Drosophila melanogaster is a cluster of homeotic genes that determine body segment identity. Expression of these genes is governed by cis-regulatory domains, one for each parasegment. Stable repression of these domains depends on Polycomb Group (PcG) functions, which include trimethylation of lysine 27 of histone H3 (H3K27me3). To search for parasegment-specific signatures that reflect PcG function, chromatin from single parasegments was isolated and profiled. The H3K27me3 profiles across the BX-C in successive parasegments showed a ‘stairstep’ pattern that revealed sharp boundaries of the BX-C regulatory domains. Acetylated H3K27 was broadly enriched across active domains, in a pattern complementary to H3K27me3. The CCCTC-binding protein (CTCF) bound the borders between H3K27 modification domains; it was retained even in parasegments where adjacent domains lack H3K27me3. These findings provide a molecular definition of the homeotic domains, and implicate precisely positioned H3K27 modifications as a central determinant of segment identity.The bithorax complex (BX-C) in DOI:http://dx.doi.org/10.7554/eLife.02833.001 Like other insects, the body of the fruit fly is divided into three main parts—the head, the thorax and the abdomen—and each part, in turn, is made up of several smaller segments. The bithorax complex is a cluster of three genes that together control the identity of the segments that make up the back half of the fruit fly's body. This gene cluster has been studied for several decades and these studies have helped to further our understanding of how genetic information is accessed and used to make an animal’s body plan.Early on in a fruit fly embryo, stretches of DNA within the bithorax complex regulate where the complex's genes are switched on, and where they are switched off. Proteins called Polycomb group proteins then keep the silenced genes off, in part by adding small chemical marks to other proteins called histones. Most DNA in a cell is wrapped around histones, and the addition of such chemical marks causes the DNA to become more tightly packed. This prevents the bithorax complex genes from being accessed and switched on. It had previously been suggested that each segment might have a unique pattern of chemical marks on the bithorax complex histones, but evidence to support this idea was lacking.Bowman et al. have now undertaken the technically challenging task of purifying the DNA and its histones from individual segments of fruit fly embryos. This revealed that segments closer to the embryo's head contain larger stretches of bithorax complex DNA covered with histones marked by the Polycomb group proteins. Bowman et al. also found that the coverage of chemical marks on the histones changed dramatically when one segment was compared to its neighboring segments. These sharp boundaries clearly outline which regulatory regions of the DNA are switched on and which are switch off; however the same pattern is not seen for the Polycomb group proteins themselves. Instead, within the bithorax complex, the pattern of these proteins is almost identical in different segments.The challenge now is to understand how the chemical marks and the Polycomb group proteins work together to restrict access to DNA in such precise patterns. Also—since similar gene clusters control the development of the body plans of mammals—this, in turn, might help us to understand how the Polycomb group proteins perform similar functions in human development and disease.DOI:http://dx.doi.org/10.7554/eLife.02833.002 Cells in higher organisms choose among developmental pathways in response to transitory cues. The choice must be remembered; this is often described as changing the epigenetic state. One prominent mechanism to fix such choices relies on the genes of the Polycomb Group (PcG). The members of this family have been well defined by genetic and biochemical studies . The PcGDrosophila homeotic genes that control segment identity. Indeed, a mutation in Polycomb, the founding member of the PcG, was discovered for its segmental transformations  and the bithorax complex (BX-C). The BX-C is the better-studied cluster, due largely to the effort of EB Lewis. Lewis showed that mutations that transform specific segments align on the genetic map in the order of the body segments that they affect (Ultrabithorax (Ubx), abdominal-A (abd-A), and Abdominal-B (Abd-B) (The prototypic targets of PcG action are the rmations . In Drosy affect . The evoy affect . The gen (Abd-B) . The reg (Abd-B) , and by  (Abd-B) . The molUbx transcription unit in haltere and third leg imaginal discs, tissues where Ubx is transcribed. These discs include cells of two parasegments (PS5&6), and so it was not clear how the K27me3-free regions might correlate with transcription units or with the genetic regulatory domains. The issue could be resolved if H3K27me3 patterns could be studied in single parasegments.Both PRC1 and PRC2 are required to maintain repression of homeotic genes in appropriate parasegments, so these complexes may generate segment-specific chromatin features in the regulatory domains of the BX-C. We initially evaluated H3K27me3 patterns because they reflect the central function of PRC2. The entire BX-C is heavily marked with H3K27me3 in whole embryos . This hiThe chromatin features of the BX-C have not been studied in individual parasegments because of the technical challenges of cell isolation and molecular analysis on small samples. To address this, we marked single parasegments with a combination of Gal4 and Gal80 drivers, and developed a nuclear sorting-chromatin immunoprecipitation-sequencing protocol. Gal4, a transcriptional activator, was expressed in a series of parasegments with a defined anterior boundary, and Gal80 was expressed in a pattern shifted one parasegment more posterior. Gal80 binds to and inactivates Gal4, leaving Gal4 activity in a single parasegment . Gal4 anFor isolation of marked nuclei, we used Gal4 to drive an mCherry-RanGAP fusion protein, from the INTACT system . Staged The largest differences in H3K27me3 enrichment between parasegments occurred at the BX-C . While tbithorax (bx) mutant lesions, is defined by the low level of H3K27me3 modification across the leftmost  92 kb of the complex  phenotypes  class of genetic mutations. We observed a reproducible mound of H3K27me3 in PS7 nuclei close to the Ubx promoter  show patterns with a PS6 anterior limit. Perhaps the promoters of these reporters interact with the Ubx promoter in distinctive ways. There was also prior evidence of an active BX-C domain in PS4. An enhancer trap 3 kb downstream of the Ubx start site shows a PS4 anterior border , which is thought to impose boundaries on regulatory domains in many systems . Figure Ubx transcription unit, separating Ubx from the adjacent gene (modular serine protease). In the Antennapedia complex, the other homeotic gene complex in flies, CP190 peaks often appear without coincident CTCF peaks  , and theCF peaks .Acetylation of H3K27 (H3K27ac) is correlated with active enhancer regions , and is Ubx, abd-A, and Abd-B   in whole embryos shows the transcription units for nd Abd-B . Embryonnd Abd-B  are not arrow in . There iarrow in , althougarrow in  marks thpatterns . There iarrow in , althougfrom PS7  and this, and so PRC1 localization might be expected to correspond to the stair-step H3K27me3 pattern. Published ChIP–CHIP and ChIP-seq profiles for PC across the BX-C vary from broad coverage matching the H3K27me3 profile 12, a PRC2 component , look siUbx expression in specific cells at specific developmental times, all of which are blocked in parasegments one through five, but active in parasegments 6 through 12  are only transcribed in a subset of cells. It is interesting that both PRC1 and PRC2 components have binding patterns that do not fully reflect function , indicating the possibility that function of these complexes is regulated separately from binding  gene, recovered as a 317 bp PCR fragment from genomic DNA.A P element was built containing both Gal4 and Gal80 . The P pthe BX-C  or at thswapping  is a lowAdh terminator. At a BamH1 site immediately upstream of the 5′ FRT site, a fragment was inserted containing: (1) an 841 bp fragment from the 5′ end of the gypsy mobile element (bases 189-1029), (2) an AscI site, and (3) a fragment from the engrailed gene (−595 bp to +243 bp relative to the major transcription start site). The gypsy fragment includes twelve tandem binding sites for SUPPRESSOR OF HAIRY WING (SU(HW)). The engrailed fragment includes the proximal PRE  binding sequences were intended to insulate the promoter from endogenous enhancers near the insertion site. Transformants were recognized by Gal4-driven GFP patterns in young larvae, and, again, any Gal4 producer could be converted to a Gal80 producer. Sequences of these P elements are available on request.We have also taken known embryonic enhancers from the BX-C and inserted them into a second vector , which w (‘PSE2’ ), and isAntennapedia P1 promoter , and a ,825,205 ). Other abx enhancer (for PS5 anterior limit) 3R: 