease digestion patterns of bulk chromatin are not affected in a chd1 mutant, suggesting that Chd1 affects the positioning of nucleosome arrays primarily over the transcribed body of genes, rather that the precise spacing between any given pair of nucleosomes chd1 mutations have modest effects on gene expression in yeast, and are virtually indistinguishable from wild type strains in phenotypic assays, they do cause a cryptic initiation phenotype, consistent with the loss of nucleosome organization over the body of genes A recent high-resolution genome-wide nucleosome mapping study in budding yeast points to an in vivo, the mechanism underlying its in vivo function and its relationship to transcription remains unclear. In this study, we examine the role(s) of Chd1 in governing the replication-independent exchange of newly-expressed histone H3 onto chromatin in budding yeast and Drosophila using genome-wide methodologies. Chd1 mutants have dramatic defects in the localization of the replication-independent histone variant H3.3 in flies, while in Saccharomyces cerevisiae, chd1Δ mutants exhibit dramatic defects in H3 turnover in coding regions. Surprisingly, Chd1 predominantly affects histone H3 exchange at the 3′ ends of coding regions, and this effect on turnover depends on gene length – H3 turnover at 3′ ends is fairly concordant between wild type and chd1Δ strains for genes 1 kb and shorter, whereas Chd1 appears to specifically stabilize nucleosomes over the 3′ ends of longer genes. Finally, we show that loss of Chd1 globally alters histone modification patterns related to active transcription, with H3K36me3 in particular shifting in concert with the changed patterns of H3 replacement. Together, our results show that Chd1 plays a key role in histone H3 dynamics, and surprisingly, that yeast Chd1's influence on H3 dynamics is most apparent at the 3′ ends of genes.Although these data clearly demonstrate a role for Chd1 in nucleosome positioning Drosophila syncytial blastoderm and only observed a modest defect in H3.3 distribution in chd1 null mutants Previously, Fyodorov and colleagues examined the distribution of epitope-tagged, full length H3.3 in the core in live salivary glands from chd1 mutant larvae. We utilized a transgenic fly expressing an AB1-GAL4 driver and a Gal inducible histone H3.3core-GFP core protein encoded by the transgene lacks the N-terminal tail, it is only incorporated into chromatin via the replication-independent pathway core-GFP was deposited into the polytene chromosome arms of salivary glands . These results do not appear to be due to any peculiarity of the AB1-GAL4 driver as we observed similar fluorescence patterns when we used sgsGAL4 and eyelessGAL4 drivers (data not shown). Furthermore, we did not observe obvious differences in the strength of H3.3core-GFP signals between flies with the three analyzed genotypes , nor between nuclei with the three observed staining patterns   , suggest of Chd1 , consistin vivo, we turned to budding yeast. To test the idea that Chd1 may modulate replication-independent nucleosome assembly or dynamics, we took advantage of the observation that the yeast H3 N-terminal tail is important for normal chromatin structure Drosophila and Physarum polycephalum, +CHD1 and chd1Δ yeast strains expressing either wild type histone H3 (H3WT) or a histone H3 N-terminal deletion mutation, H3Δ4-30. Consistent with prior observations, the chd1ΔWT H3 strain grew indistinguishably from wild type cells, and the CHD1 H3Δ4-30 strain exhibited a moderate growth defect . Among the suppressor mutations obtained in that screen, we isolated a mutation, H3-S87P/G90S, which simultaneously alters two of the four residues that distinguish histone H3.1 from H3.3 in other eukaryotes. Yeast expressing the S87P/G90S form of histone H3 from the normal HHT2 locus are viable, indicating that this mutation is unlikely to strongly perturb replication coupled chromatin assembly. As with several other of the mutations that suppress spt5Cs- , the H3-S87P/G90S mutant caused cryptic initiation of transcription  analysis, to follow the incorporation of newly-synthesized H3 genome-wide in cells arrested in the cell cycle chd1Δ (red) strains. The wild type profile recapitulates previous results from multiple labs chd1Δ mutants exhibit H3 turnover patterns in which genes appear to effectively reverse polarity. Turnover is still lowest over coding regions, but the trough of minimal turnover has shifted 5′ along coding regions. Promoter and 5′ turnover are slower in chd1Δ cells, whereas maximal H3 replacement is instead observed at the 3′ ends of genes. This behavior is highly unusual, as several published Conversely, chd1Δ on transcription chd1 cells could be due to a large shift in cellular transcription. However, we observed strong concordance of ChIP on chip of RNA Pol2 signals for wild type and chd1Δ cells  genes in budding yeast. We therefore systematically analyzed the effects of gene length on Chd1's role in H3 replacement.We sought to understand what factors might contribute to Chd1 recruitment or function at gene ends. To this end, we first examined the genes with the greatest changes in H3 replacement at their 3′ ends in chd1Δ yeast cells at gene ends (the first and last 500 bp of coding regions) as a function of gene length. At both gene ends there is strong length dependence for H3 turnover in wild type yeast cells, with turnover decreasing as a function of gene length. Notably, for both 5′ end and 3′ end H3 turnover, Chd1's effect on H3 turnover was greatest at unusually long genes. In addition, we found that Chd1's effect on 3′ turnover was greater at highly transcribed genes . Thus, at present we have no additional evidence that supercoiling per se mediates the length dependence of Chd1 on H3 turnover, although given the ability of