Li et al. show in a time-resolved analysis that EBF1 occupancy coincides with EBF1 expression and precedes the formation of chromatin accessibility. They observed dynamic patterns of EBF1 target gene expression and sequential up-regulation of transcription factors that expand the regulatory network at the pro-B-cell stage. Ebf1−/− pre-pro-B cells to allow precise experimental control of EBF1 expression in the genomic context of progenitor cells. Consistent with the described role of EBF1 as a pioneer transcription factor, we show in a time-resolved analysis that EBF1 occupancy coincides with EBF1 expression and precedes the formation of chromatin accessibility. We observed dynamic patterns of EBF1 target gene expression and sequential up-regulation of transcription factors that expand the regulatory network at the pro-B-cell stage. A continuous EBF1 function was found to be required for Cd79a promoter activity and for the maintenance of an accessible chromatin domain that is permissive for binding of other transcription factors. Notably, transient EBF1 occupancy was detected at lineage-inappropriate genes prior to their silencing in pro-B cells. Thus, persistent and transient functions of EBF1 allow for an ordered sequence of epigenetic and transcriptional events in B-cell programming.B-cell fate determination requires the action of transcription factors that operate in a regulatory network to activate B-lineage genes and repress lineage-inappropriate genes. However, the dynamics and hierarchy of events in B-cell programming remain obscure. To uncouple the dynamics of transcription factor expression from functional consequences, we generated induction systems in developmentally arrested  Howeve-B cells C. These + pro-B-cell stage. During this time course, we detected 7963 EBF1-binding sites that gained occupancy by EBF1 at various time points and remained occupied in pro-B cells. Among these persistently occupied sites, 4200, 1546, and 2217 sites were sequentially gained at 24 h, 72 h, and the pro-B stage, respectively . In addition, while performing a similar clustering approach based on ATAC-seq peaks, we noted that the frequencies of EBF1-occupied sites decreased from ∼90% in the cluster of ATAC-seq peaks at 24 h to ∼42% in the cluster of ATAC-seq peaks that appeared in pro-B cells . This reduced frequency of EBF1-occupied sites in the accessible chromatin regions of pro-B cells suggests a contribution of other transcription factors to the chromatin landscape at late stages of B-cell programming.To assess the dynamics of chromatin accessibility during B-cell programming, we performed ATAC-seq (assay for transposase-accessible chromatin [ATAC] using sequencing) analyses at similar time points after EBF1 induction and interrogated the accessible chromatin regions for the presence of EBF1 peaks identified in the ChIP-seq analysis. Consistent with our previous analysis of DNase I hypersensitivity in pre-pro-B and pro-B cells , we deteSupplemental Fig. S1C). In the cluster of EBF1-binding sites that gains chromatin accessibility at 24 or 72 h after EBF1 induction, we observed a sequential increase of H3K4me2 marks that was even more pronounced at the pro-B-cell stage. We also noted that the overall increase in H3K4me2 intensity was accompanied by a reduction of H3K4me2 as well as H3K27ac marks at the center of EBF1-occupied sites, suggesting that EBF1 binding may involve a local decrease of nucleosome density . In contrast, no obvious active enhancer marks were detected at EBF1-binding sites associated with the “gain pro-B” and “transient” clusters. We also analyzed changes in nucleosome positioning at EBF1-binding sites in pre-pro-B versus pro-B cells. To this end, we analyzed MNase-seq  data of Tcf3−/− pre-pro-B cells and Rag1−/− pro-B cells . With the exception of sites residing in the accessible chromatin, impaired MNase digestion was observed at EBF1-binding sites in pre-pro-B cells but not in pro-B cells, suggesting that EBF1 binding coincides with nucleosome remodeling.We also analyzed the dynamics of H3K4me2 and H3K27ac modifications and DNA methylation. The cluster of EBF1-binding sites that resides in domains of pre-existing chromatin accessibility showed abundant H3K4me2 and H3K27ac marks prior to and after EBF1 induction . Taken together, these data indicate that EBF1 occupancy and formation of an accessible chromatin domain precedes the loss of DNA methylation.To examine the temporal dynamics of DNA demethylation, we performed whole-genome bisulfite sequencing (WGBS) prior to and after EBF1 induction. In particular, we analyzed the CpG methylation status of genomic regions around EBF1-binding sites that are associated with low methylated regions (LMRs). These regions are enriched for enhancers and undergo transcription factor-mediated changes in DNA methylation . The cluSupplemental Fig. S2A). Consistent with the time course of Pax5 protein accumulation, the occupancy of Pax motifs was significantly increased at the pro-B-cell stage. A similar increase in the occupancy of Oct motifs was observed in pro-B cells, whereas E-box and FoxO motifs were consistently footprinted throughout the entire time course. As a control, the CTCF motif, known to be largely invariant across cell types . Additional co-occurrence of EBF1 and STAT footprints was detected at 72 h, and EBF1 and IRF co-occurring footprints were observed at the CD19+ pro-B-cell stage . In contrast, footprints of Pax and Oct motifs were found predominantly at the pro-B-cell stage, and, notably, these footprints did not co-occur with other motifs examined. As expected, the co-occurrence of CTCF footprints with other footprints was not observed during the time course. Taken together with dynamic expression of transcription factors, these results suggest that the regulatory network in which EBF1 operates involves initially a limited repertoire of transcription factors that is gradually expanded by the sequential expression and chromatin binding of additional transcription factors, including IRF4 and Pax5.We also sought to investigate whether EBF1 can recruit other transcription factors to the chromatin after its induction. To determine the co-occurrence of the footprinted motifs with EBF1-occupied motifs or each other in 200-base-pair regions, we performed pairwise co-occurrence enrichment clustering between two time points of EBF1 induction . This anSupplemental Fig. S3A). In contrast, no significant change in the transcriptome was observed between 24 and 72 h after EBF1 induction. During pre-pro-B-to-pro-B-cell differentiation, the abundance of transcripts from 2425 genes was altered by more than twofold, whereby 1148 of these genes contained persistently or transiently EBF1-occupied sites as determined by ChIP-seq analysis . We also analyzed a previous data set of dynamic gene expression in progenitor cells in which differentiation was induced by inhibition of Id2 . We subjected both groups of genes to clustering and found multiple clusters with distinct patterns of dynamic gene expression . A stringent cutoff of >10-fold altered gene expression yielded five and four clusters of up-regulated and down-regulated genes, respectively . A less stringent cutoff of twofold to 10-fold altered gene expression yielded a total of 12 clusters . For further analysis, we focused on the stringent cutoff gene clusters. Cluster U1 includes Igll1 and VpreB2, which were markedly up-regulated at 24 h after EBF1 induction and showed no significant further increase at the pro-B-cell stage  analysis at different time points after EBF1 induction. Principal component analysis of two biological replicates indicated a marked variance between the samples of uninduced (0 h) and 24-h-induced cells as well as between samples of 24-h-induced and pro-B cells . The down-regulation of transcript levels was generally quite modest at 24 and 72 h. However, in virtually all down-regulated gene clusters, the transcript levels decreased sharply at the pro-B-cell stage. In cluster D4, genes were even up-regulated prior to their down-regulation at the pro-B-cell stage. The clusters of down-regulated genes included EBF1-bound and EBF1-regulated genes that are normally expressed in hematopoietic progenitors, such as Hlx and Gfi1b, or genes that function in alternative cell lineages, including important regulators of T-cell identity (Bcl11b) and myeloid cell identity (Cebpb and Irf7) .Likewise, we identified various temporal patterns of down-regulated genes . The temporal dynamics of chromatin accessibility in the clusters of up-regulated genes revealed that the patterns of genes expression mostly paralleled those of chromatin accessibility at EBF1-occupied sites . This analysis indicated that EBF1-binding frequency and the distance to the TSS are similar in down-regulated genes and genes with unchanged expression. However, up-regulated genes were enriched for multiple EBF1-binding sites and a close distance between the TSS and the nearest EBF1-occupied site.We also interrogated the clusters of gene expression with the data sets of EBF1 occupancy and chromatin accessibility. We observed EBF1 occupancy at most deregulated genes at 24 h C,D. Notaed sites C,E. In ped sites C,E. Clused sites D,F. We aSupplemental Fig. S5A). Conversely, repressive H3K27me3 marks at TSSs were lost at 72 h after induction . Down-regulated genes showed no significant change in H3K4me2 levels and a modest decrease of H3K27ac levels at EBF1-binding sites throughout differentiation to the pro-B-cell stage . Consistent with gene down-regulation, H3K27me3 modification at TSSs was increased at the pro-B-cell stage .Finally, we interrogated the clusters for the dynamics of histone modifications. The clusters of up-regulated genes gained modest levels of H3K4me2 and H3K27ac marks at EBF1-binding sites at 24 h after EBF1 induction, and the levels were markedly enhanced at the pro-B-cell stage (Igll1 (λ5) locus, we observed a robust gain of EBF1 occupancy and chromatin accessibility at the EBF1-bound site already at 24 h . The Cd79a locus as a representative of cluster U4 was also occupied by EBF1 already at 24 h . Although Irf4 showed a temporal pattern of activation similar to that of Pax5, we observed EBF1 occupancy at the Irf4 locus only at 72 h . As representative down-regulated genes, we analyzed Pdgfrb (cluster D3) and Cebpb (cluster D4), which were down-regulated in pro-B cells 73-fold and 9.8-fold, respectively . In both genes, we detected a transient EBF1 occupancy and chromatin accessibility at 24 and 72 h . As an example for an EBF1-independent Pax5-binding site, the Pnn promoter is already accessible in pre-pro-B cells and can be occupied after Pax5 induction in the absence of EBF1 .The time-resolved analysis of B-cell programming allowed us also to gain insight into the order of changes in the chromatin landscape. We