12,508,951-12,513,096pbx enhancer (for PS6 anterior limit) 3R: 12,598,546-12,600,177iab-2 enhancer (for PS7 anterior limit) 3R: 12,636,236-12,639,140iab-3 enhancer (for PS8 anterior limit) 3R: 12,664,301-12,666,886Fragments used as enhancers inserted into the vector of Ubx transcription start site . This gave a strong pattern with a sharp PS6 anterior limit, and was used as the Gal4 source for PS6 nuclei. Insertions with the abx and pbx enhancers were subjected to P transposase to adjust the levels of Gal4 and Gal80. D. melanogaster genome coordinates are from release 5.57.Other fragments tested covered the ‘bx’ and ‘bxd’ enhancers, but these did not give sufficiently strong or uniform expression patterns. One insertion of a P element with the iab-3 enhancer fortuitously inserted 1.4 kb upstream of the 1118; p6) were generously provided by Paul Talbert and Steve Henikoff. This P element was crossed to a P transposase source (P{ry[+t7.2] = Delta2-3}99B), and offspring were screened initially for darker eye color, most likely due to multiple copies of the INTACT element. Such stocks with INTACT insertions on the second or third chromosome were then crossed to a Gal4 source, and those with brightest mCherry expression were used for subsequent efforts to mark single parasegments. The higher copy INTACT chromosomes are designated ‘INTACTn on II’ and ‘INTACTn on III’.Flies with the INTACT reporter in a P element on the third chromosome .2HPO4, 3 mM KH2PO4, 0.1% Triton X100, pH 8.0) for 2 min at 25°C, and then briefly with PBS  plus 0.1% Triton X100. The washed embryos were collected on a nytex filter, and examined under an epifluorescence stereoscope. Embryos that appeared too old, with fluorescent nuclei anterior to the desired parasegment, were manually removed. The remaining eggs were collected and weighed in a 1.5 ml microfuge tube, flash frozen in liquid nitrogen, and stored at −75°C.Agar plates with yeast paste were put on the PS4, PS5 or PS7 laying cages for 8 hr at 25°C (or 16 hr at 18°C), and then the eggs were aged for another 5 hr at 25°C (or 10 hr at 18°C). For PS6, a 6 hr collection was aged for a further 4 hr. The eggs were dechorionated (2 min in 50% bleach) and fixed in fixation buffer  under heptane, on a rotator for 15 min at 25°C. The fixed embryos were washed with stop buffer , 0.01% Triton X-100, pH7.0) plus 100 µl protease inhibitor cocktail , and disrupted with 10 strokes in a Dounce homogenizer with a loose pestle, then 10 strokes with a tight pestle. The homogenate was spun at 275 × g for 1 min to remove large debris. The resulting supernatant was spun at 1000 × g for 10 min to pellet the nuclei. The pellet was resuspended in 2 ml BBT buffer plus 40 µl protease inhibitor, and homogenized again, 20 strokes with the tight pestle. The homogenate was passed through a cell strainer cap with a 40 µm nylon mesh and stored at 0°C. Hoechst 33,342 dye was added (2 µl of 10 mg/ml solution) before sorting.For the isolation of nuclei, ∼100 mg of embryos were suspended in 5 ml of BBT buffer  embryos. Recovered nuclei constituted between 1 and 6% of total single nuclei  FACSAria IIu instrument. The fluorescence from Hoechst 33342  was used to select for a 2N DNA content, thus avoiding clumps of nuclei. These single nuclei were secondarily selected for mCherry fluorescence  stronger than 99.9% of nuclei from control  was determined with a hemacytometer, and 50–200 K nuclei were used for each ChIP. Chromatin fragmentation was accomplished either with micrococcal nuclease (MNase) or bath sonication. For MNase fragmentation, nuclei in sort buffer were supplemented with PBS containing 0.1% Triton-X100 (PBS-Tx) to a volume of 400 µl, and CaCl2 to 1 mM. Nuclei were pre-warmed to 37°C and digested with 12U MNase  for 3 min. Digestion was stopped by moving the tubes to ice and adding 10 µl of 250 mM EDTA, 250 mM EGTA, and nuclei were briefly sonicated in a Diagenode Bioruptor  . For bath sonication, the nuclear suspension in sort buffer was adjusted to ChIP buffer conditions  in a volume of 500 µl. Nuclei were sonicated for 30 min at high intensity  in a Bioruptor. Fragmented chromatin was snap frozen and stored at −80°C prior to ChIP.MNase fragmented chromatin was adjusted to ChIP buffer conditions in a final volume of 500 µl, and incubated on ice for 5 min. Prior to ChIP, protease inhibitors  were added to either MNase fragmented chromatin or sonicated chromatin. Chromatin was subjected to a high speed spin at 4°C for 10 min. Supernatant was moved to a new low retention tube, 1% input was removed and stored at 4°C, and and the remaining chromatin was incubated overnight with the appropriate antibody at 4°C. After a high speed spin at 4°C for 10 min, supernatant was moved to a low retention tube with 10 µl prewashed protein A dynabeads (Life Technologies), and incubated at 4°C for 1–2 hr with gentle rotation. After six rinses with ice cold ChIP buffer, immunoprecipitates were eluted from the beads with two successive additions of 125 µl freshly made elution buffer  incubated at 65°C for 10 min. Eluates were combined, and adjusted with Tris and EDTA to a final concentration of 10 mM Tris pH8 and 2 mM EDTA. 250 µl elution buffer was added to the reserved input and likewise adjusted. DNA cleanup began by incubating with RNase  at 37°C for 30 min, proteinase K  at 55°C for 1 hr, and crosslinks were reversed by incubating at 65°C for 1 hr. After two phenol-chloroform extractions and one chloroform extraction, purified DNA was ethanol precipitated in the presence of sodium acetate and Glycoblue (Ambion). Sequencing library construction was performed according to . Librarianti-K27me3 (Active Motif 39155), anti-K27ac , anti-K4me3 , anti-CTCF , anti-Phnnorm = [(n+1)/(ni+1)] * [Ni/N], where n, ni, N, and Ni are positional coverages in experiment and input, and total genome coverages in experiment and input, respectively.ChIP-seq reads were aligned to the dm3 genome using BWA and allowing up to one mismatch. Potential PCR duplicates, tags with multiple alignments, and paired-end reads with insert size greater than 1 kb were removed from further analysis. We calculated normalized positional coverage as previously described . BrieflyTo determine regions of enrichment, we summed the coverage within sliding windows of size 1 kb and step 50 bp across the entire genome. The same was calculated for tags assigned a random location within the chromosome. The resulting distribution of random window coverages was then used to determine the significance of observed window coverages. Individual p-value cutoffs were selected based on manual inspection of each experiment. For samples with replicates, only regions significant in both replicates were used.For genome-wide analysis of coverage difference between two parasegments for H3K27me3, we first merged the significant regions of enrichment from the different parasegments to create an aggregate set of regions of interest. Regions greater than 50 kb were split into equally sized sub-regions. For each region we calculated the density, or total normalized coverage divided by region length. Replicate density values were averaged. To find differences, region densities for two parasegments were scatter plotted against one another, and a line of best fit calculated. Points located furthest away from this fit line were identified as the candidate differing regions. This approach allows us to account for the variation in sequencing coverage and noise for each sample and parasegment.SPP  was used review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent  and 3 reviewers, one of whom is a member of our Board of Reviewing Editors.Thank you for sending your work entitled “H3K27 modifications define segmental regulatory domains in the Drosophila bithorax complex” for consideration at The Reviewing editor and the other reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.We think that the study is solid and has its value. However, we collectively believe that the current data set (H3-K27me3/ PH/ CTCF) is too preliminary compared to earlier studies but that it might become suitable if the analysis is expanded as suggested below.Ubx (i.e. in Ubx OFF and Ubx ON cells) that was reported back in 2006 is a general principle. Similarly, it was shown that several components of the general transcription machinery are bound at Ubx in the OFF state, and so it would be good to know whether assembly of the RNA Pol II machinery even at permanently repressed Polycomb target genes is a general principle.The authors should look minimally at PRC2 binding and RNA Pol II occupancy. These two profiles would help to clarify whether the 'constitutive' binding of PRC2, PRC1 etc. at the HOX gene We also noticed that the citation of relevant scientific literature is missing.Reviewer #1:Can the authors computationally overlay the different parasegments that are labeled at each time point? Currently, it is visually difficult to assess the level of overlap or lack thereof between each of the labeled parasegments.Is it possible to label any upstream parasegments? The question of contamination of PS4 by