R-loops are involved in transcriptional regulation, DNA and histone post-translational modifications, genome replication and genome stability. To what extent R-loop abundance and genome-wide localization is actively regulated during metazoan embryogenesis is unknown. Drosophila embryogenesis provides a powerful system to address these questions due to its well-characterized developmental program, the sudden onset of zygotic transcription and available genome-wide data sets. Here, we measure the overall abundance and genome localization of R-loops in early and late-stage embryos relative to Drosophila cultured cells. We demonstrate that absolute R-loop levels change during embryogenesis and that RNaseH1 catalytic activity is critical for embryonic development. R-loop mapping by strand-specific DRIP-seq reveals that R-loop localization is plastic across development, both in the genes which form R-loops and where they localize relative to gene bodies. Importantly, these changes are not driven by changes in the transcriptional program. Negative GC skew and absolute changes in AT skew are associated with R-loop formation in Drosophila. Furthermore, we demonstrate that while some chromatin binding proteins and histone modifications such as H3K27me3 are associated with R-loops throughout development, other chromatin factors associated with R-loops in a developmental specific manner. Our findings highlight the importance and developmental plasticity of R-loops during Drosophila embryogenesis. R-loops are essential for initiation of replication in plasmids and promote mitochondrial genome stability.20 In contrast, R-loops can block replication fork progression and promote genome instability in an orientation-specific manner.22 While potentially causing double-strand breaks at head-on replication-transcription conflicts, R-loops can promote recombination and double strand break repair.24 R-loops also have diverse roles in transcription and chromatin function. In mammalian cells, R-loops have been shown to regulate both histone and DNA methylation at promoter regions.14 While R-loops are often associated with histone modifications correlated with active transcription, recent work has shown that R-loops can help recruit the Polycomb complex to target loci to promote transcriptional silencing.26 Genome-wide R-loop mapping studies in yeast, plants and mammalian cultured cells have identified factors such as DNA sequence, DNA topology and histone modifications associated with R-loop formation.28 R-loop mapping studies in plants and mammalian cells have further revealed that R-loop formation can be dynamic as a function of development.29 The extent of R-loop plasticity in other metazoans has yet to be defined. Studying R-loops in the context of development could provide insight into the functional roles R-loops play in establishing developmental-specific changes in chromatin structure, function and transcriptional programs.R-loops are found in all domains of life and their formation is often conserved across cell types and even species.30 Approximately two hours after fertilization, zygotic genome activation is triggered and the transcription of over 3000 genes necessary for growth and differentiation are induced in a process known as the maternal-to-zygotic transition (MZT).32 Prior to the MZT, cells are largely undifferentiated and have abbreviated cell cycles.33 After the MZT, however, the cell cycle slows and cells become differentiated as morphogenesis proceeds.34 The changes in cell cycle programs, the onset of zygotic gene activation and cell differentiation during embryogenesis provide a unique system to interrogate whether R-loop formation or resolution impacts embryogenesis and the extent to which, if any, R-loop position and properties change as a function of development.Drosophila provide a well-established developmental system to interrogate R-loop plasticity during development. At the earliest stages of Drosophila embryogenesis, rapid cell proliferation is driven by maternally stockpiled proteins and RNA.In this study, we measured R-loop abundance and position in Drosophila embryos and cultured cells. We show that absolute R-loop levels change during embryogenesis and resolution of R-loops is essential for embryogenesis. We mapped R-loops at near base-pair resolution in 2–3 hour embryos (immediately after the MZT), late-stage embryos (14–16 hours after fertilization) and cultured S2 cells, which are derived from late-stage embryos. We show that, while some sites of R-loop formation are constant during development, there is extensive R-loop plasticity during Drosophila development. Furthermore, we were able to demonstrate changes in the localization of R-loops across gene bodies and the role AT and GC skew play in Drosophila R-loop formation. By leveraging data available through modENCODE and other publicly available datasets, we were able to identify specific histone modifications and chromatin binding proteins associated with R-loop formation in Drosophila and the role active transcription has on R-loop formation. Importantly, developmental-specific R-loops are not driven by transcriptional changes, emphasizing the role that chromatin and R-loop binding proteins play in regulating R-loop formation. Our work establishes Drosophila as a powerful developmental model system to study R-loop biology.35 This time point represents the onset of zygotic transcription and allows us to draw upon the wealth of scientific literature that has previously been published, including time-matched modENCODE datasets. The wide-scale activation of zygotic transcription at this time point should provide the first opportunity for R-loop formation during development. To