found that the formation of accessible chromatin domains around EBF1-binding sites occurred after the detection of EBF1 occupancy. With the exception of a relatively small cohort of EBF1-binding sites that resides in the accessible chromatin in pre-pro-B cells, the majority of EBF1-binding sites acquires accessibility at 24 h or even later. PU.1 has been shown previously to act as a pioneer transcription factor in progenitor cells ; therefo-B cells , we founChanges in the epigenetic landscape after transcription factor binding was also observed for the doxycycline-mediated induction of the basic helix–loop–helix protein NeuroD1 in murine embryonic stem cells . InducedThe question arises of which mechanisms underlie the generation of accessibility after binding of EBF1. We found that two sets of EBF1-binding sites that are occupied by 24 h of EBF1 induction also gain chromatin accessibility see B. At thiOur analysis also indicated that DNA demethylation is a later event in B-cell programming and is detected at the pro-B-cell stage. A role of EBF1 in DNA demethylation has been proposed . HoweverBcl11b gene , Ebf1, Pax5, and Foxo1 indicated that all of these transcription factors are required for early B-cell differentiation  by the Ikaros-related protein Aiolos at the transition of the pre-BI to the pre-BII stage  and purified by streptavidin bead-mediated magnetic-activated cell sorting (MACS) (Miltenyi Biotec). OP9 feeder cells were used to maintain the progenitors in OptiMEM medium supplemented with 4% FCS, 1% PSG, 50 µM β-mercaptoethanol, 10 ng/mL SCF, 10 ng/mL Flt3L, and 5 ng/mL IL-7.38B9 pro-B cells were cultured in RPMI-1640 medium supplemented with 10% FCS, 1% PSG, and 50 µM β-mercaptoethanol. To isolate c-KitCd8a and Ebf1, respectively. Retroviral transductions of these plasmids were performed as described  by tailless escribed .For intracellular staining, cells were fixed and permeabilized with eBioscience transcription factor staining buffer set . Cells were stained with anti-EBF1  or normal rabbit IgG . Alexa fluor 488-conjugated anti-rabbit IgG  was used to label the primary antibodies. Cells were analyzed using BD LSRII. The data were processed and visualized with FlowJo. CD19 staining was performed as described  with APCImmunoblotting was performed with the following antibodies: anti-EBF1 (BioGenes), anti-Pax5 , anti-IRF4 , anti-GAPDH , anti-FoxO1 , anti-E47 , anti-PU.1 , anti-Ikaros , and anti-H3 .ChIP was performed as described  with the2, 0.1% [v/v] Igepal CA-630) and incubated for 15 min on ice. The supernatant was discarded after centrifugation, and nuclei were used for transposition reaction immediately. The data were analyzed in the same way as ChIP-seq data. Formaldehyde-assisted isolation of regulatory elements (FAIRE) was performed as described and analyzed with qPCR . The gene list was further filtered by removing genes with low expression levels (fragments per kilobase per million reads [FPKM] < 1) in both 0-h and pro-B-cell samples. The genes bound by EBF1 within ±25 kb of TSSs were identified as EBF1-regulated genes. Short Time-series Expression Miner (STEM) was used for the clustering of EBF1-regulated genes . mRNA was enriched by using oligo dT magnetic beads for library preparation. The paired-end reads were mapped to the mouse reference genome (mm9) using Bowtie2 (version 2.3.2) and TopHat2 (2.0.13) . Cufflined genes . To idenGuide RNAs (gRNAs) were designed with an online tool  and cloned into pSpCas9 (BB)-2A-GFP . For knock-in of mutations, we transfected the template DNA (Ultramer DNA oligonucleotides purchased from Integrated DNA Technologies) with the desired mutation together with the plasmid that could express Cas9 and single gRNA into 38B9 pro-B cells via electroporation. One day after electroporation, GFP-positive cells were sorted and plated into 96-well plates (one cell per well). These single-cell colonies were sequenced, and the homozygous knock-in clones were used for subsequent experiments.All high-throughput sequencing data presented in this work were uploaded to the Gene Expression Omnibus under one superseries with accession number GSE107242. Individual series can be obtained as follows: GSE107234 (ATAC Cre), GSE107235 (ATAC Tet-on), GSE107236 (EBF1 ChIP Cre), GSE107237 (EBF1 ChIP Tet-on), GSE107238 (Histone ChIP Cre), GSE107239 (Pax5 ChIP Tet-on), GSE107240 (RNA-seq Cre), and GSE107241 (WGBS Cre).


The protein Zelda was shown to play a key role in early Drosophila development, binding thousands of promoters and enhancers prior to maternal-to-zygotic transition (MZT), and marking them for transcriptional activation. Recently, we showed that Zelda acts through specific chromatin patterns of histone modifications to mark developmental enhancers and active promoters. Intriguingly, some Zelda sites still maintain these chromatin patterns in Drosophila embryos lacking maternal Zelda protein. This suggests that additional Zelda-like pioneer factors may act in early fly embryos.We developed a computational method to analyze and refine the chromatin landscape surrounding early Zelda peaks, using a multichannel spectral clustering. This allowed us to characterize their chromatin patterns through MZT (mitotic cycles 8–14). Specifically, we focused on H3K4me1, H3K4me3, H3K18ac, H3K27ac, and H3K27me3 and identified three different classes of chromatin signatures, matching “promoters,” “enhancers” and “transiently bound” Zelda peaks. We then further scanned the genome using these chromatin patterns and identified additional loci—with no Zelda binding—that show similar chromatin patterns, resulting with hundreds of Zelda-independent putative enhancers. These regions were found to be enriched with GAGA factor  and are typically located near early developmental zygotic genes. Overall our analysis suggests that GAF, together with Zelda, plays an important role in activating the zygotic genome.As we show, our computational approach offers an efficient algorithm for characterizing chromatin signatures around some loci of interest and allows a genome-wide identification of additional loci with similar chromatin patterns.The online version of this article (doi:10.1186/s13072-017-0141-5) contains supplementary material, which is available to authorized users. The process of transcription is vital to all living organisms and is tightly regulated by multiple mechanisms, including the packaging of DNA into chromatin. In eukaryotic cells, the DNA is wrapped around nucleosomes to form chromatin. This packaging is used differentially to control in what conditions and cell types a gene is more accessible—and active—and in which conditions it is tightly packed and silenced. This packaging of DNA was shown to be mediated by various mechanisms, including the deposition of covalent modifications  at different residues of the core histone proteins that are assembled into a nucleosome . These hThe packaging of DNA into chromatin, including nucleosome positioning and their histone modifications, are fundamental to the proper activity of regulatory regions, by controlling which regions of the genome are accessible for protein binding and which are not –7. Of spComparison of tissue- and condition-specific chromatin data highlights the tight regulation of gene expression by chromatin, determining which genomic regions are active and which are not, and as a consequence which genes will be transcribed. This raises the question of causality. Who regulates packaging? Or how does the genome get packed initially in the proper architecture, e.g., to drive early developmental expression?In practically all animals, early developmental stages begin with maternal proteins and RNA that control the first hours in the fertilized egg. At this stage, these proteins control the first wave of zygotic expression and direct the first mitotic divisions . After tDrosophila melanogaster, embryonic development is characterized by a series of 13 rapid replication cycles, occurring during the first 2 h after fertilization. The division of cells slows at the 14th mitotic cycle, and zygotic transcription initiates. This marks the end of the D. melanogaster maternal-to-zygotic transition. This process is crucial for the normal development of the embryo and is tightly controlled, in both time and space, by the gradual activation of a cascade of transcription factors [In the fruit fly  factors , 13. ThiPrevious research showed that many of the early transcribed genes in Drosophila embryos contain a specific DNA motif of 7 bp, CAGGTAG, that occurs within their regulatory regions, including both promoters and enhancers –16. ThisComputational and experimental studies, by us and others, suggested that Zelda acts as a pioneer factor, binding mostly inaccessible DNA regions and making the chromatin accessible for other transcription factors to bind, thus marking thousands of genes and regulatory regions for activation which drives the first transcriptional program of the developing embryo , 16, 19.Indeed, experimental studies showed that early Zelda binding is a predictor for open chromatin and transcription factor binding at mitotic cycle 14 –17, 20. D. melanogaster, most of which in cells or during very broad temporal windows [Several studies mapped the chromatin landscape in  windows –24. Of p windows , 16. As  windows , 25.M−) showed reduced accessibility for many, but not all, distal enhancers regulating early fly development, while maintaining near-normal promoter accessibility [Intriguingly, the causal role of Zelda in proper establishment of chromatin domains and normal gene expression was also examined. Embryos lacking zygotic Zelda expression as well as embryos with mutations in specific Zelda binding sites (near early genes) showed developmental abnormalities , 17. Embsibility , 20.The latter results suggest that perhaps, in addition to Zelda, there might be another protein that marks early developmental genes for activation. To answer this question, we revisited the chromatin patterns surrounding Zelda binding sites and developed a computational statistical model to characterize histone modifications and their dynamics. Several previous methods faced a similar situation, where a set of genomic loci are given, and unsupervised machine learning techniques should be used to re-orient them    and H3K36me3 (gene body) show strong asymmetric bias, consistent with the orientation of the underlying gene  between objects within each cluster, rather than their spatial shape.Our initial chromatin ChIP-seq data consisted of nine histone modifications  in four time points throughput MZT  —and for xim: one for each of the five histone modifications at each of the two time points (marked by m). We have down-sampled the chromatin data to 10 bp resolution, resulting in vectors of length 1000. To calculate the distance disti,jm between the two