other topoisomerases to compensate for loss of Top1 we still consider this an appealing hypothesis.Chd1's effects on H3 turnover at the 3′ end of genes depended strongly upon gene length , and waschd1Δ mutations lower the nucleosomal barrier to RNA Pol2 elongation chd1Δ mutants could be unusually susceptible to eviction by RNA Pol2. This model is consistent with a recent suggestion that elongating polymerases could cause collisions and eviction of adjacent nucleosomes if they are spaced inappropriately chd1 mutants Previous results show that Chd1 has dramatic effects on nucleosome positioning over coding regions Taken together, our results identify an evolutionarily conserved role for Chd1 in histone turnover in yeast and flies. Most surprising is our finding that the major site of Chd1 function appears to be at the 3′ ends of genes, suggesting that this enzyme may be recruited or regulated by 3′ histone marks such as H3K36me3. Finally, we find that Chd1 largely affects H3 turnover over longer coding regions, raising the question of whether resolving superhelical tension could be a key role for Chd1 in maintaining wild type chromatin architecture.core-GFP]P/TM6B Tb Huchd1 flies were crossed to 5 b c sp/BcGla; P{GawB}AB1/TM6B Tb Huchd1 flies at 18°C. Flies with 5chd1 balanced by CyO Kr-GFP instead of BcGla were also analyzed and yielded similar results. Salivary glands were dissected and imaged from heterozygous and homozygous 5chd1 third instar larvae. For control nuclei, P{GawB} AB1-Gal4 flies were crossed to core-GFP]/TM6B Tb HuP[UHS-H3.3 flies. H3.3core-GFP expression was quantitated by calculating sum pixel intensity in polytene nuclei using the Volocity software package as described previously Live analysis of polytene chromosome phenotypes was performed as described previously Polytene chromosomes were prepared and fixed as described S. cerevisiae strains used in this study . ChIP assays were preformed as described previously Strains transformed with pGAL-H4-FlagH3 were grown to ∼1.2×10InputCt - IPCt). H3 turnover rates were measured as the final ratio between Flag-tagged H3 and total H3 . The sequences of oligonucleotides used in these PCR reactions are listed in Initial characterization and confirmatory analyses of ChIP samples were performed by qPCR in a Corbett Life Science Rotor Gene 6000 machine using SYBR Green as the detection dye . The fold difference between immunoprecipitated material (IP) and total Input sample for each qPCR amplified region was calculated as described in The immunoprecipitated DNA was initially PCR amplified using random hexamer primers as described in chd1Δ cells were grown to log phase and fixed with 1% formaldehyde. Cell pellets (from 100 mL cells) were resuspended in 8.8 ml Buffer Z , with addition 6.5 µl of ß-ME  and 350 µL of zymolyase solution , and the cells were incubated at 30°C shaking at 220 rpm. After spinning at 4000× g, 10 min, 4°C, spheroplast pellets were resuspended in 600 µl NP-S buffer  per 100 ml cell culture equivalent. 25–40 units (depending on yeast strain and cell density) of micrococcal nuclease  were added and spheroplasts were incubated at 37°C for 20 minutes. The digestion was halted by shifting the reactions to 4°C and adding 0.5 M EDTA to a final concentration of 10 mM.Wt and All steps were done at 4°C unless otherwise indicated. For each aliquot, Buffer L  components were added from concentrated stocks (10–20×) for a total volume of 0.8 ml per aliquot. Each aliquot was rotated for 1 hour with 100 µl 50% Sepharose Protein A Fast-Flow bead slurry (Sigma) previously equilibrated in Buffer L. The beads were pelleted at 3000× g for 30 sec, and approximately 100 µl of the supernatant was set aside for the input sample. With the remainder, antibodies were added to each aliquot  in the following volumes: 10 µl anti-H3K36me3 , or 7 µl anti-H3K4me3 . Immunoprecipitation, washing, protein degradation, and DNA isolation were performed as previously described www.microarrays.org. Labeled probes  were hybridized onto an Agilent yeast 4×44 whole genome array. The arrays were scanned at 5 micron resolution with the Agilent array scanner. Image analysis and data normalization were performed as previously described 2.5 µg of aRNA produced from the linear amplification were labeled via the amino-allyl method as described on Microarray data have been deposited in GEO (Accession #GSE38540).Figure S15chd1 mutation does not effect H3.3core-GFP expression. Total fluorescent intensity and volume of polytene nuclei were determined by analysis of confocal z-stacks using Volocity software. Average fluorescent intensity/nucleus  are shown for (A), the indicated genotypes and (B), the indicated phenotypes. In (A), non-Bc and non-Kr refer to the balancer chromosomes in the parental chd1 strains used to generate the homozygous nulls.The (TIF)Click here for additional data file.Figure S2coreGFP are reduced on polytene chromosomes of RNAi-chd1 expressing larvae  as compared to those from control larvae. Chd1 (red), Pol IIoser2 (green), GFP (blue), DAPI . (B) Levels of H3.3coreGFP are reduced on polytene chromosomes derived from chd1 mutant larvae as compared to control larvae. (C) In contrast, levels of full length H3.3-GFP remain similar on polytenes derived from chd1 mutant larvae as compared to control larvae. Both 5chd1 and 4chd1 are null alleles Loss of Chd1 affects localization of H3.3core-GFP but not H3.3-GFP. (A) Levels of H3.3(TIF)Click here for additional data file.Figure S3GAL1-FLO8-HIS3 reporter gene. Transcription initiation from the normal FLO8 start site produces a transcript in which HIS3 in out of frame and not translated. Internal initiation from within FLO8 produces in frame transcripts and a His+ phenotype as indicated by growth on media lacking histidine. Strain GHY2010 was transformed with CEN LEU2 hht2 