upstream signal may thereby be resolved.The authors should provide antibody stainings of genes within the BX-C cluster for us to align against the labeled parasegments.What about the Antp-C locus? Does H3K27me3 profiling parallel what is seen in the BX-C locus? Does dCTCF localize to boundaries here as well?As there are many characterized insulator proteins in the fruit fly, how does dCTCF co-localize with these factors? I.e. CP190, BEAF-32, Su(Hw)?How is a given constitutive CTCF site chosen to be “active” in performing a boundary function? For example, in PS7, why are activation signals able to push through the most anterior dCTCF sites? Is specificity achieved through association with distinct insulator partners at different CTCF sites?How does the H3K27me3 pattern compare to the distribution of E(Z) or other PRC2 components? Does PRC2 only sit at PREs, from which the modification spreads? Is PRC2 absent from upstream PREs present within an activated region of BX-C? If present, what inhibits PRC2 activity at these sites?Since the authors are able to purify nuclei from distinct parasegments of the fly, they should be able to provide RNA-seq or Pol-II occupancy tracks to correlate with presence of repressive H3K27me3 and active H3K27ac.The genetic system employed nicely allows for the isolation of parasegments during time points of development. What are the dynamics of the activation of the BX-C locus? Is the transition gradual or immediate? Is it possible to ChIP some of the transcription factors thought to activate these genes?What is the 3D conformation of the parasegments within the BX-C cluster? How do they compare to each other? Are active and repressive domains spatially partitioned?How is the expression and H3K27 distribution altered in mutants (null or hypomorphs) of Polycomb genes? Is there simply a decrease in H3K27me3, or is the H3K27me3 boundary shifted as well?How does the distribution of H2AK119Ub correlate with Polyhomeotic? Does it form blocks within transcriptionally repressed chromatin, as is the case for PRC2?Reviewer #3:1 )The authors should analyze the PRC2 binding profile (e.g. ChIP with antibody against E(z))in PS4, 5, 6 and 7, or at the minimum, just in PS7 as they did for Ph, CTCF etc.. This would address the conceptually important point whether the binding of PRC2 at PREs is constitutive  and that the control takes place at the level of deposition of the modification. Given that the authors may already have chromatin from PS7 nuclei or can generate it as they did for several of their experiments in the manuscript, I do not think that it is asking too much to perform such a ChIP experiment.Ubx in wing imaginal disc cells where Ubx is inactive and in haltere and third leg discs where the gene is active. One key conclusion from that work was that PRC2 (as well as PRC1 and PhoRC) is bound at Ubx both in the inactive and in the active state (with subtle differences) but that the domains of H3-K27me3 are different in the two states, with H3-K27me3 covering the entire upstream, promoter and coding region in wing disc cells but being absent from the promoter and coding region in haltere and third leg disc cells. So the observation that H3-K27me3 domains correlate with inactivity of regulatory regions in the bithorax complex is not new but the nice data shown here in embryos (with modern genomic techniques) extend and refine these findings. From the above it also directly follows that it is key to find out about PRC2 binding at the bx, bxd and abd-A PREs in PS7 (Point 1).2) This point is directly connected to the first point. The authors must discuss their findings in the context of the findings from earlier studies  nuclei are contaminated by anterior cells.:What about the Antp-C locus? Does H3K27me3 profiling parallel what is seen in the BX-C locus? Does dCTCF localize to boundaries here as well?Response: New figure depicting H3K27me3, CTCF, and CP190 in the Antp-C.Location in the revised manuscript:H3K27me3 is depleted over the Antp transcription unit, as expected. CTCF is less prevalent in the Antp-C than in the BX-C. In the Antp-C, CP190 predominates.As there are many characterized insulator proteins in the fruit fly, how does dCTCF co-localize with these factors? I.e. CP190, BEAF-32, Su(Hw)?Experimental response: ChIP-seq for CP190 in mixed cells and parasegment 7.Location in revised manuscript:Figure 2Since CP190 is in a protein complex with CTCF and can alter local chromatin structure in a way that may promote boundary function, we thought it would be a worthwhile target of investigation. It co-occupies many sites with CTCF, but also binds to the BX-C and the ANTP-C at locations where we do not detect CTCF.How is a given constitutive CTCF site chosen to be “active” in performing a boundary function? For example, in PS7, why are activation signals able to push through the most anterior dCTCF sites? Is specificity achieved through association with distinct insulator partners at different CTCF sites?Our results suggest that boundaries are constitutive. They may have nothing to do with whether an adjacent domain is active or repressed; they may only prevent the spread of activation or repression. The comparison of CTCF and CP190 profiles suggests that each boundary may be distinctive, which is borne out by sequence comparisons and swapping experiments from other labs, but this is no reason to suggest that the components of any boundary vary from one parasegment to another.How does the H3K27me3 pattern compare to the distribution of E(Z) or other PRC2 components? Does PRC2 only sit at PREs, from which the modification spreads? Is PRC2 absent from upstream PREs present within an activated region of BX-C? If present, what inhibits PRC2 activity at these sites?Experimental response: ChIP-seq for Su(z)12 in mixed cells and parasegment 7.Location in revised manuscript:Figure 3In mixed cells, Su(z)12 is present in sharp peaks at PREs and transcription start sites. In PS7, Su(z)12 peaks are somewhat diminished at the PREs in the active domains, while the peaks at the transcription start sites are strengthened. The reasons for these subtle changes will require further investigation.Since the authors are able to purify nuclei from distinct parasegments of the fly, they should be able to provide RNA-seq or Pol-II occupancy tracks to correlate with presence of repressive H3K27me3 and active H3K27ac.Experimental response: ChIP-seq for Pol-II in mixed cells and parasegment 7.Location in revised manuscript:Figure 3Ubx and Abd-A in PS7, consistent with the expression of these genes. Pol-II is also enriched at the major promoter of Abd-B, even though this gene is transcriptionally silent in PS7 and covered by H3K27me3.Transcriptionally engaged Pol-II is present at the promoter and throughout gene bodies of The genetic system employed nicely allows for the isolation of parasegments during time points of development. What are the dynamics of the activation of the BX-C locus? Is the transition gradual or immediate? Is it possible to ChIP some of the transcription factors thought to activate these genes?Unfortunately, the transgenes marking single parasegments do not turn on until at least 4 hours of development, well after repression via Pc-G has been established. These technical limitations make addressing this interesting developmental question in single parasegments quite challenging, and outside the scope of the current manuscript.What is the 3D conformation of the parasegments within the BX-C cluster? How do they compare to each other? Are active and repressive domains spatially partitioned?Abd-B promoter, the Ubx promoter, and the border between PS7 and PS8 regulatory domains). We have not observed contacts changing in an obvious manner that correlate with domain activation and repression. Given the significant amount of sorted material required for the experiments and the absence of robust differences between mixed cells and PS7, we have chosen to save a more thorough investigation of these data for another manuscript.Members of our lab have performed 4C with three different baits in PS7  of Polycomb genes? Is there simply a decrease in H3K27me3, or is the H3K27me3 boundary shifted as well?We have developed the techniques to perform ChIP-seq from small numbers of precisely staged mutant embryos, and have begun to generate data that answer these questions. An analysis as thorough as this topic deserves is outside the scope of this work, and will be featured in a future manuscript.How does the distribution of H2AK119Ub correlate with Polyhomeotic? Does it form blocks within transcriptionally repressed chromatin, as is the case for PRC2?We are also interested to the answer to this question. Unfortunately, the two commercially available antisera for mammalian H2AK199Ub  failed to enrich BX-C chromatin under our experimental conditions. This may be because the amino acid residues flanking the ubiquitinated lysine residue are different in mammalian and Drosophila H2A , and a fly-specific antibody is necessary for ChIP.Reviewer #3:1) The authors should analyze the PRC2 binding profile (e.g. ChIP with antibody against E(z))in PS4, 5, 6 and 7, or at the minimum, just in PS7 as they did for Ph, CTCF etc..Experimental response: ChIP-seq for Su(z)12 in mixed cells and PS7.Location in revised manuscript:We found that PRC1 components bind to PREs regardless of their activation state, and the PRC2 component Su(z)12 looks similar.This would address the conceptually important point whether the binding of PRC2 at PREs is constitutive  and that the control takes place at the level of deposition of the modification. Given that the authors may already have chromatin from PS7 nuclei or can generate it as they did for several of their experiments in the manuscript, I do not think that it is asking too much to perform such a ChIP experiment.2) This point is directly connected to the first point. The authors must discuss their findings in the context of the findings from earlier studies .Thanks for pointing out this oversight. In the revised manuscript, we have included additional references to