vectors representing the landscape of the modification m surrounding two loci i and j, we first used the root mean squared deviation , using a Gaussian kernel with a modification-specific parameter σm  was chosen using the eigengap heuristic , while cluster 2 peaks are more associated with transcription factors (p < 5.6e−45) and patterning  and enhancer-like (cluster 2) signatures Fig. . We usedAs a trade-off between shape and strength consideration, we added a regularization term that combined the correlation coefficient obtained for each genomic locus Eq.  by the eD. melanogaster genome at both orientations and extracted genomic loci with high local score. As shown in Fig. We then scanned the p < 1e−40), the known recognition element of Zelda [p value of 1e−18, is TTATGA and is similar to the known recognition site of two Drosophila proteins, Caudal (Cad) and Abdominal-B (Abd-B). The latter shows very low expression levels, and only toward the end of cycle 14 [p value of 1e−17, is a poly(GA)-rich motif and is similar to the known recognition site of the protein GAGA factor .To further examine the functional role of the newly discovered enhancer-like regions, we ran a de novo motif analysis on the top 1600 enhancer-like peaks, using HOMER . Our motof Zelda –17. Thisof Zelda , with a cycle 14 , while tTo determine whether Caudal or GAGA factor (GAF) may act as pioneer factors independently of Zelda, we examined ChIP-seq and ChIP-chip data for Caudal and GAF and calculated their overlap with Zelda peaks , 16, 24.p < 1e−1645), Caudal (p < 1e−36), bicoid (p < 1e−35), Kruppel (p < 1e−17) and GAF . Similar analysis for 5927 GAF binding sites , as well as the recognition motifs for hkb (p < 1e−61), Aef1 (p < 1e−52), btd (p < 1e−52).For comparison, similar motif analysis Fig. b for theD. melanogaster development [As our motif analysis showed, the GAGA motif (to which GAF binds) is enriched among the putative enhancer-like loci, with about half of GAF sites showing no Zelda binding. To test how prevalent GAF binding is at those enhancer-like loci, we also analyzed GAF ChIP data from hours 0–8 of elopment . Indeed,M−).Finally, we turned to directly test the ability of GAF to act as a pioneer factor with or without Zelda. Recently, we measured DNA accessibility via formaldehyde-assisted isolation of regulatory elements (FAIRE) experiments . The datM− mutants.To test the hypothesis that GAF and Zelda could both act as a pioneer factors, we decided to analyze ZLD and GAF binding to every putative enhancer-like region and to compare these data with their overall DNA accessibility data (as measured by FAIRE) in WT and in zldM− mutants. In addition, we expect to see that enhancers with strong GAF binding and weak Zelda binding are less affected by the deletion of zld, namely to show strong accessibility in both WT and zldM− mutants FAIRE data.For ZLD, we expected to see a positive correlation between ZLD binding and DNA accessibility  in WT, but not in zldM− mutants is less sensitive to ZLD levels, especially on the left hand side, where GAF levels are very low, yet it is almost unaffected by the deletion of zld among the strong GAF sites , we only show the average FAIRE accessibility for each group. As Fig. D. melanogaster), refined it by clustering into sub-groups of unique characteristics, and then scanned the entire genome for novel regions showing similar chromatin characteristics.In this work, we developed a computational method aimed to analyze the chromatin landscape surrounding some regions of interest  at different time points, while maintaining the spatial signature of each modification. Such models are more compelling than naive combinatorial models that only consider the heights of the various modifications but ignore the overall shape. As we have shown, many of the observed signals come in unique shapes, including a dip at the ZLD site with two flanking nucleosomes marked with one modification, all within some domain of different modification.We have analyzed early Zelda peaks throughout the Drosophila genome and identified three typical chromatin signatures they are associated with, including (1) active promoters, (2) active enhancer regions, and (3) genomic regions that were transiently bound by Zelda. We then characterized each of the classes and scanned the genome for additional regions matching the enhancer-like chromatin signature. As we have shown, many of the retrieved regions were shown to be bound by either Zelda or GAF, suggesting both act as pioneer factors by making these genomic regions accessible and depositing the appropriate histone modifications, thus marking them for activation during the maternal-to-zygotic transition. The entire role of GAF in MZT as well as the possible role for additional factors is yet to be studied.D. melanogaster, the method presented here is more general and would allow—given a query set of input loci—the identification of similar elements in any genome. In that sense, we believe that our method could be of great interest to any genome-wide research project.While these questions are key to understanding the processes that initiate and shape embryonic gene expression in the fruit fly As more genome-wide chromatin data accumulate in multiple conditions, computational methods emerge as a possible means to characterize key loci and identify novel genomic regions with similar chromatin signatures. Our work addresses these tasks by re-orienting and clustering the chromatin landscape surrounding regions of interest and then scanning the entire genome for novel regions showing similar characteristics. Using pre-MZT regions bound by the pioneer factor Zelda, we identify thousands of additional Zelda-like enhancer regions and mark the protein GAF (Vfl) as an additional factor in regulation of early developmental genes. These results are significant as they untangle the complexity in chromatin signatures near Zelda sites, and in linking GAF to chromatin accessibility and gene regulation, as well as developing a novel end-to-end method for studying genomic data.To characterize the chromatin signature around Zelda sites, we considered the top 2000 Zelda peaks in mitotic cycle 8  and lookThese 2000 Zelda peaks (namely a 3D matrix of 2000 × 10 × 1000) was subjected to an Expectation Maximization (EM)-like iterative orientation algorithm , 29. ForPeaks were associated with nearest genes and annotated with HOMER , using “xi and xj, we begin by focusing on one histone modification m:To define a distance function between two loci σm is a modification-specific parameter (set below). We then sum the adjacency values over all 10 modifications to obtain one value:We then use a standard Gaussian kernel to translate distances into weights in the spectral clustering adjacency matrixN < 10 × 1000 > matrices {xi} that correspond to the histone modification data for each peak, we defined the graph Laplacian:W is the adjacency matrix  to L2 norm of 1 . Finally, we apply a standard k-means clustering algorithm .Following Ng, Jordan, and Weiss , we trank, we applied a standard eigengap  heuristic, namely calculating the difference between successive eigenvalues (λk and λk+1) and choosing the first to surpass some threshold.To choose σm. For this, we calculated the empirical distribution of  pairwise distances for each modification m  versus the chromatin signature for cluster 2 peaks (denoted by cl2m).Following the spectral clustering of Zelda peak into three classes, we wished to identify novel enhancer regions with similar chromatin signature to that of Zelda cluster 2 peaks. For this, we scanned the genome in 10 Kb windows (stride of 100 bp) and cross-correlated the chromatin signature at each window to the average histone modification pattern of cluster 2 (enhancer-like regions). We scored each locus i by first calculating the Pearson correlation between its surrounding This approach, as opposed to just comparing the maximal peak height within each window, gives spatial resolution and identifies the unique shapes we observe for the different modifications .P:heightim denotes the average ChIP-seq signal modification m within its 10 Kb surrounding locus i, and Pm(heightim) denotes the empirical probability function (or relative frequency) of such mean ChIP-seq signal for modification m among bona fide Zelda peaks, as calculated using the chromatin signatures around the initial set of 2000 early Zelda peaks within cluster 2. This penalizes loci with similar shape  but in different ChIP magnitude.To incorporate prior knowledge and capture the overall complex shape of enhancer-like peaks, namely the typical combination of heights for each modification/time point, we also included a “prior” term Finally, we calculated the overall similarity score for each locus, by summing over the modification-specific scores:Additional file 1: Figure S1. Chromatin signatures around clustered early Zelda peaks. Heatmap and average ChIP-seq signal for five histone modifications at two time points  and 14 (right) around top 2,000 early Zelda peaks. Peaks were divided into three clusters, including cluster 1 , cluster 2 , and cluster 3 .Additional file 2: Figure S2. Average ChIP-seq binding of Zelda. (A) Average ZLD ChIP-seq signal at three time points, including mitotic cycle 8 (1 h after fertilization), mitotic cycle 11 (2 h), and mitotic cycle 14 (3 h) for clustered Zelda peaks. (B) Same, shown as metaplot (top) or heatmaps .Additional file 3: Figure S3. Functional annotation of Zelda peaks. Early 2,000 Zelda peaks (in three clusters) were annotated using HOMER into several classes including promoter/TSS, Exonic, Intronic, and Intergenic loci.Additional file 4: Figure S4. Gene expression and GO term annotations. (A) Transcription levels of gene associated with early 2,000 Zelda peaks (in three clusters), along eight time points throughout the maternal-to-zygotic transition from mitotic cycle 10–14D [e 10–14D . (B–D) GAdditional file 5: Figure S5. Analysis of chromHMM states. Chromatin data were analyzed by chromHMM [chromHMM  by firstAdditional file 6: Figure S6. Chromatin signatures and functional annotations of GAF peaks. 5,927 GAF peaks from in vivo GAF binding in Drosophila melanogaster embryos (hours 0–8 of development) [lopment)  were anaAdditional file 7: Figure S7. Distance–weight functions for various histone marks. Plotted are the empirical pairwise distance distribution (purple line) of various histone modifications at mitotic cycles 13 (bottom) and 14 (top), over pairs of early Zelda peaks <xi, xj>. Vertical red lines correspond to the 10th percentile in each distance distribution. This value is assigned as σm . The blue line shows the matching Gaussian kernel function (based on each σm), used to transform pairwise distances (X-axis) to weights (Y-axis) when building each Laplacian matrix of spectral clustering.