complement this developmental stage, we chose 14–16 hour AEL embryos to understand how R-loop formation might differ in differentiated cells with a more mature chromatin environment and a transcription program characterized by cell-type-specific maintenance.38 S2 cells, an established Drosophila cell culture line derived from late-stage embryos, were used to determine how R-loops might differ between embryos and cultured cells, where the majority of R-loop research has been conducted.39To determine if R-loop abundance and genomic location are regulated throughout development, we turned to the powerful Drosophila embryogenesis system. For our analysis, we chose embryos at two distinct time points: 2–3 hours after egg laying (AEL) and 14–16 hours AEL  . The 2–40 S2 cells and 2–3 h embryos showed similar amounts of S9.6 signal, while DNA from 14-16 h embryos showed a significant decrease in S9.6 signal . To ensure that the RNase H1 proteins were maternally deposited and present at the earliest stages of embryogenesis, we used the pUASz expression system coupled with the maternal triple driver.44 After confirming that the GFP was observable by Western blot  in S2 cells, 2–3 h and 14–16 h embryos .18 When conducted in Drosophila, however, we found high levels of RNA contamination in the final sequencing results (data not shown). Even with the ssDRIP-seq method, it was necessary to pre-treat genomic DNA preps with RNase A and RNase III as Drosophila embryos are stockpiled with RNA.While the absolute abundance of R-loops changes during development, we wanted to determine how R-loop position throughout the genome changes during Drosophila development. Genome-wide R-loop mapping during Drosophila development would allow us to ask if R-loop formation is hardwired into the genome and driven only by cell-type-specific transcription, or, more interestingly, is R-loop formation plastic during development changing independent of sequence composition and transcription status. To address this question, we performed  embryos .9 We ini45 Furthermore, our S2 data sets were highly correlated with two R-loop mapping studies performed in cultured cells, but not correlated with an R-loop data set generated for a spike-in control and not used in mapping studies .  studies -D).13,46 results -G). TakeTo map the precise location of R-loops throughout the genome and allow us to compare both quantitative and qualitative properties of R-loops, we used MACS to define R-loop peaks. Peaks were called separately against the input samples and RNase H1 treated controls, and only overlapping peaks were kept for analysis. Using this criterion, we identified 27,646, 22,581 and 29,801 peaks in S2 cells, 2–3 h and 14–16 h, respectively, which occupied between 8.3 and 12.5% of the genome. The overlap of sense and antisense R-loops had similar ratios . R-loop 47 In all samples, we found that ~50% of R-loops mapped to introns or exons , gene bodies and transcription termination sites (TTS).ene body , which i embryos . Interes embryos . The 14– embryos . The enr embryos . In the  embryos . Taken t14 To test if GC or AT skew is associated with R-loop formation in Drosophila, we measured the AT/GC skew directly over all identified R-loops. This analysis revealed a striking transition from positive to negative AT skew at the center of our combined R-loop signal. This is mirrored by a less dramatic transition from negative to positive GC skew centered at the combined R-loop signal  and 14–16 h embryos. To quantitatively determine if chromatin marks were positively or negatively associated with R-loops, we evaluated the probability of R-loops overlapping a variety of histone modifications and chromatin-associated proteins by chance using a peak shuffling bootstrap procedure  across development and which factors are developmental specific. We found that the repressive mark H3K27me3 was positively associated with R-loops in all developmental samples, highlighting the link between R-loops and transcriptional repression . Interes51 Unfortunately, time-matched GRO or PRO-seq datasets do not exist for 14–16 h embryos. We converted GRO-seq signal to FPKM for each annotated transcript in the Drosophila transcriptome. Then, we compared the GRO-seq value of all R-loop-containing genes to genes devoid of R-loops. In S2 cells, R-loop positive and negative genes had a similar median FPKM value by GRO-seq . In One explanation for developmental-specific R-loop formation is that specificity is driven by developmental-specific transcription status. To test this, we compared expression level of genes that exhibit R-loops only in S2 cell or only in 2–3 h embryos . If acti52We asked if the chromatin signature of R-loops in highly expressed genes differs from the signature of R-loops in transcriptionally repressed genes. To this end, we selected R-loops in the highest expression quartile and lowest expression quartile from S2 cells . Next, w53 Delaying the onset of zygotic transcription delays the activation of ATR (Mei41 in Drosophila), indicating that replication-transcription conflicts drive the activation of the DNA damage response at the MZT.54 It is unknown, however, what aspect of the replication-transcription conflict triggers ATR activation at the MZT. If genome instability at the MZT was at least partially due to R-loops, we would predict to see an enrichment of RPA at R-loop forming sequences in 2–3 h embryos. Qualitatively, we see overlap between RPA and R-loops in 2–3 h embryos . The cell line was grown under recommended conditions. The S9.6 antibody was purified on a protein G column using the GE aKTA system and run over a desalting column for buffer exchange into PBS to obtain a final concentration of 1 