HHF2 plasmids carrying the indicated mutations and plated to 5FOA to select for cells that had lost the wild type CEN URA3 HHT2 HHF1 plasmid. These cells were subsequently grown in liquid culture, adjusted to 1×107 cells/ml and 5-fold serial dilutions were spotted to SC-His+Gal media and incubated at 30°C for 3 days. (B) The H3-S87P/G90S mutation causes a cryptic initiation phenotype. (C) Positive and negative controls. As demonstrated previously Δ4-30 and H3K36R mutations cause a cryptic initiation phenotype whereas H3K4R mutation does not.The histone H3-S87P/G90S mutation causes a cryptic initiation phenotype. (A) Diagram of the p(TIF)Click here for additional data file.Figure S4chd1Δ yeast, as indicated. As found using the pGAL-based turnover system, loss of Chd1 resulted in increased 3′ turnover over these long coding regions.Chd1 affects H3 turnover in an independent turnover assay. (A) Schematic showing the locations of 5′ and 3′ q-PCR primers within the indicated genes. (B) H3 turnover was measured using the system described in (TIF)Click here for additional data file.Figure S5chd1Δ, and RNA Pol2 enrichment was averaged for all genes. Scatterplot shows average RNA Pol2 enrichment for all genes, comparing wild type (x axis) and chd1Δ (y axis). (B) 5′ bias in RNA Pol2 (defined as the 5′ RNA Pol2 enrichment over the 3′ RNA Pol2 enrichment) is scatterplotted for wild type and mutant as indicated. Note strong correlation with slope = 1, indicating no systematic bias in RNA Pol2 localization patterns in chd1Δ mutants.Chd1 has minimal effects on global transcription. (A) RNA Pol2 was mapped genome-wide in wild type and (TIF)Click here for additional data file.Figure S6chd1 expression microarray data to transcript length. (B) Scatterplot comparing a 20 gene moving average of Log2 (mut/WT) chd1 expression microarray data to gene transcription frequency in wild type cells.Lack of correlation between transcript length, transcription frequency and changes in mRNA expression in chd1 mutant. (A) Scatterplot comparing a 20 gene moving average of Log2 (mut/WT) (TIF)Click here for additional data file.Figure S7chd1Δ yeast was calculated, and this value is scatterplotted against gene length. Within this scatterplot, we generated 80 gene running window averages for those genes transcribed at low (green), middle (blue), and high (red) levels in wild type cells based on genome-wide Pol2 ChIP-chip Chd1 effects on 3′ histone replacement are greater at highly transcribed genes. The change in histone replacement at the 3′-CDS (last 500 bp of coding regions) between wild type and (TIF)Click here for additional data file.Figure S8Histone modification changes correlate with gene length. (A) H3K36me3 levels at the 3′ 1 kb of genes is shown for wild type and mutants, as indicated. Lines show a 50 gene running window average. (B–D) Averaged H3K4me3 and H3K36me3 data for wild type and mutant yeast is shown for short (B), long (C), and extremely long (D) genes. (E) Average gain in H3K36me3 levels at the 3′ 500 bp of genes was averaged for the three indicated gene length classes.(TIF)Click here for additional data file.Table S1Yeast strains used in the study.(DOC)Click here for additional data file.Table S2Plasmids used in this study.(DOC)Click here for additional data file.Table S3Oligonucleotides used for qPCR Analysis of Chromatin Immunoprecipitates. The indicated oligonucleotide pairs were used in initial analyses and verification of histone turnover ChIP experiments.(DOC)Click here for additional data file.


Our data from the analysis of ∼50 sequences with high Tinman occupancy show that the majority of such sequences act as enhancers in various mesodermal tissues in which Tinman is active. All of the dorsal mesodermal and cardiac enhancers, but not some of the others, require tinman function. The cardiac enhancers feature diverse arrangements of binding motifs for Tinman, Pannier, and Dorsocross. By employing these cardiac and non-cardiac enhancers in machine learning approaches, we identify a novel motif, termed CEE, as a classifier for cardiac enhancers. In vivo assays for the requirement of the binding motifs of Tinman, Pannier, and Dorsocross, as well as the CEE motifs in a set of cardiac enhancers, show that the Tinman sites are essential in all but one of the tested enhancers; although on occasion they can be functionally redundant with Dorsocross sites. The enhancers differ widely with respect to their requirement for Pannier, Dorsocross, and CEE sites, which we ascribe to their different position in the regulatory circuitry, their distinct temporal and spatial activities during cardiogenesis, and functional redundancies among different factor binding sites.The NK homeodomain factor Tinman is a crucial regulator of early mesoderm patterning and, together with the GATA factor Pannier and the Dorsocross T-box factors, serves as one of the key cardiogenic factors during specification and differentiation of heart cells. Although the basic framework of regulatory interactions driving heart development has been worked out, only about a dozen genes involved in heart development have been designated as direct Tinman target genes to date, and detailed information about the functional architectures of their cardiac enhancers is lacking. We have used immunoprecipitation of chromatin (ChIP) from embryos at two different stages of early cardiogenesis to obtain a global overview of the sequences bound by Tinman  Drosophila homeodomain protein Tinman was the first transcription factor found to control the development and differentiation of the heart in any species. In spite of that, our knowledge of the number, identities, and mode of regulation of the downstream target genes of Tinman that are necessary to exert its cardiogenic functions is still very incomplete. To address these