PC12 cells are a well-established model to study how differences in signal transduction duration can elicit distinct cell behaviors. Epidermal growth factor (EGF) activates transient ERK signaling in PC12 cells that lasts 30–60 min, which in turn promotes proliferation; nerve growth factor (NGF) activates more sustained ERK signaling that lasts 4–6 h, which in turns induces neuronal differentiation. Data presented here extend a previous study by Mullenbrock et al. (2011) that demonstrated that sustained ERK signaling in response to NGF induces preferential expression of a 69-member gene set compared to transient ERK signaling in response to EGF and that the transcription factors AP-1 and CREB play a major role in the preferential expression of several genes within the set. Here, we examined whether the Egr family of transcription factors also contributes to the preferential expression of the gene set in response to NGF. Our data demonstrate that NGF causes transient induction of all Egr family member transcripts, but a corresponding induction of protein was detected for only Egr1 and 2. Chromatin immunoprecipitation experiments provided clearest evidence that, after induction, Egr1 binds 12 of the 69 genes that are preferentially expressed during sustained ERK signaling. In addition, Egr1 expression and binding upstream of its target genes were both sustained in response to NGF versus EGF within the same timeframe that its targets are preferentially expressed. These data thus provide evidence that Egr1 contributes to the transcriptional program activated by sustained ERK signaling in response to NGF, specifically by contributing to the preferential expression of its target genes identified here. PC12 rat pheochromocytoma cells are an established model to study molecular events associated with neuronal differentiation. Following treatment with nerve growth factor (NGF), PC12 cells differentiate into neuron-like cells –5, whereOne key mechanistic link between ERK signaling and PC12 differentiation is the duration for which ERK remains active following stimulation. Stimulation with NGF induces rapid ERK activation that peaks within minutes and then gradually declines to basal levels through 3–4 h. Sustained ERK signaling through this timeframe is essential for differentiation, demonstrated in part by observation that some growth factors induce more transient ERK signaling and do not cause differentiation. The most well studied example is epidermal growth factor (EGF), which similarly stimulates ERK activation within minutes, but activity declines to basal levels more rapidly within 30–60 min. Rather than inducing differentiation, this more transient ERK signal promotes proliferation .One potential basis for the distinct biological response of PC12 cells to NGF is that NGF elicits a transcriptional program that is dependent on sustained ERK signaling through 2–4 h after stimulation. To investigate this, DNA microarray analysis was used to compare global gene expression profiles of PC12 cells treated with either NGF or EGF for 2 or 4 hours, which identified a set of 69 genes preferentially up-regulated in response to NGF at those time points  . SubsequThe Egr family comprises five members—Egr1-4 and Wilms Tumor 1 (WT1)—which share highly homologous DNA-binding domains at their C-termini composed of three zinc finger motifs that bind similar GC-rich, 9-nucleotide response elements . Their NEgr proteins are also immediate early genes (IEGs) induced rapidly and transiently in response to several stimuli including growth factors, cytokines, neurotransmitters, and multiple cellular stressors  –32. UponA role for Egr proteins in PC12 differentiation is also established. Levkovitz et al. (2001) determined that NGF-induced neurite outgrowth is inhibited by transfection with a truncated Egr3 consisting of its DNA-binding domain that acts as a dominant-negative on Egr-mediated transactivation . Transfe2. For gene expression and Western blot experiments, 106 cells were plated per 60-mm dish in growth medium and incubated for 24 h before medium was replaced with reduced serum medium (DMEM + 0.5% HS). For ChIP experiments, 6 x 106 cells were plated per 150-mm dish in growth medium and incubated for 72 h before medium was replaced with reduced serum medium. After placing in reduced serum medium, cells were incubated another 24 h before treatment with 50 ng/ml NGF  or 25 ng/ml EGF .PC12 cells  were maiFor gene expression experiments, total RNA was extracted using TRIzol Reagent  as follows. Cell cutlures were washed once with PBS and lysed in 500 μl of TRIzol, 100 μl of chloroform were added to each lysate, and samples then incubated for 3 min at room temperature. Samples were subjected to centrifugation at 12,000 x g for 15 min at 4°C and the aqueous phase collected. Two hundred μl of 2-propanol were added to each sample and samples incubated for 10 min at room temperature. Samples were subjected to centrifugation at 16,000 x g for 10 min at 4°C and the supernatants removed. RNA pellets were washed by adding 1 ml of 70% ethanol, vortexed briefly, and then subjected to centrifugation at 16,000 x g for 15 min at 4°C. The supernatants were removed and RNA pellets air dried for approximately 10 min. RNA pellets were then suspended in 50 μl of nuclease-free water and incubated at 60°C for 10 min before spectrophotometry to determine nucleic acid concentrations.Reverse transcription reactions were conducted on 900 ng of nucleic acids from each sample using TaqMan Reverse Transcription kit , according to the manufacturer’s instructions. Real-time PCR reactions where then conducted using SYBR Select Mater Mix , according to the manufacturer’s instructions. Primer sequences used for real-time PCR are listed in Following treatment with NGF or EGF, cells were washed twice with phosphate-buffered saline (PBS), lysed directly in 300 μl of 2X Laemmli buffer, and heated to 95–100°C for 5 min. Twenty μl of each lysate were loaded and electrophoresed through SDS-polyacrylamide gels , transferred to nitrocellulose membrane, and immunoblot conducted to detect Egr1 , Egr2 , Egr3 , Egr4 , WT1  and β-actin .Membranes were blocked in 5% nonfat dried milk in TBS + 0.2% Tween-20 (blocking buffer) for 30 min at room temperature and then incubated with primary antibody diluted in blocking buffer for 12–18 h at 4°C. Membranes were then washed three times for 15 min in TBS + 0.2% Tween-20 and incubated with secondary antibody  diluted in blocking buffer for 1–2 h at room temperature. Membranes were washed three times for 15 min in TBS + 0.2% Tween-20 after which bands were detected by chemiluminesce .ChIP assays were performed as previously described . For eacMullenbrock et al. (2011) identified 69 genes preferentially up-regulated in PC12 cells 2–4 h after NGF treatment as compared to EGF treatment; 21 of those genes contained putative Egr binding sites (identified using the V$KROX_Q6 position weight matrix) within 5 kb upstream of their transcription start sites that were conserved in human, mouse and rat . Given tEffects of NGF on Egr expression levels were evaluated first. Consistent with their characterization as IEGs, NGF induced a robust increase in Egr1-4 transcripts at 30 min post-treatment, followed by a gradual decline through 4 h . TranscrOur data for Egr1 are consistent with previous studies that also demonstrated induction of Egr1 transcript and protein in PC12 cells with near identical kinetics following NGF treatment , 61. IndRdh10 for which primer design was limited due to high GC content  contained multiple dispersed Egr binding site candidates, for which multiple primer sets were used  to evaluate Egr binding within the respective regions. Primers to amplify a region approximately 100 bp upstream of the Myog gene, which lacks an Egr binding site, were used as a negative control for Egr binding.Binding of Egr family members to upstream regions of genes with putative Egr binding sites was examined next in PC12 cells by ChIP. Primers were designed within 250 bp of each predicted Egr binding site to quantify their recovery by real-time PCR, except for the three putative sites upstream of Myog following Egr1 ChIP from untreated cells, whereas % input for Sik1/Snf1lk was 7-fold greater, indicating Egr1 is bound upstream of only Sik1/Snf1lk in the absence of NGF. After 1 h of NGF treatment, % input values increased well above that of Myog for several targets. Of the 20 genes examined, 15 exhibited average values greater than 2-fold above Myog, of which 12 were statistically significant (p < 0.05) and 2 approached significance . To our knowledge, 10 of these genes  are novel Egr1 targets, whereas 5  were previously identified targets [Sik1/Snf1lk. The molecular explanation for Egr1’s differential binding to Sik1/Snf1lk versus the other targets was not examined.Binding of Egr1 was examined first due to its established role in PC12 differentiation , 59. Sin targets –74. For Sik1/Snf1lk. After NGF treatment, 11 targets exhibited % input values greater than 2-fold above Myog, which were the same targets bound by Egr1 after NGF treatment excluding Mafb. However, values for those 11 targets were 3- to 8-fold lower than % input values from Egr1 ChIP. Whether these differences reflect differential binding of Egr1 versus Egr2 upstream of target genes or differential effectiveness of the respective antibodies in ChIP was not determined. ChIP with anti-Egr3, -Egr4 or -WT1 antibodies did not demonstrate binding of these family members upstream of any putative targets before or after NGF treatment (data not shown).ChIP with anti-Egr2 antibody yielded similar results to Egr1, but with notable differences . In untrAltogether, these ChIP data demonstrate that NGF induces Egr1 binding upstream of several genes that are preferentially up-regulated during sustained ERK signaling, indicating it may contribute to their expression at 2–4 h. The data provide more modest evidence for Egr2 binding, suggesting it may also play a role. Given the more robust data for Egr1, additional experiments focused on further examining its role in the network.To begin examining how Egr1 might contribute to the preferential expression of its target genes 2–4 h post-NGF versus -EGF treatment, Western blot analysis and densitometry were conducted to compare its levels 0–4 h after treatment with each growth factor. Both NGF and EGF induced strong up-regulation of Egr1 at 1h followed by a decline to near basal levels through 2–4 h ; howeverMyog after Egr1 IP from untreated cells, whereas values increased above Myog after EGF treatment. Comparison of % input values for each gene independently after 1 h NGF versus 1 h EGF treatment showed no significant difference  are novel Egr1 targets and three  were previously identified. The kinetics of Egr1 binding to a subset of these targets were examined 0–4 h after NGF treatment; consistent with its expression pattern, binding peaked 1 h after NGF treatment and then gradually declined through 4 h. EGF induced similar effects on Egr1 expression and binding upstream of target genes, but with notable differences. Both Egr1 protein levels and binding upstream of targets peaked 1 h after EGF treatment; however, both then declined more rapidly through 2–3 h compared to NGF treatment. These data collectively demonstrate that, compared to EGF, NGF induces sustained Egr1 expression and binding upstream of several target genes during the same timeframe in which those genes are preferentially expressed, which supports our hypothesis that Egr1 contributes to the transcriptional program activated by sustained ERK signaling. Given Egr1’s transactivation activity, the simplest extrapolation is that Egr1 contributes to the program through sustained transactivation of its target genes. However, since Egr1 can also act as a transcriptional repressor, a more complex mechanism for Egr1’s contribution cannot be ruled out.As expected based on its previous characterizations as an IEG, NGF induced strong, but transient Egr1 expression; protein levels peaked 1 h after treatment and then declined to near basal levels through 6 h. ChIP experiments demonstrated that 1 h of NGF treatment induced Egr1 binding upstream of 11 of the 21 genes with predicted Egr binding sites, whereas Egr1 was bound upstream of one target, The Venn diagram in Sik1/Snf1lk in the untreated cells, especially given its low expression levels compared to 1 h post-NGF treatment. AP-1, whose expression and binding upstream of target genes are induced by NGF similarly to Egr1 [Sik1/Snf1lk. However, in contrast to Egr1, AP-1 binding to Sik1/Snf1lk was induced similarly to AP-1 binding to its other targets. Nevertheless, selective binding of transcription factors to specific target genes when at basal levels is not unprecedented. For example, transcription factors c-Myc, REST/NRSF, and Ste12, have been shown to selectively bind target genes when at basal levels [Sik1/Snf1lk.One surprising result from the Egr1 ChIP experiments discussed here was the selective binding of Egr1 upstream of only  to Egr1 , also bil levels –77. Enril levels ; howeverVgf and Hbegf both encode neurotrophic factors expressed throughout the nervous system and have been implicated in several aspects of its development and maintenance [Kdm6b/Jmjd3 encodes an H3K27 demethylase and Kctd11 encodes an adaptor for the ubiquitin ligase Cullin3, both which contribute to differentiation of embryonic and neural stem cells to neurons [Sema6a encodes the transmembrane protein semaphorin6a, which regulates axon guidance and dendritic growth during development [Arc encodes a post-synaptic protein that plays important roles in synaptic plasticity associated with learning and memory [Mafb encodes a transcription factor that controls hindbrain segmentation and development at least in part via regulation of Hoxa3 and Hoxb3 expression [Tph1 encodes tryptophan hydroxylase-1, which catalyzes the rate-limiting step in serotonin synthesis; it is expressed in mouse brain during late stages of development and then restricted to the pineal gland in adulthood. Polymorphisms within the Tph1 gene have been implicated in multiple psychiatric conditions [Of the 69 genes preferentially induced by NGF, approximately 50% have been implicated in neuronal development and/or function , which intenance , 78–81.  neurons –86. Semaelopment –89, wherd memory , 91. Mafpression –94. Tph1nditions –97.Vgf, Trib1, and Phlda1) also yielded highest values from Egr2 ChIP. However, the Egr2 ChIP values were overall much lower (3- to 8-fold) than those from Egr1 ChIP, which could be due either to lower levels of Egr2 binding to the Egr binding sites or lower efficiency of the Egr2 antibody for ChIP in general. Given the modesty of the Egr2 ChIP results, additional experiments focused on Egr1.Examination of Egr family members other than Egr1 provided more modest evidence for contribution of Egr2 to the transcriptional program. Egr2 transcript and protein levels mirrored those of Egr1 in response to NGF. ChIP experiments suggested induction of Egr2 binding to predicted targets after NGF treatment with an overall pattern similar to Egr1. Target genes that yielded highest % input values from Egr1 ChIP after 1 h of NGF treatment  that characterized a transcriptional program in PC12 cells whose activity is associated with sustained ERK signaling and neuronal differentiation. Mullenbrock et al. identified a set of 69 genes preferentially up-regulated during sustained ERK signaling and provided evidence that transcription factors AP-1 and CREB cooperatively contribute to the preferential expression of 25 genes within the set. Data shown here provide evidence that transcription factor Egr1 also plays a cooperative role through regulation of 12 genes within the set, nine of which are also targeted by AP-1 and/or CREB during sustained ERK signaling see ; we alsoS1 FigMyog gene were used as a negative control for Egr1 binding. (a) Data from Egr1 ChIP are plotted as % input and are averages from three to four independent experiments ± S.E. *, Student’s t tests were conducted comparing the % input value for Myog after Egr1 IP to the % input values for each predicted Egr target after Egr1 IP, which yielded p values ≤ 0.05 for several primer sets. (b) Data from Egr2 ChIP are plotted as % input and are averages from two independent experiments ± S.E.PC12 cultures were treated with or without 50 ng/ml NGF for 1 h and subjected to ChIP assay using antibodies against Egr1, Egr2, or an IgG control. Real-time PCR was then conducted on the immunoprecipitated DNA using primers within 250 bp of each predicted Egr binding site (see (TIFF)Click here for additional data file.S1 TableTranscript levels of Egr1-4 and WT1 were evaluated by real-time RT-PCR using the forward and reverse primers listed within this table.(XLSX)Click here for additional data file.S2 TableThis table provides the locations and sequences of putative Egr binding sites identified within the rat genome. Locations and sequences for forward and reverse primers used in real-time PCR following ChIP are also listed, alongside their Figs (XLSX)Click here for additional data file.S3 Tablet test. p values for all Egr1 targets were ≥ 0.25.Percent input values for several Egr1 target genes after Egr1 ChIP from cells treated with 50 ng/ul NGF or 25 ng/ul EGF for 1 h are listed and compared by Student’s (XLSX)Click here for additional data file.