mg/mL. The antibody was aliquoted and stored at −80 °C. A fresh aliquot was used for every ssDRIP-seq experiment.43 The A isoform was chosen as the isoform B isn’t detected in Drosophila tissues.61 The mitochondrial localization start site was converted to AAA to ensure RNase H1-GFP would only be present in the nucleus. The catalytically dead version of RNase H1 (D201N) was made by site-directed mutagenesis (Agilent QuickChange Lightning). Plasmids were injected into an attP2 containing stock (BestGene) for site-specific integration.Drosophila RNase H1 was cloned from RNA derived from Oregon R embryos. RNA was converted into cDNA, PCR amplified, and cloned into the pUASz vector with a C-terminal GFP tag.For the overexpression experiments, homozygous RNase H1 males were crossed with unmated female homozygous for the maternal triple driver  to drive expression early in embryogenesis. Male Oregon R flies were crossed with MTD females as a control. Progeny were transferred to bottles with a grape juice agar plate with wet yeast for embryo collection. 100 unhatched embryos were carefully moved to a fresh grape juice plate and incubated overnight at 25 °C. After 36 hours, unhatched embryos were counted. This was repeated three times each from two separate crosses.S2 cells were obtained directly from the Drosophila Genomic Resource Center (DGRC). Cells were confirmed negative for mycoplasma contamination via PCR. Cells were grown at 25 °C in Schneider’s Drosophila Medium with 10% heat-inactivated FBS (Gemini Bio Products) and 100 U/mL of Penicillin/Streptomycin (Fisher Scientific).Oregon R flies were expanded into population cages containing grape juice plates supplemented with wet yeast. Population cages were kept at 25 °C in a humidified room and plates were changed daily. Before embryo collections, flies were precleared for at least one hour to minimize the number of late-stage embryos. Embryos were collected and aged at 25 °C to obtain embryos that were 2–3 or 14–16 hours old. After aging and collection, embryos were dechorionated in 50% bleach for 2 minutes and thoroughly rinsed in water. Embryos were flash frozen in liquid nitrogen and kept at −80 °C until ready to use. An aliquot of embryos was taken from each batch before freezing to verify staging. For this, embryos were fixed in heptane and 2% paraformaldehyde for 20 minutes with shaking, devitellinized in methanol, washed with methanol and rehydrated in PBS + 0.1% Triton X-100 overnight. Embryos were stained with DAPI and mounted in Vectashield medium (Vector Labs). Images were acquired on a Nikon Ti-E inverted microscope with a Zyla sCMOS digital camera.26 For genomic DNA isolation from S2 cells, cells were collected at 70–80% confluency, washed once in PBS, resuspended in TE with 0.5% SDS and 100 μg/mL proteinase K and incubated at 37 °C overnight. Embryos were devitellinized in heptane and methanol, rinsed thoroughly in PBS and incubated in 50 mM Tris-HCl pH 8.0, 100 mM EDTA, 100 mM NaCl, 0.5% SDS, and 5 mg/ml proteinase K for 3 hours at 50 °C. At this point, cells and embryos were processed the same. Extracts were purified with phenol:chloroform, and DNA was precipitated with sodium acetate and ethanol. DNA was spooled using a glass pipette and transferred to 70% ethanol. After several washes in ethanol, the DNA was air dried and resuspended in TE. To degrade free RNA, samples were incubated with 100 μg of RNase A with 500 mM NaCl for 1 hour at 37 °C. RNase A was degraded by spiking in 100 μg/mL proteinase K and incubated for an additional 45 minutes. Samples were cleaned with phenol:chloroform, precipitated with sodium acetate and ethanol, and resuspended in TE. Samples were diluted to 100 ng/μL and sonicated in a Bioruptor Plus for 8 cycles (30″ on/90″ off) on low power. 10 μg of nucleic acid was digested with 5 μL RNase H1 (NEB) at 37 °C for 16 hours and 10 μg was mock digested without RNase H1. Both samples had 1 μL of RNase III added (Thermo Fisher). After phenol: chloroform purification and precipitation, samples were immediately used for DRIP or slot blot experiments.Genomic DNA purification is based on Alecki et al., 2020.62 and normalized intensity was obtained by dividing the S9.6 signal by the dsDNA signal.63 A standard plot was made for each sample and antibody, and samples were chosen for analysis when their intensity was linear.Hybond Nylon membrane (Amersham) was pre-soaked in TE and a slot blot apparatus was assembled according to manufacturer’s instructions (Bio-Rad). Samples with matching RNase H1-digested controls were added to the blot in decreasing amounts, and nucleic acids were crosslinked to the membrane with a Strategene UV Stratalinker 1800 using the auto crosslink setting. Blots were blocked in milk, incubated with S9.6  followed by mouse-HRP and imaged in a Bio-Rad Chemidoc MP. After imaging the R-loops, blots were stripped and re-probed using a dsDNA-specific antibody (Abcam ab27156) at 1:20,000. Intensities were measured with ImageJ,14 Briefly, 4.4 μg of DNA was resuspended in 500 μL of TE. 10% was taken for the input sample. DRIP binding buffer was added to each sample  and 20 μL of 1 mg/mL S9.6 was added to each DRIP reaction. After overnight incubation at 4 °C, 50 μL of pre-washed protein G Dynabeads (Life Technologies) were added to the extract. After 2 hours at 4 °C, beads with captured nucleic acid were washed in 1x DRIP binding buffer 5 times and eluted in 50 mM Tris, 10 mM EDTA, 0.5% SDS with proteinase K at 50 °C for 45 minutes. Nucleic acid in the eluate was purified with phenol:chloroform, precipitated and resuspended in 10 mM Tris. For DRIP-qPCR, 1 μL of nucleic acid was diluted 1:10 in water and mixed with 10 μL SSoAdvanced Universal Sybr (Bio-Rad). Primers