issues, we have performed a genome-wide analysis of DNA regions associated with Tinman-binding in embryos and the genes linked to them. The combined data from our in-depth in vivo assays of sequence elements with high Tinman occupancy allow the following general conclusions: (1) The majority of such sequences are active as regulatory elements  in mesodermal tissues that include Tinman-expressing cells. (2) The enhancers active in the heart progenitor cells and the heart generally are dependent on tinman gene activity, whereas those active in non-cardiac mesoderm are often bound neutrally by Tinman. (3) Tinman binding motifs in most cases are essential for cardiac enhancer activity, but in some cases they can be functionally-redundant with those of other cardiogenic factors. (4) Tinman-occupied cardiac enhancers are enriched for a newly discovered binding motif for an unknown factor that is functional in vivo.The  Drosophila relies on a regulatory network of evolutionarily conserved transcription factors and closely integrated signalling events. The key cardiogenic transcription factors include the NK homeodomain factor Tinman (Tin), the GATA factor Pannier (Pnr), and the Dorsocross T-box factors . Loss of function of either of these factors results in a complete failure of heart formation .Early cardiogenesis in tin fulfills functions in addition to cardiogenesis during mesoderm patterning and mesodermal tissue development  in specific dorsal muscle founders as well as pericardial progenitors and bagpipe (bap) in the trunk visceral mesoderm primordia. Both eve and bap are direct targets of tin that contain essential Tin binding sites within their respective mesodermal enhancers. In addition, however, the eve enhancer (MHE/EME) requires sites for the pan-mesodermal regulator Twist, Smad binding sites targeted by Dpp, TCF/Pan sites that mediate both derepression and activation in cells receiving Wg signals, and Ets domain binding motifs likely mediating inputs from receptor tyrosine kinases bap enhancer additionally requires Smad sites for its activation  and sites for the forkhead domain repressor Sloppy paired (Slp) for blocking its activation by Tin and Smads  In accordance with its early and more widespread expression, first in almost the entire mesoderm and then in the whole dorsal mesoderm, tin target genes have been defined by using candidate approaches based upon genetic and expression data, particularly with respect to heart development. These include genes encoding various cardiac transcription factors  Sur) b3-tubulin) Toll) tin mutant backgrounds, Tin binding to a linked cardiac enhancer element, and loss (or strong reduction) of cardiac enhancer activity upon mutation of the Tin binding motifs. In most of these cases, only the role of Tin has been defined at the level of the respective enhancers, but their detailed functional architectures have not been examined. However, in a few cases additional direct and obligatory co-regulators were described, in particular Smads (for the tinD enhancer of tin) Hand) Toll) A number of additional direct tin, for a more complete picture of the roles of tin it is necessary to recover functionally important tin target genes at a more global level. A profitable starting point towards this goal can be the genome-wide identification of the DNA sequences to which Tin is bound in vivo. In our present study, we have taken this directed approach by performing chromatin immunoprecipitations (ChIPs) of Tin-bound sequences from embryos during early cardiogenesis, analyzing them globally, and dissecting selected examples of them functionally. Similar approaches have been initiated by others in parallel to ours Although the candidate approach-based studies were successful in providing a basic framework of the regulatory circuits during heart development downstream of in vivo analysis of ∼50 Tin-bound sequences. The overwhelming majority of these sequences, which were selected largely among the ∼250 regions with highest levels of Tin occupancy , showed enhancer activities in various mesodermal tissues, including the entire early mesoderm, dorsal and cardiac mesoderm, heart, visceral mesoderm, somatic mesoderm, and Dorsal Median cells. To test whether Tin binding is functionally relevant, we focused on the subset of enhancers active in the dorsal and cardiac mesoderm and/or the heart and demonstrate that all enhancers of this class require functional tin for being active. For a representative subset, we tested the in vivo requirements for their Tin binding motifs, as well as the requirements for the Pnr and Doc binding motifs that were also present in these enhancers. Further, by using machine learning approaches we identified a novel motif as a classifier of cardiac enhancers and show that in three out of eight tested enhancers this motif is essential for their cardiac activity. Altogether, our data indicate that high-level Tin binding signifies mesodermal enhancer activity, and at least with respect to highly-occupied cardiac enhancers, in vivo binding of Tin is almost always essential, although it can be functionally-redundant with other bound cardiogenic factors. We discuss the large architectural diversities of the Tin-bound cardiac enhancers in terms of the arrangement and requirement of the binding motifs for the various cardiogenic factors as well as more general aspects regarding the interpretation of genome-wide binding data from ChIP analyses.Herein, we present an analysis of the Tin-bound regions and their linked genes in embryos during mesoderm patterning (3–5.5 h after egg laying) and during specification of heart, visceral and somatic muscle progenitors (5–8 h AEL). After their global characterization, which shows that the linked genes are associated preferentially with expression and functions in various mesodermal