Single nucleotide polymorphisms (SNPs) in the human angiotensinogen (hAGT) gene may modulate its transcription and affect the regulation of blood pressure via activation of the renin-angiotensin aldosterone system (RAAS). In this regard, we have identified polymorphisms in the 2.5 Kb promoter of the hAGT gene that form two haplotype (Hap) blocks: -6A/G  and -217A/G . hAGT gene with Hap -6A/-217A (Hap I) is associated with increased blood pressure whereas, Hap -6G/-217G (Hap II) is associated with normal blood pressure in human subjects. Since RAAS over activity contributes to hypertension in obesity, we have made transgenic mice (TG) containing either Hap I or Hap II of the hAGT gene to understand the role of obesity on its transcriptional regulation. Although, a high-fat diet  elevates hAGT and mAGT regardless of haplotype, this effect is significantly (p<0.05) accentuated in Hap I mice, in both adipose and liver tissues. Chromatin Immuno- precipitation (ChIP) assay shows an increased binding of transcription factors including, GR, CEBPβ and STAT3 to the chromatin of the Hap I TG mice after high-fat diet as compared to Hap II TG mice (p<0.05). Differential plasma levels of hAGT in Hap II and I mice, after high-fat diet, further corroborate the variable transcriptional regulation of the hAGT, governed by gene-haplotypes. Taken together, our results show that SNPs in the Hap-I of the hAGT gene promote high-fat diet-induced binding of transcription factors GR, CEBP-β and STAT3, which lead to elevated expression of the hAGT gene in hepatic and adipose tissues. Hypertension is a complex disorder arising from intricate crosstalk between environmental factors and genetic predispositions , 2. Impoin vivo setting.AGT is the sole substrate of the renin-angiotensin system (RAS), which is central to mammalian blood pressure regulation –8. RAS oThe human AGT gene contains multiple SNPs in its 2.5 Kb promoter –16. ManyWe tested the effects of the HFD on the transcriptional regulation of different haplotypes of the hAGT gene. High-fat diet up regulated the hAGT expression in both groups; however, change in the hAGT expression is significantly (p<0.05) greater in adipose  and liveIn silico Transfac analysis shows that nucleotide sequence present in the promoter of Hap I of the hAGT gene has stronger homology with consensus binding sites of transcription factors GR, CEBPβ, HNF3 and STAT3 as compared to Hap II of the hAGT gene  increased in Hap I TG mice as compared to Hap II . MoreoveIn the present study, we have examined the HFD-mediated, allele-specific, transcriptional regulation of the hAGT gene in adipose and liver tissues. Previous studies have suggested that the hAGT gene contains 4 major haplotypes. Hap I  has the highest risk for increased blood pressure whereas, the Hap II  confers the lowest risk for increased blood pressure . To undeThe first key finding of this study is that HFD positively regulates the hAGT expression, which is greater in mice with HAP I. Higher hAGT expression in adipose and liver tissue is observed after HFD in both Hap I and Hap II TG mice. However, difference in hAGT expression was much higher in the Hap I as compared to the Hap II TG mice. These results are exciting but not unexpected since other investigators have previously reported diet-induced induction of the AGT in liver and adipose tissues [RAAS over-activity and hypertension are frequent accompaniments of diet-related pathologies like, the metabolic syndrome. It is well accepted that the liver and the adipose tissues are the primary source of systemic AGT in rodents and humans. We have previously shown that the hAGT gene expression is higher in liver and fat of the Hap I TG mice as compared to the Hap II .The firs tissues –24. This tissues .The second key finding of this study, in agreement with published reports, is the diet-induced increase in levels of transcription factors in the liver and the adipose tissues. Expression of glucocorticoid receptors, STAT3 and CEBPβ is significantly increased by HFD. It is noteworthy that this increase in transcription factors is haplotype-independent. Transcription of a gene is regulated by a plethora of transcription factors, binding to their cis-acting DNA elements in the promoter and enhancer regions of a gene. SNPs in the promoter and/or enhancer regions of the gene alter this binding and thus, have the potential to modulate gene transcription. Nucleotide sequence of the hAGT gene promoter containing -217A (Hap I) has stronger homology with the consensus C/EBPβ binding site as compared to -217G. C/EBP plays an important role in the expression of a gene in the hepatocytes [MeG will inhibit the binding of the transcription factors to this site in the Hap II TG mice. On the other hand, nucleotide sequence of the promoter region of the hAGT in the Hap I TG mice contains CCAG . Interestingly, variant -1074T (present in the Hap I) makes stronger HNF3 binding site as compared to variant -1074G (present in the Hap II). Transcription factor HNF3 plays an important role in the liver-specific expression of a gene. In addition, HNF3 has been termed as the “pioneer” transcription factor and opens up the chromatin for binding with the estrogen, androgen and mineralocorticoid receptors , 34. Addthird important finding of this study; HFD-altered transcriptional milieu modulates the human AGT expression in a haplotype-dependent manner via enhanced transcription factor binding to the chromatin of the hAGT in the HAP I mice. Expression of these transcription factors is independent of haplotype but, we have found an increased binding of GR and CEBPβ to the chromatin obtained from the adipose tissue of the Hap I TG mice as compared to Hap II. As discussed earlier, SNPs in the promoter of Hap I of the hAGT gene have stronger binding with HNF3 and C/EBP-β [This leads to the  C/EBP-β . Importa C/EBP-β , 35–37. Diet-induced obesity is a chronic inflammatory state , 25. Thiet al. have measured the expression of different genes in the adrenal gland of hypertensive rats and mice  by microarray and found that CEBPβ expression is increased in hypertensive animals [We observe an increased binding of CEBP-β and STAT3 after HFD diet in chromatin obtained from the adipose tissue of Hap I TG mice. Since endogenous mAGT gene does not contain SNPs to modulate GR, or C/EBP- β binding, we did not observe differential regulation of the mAGT gene by HFD, which lends credibility to the hypothesis that HFD promotes haplotype-mediated transcription of the hAGT gene. Thus, these results strongly suggest that the transcriptional milieu of liver and adipose tissues is altered after HFD, which then affect the hAGT expression in a haplotype-dependent manner due to, increased affinity of the chromatin of the Hap I TG mice to transcription factors such as CEBP-β, GR, HNF-1 and STAT3. It is important to mention in this context that Friese  animals . Earlier animals , 43. ThiSystemic implications of increased transcription of the hAGT gene by HFD (HFD) in Hap I TG mice were confirmed by the assessment of plasma AGT levels. These observations are supported by previous studies that demonstrated the activation of the systemic RAAS in experimental models of diet-induced obesity –46. It wTo summarize, pathophysiological variables like the HFD alter the cellular transcriptional milieu that ultimately results in the modulation of the hAGT gene expression. HFD increases the expression of transcription factors such as GR, HNF-1, STAT-3 and C/EBPβ in the liver and adipose tissues, which results in increased binding of these transcription factors to the chromatin and increased expression of the hAGT gene in animals containing Hap I of this gene. This would potentially make subjects with Hap I more susceptible to HFD-induced, AGT-mediated complications such as hypertension. Clinical significance of this study is that it will help identify individuals with risk haplotype undergoing diet induced metabolic syndrome. In this setting, identification of “at-risk” haplotypes for the hAGT will benefit patients by providing timely and targeted therapy so as to prevent long-term complications. It is worth noting that the sample size in this study is small due to low-breeding of the TG lines but, the data remains significant nonetheless.ad libitum. Animals were maintained in a 22°C room with a 12-h light/dark cycles and received drinking water ad libitum.All animal experiments were performed according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the institutional ethical animal care and use committee at the University of Toledo College of Medicine (UTMC). The human AGT (hAGT) TG mice utilized in this study were generated in our laboratory as described previously . GenotypPlasma hAGT levels were determined by an ELISA kit purchased from Ray Biotech, Inc in male TG-mice. The hAGT concentration in the samples was determined directly from the standard curve according to the manufacturer’s protocol .-ΔΔCT method [Adipose and liver tissue were harvested at the end of the experiments and snap-frozen in liquid nitrogen. The extracted tissues were stored at -80°C until utilized for further experiments. RNA was isolated using RNeasy Plus mini kit (Qiagen). RNA (1ug) was reverse-transcribed into cDNA using the Revert Aid first strand cDNA synthesis kit (Fermentas), as described in the protocol. Following a 95°C incubation for 10 min, 40 cycles of PCR , were then performed using 1μl of cDNA, 50 nM PCR primers, and 12.5 μl of SYBR Green PCR Master Mix in 25μl reactions. Threshold cycles for three replicate reactions were determined using MxPro-Mx3005P software (version 4.10), and relative transcript abundance was calculated following normalization with mouse GAPDH. Primers for human and mouse AGT and mouse GAPDH were obtained from Super Array Bioscience Corp.  or from Integrated DNA Technologies . In general, relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample, such as untreated control. This is a widely used method to present relative gene expression by the comparative CT method, also referred to as the 2T method –49.CTCAGGCTGTCACACACCTA) as a forward and -6AGT rev (TCTTCCCCTGGCCGGGTCACGAT) as a reverse primer, when GR and CEBPβ antibodies were used to examine their binding at the -217 position of the hAGT gene, or (b) -1757 AGT for (CAGGCACAGTGGAAACTCTCC) as a forward primer and -1554 AGT rev (AGTAACAAGTCCACCTGGAC) as a reverse primer, when GR and CEBPβ antibody were used to examine the binding of GR across the -1561 and -1562 polymorphic sites of the hAGT gene. The PCR-amplified products