were added to a final concentration of 250 nM each. A list of primers used in this study can be found in DRIP was carried out as described in Ginno et al. 2012.64 Each paired read was trimmed 10 base-pairs at the 3′ end to eliminate the additional low complexity from the library preparation kit. Reads for sequencing were mapped to the Drosophila genome (dm6) using bowtie2 version 2.3.4.1 using the –very-sensitive-local setting.65 Duplicates were marked using picard MarkDuplicates v2.17.10, and stranded bam files were created using samtools as described in Xu and Sun et al. 2017.66 Stranded bam files were used to generate ssDRIP peaks with callpeaks from MAC2 v2.1.2.67 The RNase H1 pretreated DRIP file was used as control, peak calling was done on the 2 replicates in paired-end mode, with −keep-dup = auto and effective genome size for Drosophila dm6. A small number of peaks mapped to both strands as determined with bed-tools.68 Peaks that mapped to both strands and had reciprocal overlap of 90% as determined by bedtools intersect were removed from down-stream analysis. Bam files were combined and 50 million reads were randomly selected for visualization. Stranded reads were visualized using deeptools bamCoverage using --binSize 50 bp, --ignoreForNormalization chrY chrM, and --normalizeUsing RPKM.69 Pearson correlation plots were created using deeptools multiBamCoverage and plotCorrelation with default settings, 1 kb windows and the mitochondrial genome excluded.Fastq files were initially trimmed of adapters using Trimmomatic v0.3.8.47 Overall sense and antisense R-loops were determined via bedtools intersect with strandedness against the Refseq Drosophila transcriptome, downloaded from UCSC genome browser. Metagene plots were made with the Deeptools software package, using computeMatrix and plotProfile. For computeMatrix, scale-regions or reference-point as appropriate, with a 1 kb region size and 500 bp up- and down-stream of the start and end site, respectively. For options -binSize was 50 and the mean was plotted. For plotProfile, ‘add standard error’ was added to Plot type. –yMin and –yMax were chosen to be the same for both sense and antisense to aid in visualization.Peak annotation was performed using Pavis to the dm6 genome with up- and downstream regions set to 5 kb.72Gene Ontology enrichment analysis of R-loop containing genes was performed with PANTHER, with Fisher’s exact test and using the Bonferroni correction for multiple testing.73 R-loops peaks were split into 2 files containing their + and – peaks and annotation of R-loop peaks was done with HOMER software package using annotatePeaks.pl against dm6 and requiring the appropriate strandedness.73 R-loops mapping to transcripts were extracted from the HOMER annotation, and GROseq values for these transcripts was determined using custom R scripts. Plots summarizing these data were created in Prism 9.GRO-seq FPKM counts were determined with HOMER analyzeRepeats.pl using S2 datasets from Core and Lis et al. 2012 and GRO-seq data on 2–2.5 embryos from Saunders and Ashe et al. 2013.74 We considered samples assayed in S2 cells and at two developmental time-points . These were chosen to match the ssDRIP timepoints.We downloaded histone modification peaks and transcription factor binding sites identified by ChIP-chip or ChIP-seq in Drosophila from ModENCODE .74 We cop value for the observed amount of overlap by comparing the number of overlapping bp to a null distribution. We obtained the null distribution by randomly shuffling length-matched regions throughout the genome and calculating the amount of overlap in each permutation. The p-values are adjusted for multiple testing using the Bonferroni method.For each ChIP-chip or ChIP-seq marker with a matching DRIP timepoint, we calculated the number of overlapping base-pairs (bp) between the marker and the R-loop peaks. We used permutation-based approach to determine whether the observed amount of overlap was more or less than expected by chance. Briefly, we calculated an empirical 75 Peaks called from DRIP were lifted over to dm3 for this analysis. For peaks obtained from ChIP-chip data, we required that the shuffled peaks maintained both the overall length distribution and the probe density of the original peak. We reshuffled any peaks that fell more than 2 standard deviations (approx. 0.03) away from the original probe density until at least 99% of the original peaks were appropriately matched. We performed 1000 permutations for each marker and R-loop pair.When permuting, we matched the length distribution of the shuffled peaks to the original set of peaks, and excluded all gap and blacklisted regions from consideration .For the general analyses, we maintained the location of the R-loop peaks and shuffled the locations of the histone modification or transcription factor binding peaks. For a secondary analysis, we examined a subset of R-loops quantified specifically in the TTS and 3′ UTR. For this set of R-loops, we maintained the R-loop location within the TTS/3′ UTR and shuffled the chromatin markers.76We calculated GC and AT skew over the entire Drosophila genome (dm6). GC skew was calculated for 50 bp windows tiled across the annotation regions as iG repressnts the frequency of guanine nucleotides and iC represents the frequency of cytosine nucleotides in the window i. The range of GC skew for a window (iS) spans from −1 to 1. AT Skew was calculated in the same way. The resulting GC and AT skew was converted to a bigwig file, and the value across each set of genomic regions was calculated using the computeMatrix function from deeptools and visualized using plotProfile .In the equation, Supplemental materialSupplemental Table 1Supplemental Table 2