Further, genes retain penta-acetyl H4 and H2A.Z, suggesting that even in the absence of UBF the rRNA genes can maintain a potentially active state. In contrast to canonical histone H1, binding of H1.4 is dependent on UBF, strongly suggesting that it plays a positive role in gene activity. Unexpectedly, arrest of rRNA synthesis does not suppress transcription of the 5S, tRNA or snRNA genes, nor expression of the several hundred mRNA genes implicated in ribosome biogenesis. Thus, rRNA gene activity does not coordinate global gene expression for ribosome biogenesis. Loss of UBF also unexpectedly induced the formation in cells of a large sub-nuclear structure resembling the nucleolar precursor body (NPB) of oocytes and early embryos. These somatic NPBs contain rRNA synthesis and processing factors but do not associate with the rRNA gene loci (NORs).Upstream Binding Factor (UBF) is a unique multi-HMGB-box protein first identified as a co-factor in RNA polymerase I (RPI/PolI) transcription. However, its poor DNA sequence selectivity and its ability to generate nucleosome-like nucleoprotein complexes suggest a more generalized role in chromatin structure. We previously showed that extensive depletion of UBF reduced the number of actively transcribed ribosomal RNA (rRNA) genes, but had little effect on rRNA synthesis rates or cell proliferation, leaving open the question of its requirement for RPI transcription. Using gene deletion in mouse, we now show that UBF is essential for embryo development beyond morula. Conditional deletion in cell cultures reveals that UBF is also essential for transcription of the rRNA genes and that it defines the active chromatin conformation of both gene and enhancer sequences. Loss of UBF prevents formation of the SL1/TIF1B pre-initiation complex and recruitment of the RPI-Rrn3/TIF1A complex. It is also accompanied by recruitment of H3K9me Upstream Binding Factor (UBF) is multi-HMGB-box protein found in all vertebrates. Although this protein has been implicated in transcription of the ribosomal RNA (rRNA) gene in vitro, little is known of its function in vivo. We previously found that UBF creates a nucleosome-like structure on DNA, and that this structure is remodeled by MAP-kinase phosphorylation. Using conditional gene deletion in mouse and mouse cells we show that UBF defines the active chromatin domains of the rRNA genes and is essential for transcription of these genes. Using this system we show that, contrary to expectation, rRNA gene activity does not coordinate ribosome production. We further show that in the complete absence of rRNA synthesis a somatic nucleolar precursor body is formed. Our data show that UBF determines a dynamic transition between the active and inactive rRNA gene states that is independent of changes in DNA methylation. The nucleolus is the largest visible structure in the mammalian cell nucleus and the site of ribosome biogenesis. As such, its activity is a key determinant of a cell's capacity to grow and proliferate, and its size and morphology are used as clinical markers of cancer The rRNA genes, also known as the rDNA, are transcribed by RNA polymerase I (RPI or Pol1) with the aid of the pre-initiation factors SL1/TIF1B and Rrn3/TIF1A. Recruitment of SL1 to the RPI promoter in vitro was originally shown to require Upstream Binding Factor (UBF), a multi-HMGB-box protein found in all vertebrates Ubf gene, and in so doing have made several unexpected findings. We find that the mouse Ubf gene is indeed essential for rRNA gene activity, cell proliferation and embryo development. Elimination of UBF causes large-scale changes in rRNA gene chromatin consistent with a transition from the active state to a potentially active resting state, but not heterochromatinization. Unexpectedly, inactivation of rRNA gene activity has no effect on the activity of the hundreds of RPII and RPIII genes implicated in ribosome biogenesis, showing that rRNA gene activity does not coordinate the gene expression required for ribosome assembly. Finally, elimination of UBF reveals somatic nucleolar precursor bodies that are spatially distinct from chromosomal rDNA loci.MAP-kinase phosphorylation of the UBF was found to regulate RPI transcription elongation rates in vitro and in vivo in the response to growth factors Ubf allele in which Lox recombination sites were placed in intron 2 and intron 5, and a neo selective marker gene flanked by FRT sites was inserted within intron 5 , suggesting that the Ubf gene was inactivated by the insertion and hence was essential. The mice were then crossed with FLPeR (Flipper) and Cre expressing mice to generate both flUbf and UbfΔ alleles, and subsequently Cre and Flipper transgenes removed by backcrossing , at or during the compaction phase  layer, inner cell mass (ICM) and blastocoel cavity. When the UbfΔ/Δ embryos were cultured in vitro they failed to develop further, while UbfwtΔ/ and wt/wtUbf litter-mates developed to form late blastocysts  cells carrying a potentially conditional “flox-neo” intron 5  & B. Miccrossing  and S1B.entified . Analysion phase  and S1E.stocysts  and S1F.fl/flUbf mice . Short-term, induction of ER-Cre activity by a 4 h treatment with 50 nM 4-HT induced near complete excision of the floxed UBF exons by 12 h, and excision was complete by 24 h post 4-HT (pHT)   and S2A.HT (pHT) , as obseHT (pHT) . Inhibitfl/fl/Er-cre+/+Ubf cells, UBF associated with the rDNA at the 47S promoter (T0/Pr), throughout the transcribed region and at the Spacer Promoter (SpPr) and Spacer Terminator (Tsp) lying ∼2 kbp upstream of the 47S promoter, but not across the rest of the Intergenic Spacer (IGS) . As expected, in the untreated er (IGS) . By 48 hConsistent with the near complete shutdown of rRNA synthesis , by 48 h0). In contrast, its association with the 47S termination sites (T1–3) was strongly enhanced. This suggests that TTF1 is predominantly a constitutive factor, found on both active and inactive genes. Its partial displacement from the binding sites upstream of the 47S transcribed region to the downstream termination sites may be related to its function in looping the rDNA In contrast to the other RPI transcription factors, association of the Transcription Termination Factor (TTF1) with the rDNA was clearly not dependent on UBF . Howeverin cis as enhancers of gene transcription 0 or the formation of an upstream barrier to the spread of repressive chromatin.The rDNA sequences between the Spacer Promoter and the 47S promoter act 0/Pr) and within the IGS. Association of the heterochromatic HP1α with the rDNA was also enhanced but unlike H1 this occurred over the enhancer and transcribed regions previously occupied by UBF ) covering exon 2 (coordinates 102307100–102308992), exons 3, 4 and 5 (102308989–102310581), and exons 6 and 7 (102310611–102313115) were isolated from isogenic ES cells  and cultured for one day in 5% CO2 at 37°C. Embryos were collected in 8-wells plate (Ibidi) and imaged by bright-field microscopy. DNA from the individual blastocysts was then amplified using the REPLI-g Mini kit (QIAGEN) and individual embryos were genotyped by PCR using the primers: A; 5′TGATCCCTCCCTTTCTGATG, E; 5′ATCTAACCCCGCTTTCCTGT, C; 5′CACGGGAAAACAAGGTCACT, see Heterozygous fl/fl/Er-cre+/+Ubf and isogenetic wt/wtEr-cre+/+Ubf embryos were prepared as previously described Primary mouse embryo fibroblasts (MEFs) from E14.5 6 cells each) for 18 hours in DMEM, high glucose, 10% fetal bovine serum. To activate ER-Cre, 4-hydroxytamoxifen (4-HT) was added to a final concentration of 50 nM, and after 4 hr incubation the medium replaced with fresh medium without 4-HT and cells harvested for analysis at various time points. This minimal 4 h ER-Cre induction generates full recombination of Lox sites while avoiding the non-specific effects of more common treatments with 0.6–2 µM 4-HT for 24–48 h 5′TGATCCCTCCCTTTCTGATG, B; 5′TGGGGATAGGCCTTAGAGAGA, C; 5′CACGGGAAAACAAGGTCACT, (3H]-uridine (PerkinElmer) to the culture medium and incubation for a further 3 h. RNA was extracted with Trizol (Invitrogen) according to the manufacturer's