were analyzed on 2% agarose gel. The relative band intensities by densitometry were measured using Quantity One quantitation software from Bio-Rad, and the results were normalized to the band intensities from the respective input DNA. The fraction enriched by rabbit IgG was used as a negative control for nonspecific binding. A primer set that amplified the DNA fragment of the hAGT gene with no known glucocorticoid response element was used as a nonspecific control for GR-enriched DNA fragments. In another set of experiments, 900ul of chromatin solution was immunoprecipitated with pSTAT3 antibody. Immunoprecipitated DNA was then amplified using CTCAGGCTGTCACACACCTA as a forward primer and (CTTTATGTTTTTGGCGTCTTCCA) (Promega Biotech) a reverse primer to amplify APRE-1 regions. Oligonucleotide TATGCTAGGGCTCACTCTGTTCAGCAGTGAAACTC was used as a forward primer and the reverse primer for amplification of APRE-3 (STAT binding site) region in the hAGT proximal promoter region. Oligonucleotide GAGGTATTTGTGTGTTTGTTGATTGT and ACAGGGCATGACAGAGACCTTGG were used as forward and reverse primers to amplify 320 bp  of the upstream region of the hAGT promoter.The ChIP assay was performed using the EZ-ChIP assay kit from EMD Millipore as described previously . Mice wewww.gene-regulation.com/pub/programs.html for analysis of the transcription factor binding sites.In silico analysis of the hAGT promoter and transcription factor binding sites is performed using TRANSFAC (TRANScription FACtor database) analysis. The Transfac web site was used in our analysis mentioned in this paper but this site has been dis-continued. We are now using All experiments were conducted with four animals in each group. Data are expressed as the means S.E. Statistical significance was assessed using two-way analysis of variance with a Tukey-Kramer post hoc analysis. Two factors for the two-way ANOVA are, haplotype and diet. The significance level was set at (p≤ 0.05). For Figs S1 FigHuman AGT expression was significantly elevated after high fat diet (HFD) in TG mice with Hap I compared to Hap II in adipose and liver tissue . Figure shows the quantitative RT-PCR analysis of hAGT and mAGT mRNA level in adipose  and in liver  in TG mice after 12 weeks of control diet or HFD. n = 4 per group in both CD and HFD groups.(TIF)Click here for additional data file.S2 FigRelative mRNA expression is calculated for each group compared with its respective control diet group (n = 4).(TIF)Click here for additional data file.S3 FigQ-PCR was performed to quantify the relative binding at -217 region. ChIP assay was performed from the chromatin obtained from the adipose tissue of HFD treated TG animals (n = 4).(TIF)Click here for additional data file.S4 FigQ-PCR was performed to quantify the relative binding at -1329 region. ChIP assay was performed from the chromatin obtained from the adipose tissue of HFD treated TG animals (n = 4).(TIF)Click here for additional data file.S5 FigPlasma AGT levels were determined by an ELISA (n = 4).(TIF)Click here for additional data file.S1 File(XLSX)Click here for additional data file.


Tribolium castaneum is described for the first time, applying an approach that combines an in silico gene characterisation with an in vivo immune priming experiment using the Gram-positive entomopathogen Bacillus thuringiensis. We identified an unusually high number of three different transcripts for extracellular SOD and found that priming leads to a fine-tuned modulation of SOD expression, highlighting the potential of physiological co-adaptations for immune phenotypes.The production of reactive oxygen species (ROS) is a normal consequence of the aerobic cell metabolism. Despite their high and potentially detrimental reactivity with various biomolecules, the endogenous production of ROS is a vital part of physiological, immunological, and molecular processes that contribute to fitness. The role of ROS in host–parasite interactions is frequently defined by their contribution to innate immunity as effectors, promoting parasite death during infections. In vertebrates, ROS and antioxidant system enzymes, such as superoxide dismutase (SOD) are also involved in acquired immune memory, where they are responsible for T-cell signalling, activation, proliferation, and viability. Based on recent findings, ROS are now also assumed to play a role in immune priming, i.e., a form of memory in invertebrates. In this study, the potential involvement of Cu,Zn SODs in immunity of the red flour beetle  One of the main sites of formation are mitochondria, where ROS are produced by interaction between molecular oxygen  formation  , 18–20. ormation . In summTribolium castaneum. In this model organism, enhanced protection after repeated exposure to the same bacterial pathogen was demonstrated for septic as well as oral infection routes  is mainly considered a hallmark of vertebrate species. However, recent studies demonstrated that invertebrates are also capable of mounting alternative forms of adaptive immune responses despite lacking features of classical vertebrate immunity , 25. Amon routes , 27, andn routes , 29. Thin routes ], includn routes . Althougn routes , 31–33. llonella –37.T. castaneum, the analysis of transcriptomic data from two populations of T. castaneum, infected with the entomopathogen Bacillus thuringiensis by septic wounding, emphasised the importance of an up-regulation of transcription in genes with a predicted oxidative function for the systemic immune response  is a ubiquitous family of metallo-enzymes that catalyse the dismutation of •O history , 40. SOD history , 42. In T. castaneum were obtained from our laboratory strain CRO1 ]. For the challenge, we used live bacteria at the concentration of 5 × 105 cells × mL−1.For the immune priming, the bacteria suspension was first adjusted to a concentration of 1 × 10The appropriate bacteria concentrations used in this study were determined on the basis of preliminary experiments conducted in our laboratory, aiming at a priming concentration that leads to enhanced survival upon following challenge, and the lowest concentration that still induces mortality upon challenge, respectively (data not shown).T. castaneum upon challenge with Bt1, in order to ensure that the infected individuals were not moribund , which is known to respond to septic infection with B. thuringiensis  as described in previous experiments (®) and sequenced with the 3130xl Genetic Analyzer (Applied Biosystems) to confirm the specificity of the designed primers.Quantitative real-time PCR analyses were performed in a final volume of 10 µL, with 1–10 ng of cDNA, 1 × KAPA™ SYBR Green PCR Master mix, 0.5 µM for each primer. A dissociation curve was used to confirm the specificity of the amplicon. We verified the efficiency of the primers by drawing standard curves for target genes (amplification efficiency >1.9). qRT-PCR reactions were performed in duplicates in a PCR system LightCyclereriments . Thermaleriments  with resT. castaneum were aligned to other confirmed metazoan sequences, using the Muscle programme in order to study their level of conservation . In this work, homology protein modelling was carried out with SwissModel bioinformatic toolssod genes was analysed with a handmade pipeline designed in Python 3.0 to identify putative transcription factor binding sites such as antioxidant responsive element (ARE), metal responsive element, and xenobiotic responsive element (XRE) sites, according to previous studies .Survival data were analysed using multiple pairwise Wilcoxon tests across treatments in R Version 3.3.2.T. castaneum Genome Database , we identified three sequences putatively coding for members of the Cu,Zn SOD family. We named the respective genes tc-soda (OGS-ID TC007011), tc-sodb (OGS-ID TC011676), and tc-sodc (OGS-ID TC011675).In the In silico analysis of tc-soda indicates that the open reading frame (ORF) includes 462 nt and that it encodes a putative protein of 155 aa, with a deduced molecular weight of 15.7 kDa. This gene contains two exons and one intron, the latter located in the 5′untranslated (UTR) region  is located in the 5′ UTR. Tc-sodb2 is a truncated form of tc-sodb1, consisting of three exons and two introns  is characterised by a 684 nt ORF that encodes a 217 aa protein with a predicted molecular weight of 24.3 kDa. The 5′-UTR region for this gene is 662 nt and the 3′-UTR region is 36 nt long. tc-sodc consists of three exons and three introns: one of these introns is located in the 5′ UTR  in Tc-SODb1 and 2, as well as between residues 19 and 20 (VQS-AS) in Tc-SODc, but not in Tc-SODa .The deduced amino acid sequences of the four identified Cu,Zn SODs from Xenopus laevis (NCBI NP_001080933.1), Homo sapiens (NCBI ABL96616.1), and Bos taurus (PDB 1CB4), while Tc-SODb1, Tc-SODb2, and Tc-SODc were compared with SOD3 from Homo sapiens only, as this is the only metazoan species with a confirmed crystallographic structure for the EC-SOD (PDB 2JLP). The copper and zinc coordination environment and the active site are conserved in all compared sequences for SOD1 and 3. The 3/10 α-helix in the C-terminus is missing from Tc-SODb2 and Tc-SODc.Following our initial analysis, the predicted protein sequences of the considered SOD isoforms were aligned with the respective orthologues from other metazoan species with a confirmed crystallography structure . Tc-SODa was compared with SOD1 sequences from Tc-soda and extracellular isoform Tc-sodc with their respective orthologues from other species in taxonomic order . Most intriguing, the two variants of Tc-sodb do not fall into the group of Tenebrionidae, but seem to be more closely related to the EC-sod from the leaf beetle Phaedon cochleariae suggesting a gene duplication event of an ancestral EC-sod gene before the split of the recent coleopteran families , but not in the wounding control (priming with PBS), while sodb only shows a statistically significant increase of mRNA expression when primed with hkBt1 in the non-challenged treatment, and only if compared with the wounding control . In contrast, the other gene that codes for SOD3, sodc, is down-regulated in all primed or challenged treatments, compared with the untreated control (naive/naive) . In order to quantify the immune activation due to the priming and/or challenge treatment, we also measured the mRNA expression of the gene for the AMP attacin (atta2), which was shown to strongly respond to immune insults. Indeed, atta2 mRNA levels remained low when the animals were unchallenged, but showed increased expression after both repeated wounding and