tissues and, unexpectedly, also in ectodermal tissues, we present data from our functional Genome-wide binding sites for Tin in embryos were determined for two developmental windows, 3–5.5 h and 5–8 h after egg laying. During the 3–5.5 h window (herein termed “Early”), Tin is expressed initially in the entire mesoderm (except for the hematopoetic mesoderm) and then becomes restricted to bilateral dorsal mesodermal areas under the influence of Dpp signals . During Chromatin immunoprecipitations were performed with our high quality, immunopurified Tin antibody We compared our binding regions with those obtained in an independent study of Tin in comparable developmental windows  regions, which are regions displaying a binding complexity of >8 transcription factors of diverse functions during blastoderm stages, as determined by the ModENCODE Consortium tin ranked among the top 25 in both lists. An analysis for encoded protein domains that are enriched among the Tin-bound genes showed a preponderance of various types of DNA-binding domains at the top of both datasets. In addition, genes likely encoding components of signalling cascades such as protein kinases, immunoglobulin domains, EGF-like regions, and leucine-rich domains ranked near the top of the lists  terms. As shown in he lists .tin is expressed and is known to exert its genetic functions. For this purpose, we used the temporal and spatial annotations from the 3833 genes in the BDGP embryonic in situ hybridization database that had yielded “acceptable quality” expression data + tissues is linked preferentially to expression in ectodermal and neuronal tissues, but upon omission of the genes with Tin-overlapping expression domains there is still a strong bias for ectodermal and neuronal expression. Likewise, the omission of the genes associated with Tin binding in HOT regions, which may potentially reflect non-functional  binding We also addressed the question of whether the Tin-bound genes were expressed preferentially in the tissues in which de novo motif search using Regulatory Sequence Analysis Tools (RSAT) Drosophila, the GATA-4/-5/-6-related factor Pannier (Pnr) is known to play a key role in early heart development de novo motifs presumed to bind Tin and GATA (Pnr) are located preferentially in the center of enriched binding regions , bap , mid , H15 , mir-1 , , eya , Doc genes , Poxm , zfh1 , mef2 , jeb (#E177), odd , Six4 (#E255), pnr , slou , lbe , Him , Tl (#E359), disco , btn (#E455), apt , svp , htl (#L383), lbl (#L443), and tin (#L424). Apart from these, the top-ranking genes included many that are expressed in mesodermal tissues with patterns that overlap spatially and temporally with Tin , with the assumption that on average the regions bound more strongly  would be more likely candidates for functional Tin targets with Tin .in vivo we selected 51 binding regions  Exclnau) was active in the early dorsal somatic mesoderm (39 of the 51 fragments tested (76%) were active in mesodermal domains overlapping with Tin domains. Among these, 16 (41%) were active in the cardiogenic mesoderm and/or in the developing heart, which were of particular interest to our study, and one , CG5522, CG32792 and, surprisingly, H15 and hh, two genes that are not known to be expressed in the somatic mesoderm. The other half showed activity in more restricted patterns in the somatic mesoderm, such as in large lateral cell clusters (lbeL170), segmental stripes (slpE35), and stripes restricted to ventral areas .14 of the 39 positive fragments (36%) were active in the somatic mesoderm but not in the cardiogenic mesoderm . Half ofAlk, Fas3, and H2.0, which are known to be expressed in the trunk visceral mesoderm. The enhancers of both Alk and Fas3 start being active in the entire segmental visceral mesoderm primordia during the 3–5.5 h window  in vivo analysis shows that 50 of our 51 enhancer regions tested in vivo and all ten positive control enhancers used are also positive for Tin occupancy in their screens (the exception being the low ranking wgnE1197), and are often positive for various combinations of Doc, Pnr, pMad, and dTCF (6 of the positive fragments (15%) tested positive in the primordia of the trunk visceral mesoderm and one (oddE81) in fat body primordia . These ih window , whereaser cells . The remer cells . The DM and dTCF . Perhapsand dTCF . In additin in generating the observed patterns of mesodermal enhancer activity we tested a selection of the reporters from each class in tin mutant backgrounds. As shown in tin mutant embryos. Likewise, the activity of the btn enhancer in DM cells  and the unc-5L25 and CG3638L6 enhancers in both cardioblasts and pericardial cells  to the Tin or T-box motifs, respectively, is functionally relevant but either factor alone is largely sufficient for activating the enhancer.Surprisingly, for the unc-5L25s enhancer neither mutation of the Tin motifs nor of the GATA or Doc motifs had any measurable effect on its activity in cardioblasts and pericardial cells . We can al cells , 5F8. Hede novo motif discovery using RSAT de novo motifs, which we put together with previously described motifs in Drosophila  Motif”. This motif has a bootstrap confidence of 96% and its presence is the strongest predictor of cardiac enhancers followed by the GATA motif (Drosophila gene upstream regions (1.53 CEE motif hits per kb).We used machine learning methods to test whether we can predict cardiac enhancers based on their motif content, as had previously been shown for human heart enhancers hila see . To prioTA motif . In sevede novo motif finding and model selection, which can lead to an over-estimation of the AUC. For this reason, it was important to test the predictions of the classifier (namely the CEE motif) for their in vivo relevance in transgenic animals carrying reporter constructs