protocol and analyzed by gel electrophoresis, fluoroimaging  and RNA species quantitated by scintillation counting as previously described Cells were grown in 6 cm petri dishes  large subunit (A194) and TTF1 were generated in the laboratory, anti-Rrn3/TIFIA, -TAF1C and –TAF1B were kindly provided by Ingrid Grummt and anti-H1.4 and -H1.4-S187phospho kindly provided by Craig Mizzen. All other antibodies were obtained commercially; Anti-TBP, -H2A.Z, -H3 and -H4 (Abcam), anti-H1, -H3K9met3, -H4ac5 and -HP1α (Millipore), anti-Tubulin (Sigma), and anti-Fibrillarin (Covance). The pre-immune (PI) serum was from the rabbit in which the UBF antibody was generated.6 cells. Immunoprecipitated DNA was analysed by qPCR/SYBR Green. Reactions (20 µl) were performed in triplicate with 2.5 µl of sample DNA, 20 pmol of each primer, and 10 µl of Quantitect SYBR Green PCR Master Mix (QIAGEN). Forty-five reaction cycles of 30 s at 94°C, 30 s at 58°C, and 30 s at 72°C were carried out on a Multiplex 3005 Plus (Stratagene/Agilent). The amplicon coordinates relative to the 47S rRNA initiation site (BK000964) were as follows: IGS3, 42646–42903; SpPr, 43089–43253; Tsp, 43267–43421; T0/Pr, 45133–40; ETS, 3078–3221; ITS1, 6258–6432; 28S, 10215–10411; T1–3, 13412–13607; IGS2, 25552–25783. Two to five biological replicates were analysed by ChIP for each antibody; UBF, TTF, H4 and H4ac 5 times each, RPI, H3, H3K9me3 and H1 3 times each, TIFIA, TAF1C, TAF1B, TBP, H1.4, H1.4-p187, HP1a and H2Az 2 times each. Though it was not possible to perform all ChIP analyses in parallel on all chromatin preparations, all analyses included both UBF and TTF ChIPs as reference standards.ChIP was performed essentially as previously described 2 was systematically between 0.99 and 1.0, demonstrating a near perfect fit.Data was analysed using the MxPro software (Agilent). The relative occupancy of each factor at each amplicon is given as % immunoprecipitation of the DNA input prior to ChIP. It was determined by comparison with a standard curve of amplification efficiency for each amplicon using a range of input DNA amounts and generated in parallel with each Q-PCR run. All primer pairs gave the similar amplification efficiencies (90–105%) as determined from the gradient of the curve fit. The curve fit correlation coefficient RThe methylation assay was developed by Anne Rascle and Joachim Griesenbeck and kindly made available to us before publication. Briefly, the assay is based on the fact that rDNA is in greater part fully methylated or unmethylated. 20 µg genomic DNA was digested with BamHI and subsequently with either SmaI or XmaI, and then analyzed by Southern blotting using the PflMI/BamHI rDNA fragment  labelledPsoralen crosslinking was performed and analyzed as described previously www.r-project.org) version 2.14.0. Firstly, data were normalized using RMA-sketch. wt/wt/Er-cre+/+Ubf normalized gene expression was then subtracted from the corresponding fl/fl/Er-cre+/+Ubf timepoint before all the expressions were placed relative to time T0. We required a gene to be detected in at least one time point using a cutoff of 0.001 on the detection p-values obtained from the DABG algorithm (Affymetrix Power tools) to be used in the analysis.RNA for expression microarray analysis was extracted by the acid phenol/guanidinium thiocyanate method Cells were washed with PBS, fixed in 4% PFA for 15 minutes and permeabilized with 0.5% PBS/Triton for 5 minutes. Incubation with primary antibody was performed for 1 h in PBS-5% BSA and cells were stained with AlexaFluor 488/568 conjugated anti-rabbit or -mouse secondary antibodies (Molecular Probes) and counterstained with DAPI. After mounting in 50% glycerol/50% 0.2 M Na-glycine, 0.3 M NaCl, 3D epifluorescent 3D image stacks were acquired using a Leica DMI6000B microscope equipped with an Orca C4742-80-12AG camera (Hamamatsu) and Volocity (Perkin-Elmer Improvision) and were subsequently deconvoluted . In a few cases 3D stacks were also obtained using a Leica SP5 II scanning confocal microscope. Nucleolar statistics were obtained from three independent immunofluorescence experiments in which ∼20 nuclei were analysed by a protocol established using the Volocity software. DAPI staining was used to define the nuclear volume and Fibrillarin staining to define the nucleoli and their individual volumes. 3D Immuno-FISH was performed as previously described All animal care and animal experiments were conducted in accordance with the guidelines provided by the Canadian Council for Animal Protection, under the surveillance and authority of the institutional animal protection committees of Laval University and the CHU de Québec. The specific studies described were performed under protocol #2011-054 examined and accepted by the “Comité de protection des animaux du CHU de Québec”. This ensured that all aspects of the work were carried out following strict guidelines to ensure careful, consistent and ethical handling of mice.Figure S1(m)Ubf gene is essential for mouse development beyond morula. A) Targeting vector and strategy for target insertion into the wild type Ubf gene. B) Structure of the targeted mutant Ubf gene, and the conditional −flUbf (Floxed) and deleted Ubf−Δ (Δ) alleles. C) Example of Southern analysis of targeted and deleted Ubf alleles using the 3′ probe indicated in B). D) Examples of PCR genotyping of mice tails. E) Examples of mouse embryos and genotyping at the equivalent of 3.5 dpc. Ubf null embryos arrest at the morula stage. F) In vitro development of 3.5 dpc embryos to late blastocysts  and subsequent gentotyping. In A) and B) the lettering A to E refer to PCR primers used in genotyping, and in D) to F) bracketed letters indicate the combinations of these primers used to genotype.The mouse (EPS)Click here for additional data file.Figure S2fl/fl/Er-cre+/+Ubfcells treated with 50 nM 4-HT for 4 h. B) and C) Whole protein extracts prepared from fl/fl/Er-cre+/+Ubfcells at the given times post 4-HT treatment were analyzed by Western blot to determine the levels of UBF and of 13 other relevant proteins. Tracks were loaded with equal amounts of total protein with the exception of the first three tracks in B in which the given percentages of whole extract at 0 h post 4-HT treatment were loaded.A) Time course of ER-Cre induced recombination at the UBF locus in (EPS)Click here for additional data file.Figure S3fl/fl/Er-cre+/+Ubf cells prepared at the indicated times post 4-HT treatment, as in A) Map of the rDNA repeat showing the sites (amplicons) sampled by ChIP analysis. B) ChIP analysis of the individual components of SL1 used to generate the mean distribution of this initiation factor shown in (EPS)Click here for additional data file.Figure S4fl/fl/Er-cre+/+Ubf and +/+/Er-cre+/+Ubf cells at the given times after 4-HT treatment. The upper diagram indicates the BamHI rDNA fragment that was analyzed by Southern blot and the position of the probe used. The lower panels show the uncleaved BamHI rDNA fragment (4.7 kb) and the 0.9 kb fragment generated by cleavage of all 11 internal SmaI or XmaI sites. SmaI is sensitive to CpG methylation while XmaI is not. Hence, on genes on which all SmaI/XmaI sites are methylated SmaI cleavage is prevented, leaving the 4.7 kb BamHI fragment intact, while XmaI cleavage continues to generate only the fully cleaved 0.9 kb fragment. Since the 4.7 kb fragment can be cleaved at all or any of 11 SmaI sites, even fractional changes in methylation status will be detected by this technique. However, only the 4.7 and 0.9 kb fragments were detected at all time points, showing that little if any change in methylation status occurred. B) Raw preimmune subtracted ChIP data for H3, H3k9me3, H4 and H4ac. C) Raw, unsubtracted ChIP data for HP1α and H1.4-S187p and preimmune antibodies. D) Analysis of H4ac/H4 and H3K9me3/H3 ratios at the Camk2b locus normalized to 0 h pHT.A) DNA CpG methylation assays of (EPS)Click here for additional data file.Figure S5fl/fl/Er-cre+/+Ubf cells at 72 h post 4-HT normalized to relative levels for wt/wt/Er-cre+/+Ubf cells at the same time point. The original data can be found in the GEO databank under the accession number GSE55450.Unbiased gene enrichment plots of expression microarray data corresponding to the Boxplots shown in (EPS)Click here for additional data file.Figure S6fl/fl/Er-cre+/+Ubf cells stained for UBF, RPI , Rrn3/TIF1A or TTF1 and for Fibrillarin, and counter-stained with DAPI, at the indicated times post 4-HT treatment. Images show 0.2 µm optical sections from deconvoluted image stacks.A) to D) Single optical sections from 3D IF image stacks of (EPS)Click here for additional data file.Figure S7fl/fl/Er-cre+/+Ubf cell metaphase spreads were stained for rDNA by FISH (green) and counterstained with DAPI (red). B) to D) 3D Immuno-FISH of fl/fl/Er-cre+/+Ubf cells at the indicated times post 4-HT treatment, analyzed by FISH for rDNA (green) and by IF for UBF, Fibrillarin and TTF1 respectively, and counter stained with DAPI. Images show single 0.4 µm optical sections from deconvoluted 3D image stacks.A) (EPS)Click here for additional data file.