after infection with Bt1 .The Cu,Zn-SOD mRNA levels in all specimens were estimated by RT-qPCR 1 day after the infection. The relative expression of p < 0.05). In infected specimens, the expression pattern of SOD is reversed, resulting in an increase in both wounded and primed experimental groups when compared with the unprimed controls (p < 0.05).The protein activity of SOD remained unchanged in animals without challenge compared with untreated controls (naive/naive) Figure . In PBS T. castaneum, which is an intensively used model organism for studies of immune priming, and then experimentally analysed the gene expression of these main components of the antioxidant system. The combination of in silico and mRNA expression data demonstrates the presence of one gene coding for IC-SOD (tc-soda) and two genes coding for EC-SOD (tc-sodb and tc-sodc). The two separated genomic regions containing tc-sodb and tc-sodc, might have resulted from a gene duplication event, that might have contributed to the functional specialisation of these two isoforms in T. castaneum, as previously demonstrated in unicellular eukaryotes for IC-SOD and others genes belonging to the antioxidant system . The presence of two different mRNAs that map in the same genomic region is not unexpected per se. Due to the continued refinement of RNAseq technology, we are now able to identify alternative mRNA transcription, be it as a consequence of an alternative ORF or an intronic alternative polyadenylation recognition [for review see de Klerk and ’t Hoen , offsetting the beneficial effect of an increased gene product and the negative effect of decreased overall fitness, resulting in a heritable heterozygosity  might have been under stronger evolutionary constraints than the intracellular isoform (tc-soda), as a consequence of being target sites for insecticides.We offer two hypotheses for the redundancy of gene products for EC-SODs in zygosity . Similarzygosity , 56 and zygosity . Althougzygosity  found ontc-sodb are most closely related to the SOD3 isoforms of the mustard leaf beetle P. cochlearis. Interestingly, in this coleopteran species, substantial EC-SOD activity is found in the haemolymph as well as in the defensive secretions, and SOD-deficient larvae showed increased mortality when challenged with the entomopathogenic fungus Metarhizium anisopliae  and ultimately the enzyme activity in the cytosol. In the promoter region of all tc-sods, we identified several putative hARE sites, whereas we found one ARE and XRE only in the case of tc-soda. Previous studies highlighted that, in invertebrates, a partial ARE sequence is sufficient to promote gene transcription in the presence of ROS . Moreover, we found that Tc-sod genes can be induced by such immune insults, potentially through activation via the regulatory elements described above. In fact, we detected not only basal transcription of all isoforms in the control specimens (naive/naive), but also a modulation of this transcription in response to septic priming (PBS and hkBt1) and challenge treatments (PBS and live Bt1). The transcription of the IC-SOD (tc-soda) was up-regulated after priming with hkBt1 . Notably, a simple wounding (PBS priming) did not lead to any significant up-regulation of tc-soda. In contrast, the priming-induced up-regulation of the transcription of the EC-SOD tc-sodb , was only visible in animals that were left naive for challenge. Surprisingly, the second EC-SOD (tc-sodc) showed a differing pattern of down-regulation in all priming and/or challenge treatments compared with fully naive animals. One potential scenario is that tc-soda is up-regulated to protect hosts from the infection-induced •O2− formation, while the extracellular isoforms are either down-regulated (tc-sodc) or remain mainly transcribed constitutively. This might decelerate the elimination of antimicrobial ROS from the intercellular space , while preserving cell viability during infections in T. castaneum, ultimately contributing to the overall survival of the host during bacterial infection.It has been suggested that ROS might play a role during oral priming responses in astaneum . Howeverpricking , can als1 Figure A. This patta2, which mostly showed the expected typical pattern of up-regulation after wounding or bacterial challenge, but did not respond in animals that were primed but left naive for challenge. The role of SOD’s can be viewed as regulators of ROS, and are therefore not directly comparable with immune effector molecules such as AMP’s. This result is in line with what has been reported recently by Tate et al., where atta2 expression during B. thuringiensis infection of T. castaneum was highly dependent on bacterial load, while other, metabolic pathways that seem involved in a broader priming effect are not effected by the concentration of bacteria (2O2 during infection. In fact, it is well known that high concentrations of H2O2 inhibit the enzymatic activity of Cu,Zn SODs in vivo (2O2, is an important component of the innate defence mechanism against B. thuringiensis in T. castaneum. Future studies will allow us to better understand the fate and potential role of H2O2 produced by SOD activation during immune priming, especially since this metabolite is strongly involved in signalling (It is noteworthy that the immune-regulatory signal of priming on SOD gene transcription differed from the transcription profile of the AMP gene bacteria , 65. Thebacteria . Unfortubacteria . While w in vivo –68. Suchgnalling –71 and ignalling  in verteT. castaneum. Moreover, our findings support the current idea that priming is a specific reaction that is qualitatively different from a simple low-level immune activation (In conclusion, the expression of Cu,Zn-SOD genes is affected by immune priming and challenge in tivation , 38 and tivation , that istivation , especiaThis work involved an invertebrate species. No ethical permit is needed according to German law.in silico characterisation of sod genes. RB reconstructed the phylogenetic trees. KF, DF, and GS analysed the data. All authors contributed to writing the manuscript and gave final approval for publication.DF conceived the study. KF, DF, and JK designed the experiments. KF, DF, FC, and GS performed the experiments and assays. DF performed The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer IE and handling editor declared their shared affiliation.


Here, we reveal the patterns of histone modification change and nucleosome positioning dynamics and their epigenetic regulatory roles during the in vivo glial differentiation in early Drosophila embryos. The genome-wide average H3K9ac signals in promoter regions are decreased in the glial cells compared to the neural progenitor cells. However, H3K9ac signals are increased in a group of genes that are up-regulated in glial cells and involved in gliogenesis. There occurs extensive nucleosome remodeling including shift, loss, and gain. Nucleosome depletion regions (NDRs) form in both promoters and enhancers. As a result, the associated genes are up-regulated. Intriguingly, NDRs form in two fashions: nucleosome shift and eviction. Moreover, the mode of NDR formation is independent of the original chromatin state of enhancers in the neural progenitor cells.Chromatin remodeling plays a critical role in gene regulation and impacts many biological processes. However, little is known about the relationship between chromatin remodeling dynamics and  Epigenetic factors play a critical role in many biological processes through regulating gene transcription. Chromatin remodeling is one of the key epigenetic mechanisms, mainly including nucleosome positioning and histone modification dynamics. Nucleosome is the fundamental repeating structural unit of chromatin in Eukaryotes. It regulates DNA template-based processes, such as DNA replication, DNA repair, and transcription, by controlling DNA accessibilityde novo established enhancers can predict the differentiation potential of progenyin vitro differentiated cells and failed to represent the scenarios under the proper in vivo context within the organism.There are a multiplicity of chemical modifications on histone tails of nucleosomes. Histone modifications function either by changing chromosomal state or by recruiting nonhistone proteins to chromatinDrosophila early embryos, a single transcription factor Glial Cells missing (Gcm) drives glial differentiation from the multipotent neural progenitor cellsGcm is transiently expressed in these progenitor cells and can be used as a marker geneRepo), is specifically expressed in glial cells12Drosophila central nervous system, it is feasible to explore the chromatin remodeling and its role during the in vivo glial differentiation in Drosophila embryos.Glia is a major cell type of the nervous system and known as “supporting cells” that provide support and protection for neurons. Glial cells differentiate from multipotent neural stem cells. In in vivo context. INTACT-captured cell-type specific nuclei can be used for gene expression, epigenomic, and proteomic profiling1516Gcm promoter and in glial cells under the control of Repo promoter, respectively. Thus, we isolated the multipotent neural progenitor cells and glial cells by affinity purification from early Drosophila embryos. We profiled gene expression and chromatin state (nucleosome occupancy and core histone modifications) of the two cell types through high-throughput sequencing technology. Our results reveal different change patterns of core histone modifications in promoters and extensive nucleosome remodeling. Increased H3K9ac signals up-regulate the group of genes important to gliogenesis. NDRs form in both promoters and enhancers. Moreover, the mode of NDR formation is independent of the chromatin state of enhancers in the neural progenitor cells.In this study, we employ INTACT (isolation of nuclei tagged in specific cell types) approachDrosophila line containing Gcm-Gal4 > UAS-NTF (Gcm > NTF) for affinity purification of the multipotent neural progenitor cells . The NTF gene is the nuclear targeting fusion gene introduced in INTACT approachDrosophila line containing Repo-Gal4 > UAS-NTF (Repo > NTF) for affinity purification of glial nuclei from stage 15–16 embryos (12–14h AEL)  . The pur14h AEL) . That isin situ hybridization from Berkeley Drosophila Genome ProjectWe next collected different tissue-specific genes that were determined by RNA Repo is significantly up-regulated during the glial differentiation whereas the GNP marker gene Gcm is down-regulated  genes during the glial