in which the CEE motifs were mutated. We included all enhancer fragments that were also tested for functionality of the Tin, Doc, and GATA motifs and in addition, the published cardiac mef2 enhancers I-E and II-B[S]. As shown in + cardioblasts. In rare remaining positive cardioblasts, nearly full activity was seen . Comparable results were also reported by the Furlong group in vivo binding of most factors is very promiscuous. According to this view  vnd, which is flanked by one of the most highly Tin-occupied sequences in the entire genome. Of note, it has been shown that in mesodermal cells this particular region features the presence of repressing chromatin marks (H3K27me3) and the absence of activating marks and of PolII, but contains H3K4me1 which frequently marks potential enhancers vnd locus, Tin functions in maintaining the repressed state of a potential enhancer and as a consequence excludes vnd expression from the mesoderm in order to restrict it to the ventral ectoderm  of the Tin-bound regions overlap with so-called HOT regions, which are characterized by the simultaneous binding of >8 different transcription factors during pregastrulation stages tin during various stages of cardiac development. In addition, in light of global data from other factors and the distributions of their binding motifs tin mutant backgrounds, particularly among those active in the somatic mesoderm. In some cases shown herein, as well as in the case of the Fas3L254 enhancer , mutations of the Tin binding motifs did not affect enhancer activity in the somatic, cardiac, or visceral mesoderm, respectively. We infer that also at mesodermal enhancers, transcription factors such as Tin can bind neutrally, for example if a fortuitous binding motif is made accessible by other factors, including “chromatin-opening” factors or specific histone marks. In other instances, they may be attracted via mass action solely involving protein-protein interactions, without significantly contributing to the enhancer activity. Unfortunately, laborious tests are necessary in each case to firmly distinguish between different scenarios such as, 1) binding that is biologically essential; 2) binding that makes subtle contributions to the enhancer activity, which may either be biologically irrelevant or perhaps affect the robustness of the developmental process; 3) binding that is functionally redundant with that of other factor(s); 4) promiscuous binding with neutral effects due to the absence of necessary co-factors; 5) binding that is functionally neutralized by a co-bound repressor, as has been shown in the case of some visceral mesoderm enhancers that bind both Tin and the repressor Sloppy paired (Slp) in vivo tests, global binding data and computational enhancer predictions need to be interpreted with great caution  was essential, which suggests full or partial functional redundancy between these factors when bound to this enhancer. In pericardial cells, enhancer activity was retained in the total absence of binding sites for all three factors, as was the case in cardiac-specific in vivo binding of Pnr and Doc, as well as the Dpp and Wg signaling effectors Mad and TCF, and compared their occupancies with that of Tin. They showed that a significant fraction  of the Tin-bound regions were co-occupied by all four of the other factors, which reinforces the notion that factors are often co-recruited to active enhancers in the tissues in which they are jointly expressed DrosophilaDrosophila enhancers of this type were indeed active in the dorsal and cardiogenic mesoderm, in which these factors overlap In their recent study, Junion et al. CG14888; BDGP; pnr, may argue for the possibility that it is more commonly Tin rather than Pnr and Doc that functions like a pioneer to promote the recruitment of subsequent cardiogenic factors. Tin may be bound to co-repressors prior to the recruitment of subsequent activators, which could sharpen the on/off state of enhancers in vivo occupancies of cardiogenic factors on binding site-mutated enhancers or, globally, the occupancies of embryonic enhancers in purified mesodermal cells from tin, pnr, or Doc mutant embryos.Largely based on their analysis of the global frequencies of the motifs for Tin, Pnr and Doc within the regions binding all five tested factors versus in those binding only Tin plus one other factor, Junion et al. de novo CEE motif is both constructed on and used for the classification of the same dataset. This can lead to selection bias that can produce both a high false positive rate in discovering significant motifs and an overly optimistic estimate of the classifier performance de novo motifs prioritized by the machine learning approach is required to verify their biological function.The CEE motif discovered by our machine learning approach using 24 enhancers with cardiac activities vs. 37 non-cardiac enhancers was the motif most highly predictive for the cardiac set of enhancers. The validity of the approach was supported by the fact that we also found the GATA motif as a classifier for cardiac enhancers whereas the forkhead domain binding motif was found as a classifier for somatic mesodermal enhancers. The latter was also reported in a recent study using a related approach Drosophila, the Ets domain factor Pointed (Pnt) has been identified as an important regulator of heart patterning and the induction of tin-dependent muscle founder cells in the dorsal mesoderm eve MHE enhancer tested for Pnt binding does contain CEE motifs, although they were not included in the mutational analysis by Halfon et al. in vivo during the relevant stages, and genetic as well as biochemical assays, are needed to clarify whether either Pnt or Dorsal might correspond to the presumed CEE motif binding factor or whether another factor is involved. In the near future, the