The RAS/MAPK signaling pathway can regulate gene expression by phosphorylating and altering the function of some, but not all, ETS transcription factors. ETS family transcription factors bind similar DNA sequences and can compete for genomic binding sites. However, MAPK regulation varies across the ETS family. Therefore, changing the ETS factor bound to a cis-regulatory element can alter MAPK regulation of gene expression. To understand RAS/MAPK regulated gene expression programs, comprehensive knowledge of the ETS family members that are MAPK targets and relative MAPK targeting efficiency across the family is needed.An in vitro kinase assay was used to rank-order 27 human ETS family transcription factors based on phosphorylation by ERK2, JNK1, and p38α. Many novel MAPK targets and specificities were identified within the ETS family, including the identification of the prostate cancer oncoprotein ERG as a specific target of ERK2. ERK2 phosphorylation of ERG S215 required a DEF docking domain and was necessary for ERG to activate transcription of cell migration genes and promote prostate cell migration. The ability of ERK2 to bind ERG with higher affinity than ETS1 provided a potential molecular explanation for why ERG overexpression drives migration of prostate cells with low levels of RAS/ERK signaling, while ETS1 has a similar function only when RAS/ERK signaling is high.The rank ordering of ETS transcription factors as MAPK targets provides an important resource for understanding ETS proteins as mediators of MAPK signaling. This is emphasized by the difference in rank order of ERG and ETS1, which allows these factors to have distinct roles based on the level of RAS/ERK signaling present in the cell.The online version of this article (doi:10.1186/s12964-015-0089-7) contains supplementary material, which is available to authorized users. Most transcription factors can be grouped into families based on common DNA binding domains  leading There are 28 genes encoding ETS family transcription factors in humans. All ETS factors have a winged helix-turn-helix DNA binding domain that allows monomeric binding to the sequence GGA(A/T) . HoweverKnowing that an ETS factor is a MAPK target is important for understanding regulatory mechanisms. For example, we have recently demonstrated that competition between ETS proteins for a RAS-response element consisting of neighboring ETS and AP-1 binding sites alters cancer cell migration. The ubiquitously expressed ETS protein ETS1 binds ETS/AP-1 sequences in the enhancers of cell migration genes . ETS1 isDespite the importance of MAPK signaling in the ETS family, it is unknown how many of the 28 human ETS family members can respond to this signaling pathway. Multiple studies have identified individual ETS proteins as a MAPK targets . HoweverE. coli with a 6x Histidine tag on the N-terminus. For ETS genes that express multiple transcripts, we chose versions commonly described in the literature, and biased our selections towards longer versions to include as many potential MAPK target sequences as possible. The transcripts and primer sequences used are in  was eight times higher than for ETS1 (KD = 1.79 μM), and this difference was statistically significant .This higher selectivity could be due to ERK docking with ERG with higher affinity than ETS1. To test this, the affinity of ERK for ERG and ETS1 was compared using microscale thermophoresis (MST) Figure E. The afThese in vitro data suggest that low levels of pERK could phosphorylate ERG, but higher levels would be required to phosphorylate ETS1. To test if this is true in vivo, levels of pERG and pETS1 were compared in the RWPE system. RWPE cells over-expressing ERG Figure F or ETS1The ETS family DNA binding sequence (consensus CCGGAAGT) occurs in multiple cis-regulatory motifs that mediate the gene expression changes that occur after mitogen signaling ,48. HoweComparing MAPK targeting of ETS factors provided data consistent with previous studies Tables , 2 and 3The number of PX(SP/TP) motifs correlated with targeting by all MAPKs, and particular D and DEF domain sequences correlated with specificity for p38 and ERK. However, these sequences alone could not entirely predict our findings. This is likely because of the role of tertiary structure in kinase interactions with both the docking sequence and the target residues. In the case of ETS1 and ETS2, for example, the structure of the pointed domain can mediate a specific ERK2 interaction , consistOur survey discovered ERK phosphorylation of S215 in ERG, and a specific antibody confirmed that this phosphorylation event occurs in vivo Figure . We demoThe higher affinity of ERK2 for ERG compared to ETS1 correlated with the finding that ERG S215 is phosphorylated in cells with low levels of ERK signaling, while ETS1 T38 is only phosphorylated in cells with high levels of ERK signaling. It also corresponds to the finding that ERG can promote cell migration in a low ERK signaling background, but ETS1 requires a high ERK signaling background , which activates RAS/ERK signaling, but because the RAS/ERK pathway is intact, negative feedback loops can keep ERK activity low . In a tue tumors . As the Here we characterize the MAPK specificity across the ETS family of transcription factors. Phosphorylation by all three MAPKs tested correlated with the number of PX(TP/SP) motifs, while p38α specificity correlated with LRL motifs and ERK2 specificity with FXFP motifs. A novel ERK2 phosphorylation site was identified in ERG. Phosphorylation of this site via a high affinity interaction with ERK2 was necessary for ERG function in prostate cells and indicates that low level RAS/ERK signaling may be required for ERG to drive prostate cancer.SPIB, ETV7, ELF3 25728) and ELK1 (27156) clones were obtained from Addgene or were gifts. ETV2 mRNA-ORF clone was from GeneCopoeia (EX-H1877-M02), and ETV3L from Origene (RC22031). All other ETS clones were reverse transcribed (RT-PCR) from mRNA isolated from cell lines or tissues with high expression . ETS ope728 and E32P ATP. The kinase assays shown in the left panels of Figure Kinase reactions were performed as described previously  with som4 cells were introduced to the transwell  and incubated for 60 h. Migrated cells are reported as the mean of at least three biological replicates with two technical replicates each.RWPE1 and RWPE2 cell lines were cultured in Keratinocyte SFM (Invitrogen) and 1% Penicillin/Streptomycin (100X solution – Mediatech-Cellgro). ETS proteins with N-terminal 3xFlag tags were stably expressed in RWPE1 or RWPE2 cells via retrovirus as described previously . TransweRNA levels were measured by quantitative RT-PCR as described previously  using prPrior to immunoprecipitation, cells were treated for 6 hours with the following inhibitors: MEK1/2 , JNK , P38 kinase , AKT , PKA , PI3Kinase , FRAP/mTOR , or DMSO,and cells were washed twice with ice cold 1X PBS and solubilized in RIPA buffer . Equal concentration of protein extracts were added to the anti Flag M2 magnetic beads (Sigma) and rotated overnight. Beads were washed 3 times with 1 M RIPA buffer and immunoblotted.MST analysis used a NanoTemper Monolith NT.115 (NanoTemper Technologies) as described . Briefly


To date, there has been an increasing number of drugs produced in mammaliancell cultures. In order to enhance the expression level and stability of targetrecombinant proteins in cell cultures, various regulatory elements with poorlystudied mechanisms of action are used. In this review, we summarize and discussthe potential mechanisms of action of such regulatory elements. E.coli cells. However,the production of proteins in bacteria is associated with a number oflimitations: disrupted folding of some proteins, absence of crucialmodifications, and the inability to produce larger molecules [Therapeutic proteins are major structural and regulatory molecules essentialfor a normal functioning of the human body. Part of recombinant proteins thatare small in size and do not require additional modifications are produced inthe most economical bioreactor –olecules , 2.Someolecules .Therefoolecules , 5. CHO olecules . It soonolecules . Optimalolecules , 4, 5.YThe most widely used method in industrial biotechnology is transfection with linearized plasmid DNA, 8,whicdhfr gene included in theconstruct and, as a result, of the gene encoding the target protein. Anotherfrequently used selectable marker is the gene encoding glutamine synthetase(GS). The CHO-K1 cell line, which contains mutated allele ofgs, is used when working with this marker. In this case,L-methionine sulphoximine (MSX) is used as a selective agentfacilitating the selection of the most effective clones [In order to enhance the producing capacity of clones, selective increase in thenumber of construct copies, which results in target protein expression, is usedin industrial biotechnology . This ise clones .dhfr and gs, exhibit a higherproliferative potential and viability. Among the disadvantages of usingfluorescent protein genes as selectable markers is the inability to amplify thecopy number, which in most cases would lead to increased producing capacity ofa cell clone.Clone selection can be conducted using other reporter genes, as well. The mostpromising options are the ones that do not require selection of a mutant cellline. Fluorescent protein technology, which allows one to select cells with maximumexpression of a target gene based on emission at a particular wavelength, can be considered, 18.SucRecently, the application of special robots capable of selecting individualcell clones with the most efficient expression of the target protein, which isidentified using antibodies, has become widespread .AlongsiVector constructs have also been developed based on viruses, mobile elements,bacterial anti-phage protection system, and recombination systems in phages andyeasts , 19.AppThe main challenge in obtaining producing cell lines containing multipleexpression vector copies is heterochromatin formation of repeated sequences ofvector constructs, which usually enhances upon cell proliferation. The mainrole in heterochromatin formation of a repeated DNA sequence is played by RNAinterference and noncoding RNAs that can stimulate repressive chromatin zoneformation at promoters, as well as methylation of CpG sites in promoterregions, which decreases the efficacy of transcriptional factor binding in suchregions -23.ThisStrong viral promoters, such as the cytomegalovirus (CMV) promoter and theearly promoter of SV40, as well as strong cellular promoters of housekeepinggenes, such as β-actin and factor EF1α genes, are usually used fortransgene expression .Strong viral or cellular promoters contain a minimal promoter of approximately100 bp, which serves as the transcription start site (TSS), and a strongenhancer located in close vicinity to the promoter. For instance, the mostwidely used CMV promoter bears a core region located between positions–62 and –1 bp from TSS and the enhancer (–544 through–63 bp) . There aOne of the approaches in applying effective promoters in biotechnology isidentification of strong promoters directly in the cell cultures that arefurther to be utilized for the generation of target protein-producing lines.Thus, total genome screening of the strongest promoters in CHO cells that aremost commonly used as the expression system in mammalian cells has beenperformed -35. As eEF1α gene [One of the solutions to the position effect is using long regulatory sequencesof actively transcribed housekeeping genes located on both sides of the codingregion of the gene. Thus, a high expression level of target proteins(6–35 times the level of expression from a standard CMV promoter) hasbeen obtained for vectors bearing a 12 kbp regulatory region or a 4 kbp3’-region of the Chinese hamster F1α gene . One of F1α gene .Artificial modification of promoters is another promising approach to enhancetheir activity. For instance, a strong CMV promoter has been demonstrated toundergo negative regulation resulting in methylation of GC regions attranscription factor (TF) binding sites comprising promoters and, as aconsequence, inhibition of TF recruitment to the promoter. As a result, theactivity of the CMV promoter is greatly decreased. Such a negative affect canbe avoided by integrating between the enhancer and core elements of the CMVpromoter a regulatory sequence that binds transcriptional factors suppressingthe DNA methylation process . AneffeA novel means to increasing a target protein expression is artificialrecruitment of effective transcriptionassociated complexes to the promoter, 41. ForIn order to enhance reporter gene transcription and reduce transgene expressiondependence on the surrounding chromatin, strong cellular enhancers are used, 45. OneIn order to increase the efficiency and stability of target protein expression,known insulators are used , 48-51.Recently, certain insulator proteins have been shown to participate in theorganization of specific, long-range interactions between distal regions ofchromatin -59. InsuFig. 