Ig) loci during B cell maturation. First V(D)J recombination occurs during early B cell stages followed by class switch recombination (CSR) and somatic hypermutation (SHM) which occur during mature B cell stages. Given that RAG1/2 induces DNA double strand breaks (DSBs) during V(D)J recombination and AID (Activation-Induced Deaminase) leads to DNA modifications (mutations during SHM or DNA DSBs during CSR), it is mandatory that IgH rearrangements be tightly regulated to avoid any mutations or translocations within oncogenes. Ig loci contain various cis-regulatory elements that are involved in germline transcription, chromatin modifications or RAG/AID recruitment. Ig cis-regulatory elements are increasingly recognized as being involved in nuclear positioning, heterochromatin addressing and chromosome loop regulation. In this review, we examined multiple data showing the critical interest of studying Ig gene regulation at the whole nucleus scale. In this context, we highlighted the essential function of Ig gene regulatory elements that now have to be considered as nuclear organizers in B lymphocytes.B cells undergo genetic rearrangements at immunoglobulin gene ( Ig) genes. Among these events, V(D)J recombination takes place in the bone marrow during the early steps of B cell development and occurs in an antigen-independent manner. Mature B cells migrate towards secondary lymphoid organs and continue their differentiation once stimulated by antigens. This process integrates secondary beneficial DNA remodeling events including class switch recombination (CSR) and somatic hypermutation (SHM) but can also induce B cell death through locus suicide recombination (LSR), a detrimental rearrangement that abrogates surface B cell receptor expression , supranucleosomal (chromatin domains and compartments) and nuclear (chromosome territories) scales –4. In adomosomes . Some ofreceptor  and Th2 receptor . At TAD receptor . TAD borreceptor .Ig gene loci. Most Ig gene regulation studies have so far been performed at the nucleosomal scale (epigenetic modifications and regulatory transcription of loci and gene segments). The increasing interest in understanding gene regulation at the whole nucleus scale prompted B cell scientists to revisit previous models at both supranucleosomal (DNA loops and TADs) and nuclear (chromosome territories and nuclear position) levels  levels –22. To pIg genes are encoded by three loci located on three distinct chromosomes. The immunoglobulin heavy chain (IgH) locus lies on chromosome 12. Immunoglobulin light (IgL) chain loci contain either kappa immunoglobulin light chain (Igκ) or lambda immunoglobulin light chain (Igλ) genes and are respectively located on chromosomes 6 and 16. In mice, at least 95% of B cells express Igκ light chains  among which hs4 and hs5 are CTCF binding sites (IGCR1)  core enhancer element (cEµ) flanked by two matrix attachment regions (MARs) located between the last HJ exon and the Sµ region. Between the Cγ1 and Cγ2b constant genes, two transcriptional enhancers hRE1 and hRE2 (3’Regulatory Region (3’RR), spanning more than 30 kb, is composed of four enhancers, called hs3a, hs1.2, hs3b and hs4 (hs1.2 enhancer is flanked by inverted repeated intervening sequences (IRIS) that form a 25kb-long quasi-palindrome  lie in a region containing ten CTCF Binding Elements (3’CBEs)  . The proand hRE2  form theand hs4 . The centhe 3’RR . Downstr(3’CBEs) . This reD border .IgH regulatory elements that helped elucidate Eµ and 3’RR super-enhancer functions during early and late B cell development  and hs3-6 comprises the Sis (Silencer in the intervening sequence) elements. A MAR-intronic Eκ enhancer (MiEκ)  and an intronic enhancer. The 3’Eκ enhancer region is situated downstream from the unique Cκ gene (Ed (due to its downstream location within the Igκ locus), is located distal to 3’Eκ  –43, loca Cκ gene . A third to 3’Eκ .IgH and Igκ loci, the Igλ locus is smaller (200kb) and uniquely organized. It comprises four families which contain a pair of Jλ and Cλ segments with only three Vλ segments  chain which promotes active IgA J recombination. Once engaged in the B cell lineage after expression of B cell specific transcription factors  at its surface. The pre-BCR, indispensable for B cell development J recombination, particularly for loci contraction/decontraction, nuclear positioning (center vs periphery) and heterochromatin addressing (IgH locus throughout early stages is involved in IgH accessibility in order to correctly orchestrate V(D)J recombination  remains until the immature B cell stage  . Ultimately, IgH looping plays a critical role in V(D)J recombination by forming a “recombination center” containing pDQ52, all four HJ segments and cEµ (RSS) and therefore focal RAG targeting activity during HD to HJ joining. This loop extrusion, is thought to ensure better sequence recognition by the RAG complex, avoiding off-targeting  . During traction . This cobination , 61. Thipertoire . After p-B cells  or in la-B cells  dependin-B cells  Discrepa-B cells  and Rold-B cells  reports  and cEµ . These largeting .IgH locus contraction and looping are important to permit equal usage of all HV genes, nuclear positioning of IgH loci seems more determinant than locus contraction in the orchestration of ordered V(D)J rearrangements J repertoire by decreasing distal HV usage (IGCR1 seems to work as an insulator to delimit Eμ action/function. These findings are contradicted by Busslinger’s group who carried out 4C-Seq experiments in IGCR1 deficient pro-B cells where interaction patterns remain unchanged compared to Rag-/- pro-B cells (HindIII (6pb cutter) and Sau3AI enzymes, Sen’s group used two 4pb cutter enzymes (MseI and