availability of an increasing number of enhancers active in the cardiac mesoderm and other mesodermal tissues will greatly improve the reliability of machine learning approaches to detect novel functional motifs in tissue-specific enhancers.In light of these caveats, our findings that three out of eight tested enhancers require the CEE motifs for normal cardiac activity do support the conclusion that these motifs correspond to binding sites of a crucial factor in cardioblasts and their progenitors. This putative factor likely cooperates with the other cardiogenic factors that are active at these enhancers. We can not exclude that this factor is also active when bound to the other cardiac enhancers that did not depend on its binding sites, where it may be functionally redundant. Based upon the sequence of the CEE motif it is difficult to predict the identity of the corresponding binding factor with confidence. However, we note that the CEE motif bears a striking similarity with the binding motifs of several vertebrate Ets domain factors  at the microarray core facility of Mount Sinai School of Medicine . ChIP peaks were called using Model-based Analysis of Tiling-arrays (MAT) Embryo crosslinking, chromatin isolation, chromatin immunoprecipitation and DNA amplification were carried out according to the protocol described previously in detail M{eGFP.vas-int.Dm}ZH-2A;M{RFP.attP}ZH-51C and M{eGFP.vas-int.Dm}ZH-2A;M{RFP.attP}ZH-35B346/TM3,eve-lacZtin and 346/TM3,eve-lacZtin-ABD;tinThe strains ZH-35B . For AlkE301, AopE53, AopL18, CG5522E11, EgfrE1, EgfrE63, gukhE135, meso18EE403, mipleE8, NrtL30, nvyE164, slpE35, tshL8 and wgnE1197, cloning was done into the eve.promoter-LacZ-attB vector ZH-51C . Although located closer to the genes Her and Dot, respectively, the enhancer activities of HimL47 and oddE81 clearly reflected specific aspects of the neighboring genes Him and odd and were named accordingly. The Tin and Doc sites within the genomic fragments were predicted using the web-based tool Target Explorer de novo DNA synthesis   . For Tar. The CEE motif was derived by merging a consensus motif enriched in Repeat Masked cardiac enhancers and a consensus motif discovered by oligo-diff when comparing cardiac with somatic enhancers. The somatic1 motif . We used a log-odds threshold of 4.6 to call significant motif hits with a Markov background of order 2. A LASSO classifier was trained to predict cardiac enhancers from the set of 61 experimentally verified enhancers based on the density of motif hits (defined as number of motif hits per kb of sequence). Motif hit density was used because the sequences were of different lengths. The classifier was trained on 1000 bootstrap samples of 61 enhancers and the regularization parameter picked so to minimize cross-validation error with the maximal of 50 features selected. We reasoned that if a classifier picked more than 50 features it would most certainly be over-fitting the data. The final classifier (We used a classification approach similar to assifier  was consin situ database release 3 We assigned each ChIP peak to the nearest gene boundary (5′ or 3′ exon) from the peak maximal enrichment point in either direction because a number of key mesodermal enhancers are known to be downstream of their respective target genes. If the peak was in an intron, we assigned it to the nearest transcription start site of the nested gene. We performed GO enrichment analysis using Ontologizer de novo motifs. Significant motif hits were called above log-odds threshold of 4.5 and their position recorded in percentages relative to peak region width. Thus, we used the original peak positions without trimming them to a fixed size. The motif counts were then binned in 5% bins and the resulting histogram smoothed using loess smoothing. We scaled the density so that number 1 represents the expected uniform distribution. By scanning with unrelated PWMs we verified that the expected distribution is indeed a flat line at 1 (data not shown).We analysed motif location  by takinFigure S1Analysis of peak locations and peak overlaps with other studies. (A) Peak positions in Tin Early dataset. We assigned each peak to the target gene as described in (TIF)Click here for additional data file.Figure S2commE28-GFP in lateral somatic mesoderm  and lateral muscle precursors .  Expression of fendE160-GFP in all somatic mesoderm  and somatic muscles , as well as in lateral ectoderm (ec).  Expression of H15L5-GFP in somatic muscle precursors  and muscles, as well as in amnioserosa .  Expression of hhL2-GFP in somatic mesoderm  and muscles .  Expression of lbeL170-GFP in lateral somatic mesodermal cell clusters encompassing the area around the SBM founder cells and the lateral adult muscle precursors .  Striped expression of meso18EE403-LacZ in somatic mesoderm , residual expression in the somatic mesoderm at stage 14 , and robust expression in the anterior visceral mesoderm at stage 14 .  Striped expression of PoxmE138-GFP in the ventral somatic mesoderm  and in ventral somatic muscles, especially muscles 26, 27, 29 .  Expression of PscE14-GFP in somatic mesoderm  and somatic muscles .  Expression of Six4E255-GFP in ventral and lateral somatic mesoderm  and ventral/lateral muscle precursors .  Expression of slpE35-GFP in segmental stripes in somatic mesoderm at stage 10  and stage 11 .  Expression of zfh1E141-GFP in somatic mesoderm  and muscle precursors .  Expression of CG5522E11-GFP in somatic mesoderm and ectodermal stripes .  Expression of CG10479L38-GFP in somatic muscle precursors (sm), salivary glands, and endoderm .  Striped expression of CG32792E165-GFP in somatic mesoderm  and in somatic muscle precursors .Activity patterns of Tin-bound enhancers in somatic mesoderm and muscles. Genes are ordered alphabetically, with CGs last.  