1A)[To date, insulator architectural proteins (IAP) of Drosophila remain poorlydescribed, which is largely due to the ease of generating transgenic lines offlies. The study of insulator properties in Drosophila transgenic lines showedthat each insulator binds several IAP, which in turn determine the specificityof longrange interactions , 53. As Fig. 1A).homing can be explained by the recruitment ofIAP to the regulatory element that comprises the P element upon introduction ofthe construct into an embryo. This results in specific interaction between theP element and the cognate endogenous regulatory element, with furtherintegration of transposon into a certain region at the chromosome.Usually, transgenic Drosophila lines are obtained by injecting a vector thatcontains P element ends flanking the transgenic construct alongside the geneencoding transposase essential for construct integration into the genome. Cases hin vitro [Genome-wide studies of IAP-binding site distribution unambiguously demonstratedthat insulators are not fixed boundaries between transcriptional domains, 68.Twoin vitro . Thefirin vitro ,70, andin vitro . This mein vitro .The secoin vitro , 73. In The mechanisms that determine the barrier function of insulators have beendescribed in detail in Drosophila and mammals. In particular, it was found thatIAP help to recruit the protein complexes responsible for nucleosome remodelingand modification to insulators -78, resuTo date, only a single DNA-binding insulator protein, CTCF, has been describedin vertebrates , 80, whiExcept for their key role in the formation of chromatin architecture, CTCFprotein domains remain poorly studied and the mechanism of CTCF performance inmaintaining long-range interactions has not been characterized yet. The mainpart of the protein consists of 11 C2H2-type zinc fingers (ZF), with only fourof them (4th to 7th) being essential for the recognition of the core DNA motif. The remThe cohesin complex, which associates directly with CTCF , is suggFig. 2)[The most well-studied vertebrate insulator, HS4, consisting of 1,200 bp andlocated at the 5’-end of the chick β-globin locus, is used inbiotechnology , 98.A cFig. 2). ProteinFig. 2).USF hasFig. 2), 101.ThFig. 2). TheproFig. 2).Since its discovery, insulator HS4 has been actively utilized for transgeneexpression in mammalian cell cultures . Two comMost effectively, HS4 insulator can be applied in vectors that for some reasonhave a limited size. Thus, a full-size 1.2 kbp insulator significantly reducesthe efficiency of cellular transformation with lentiviral vectors . Therefore,HS4-duplicated core element of 250 bp, which contains the binding sites of allthe identified transcription factors required for the manifestation ofinsulator activity, is used in vectors of this class , 106. InDespite the examples of successful use of a 1.2-kbp HS4 insulator or its coreregion , 108, abIn conclusion, the following basic mechanisms for the protection of transgeneexpression using the HS4 insulator can be put forth: 1) formation of anucleosome- free DNA region that can disrupt the linear spread ofheterochromatin; and 2) recruitment of protein complexes that enhancenucleosome mobility, modify histones, stimulate transcription, protect CpGsitesfrom methylation, and terminate weak transcription. It has not been determinedyet whether the HS4 insulator is capable of guiding construct integration intotranscriptionally active chromatin zones and directly interacting with thetarget gene promoter for further transcription stimulation. Apparently, themain disadvantage of HS4 and other insulators is the dependence of theiractivity on the set of particular transcription factors expressed in the cellline. in vitro, havebeen widely used from the beginning of the 90s [In order to enhance transformation efficiency and improve the stability oftransgene expression, sequences of 300-5,000 bp, usually A/T-rich, whichinteract with a fraction of the nuclear matrix , as shown in experiments the 90s -110. S/M the 90s , 112. It the 90s , 114.Based on the characteristics of the secondary structure, there is about anorder of 50,000 elements predicted for the human genome that supposedly sharethe properties of S/MAR , 116. A The structure of S/MAR elements indicates that they might serve asrecombination hotspots. Indeed, it was shown that disruption sites that occurdue to inversions associated with human diseases are often localized inS/MAR-elements , 118, anInitially, it was thought that lamina are a major component of nuclear matrixproteins . Later, SATB1 is the most well-studied matrix protein involved in many biologicalprocesses, such as differentiation of T cells and epidermis -137. SATSAF-A, another matrix protein, includes a DNAbinding (SAF) and an RNA-binding(RGG) domain. It is interesting that Xist RNA, which regulates dosagecompensation in mammals, also associates with the RGG domain. This interactiondetermines the localization of Xist RNA on the X chromosome . AccordiAccording to the most commonly used model, S/MAR elements interact with theproteins of the nuclear skeleton (matrix proteins), resulting in the formationof chromatin loops where S/MAR serves as a core element . Genes lDespite the lack of understanding of the mechanisms underlying S/MAR action,abundant experimental data has been obtained demonstrating the effectiveness ofusing these elements for enhancing the expression of target proteins inmammalian cell cultures . ForexaIn conclusion, it can be stated that S/MAR present regulatory elements that areless studied than insulators. The most probable mechanisms of S/MAR action inenhancing transgene expression are 1) site-specific integration ofS/MAR-bearing constructs into the regions of transcriptionally active chromatinand amplification of the copy number of the integrated construct, 2)association with S/MAR complex elements that guide transcriptionally activechromatin zones and thus suppress the spread of heterochromatin, and 3)immediate promoter activation by transcription factors that directly bind toS/MAR.HNRPA2B1 and CBX3or TBP and PSNB1, which are activelytranscribed in all cells of an organism [Between 2000 and 2002, a small company named CobraTherapeutics developed atechnological platform based on regulatory elements isolated from housekeepinggenes, which are actively transcribed at all stages of development and in allcells of an organism, for obtaining efficient cell lines producing recombinantproteins . These rorganism . The firorganism , 152. Thorganism . It wasorganism -159. It organism , 160.It should be noted that, unlike other regulatory elements such as LCR andenhancers, which exhibit pronounced cell specificity, promoters of housekeepinggenes can function effectively in various cell lines. In experiments withvarious UCOE that were reduced in size in order to assess the possibility ofusing UCOE in expression vectors, more compact-size variants of UCOE (1.5 to 3kbp) have been obtained. Such truncated elements completely retain theiractivity during the generation of high-producing cell lines .UCOE actively participate in the process of transcriptional regulation, whichimplies the existence of direct interactions between the promoter regulatoryelements responsible for the expression of the reporter gene located in thevector and the transcription factors associated with UCOE. Therefore, UCOE caneffectively act only on certain promoters and the functional activity of thesepromoters does not manifest itself in cell lines . Some stRPL32 promoter, integratedbefore any other of the studied promoters, can significantly increase theefficiency of stable cell clone selection. It is worth mentioning that thedirection of the RPL32 promoter should coincide with thedirection of the promoter responsible for reporter gene expression, and coreelements of the RPL32 promoter in this case are essentialcomponents of the system for providing a stimulating effect.It was also demonstrated that a combination of two strong promoters may in somecases facilitate the generation of stable cell lines and enhance transgeneexpression . AnalysiIn general, combining strong promoters is a promising way to improve theefficiency of generating cell clones producing a target protein. Strongpromoters bearing a combination of enhancer and core promoter recruit proteincomplexes, which in turn support a transcriptionally active state of chromatin.According to the data of genome-wide studies, promoters act as effectiveboundaries that are able to protect genome areas against the spread ofrepressive chromatin regions ,166.TrazeoR, which isresponsible for resistance to the antibiotic Zeocin, and the death oftransfected cells when cultured in a selective medium with the addition ofZeocin. The screening aimed at detecting DNA fragments, integration of whichbetween Lex binding sites and the zeoR promoter protects thepromoter from HP1-dependent repression, enabled to find a series of regulatoryelements 500 to 2,000 bp in length called antirepressors (STAR). Other knownregulatory elements such as the insulator HS4, MAR, and UCOE lack that ability.A comparative analysis of various regulatory elements [The Chromagenic company has developed a technological platform based on atest-system which effectively allows the identification of regulatory elementscapable of suppressing the spread of heterochromatin areas . The teselements  has showTo date, no universal regulatory element with a clear mechanism of action hasbeen found that can be effectively used in all types of vector constructsdesigned to generate cell lines producing various proteins at a high level.This is largely due to the complexity of the mechanisms that regulate promoteractivity and also the absence of actual evidence on the original concepts ofstrict organization of genes with the same expression profile intotranscriptional domains surrounded by special regulatory elements from a classof insulators or S/MAR. Clearly, some mechanisms must exist that suppressexcessive transcription even for the strongest promoters. RNA interference isone of such mechanisms. It is possible that a detailed understanding of themechanisms of transcription activation and suppression will lead to thedevelopment of artificial promoters that allow researchers to obtain stablehigh levels of target gene expression in transgenic systems.