NlaIII) that generate smaller fragments and probably offer higher resolution . To decipher the role of pDQ52 and the Eµ enhancer, Guo and colleagues generated two mouse models (-E- (both pDQ52/DQ52 and Eµ enhancers are deleted) and P-E+ (only promotor pDQ52 and DQ52 segments are deleted). Using 3D-FISH, authors demonstrated that the Eμ enhancer is required for IgH locus contraction in pro-B cells. Indeed, in P-E-, but not in the P-E+ model, the large loops between HV-D intergenic regions , Eµ and the 3’RR are drastically reduced underlining the important role of Eµ in loop formation. Moreover, ChIP-Seq experiments coupled with 3C experiments demonstrated that such large domain interactions are shaped by CTCF and cohesin proteins J recombination by regulating both long range interactions and IgH sub-nuclear positioning. While Eµ seems to facilitate IgH loops in developing B cells, the presence of this enhancer is not strictly necessary since another study showed, by 4C-Seq, that several long-range interactions remain detectable in the absence of Eµ  and IgH (trans-acting) monoallelic positioning within the nucleus and somehow promotes a diversified antibody repertoire  after initiating DNA double strand breaks and recombination. In addition, locus suicide recombination (LSR) is another rearrangement which leads to deletion of all IgH constant genes and therefore induces B cell death by abrogating surface B cell receptor expression , B cells undergo to two major genetic rearrangements initiated by AID : SHM andpression . In parapression .IgH alleles were not located in similar positions within nuclei of resting naive and activated B cells. At the resting mature B cell stage, both IgH alleles are localized in euchromatin whereas their respective nuclear position upon in vitro activation is still debated. According to Skok’s lab, the IgH allele colocalized with PCH and replicated later, suggesting that this allele is the unproductive allele  with or without interleukin (IL-4), which respectively induce CSR mostly towards IgG3 and IgG1; by acquiring additional contact between previously interacting enhancers and acceptor switch region (3Sγ or 1Sγ) involved in CSR  in plasma cells often undergo physical clustering by forming pairs or triplets. Such clusters, often composed of functional alleles, preferentially localize within the same transcription factory near the nuclear periphery  has been proposed in the context of the first-described IgH locus loop bringing the 3’RR and Eµ regions into close contact before CSR. Upon LPS-stimulation, this configuration acquires additional contact between the acceptor S region involved in CSR and previous interacting IgH enhancers  are mediated by Igk, especially by 3’Ek enhancer. Moreover, relocation and mis-cohabitation of Ig loci correlate with decreased transcription of each Ig gene, as observed in 3’Eκ-/- plasma cells in comparison with wt cells  does not impair loop formation at resting and activated B cell stages . Several AID off target genes  lie in close proximity to IgH during CSR and altogether are located in a chromosomal territory containing a high AID concentration (Ig loci could be widely considered as critical to maintain B cell genome integrity. In this way, their respective regulatory elements could play the role of B cell genome guardians thereby avoiding illegitimate events that contribute to lymphomagenesis.location ; its dislocation . Given tntration . As a coCB wrote the manuscript and prepared the figures. SN wrote the manuscript. EP provided critical feedback. All authors contributed to the article and approved the submitted version.CB was supported by a Ph.D. fellowship of the french Ministère de l’Enseignement Supérieur, de la Recherche et de l’Innovation. ANR-21-CE15-0001-01.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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Salmonella typhimurium and Escherichia coli is a common ancient multicellular behavior relevant in cell–cell and inter-organism interactions equally, as in interaction with biotic and abiotic surfaces. With the expression of the characteristic extracellular matrix components amyloid curli fimbriae and the exopolysaccharide cellulose, the central hub for the delicate regulation of rdar morphotype expression is the orphan transcriptional regulator CsgD. Gre factors are ubiquitously interacting with RNA polymerase to selectively overcome transcriptional pausing. In this work, we found that GreA/GreB are required for expression of the csgD operon and consequently the rdar morphotype. The ability of the Gre factors to suppress transcriptional pausing and the 147 bp 5′-UTR of csgD are required for the stimulatory effect of the Gre factors on csgD expression. These novel mechanism(s) of regulation for the csgD operon might be relevant under specific stress conditions.Rdar biofilm formation of  Escherichia coli. Several roles have been attributed to those proteins: (i) stimulating transcription initiation by likely facilitation of promoter escape, (ii) promoting release of stalling RNA polymerases during transcription elongation by inducing the intrinsic endoribonucleolytic activity of the RNA polymerases, and (iii) chaperone activity promoting refolding of denatured proteins [E. coli we have previously described that the Gre factors affect expression of biofilm promoting factors such as type 1 fimbriae and flagella [E. coli K-12-derivative strains allowed the conclusion that genes upregulated by GreA are mostly involved in energy metabolism and protein synthesis, whereas most of the genes downregulated by GreA belong to the cell metabolism and stress response [E. coli. It has been suggested that transcriptional pausing