Expression of (TIF)Click here for additional data file.Figure S3AlkE301-LacZ in visceral mesoderm precursors  and visceral mesoderm .  Expression of Fas3L254-GFP in visceral mesoderm precursors  and visceral mesoderm .  Expression of gukhE135-LacZ in visceral mesoderm , as well as in epidermal cells.  Expression of H2.0L95-GFP in trunk visceral muscle founders and caudal visceral mesoderm  and in trunk visceral mesoderm and longitudinal visceral muscle precursors .  Expression of NrtL30-LacZ in trunk visceral muscle founders and epidermal stripes , and in visceral musculature .  Expression of wgnE1197-LacZ in mesodermal pair-rule stripes , and in visceral musculature of proventriculus and posterior midgut .  Expression of oddE81-GFP in fat body primordia  and developing lateral fat body , an expression that reflects one aspect of the endogenous odd mRNA pattern.Activity patterns of Tin-bound enhancers in trunk visceral mesoderm and fat body precursors.  Expression of (TIF)Click here for additional data file.Figure S4tin-dependent dorsal median cells.  Expression of btnE455-GFP in dorsal median cells .  Expression of Ten-mE146-GFP in dorsal median cells and developing head CNS .Activity patterns of Tin-bound enhancers in (TIF)Click here for additional data file.Figure S5abd-AL104-GFP in striped ventral ectodermal domains  and in the stomatogastric nervous system .  Expression of AntpL15-GFP in epidermal areas of the maxillary segment and in single ectodermally-derived clusters of cells in each thoracic segment . At stage 15 (B′), AntpL15-GFP is present in head areas, maxilla, and the ring gland.  Expression of AopE53-LacZ at stage 13 in most epidermal cells .  Expression of AopL18-LacZ at stage 13 (D) and stage 14 (D′) in epidermal cells of gnathal segments and in ventrolateral segmental areas of thoracic and abdominal segments.  Expression of caupL37-GFP in the stomatogastric nervous system .  Expression of commE137-GFP largely in lateral, segmental clusters of ectodermally derived cells .  Expression of DiscoL286-GFP in endodermal cells .  Expression of DrE59-GFP in the lateral column of neuroectodermal cells  and in foregut, hindgut, and salivary gland .  Expression of EgfrE63-LacZ in lateral ectodermal cells  and the roof of the stomodeum .  Expression of mipleE8-LacZ in the endoderm  and in anterior head tissues .  Expression of nvyE164-LacZ in peripheral nervous system progenitors  and PNS .  Uniform epidermal expression of shgE33-GFP .Activity patterns of Tin-bound enhancers in non-mesodermal tissues.  Expression of (TIF)Click here for additional data file.Figure S6tin. Shown are stage 11–12 wild type embryos (A–K) and homozygous 346tin embryos (A′–K′) carrying the denoted lacZ or GFP reporter constructs and stained with anti-β-Gal or anti-GFP (red) and anti-Tin (green). (A) Tin protein and AlkE301-LacZ co-localize in trunk visceral mesoderm. (A′) In the absence of tin activity, AlkE301-lacZ is no longer expressed in the trunk visceral mesoderm. (B) Expression of btnE455-GFP in dorsal median cells after Tin has become restricted to dorsal mesoderm. (B′) Lack of btnE455-GFP expression at the normal positions of dorsal median cells. (C) Co-expression of Fas3L254-GFP with Tin in the trunk visceral mesoderm. (C′) Severe reduction of Fas3 L254-GFP expression in the absence of tin activity. (D) Co-expression of hthE54-GFP with Tin in anterior cardiogenic mesoderm (arrows). (D′) Lack of hthE54-GFP expression in anterior cardiogenic mesoderm (arrows). Somatic mesodermal hthE54-GFP is tin-independent. (E) Expression of lbeL170-GFP in somatic mesodermal clusters after Tin has been restricted to dorsal mesoderm. (E′) Near lack of lbeL170-GFP expression in the absence of tin activity. (F) Co-expression of nauL35-GFP and Tin in dorsal somatic mesoderm clusters. (F′) Lack of nauL35-GFP expression in dorsal somatic mesoderm in the absence of tin activity. (G) Co-expression of nocL7-GFP and Tin in cardiogenic mesoderm, and segmented expression in dorsal somatic mesoderm. (G′) Both cardiogenic and somatic mesodermal nocL7-GFP depend on tin activity. (H) Co-expression of NrtL30-LacZ and Tin in the trunk visceral mesoderm (arrows). (H′) Lack of NrtL30-LacZ activity in trunk visceral mesoderm in the absence of tin activity. Ectodermal GFP is unaffected. (I) Six4E255-GFP activity in segmental stripes within the ventrolateral somatic mesoderm after Tin has been restricted to the dorsal mesoderm. (I′) Slight reduction of Six4E255-GFP expression levels in the absence of tin. (J) zfh1E141-GFP expression in entire mesoderm at the time when Tin has become restricted to the cardiogenic and parts of the visceral mesoderm. (J′) In the absence of tin activity there is no significant change in zfh1E141-GFP activity.Dependence of the activities of selected Tin-bound enhancers on (TIF)Click here for additional data file.Table S1Summary of Tin peaks and their gene assignments in the Tinman Early and Late datasets.(XLS)Click here for additional data file.Table S2GO terms and protein domains enriched in genes associated with Tin binding peaks. A) GO terms. B) Protein domains.(XLS)Click here for additional data file.Table S3in situ hybridization terms. A) Terms related to Tin expression domains. B) All terms.Enrichment of BDGP (PDF)Click here for additional data file.Table S4Integrated tables of Tin binding peaks from 3–5.5 h (top 455 peaks) and 5–8 h (top 443 peaks) datasets. A) 3–5.5 h dataset. B) 5–8 h dataset.(XLS)Click here for additional data file.Table S5in vivo and in machine learning approach. Summary of all enhancer data, binding motifs present, and comparison of the in vivo occupancy of these enhancer regions by various factors tested in Junion et al. (2012) List of fragments tested (XLS)Click here for additional data file.Table S6List of mutated sequences.(XLS)Click here for additional data file.