Interferon Regulatory Factor 5 (IRF5) plays a major role in setting up an inflammatory macrophage phenotype, but the molecular basis of its transcriptional activity is not fully understood. In this study, we conduct a comprehensive genome-wide analysis of IRF5 recruitment in macrophages stimulated with bacterial lipopolysaccharide and discover that IRF5 binds to regulatory elements of highly transcribed genes. Analysis of protein:DNA microarrays demonstrates that IRF5 recognizes the canonical IRF-binding (interferon-stimulated response element [ISRE]) motif in vitro. However, IRF5 binding in vivo appears to rely on its interactions with other proteins. IRF5 binds to a noncanonical composite PU.1:ISRE motif, and its recruitment is aided by RelA. Global gene expression analysis in macrophages deficient in IRF5 and RelA highlights the direct role of the RelA:IRF5 cistrome in regulation of a subset of key inflammatory genes. We map the RelA:IRF5 interaction domain and suggest that interfering with it would offer selective targeting of macrophage inflammatory activities.  •Genome-wide function of IRF5 in LPS-stimulated macrophages was analyzed•IRF5 cistrome overlaps with RelA cistrome at multiple loci•IRF5 targets regulatory elements of highly inducible inflammatory genes•IRF5 recruitment to key inflammatory loci is assisted by RelA Saliba et al. show that the genome-wide binding of IRF5 and RelA in LPS-stimulated macrophages overlaps at multiple loci. IRF5 and RelA target regulatory elements of highly inducible inflammatory genes. IRF5 is recruited to a composite PU.1:ISRE motif, and its binding is aided by NF-κb. This study systematically defines the mechanism of IRF5 and RelA transcriptional regulation of a key subset of inflammatory genes and highlights IRF5:RelA interaction interface as a target for specific interventions. A finely tuned inflammatory response to microbial and endogenous insults is essential for host survival. During inflammation, gene programs are activated by orchestrated changes in transcription and are determined by transcription factors (TFs) binding to accessible DNA regulatory elements found in promoters and enhancers. Ubiquitously expressed TFs such as nuclear factor-κB (NF-κB), interferon regulatory factors (IRFs), and activator protein 1 (AP.1) each play a central role in eliciting an inflammatory response to extracellular Toll-like receptor 4 (TLR4) stimulation by lipopolysaccharide (LPS). These ubiquitous stimulus-inducible TFs appear to work in conjunction with lineage-restricted constitutive TFs, such as PU.1, to define lineage-specific enhancers . HoweverIL-10 /Th17-associated mediators, such as interleukin-1 (IL-1), IL-12, IL-23, and tumor necrosis factor α (TNF-α) . MoreoveIL-10 . A functnfection  and in tnfection . These dTNF gene .In this study, we used GM-CSF (granulocyte/macrophage-colony-stimulating factor)-derived macrophages (GM-bone marrow-derived macrophages [BMDMs]) to investigate whether the recruitment of IRF5 via its interactions with RelA is a common mechanism of proinflammatory gene regulation by IRF5. By intersecting the chromatin immunoprecipitation-sequencing (ChIP-seq) analysis of the individual TFs in LPS-stimulated GM-BMDMs with gene expression data and histone methylation status data sets, we show that the IRF5 and RelA cistromes target inflammatory genes. The two cistromes overlap only at a limited number of genomic regions located in the PU.1-marked regulatory elements of inflammatory genes, 70% of which are induced upon LPS stimulation, as shown by the recruitment of RNA polymerase II (PolII). Using in vivo and in vitro motif discovery analyses, we demonstrate that the IRF5:RelA cistrome is best explained by the presence of consensus NF-κB and noncanonical composite PU.1:interferon-stimulated response element (ISRE)-binding sites. We demonstrate that IRF5 genome recruitment to inflammatory genes is aided by RelA. These results reveal a genomic strategy for controlling an inflammatory gene program in GM-BMDMs via establishment of a unique IRF5:RelA cistrome to target inflammatory genes.To investigate the model of IRF5-RelA transcriptional cooperation, ChIP-seq was used to determine the genome-wide binding of IRF5, RelA, and PolII in GM-BMDMs stimulated with LPS or left unstimulated. Upon LPS stimulations, these macrophages are predominantly homogeneous IRF5-positive cells that display a distinct phenotype of cytokine and cell surface molecule expression compared to M-CSF (CSF-1) (M)-BMDMs A Fleetw. ChIP-seLta), Ltb, and Tnf gene cluster, chemokine (C-C motif) ligand 5 (Ccl5), IL-1α (Il-1a), and other immune related gene loci. In the case of the Tnf gene cluster, IRF5 and RelA show similar binding patterns as we previously reported in human monocyte-derived dendritic cells (−4). Therefore, the overlap of IRF5 and RelA binding initially observed in the Tnf gene cluster  increase in IRF5 recruitment following LPS stimulation for 2 hr  more prominently than with the rest of either RelA or IRF5 peaks (−7) or IRF5 (p < 10−12) acting independently , respectively. To do this, we intersected the data sets with H3K4me1/H3K4me3-positive regions from M-BMDMs  and GM-B−4; It has recently been shown that binding of a pioneer TF PU.1 is essential for defining macrophage-specific enhancers because it promotes the deposition of H3K4me1 . Here, w−10), such as Il-6, Il-12a, Il-1a, Fpr2, Aoah, Adam17, Cxcl2, and Saa3 (Mmp25 and Socs3) or only RelA   of the genes into three categories. These consist of genes that encompass ChIP-seq peaks present in (1) both IRF5 and RelA, (2) only RelA, or (3) only IRF5 A. We nexand Saa3 C. Moreovnd Sod2) C. In catene loci . In cateactivity . The obsactivity . In totaactivity , at manyactivity  allows fTo better understand the regulatory code at inflammatory loci, we first performed ab initio DNA motif analysis around the top 500 RelA and IRF5 peaks. Motif analysis revealed the known κB motif  in RelA Because ChIP only identifies genomic regions that interact with TFs but not necessarily individual binding sites , we usedZ scores were used to perform receiver operating characteristic (ROC) curve analyses using the IRF5 and RelA cistromes from this study to quantify whether the IRF5- or RelA-bound regions (true positives) scored higher than the unbound regions (true negatives), similar to All the IRF5- and RelAp50-binding sequences with their respective Together with the ab initio analysis above, we therefore interpret this result as evidence that at the inflammatory gene loci, IRF5 is likely to be recruited to a composite PU.1:ISRE site rather than to a canonical ISRE site, whereas RelA binds directly to the respective κB site. The dynamics of PU.1 and IRF5 binding at the PU.1:ISRE composite sites are unclear and warrant further investigation.Il-1a, Il-6, and Tnf genomic loci was analyzed by ChIP-qPCR. We observed a significant reduction in IRF5 binding to the regions of overlapping IRF5:RelA peaks , basal , and stimulus-inducible  TFs . Here, wBinding of a pioneer TF, PU.1, in macrophages is thought to be sufficient to promote the deposition of H3K4me1 and to create small open regions of accessible DNA that can be bound by stress-inducible dynamic TFs, such as NF-κB and IRFs  . Less clCan this be a mode of binding at other gene loci identified in this study as IRF5 but not RelA targets? The interactome of IRF5 is rapidly expanding ; thus, iIt is possible that IRF5 acts similarly to another member of the IRF family, IRF3, which was shown to promote transcription by removing a nucleosome barrier . IRF5 inIRF5 is a genetic risk factor for many autoimmune diseases, including systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, and inflammatory bowel disease . Anti-inIrf5−/− mice has been described by The generation of For the generation of M1 macrophages differentiated with GM-CSF, bone marrow of WT C57Bl6 mice was cultured in RPMI-1640 medium (PAA Laboratories) supplemented with recombinant mouse GM-CSF . After 8 days, cells were washed with PBS and replated, then stimulated with LPS .Nuclear lysates of formaldehyde-fixed GM-CSF macrophages were isolated as described previously by −/− GM-BMDMs with 50 bp paired-end sequencing following stimulation with LPS for 0 and 2 hr. Preliminary analysis of these data sets by MACS2 algorithm with filtering out of peaks detected in the IRF5−/− from the WT corroborated the findings reported in this manuscript. Further details of next-generation sequencing and analysis are provided in the Please note that independent IRF5 ChIP-seq data sets were recently generated by us in WT and IRF5Microarray (accession number E-MTAB-2032) data were analyzed in R/bioconductor using the beadarray (version 2.4.2; D.G.S. designed research, performed the wet lab experiments, and wrote the paper. A.H. performed bioinformatics analysis of the data and helped write and edit the manuscript.