represents a widespread regulatory mechanism aimed at modulation of transcriptional output from specific operons in response to changing environmental conditions in a Gre factor dependent manner [The ubiquitous transcription elongation factors GreA and GreB, henceforth jointly named Gre factors, small cytoplasmic proteins of <20 kDa, have been extensively studied in proteins ,2,3. In flagella ,5. Moreoresponse . Of noteSalmonella enterica serovar Typhimurium (S. Typhimurium), while explored to a lesser extent, it has been shown that the Gre factors play a major role in S. Typhimurium pathogenic lifestyle. We showed that a strain lacking the Gre factors was avirulent both in vitro and in vivo. Specifically, the expression of the SPI-1 genes, mediating host cell invasion, was importantly down regulated in the absence of the Gre factors due to a direct effect on the transcription elongation of the hilD gene, coding for the master regulator of the SPI-1 genes. A transcriptional pause in the 3′-UTR of the hilD gene was the target for the Gre factors [S. Typhimurium has the ability to form biofilm on diverse biotic and abiotic surfaces, such as interacting with epithelial cells during the infection process [S. Typhimurium during food processing [S. Typhimurium biofilm have been performed upon growth on/in rich media, low osmolarity and low temperature (25–28 °C). Under these conditions, S. Typhimurium produces a rdar colony morphotype on agar plates and a pellicle air-liquid interface biofilm [csgBAC and csgDEFG. The biosynthesis of cellulose requires proteins encoded by the divergently transcribed operons bcsQRABZC and bcsEFG. Production of c-di-GMP by AdrA, a diguanylate cyclase encoded in the adrA gene, is necessary to eventually post-transcriptionally activate biosynthesis of cellulose. CsgD, an orphan transcriptional regulator encoded in the csgDEFG operon, regulates production of both curli and cellulose by stimulating transcription of the csgBAC and adrA genes [S. Typhimurium and E. coli [In  factors . S. Typh process  and adheocessing . Most st biofilm . Two pivrA genes ,12. CsgDrA genes ,14. A 14rA genes ,16,17,18 E. coli .E. coli and S. Typhimurium indicated that Gre factors might play a role in the regulation of the ability of S. Typhimurium to form biofilms. Here, the role of the Gre factors in regulating the process of biofilm formation in S. Typhimurium has been explored. We found that biofilm production was impaired in strains lacking the Gre factors. Consistently, the production of curli and cellulose was found to be strictly dependent of the Gre factors being abolished in strains lacking both factors. Expression studies showed that the Gre factors are required for the transcriptional expression of the master regulator CsgD. Gre-mediated regulation of csgD does not occur at transcription initiation but on the level of transcription elongation within the 5′-UTR of csgD. Our data add new players to the complex regulation of the biofilm formation process in S. Typhimurium and highlight the impact of 5′-UTRs of mRNAs as key regulatory hubs, not only at the posttranscriptional level but also at the level of Gre-mediated transcriptional elongation.The putative widespread role of Gre factors on transcription elongation and its previously described key role in fundamental regulatory pathways for both Salmonella enterica serovar Typhimurium (S. Typhimurium) wild-type strains used in this work are UMR1, a rdar28+ nalidixic acid resistant derivative of ATCC14028 [+ derivate of SL1344 [0  or CFA plates . When required, ampicillin (50 μg/mL), kanamycin (50 μg/mL), chloramphenicol (25 μg/mL), spectinomycin (25 μg/mL), arabinose (0.02%) and calcofluor  (0.2%) were added.The TCC14028 , and SV5f SL1344 ,22. All lacZ fusions were generated in two subsequent steps. First, homologous recombination by one-step gene replacement [csgD gene from different positions (+9 and +147). Subsequently, a promoter-less lacZ gene was integrated by site-specific recombination via a suicide plasmid [S. Typhimurium SV5015 was performed by the standard approach [lacZ gene, was electroporated into the resulting strains transformed with plasmid pCP20. The pKG136 plasmid was integrated into the chromosome by FLP-mediated recombination aided by plasmid pCP20. Recombinant clones were selected on LB agar containing kanamycin and X-Gal. Primers used in this work are listed in Chromosomal lacement  was appl plasmid  (see Filapproach . Construw/v) glucose, 0.25% (w/v) K2HPO4, 0.0125 g/L Evans blue  and 0.0250 g/L fluorescein ) [Mutant alleles were transduced into a novel strain background by phage P22 HT/int4 . TransduO, USA))  supplemecsgD was PCR amplified using primers csgD UTR Fw/csgD UTR Rv carrying restriction sites for NcoI and SalI, respectively. The amplified fragment was cloned in pGEM-T easy . The cloned fragment was sequenced and subcloned into the NcoI and SalI restriction sites of the pTT68 plasmid.The 5′UTR of S. Typhimurium suspension to an OD600nm of 0.02. Plates were incubated for 72 h at 28 °C in static conditions inside a plastic bag with wet cellulose paper to maintain the humidity levels. Liquid culture was removed and the OD600nm determined. Quantitative measurement of the biofilm biomass was performed by crystal violet (CV) staining as described [w/v) CV solution , was added to the wells and incubated for 15 min, rinsed with water and air-dried. 