differentiation, 725 down-regulated and 1562 up-regulated in glia . For exaegulated . GO termegulated . In contegulated . This imChromatin state in the promoter regions plays a critical role in gene activity. Profiles of the core histone modification signals in the promoter regions show three major chromatin states: H3K4me3+/H3K9ac+, H3K27me3+, and none . There aDrosophila Genome Project20We further investigated how chromatin state dynamics functions in the glial differentiation. A total of 368 genes closely related to glial development and differentiation were collected from Berkeley To gain insights into nucleosome remodeling and its role during the glial differentiation, we first scanned the genome using a 200-bp window and calculated nucleosome occupancy in each window for the two cell types. Comparison analysis show that extensive nucleosome remodeling occurs during the glial differentiation and it results in a relative more closed chromatin structure . This isWe next predicted genome-wide nucleosome positions using the tool GeneTrackcis regulatory elements beyond promoters and play a critical role in establishing and maintaining cell identity24Enhancers are distal Nucleosome remodeling in enhancers changes DNA accessibility and regulates gene activity. Thus, we scanned enhancers and identified regions of at least 150 bp (~length of a nucleosome). These regions are defined as enhancer NDRs. Their length ranges from 154–417 bp and peaks at 163 bp . This inDrosophila embryos show a low level of average H3K9ac signals in promoters in glial cells compared to GNP cells. However, H3K9ac signals are still increased in the group of genes that are involved in glial functions and whose expression levels are increased during the glial differentiation. Nucleosome positioning facilitates the glial differentiation through NDR formation in both promoters and enhancers. NDRs form in enhancers in two modes: nucleosome shift and eviction. These findings shed new light on patterns of chromatin remodeling and its epigenetic regulatory role during in vivo neural progenitor cells differentiating into glial cells.Chromatin structure is the underpinning of gene regulation. Nucleosome occupancy and post-translational histone modifications are the key two chromatin remodeling factors and play a critical role in cell differentiation. Profiling analyses of gene expression and core histone modification signals in affinity-purified neural progenitor cells and glial cells from early Gcm regulates both glial cell differentiation and the development of the plasmatocyte/macrophage lineage of hemocytes262729Gcm expression rapidly disappears after stage 11Histone modifications play an important role in cell fate determination. It has been reported that low levels of H3K9ac signals are a prerequisite for glial differentiationcis regulatory elements and also play a critical role in lineage commitment3132Enhancers are distal in vivo nucleosome positioning during Drosophila embryonic developmentDrosophila line generation, knockdown etc. assays are needed to address this question. This will largely improve our understanding of in vivo chromatin remodeling and its role during the glial differentiation.ATP-dependent chromatin remodeling enzymes are a key factor regulating nucleosome positioning. Different families of chromatin remodelers impact nucleosome organization in distinct fashions. For example, chromatin remodeler ISWI facilitates nucleosome placement in DNA sequences favoring nucleosome formation whereas remodeler families (P)BAP, NURD, and INO80 help nucleosome placement in DNA sequences disfavoring nucleosome formationw1118; p[UASRG]6 (III)3xFLAG-BLRP-mCherry-RanGap and BirA under the control of GAL4. Other Drosophila lines Gcm-Gal4, UAS-mCDGFP (II), Repo-Gal4/Tm3 (III), Sp/Cyo; Dr/Tm6B , and Tm3/Tm6B (III) were obtained from the Bloomington stock center.The transgenic line Drosophila line used to collect Gcm-expressed neural progenitor nuclei is generated as follows: Gcm-Gal4, UAS-mCDGFP virgin flies were crossed with Sp/Cyo; Dr/Tm6B male flies to generate F1 offspring Gcm-Gal4, UAS-mCDGFP/Cyo; +/Tm6B . w1118; p[UASRG]6 virgin flies were crossed with Sp/Cyo;Dr/Tm6B male flies to generate F1 offspring +/Cyo; p[UASRG]6/Tm6B . These two types of F1 female virgins were crossed with Sp/Cyo; Dr/Tm6B male flies to create F2 offspring Gcm-Gal4, UAS-mCDGFP/Cyo; Dr/Tm6B  and Sp/Cyo; p[UASRG]6/Tm6B , respectively. The F2 fly cross produces F3 offspring Gcm-Gal4, UAS-mCDGFP/Cyo; p[UASRG]6/Tm6B. The selfcross of F3 flies generates F4 offspring Gcm-Gal4, UAS-mCDGFP; p[UASRG]6. The embryos of F4 flies by selfcross were collected to isolate the neural progenitor cells.The Drosophila line used to collect glial nuclei is generated as follows: Repo-Gal4/Tm3, Ser, Twi-GFP virgin flies were crossed with w1118; p[UASRG]6 male flies. The male F1 offspring  was retained by checking mcherry fluorescence in the third larva by fluorescence microscope, and crossed with Tm3/TM6B virgin flies to generate F2 offspring Repo-Gal4, p[UASRG]6/Tm3 flies. The embryos of F2 flies by selfcross were collected to isolate the glial cells.The Antibodies for ChIP assays include H3K4me1 , H3K4me3 , H3K27ac, H3K27me3 , H3K9ac . Antibodies for immunostaining are mouse-Repo from Developmental Studies Hybridoma Bank (DSHB), donkey anti-mouse IgG H&L . Anti-Flag-coated M2 magnetic beads (Sigma-Aldrich) were used for affinity purification.2HPO4, 1.4 mM NaH2PO4) + 0.1% Triton-X-100), and were rinsed with tap water to remove the yeast. Then embryos were dechorionated with 50% solution of bleach for 3 minutes and were cross-linked in a 1:3 mix of ChIP-fixed buffer  with 1.8% formaldehyde and heptane for 15 min on a shaker with speed at 300 rpm. The aqueous and organic phase was replaced PBST with 0.25 mM glycine to terminate cross-linked reaction, and fixed embryos were rinsed by PBST for 3 times, and were store at −80 °C.Embryos were collected on grape juice plates with yeast paste from embryo collection cages for 2 hr, and allowed to develop for 5 and 12 additional hours at 25 °C. Then embryos are 5–7h-old and 12–14h-old, respectively and collected for cross-linking. Embryos were transfered onto the mesh with PBST  with 12U MNase , and then incubated at 37 °C for 20 min. Terminated the digestion on ice for 10 min by adding EDTA to a final concentration of 10 mM. Supernatant was discarded and pellet was washed with A2 buffer , and resuspended in A2 buffer with 0.1% SDS. The pellet was dissolved through sonication with 3 cycles of 20 s duration with at least 40 s pause between cycles at the power setting of 6 (out of 20) on Misonix sonicator XL-2000. The supernatant was retained for next ChIP assay, or reversal of cross-linking to harvest nucleosomal DNA as follow: the supernatant was treated with RNase A at 37 °C for 0.5–1 hours and Proteinase K at 65 °C for 2 hours, respectively. Next, the nucleosomal DNA mixture was extracted by phenol-chloroform and precipitated with a 1:10:100:200 mix of 20 mg/mL glycogen, 3 M pH 5.3 NaOAc, nucleosomal DNA mixture and cold 100% ethanol.Affinity-purified nuclei were collected by centrifugation at 1000 g for 5 min, suspended in 500 μL 37 °C pre-heated MNase digestion buffer  reaction with histone modification antibodies of appropriate doses as indicated in the specification. Mixture contained chromatin, antibody, and ChIP buffer  were incubated on a rotator overnight at 4 °C. Then 20 μL of ChIP Grade Protein G Magnetic Beads  was added to each IP reaction. The mixture was incubated for 2 hr with rotation. Next, beads were washed three times with low salt wash buffer  and once with high salt wash buffer , 5 min for each wash. Beads were suspended in 150 μL of ChIP elution buffer  at 65 °C for 45 min. Finally, we reversed cross-linking to harvest ChIP’ed DNA fragments and sequenced them on Illumina HiSeq2000 platform using 49 bp single end protocol.We dechorionated embryos in 50% solution of bleach and then fixed them in a 1:1 mix of 4% formaldehyde in PBS with 0.3% Tween 20 and heptane for 20 min on a shaker. The aqueous phase was discarded and replaced with methanol, and embryos were shaken for 3~5 min at 300 rpm to burst vitelline membranes. Embryos were rinsed three times in methanol and rinsed three times in PBS with 0.3% triton X-100. Embryos were blocked with PBST supplemented by 5% normal donkey serum (NDS) for 30 min, then incubated with primary antibodies of various dilutions in PBST containing 5% NDS overnight at 4 °C. The embryos were washed as above described, followed by incubation with the secondary antibody for 1–2 hours, then were washed three times again to avoid non-specific binding. The stained embryos were mounted on slides in PBS with additional 50% glycerol. Slides were examined on Zeiss Imager M2 microscopy. To evaluate the purity of affinity-isolated nuclei, DAPI-stained total and affinity-purified nuclei were counted on a hemacytometer, respectively.Drosophila transcripts (FlyBase r5.43) using Tophat (v1.3.1) with default parameters settingNuclear RNA was extracted from affinity-purified tagged nuclei using the RNeasy Micro Kit (Qiagen). Genomic DNA was removed with Turbo DNA-free kit (Ambion). The cDNA library for RNA sequencing was constructed using standard Illumina libraries prep protocols. Sequencing was conducted on Illumina HiSeq2000 platform using 49 bp single end protocol. Sequencing reads were mapped to Gene ontology analysis of gene set was performed using the functional annotation tool DAVID (v6.7)Drosophila reference genome (dm3) with up to two mismatches using BowtieHistone modification sequencing reads were mapped to the Enhancers were H3K4me1 enriched peaks that were identified by the tool HOMER using a 1000-bp sliding window with a false discovery rate of 0.1%Drosophila reference genome (dm3) with up to two mismatches using BowtieNucleosomal DNA sequencing reads were aligned to the Nucleosome organization within a region was shown in heatmap as follows: we located nucleosomal reads in the genome that comprise nucleosomes predicted by GeneTrack. Then we summed read counts nucleosome occupancy using a 5-bp bin and smoothed with 5 bins. We further normalized the nucleosome occupancy using the total reads within the region.Accession codes: The RNA-seq, MNase-seq and ChIP-seq data sets have been deposited in the Gene Expression Omnibus (GEO) under accession number GSE83377.How to cite this article: Ye, Y. et al. Chromatin remodeling during the in vivo glial differentiation in early Drosophila embryos. Sci. Rep.6, 33422; doi: 10.1038/srep33422 (2016).