0.2 mL of acetic acid 30% (v/v) was added to the wells. The OD570nm of the resulting solution was determined. The signal detected in control wells containing only culture media was subtracted from the values obtained for all the samples. The data are mean values with standard deviations of six independent cultures.For biofilm on glass, qualitative assays were performed as described . For qua0 plates at 28 °C for 24 or 48 h. After incubation, bacterial suspensions in LB were obtained, its OD600nm determined and kept on ice until β-galactosidase assay was performed, as described by Miller [Cultures were grown on LBy Miller . Enzymat0 plates supplemented with calcofluor. Plates were incubated at 28 °C for 48 h and fluorescence was observed under a UV light source. Qualitative assessment of bacterial cellulose production was determined on LB0 agar plates supplemented with Congo red  (40 mg/L) and Coomassie brilliant blue G-250  (20 mg/L)). Five microliters of a bacterial suspension in water (OD600nm of 5.0) from a fresh LB-agar plate were spotted onto the plate and incubated at 28 °C.The rdar morphotype was judged visually on Congo red agar plates  strain and its mutants ∆greA, ∆greB and ∆greA∆greB (from now referred as ∆greAB) to form biofilm on glass and plastic. Both the qualitative biofilm assay on glass and quantitative determination of biofilm formation on plastic indicated that biofilm production was impaired in the absence of both Gre factors  morphotype, indicating that they do not produce curli and cellulose. The requirement of the Gre factors for the production of the biofilm-promoting factors was further characterized. The transcriptional expression of csgB, coding the major subunit of curli, was monitored using a chromosomal csgB::lacZ fusion  comprising the 41 and 44 residues , whereas no change in expression of the csgD fusion lacking the 5′-UTR (csgD+9::lacZ). Remarkably, no induction of the csgD+147::lacZ construct was detected when GreAD41AE44Y was expressed, indicating that the reported effect of the Gre factors on csgD expression can be due to the resolution of a transcriptional pause occurring in the 5′-UTR of the csgD gene. Although, in vitro transcription experiments will be required to confirm the direct involvement of the Gre factors in csgD transcription, our data suggest that GreA and GreB alleviate transcriptional arrest in the 5′-UTR of csgD mediated by the residues participating in transcript cleavage by the RNA polymerase. Whether additional GreA and GreB checkpoints exist upon transcription of the csgD open reading frame and/or of the csgDEFG operon needs to be investigated in future studies. Gre factors promote transcription elongation by resolving backtracking of paused transcription complexes . In E. cl pauses . E. coliresidues . Previoudentity 9% comprisnstructs B. As expS. Typhimurium, the activity of the CsgD regulator is pivotal in the stimulation of rdar biofilm formation including modulation of the synthesis of two biofilm promoting factors, curli and cellulose. The synthesis of those biofilm matrix components requires a relevant number of the assets available for the cells. Different environmental stresses including nutrient starvation trigger biofilm formation in S. Typhimurium. Rdar and alternative modes of biofilm formation are delicately regulated. The switch between planktonic and sessile (biofilm) cells denotes not only a radical change in the single cell versus population behavior, but also in the physiology and metabolism of the organism. In csgD expression. A potential role for the Gre factors in bacterial adaptation to different environmental stresses has been proposed [greA and greB occurs under different environmental conditions in E. coli [greA gene is σE-dependent in E. coli and S. Typhimurium, is induced under hypoxia in Mycobacterium avium and under acid stress in Streptococcus mutans [Deinococcus radiodurans [In this report, we describe the Gre factors as new players in the array of regulators affecting proposed ,7. Hence E. coli . The exps mutans ,36,37,38s mutans ,40,41. Aiodurans . S. Typhimurium that the Gre factors are required for the expression of hilD encoding the main regulator of the SPI-1, a genomic island encoding the type three secretion system 1 needed for invasion of epithelial cells. The the 3′-UTR of the hilD gene was identified as the target of the Gre factors [csgD transcript through nucleolytic cleavage. Such mRNA processing might even influence the effect of additional known post-transcriptional regulators that target the 5′-UTR of csgD and can constitute an additional regulatory mechanism in the multilayered regulation of the csgD gene. Transcriptional pausing causes gene repression that can be alleviated by the Gre factors as a response to specific environmental stimuli. In a previous report, we described in  factors . Here, wcsgD operon to negatively or positively affect transcription [csgD 5′-UTR and the impact of the Gre-dependent pausing on the regulatory pathways that convey on the 5′-UTR of csgD.Long 5′UTR are regulatory motifs often involved in post-transcriptional regulation by very diverse mechanisms and targets for regulation at the level of transcription elongation . The RNAcription ,16,18. Icription , therefo


Multisubunit RNA polymerases (RNAPs) carry out transcription in all domains of life; during virus infection, RNAPs are targeted by transcription factors encoded by either the cell or the virus, resulting in the global repression of transcription with distinct outcomes for different host–virus combinations. These repressors serve as versatile molecular probes to study RNAP mechanisms, as well as aid the exploration of druggable sites for the development of new antibiotics. Here, we review the mechanisms and structural basis of RNAP inhibition by the viral repressor RIP and the crenarchaeal negative regulator TFS4, which follow distinct strategies. RIP operates by occluding the DNA-binding channel and mimicking the initiation factor TFB/TFIIB. RIP binds tightly to the clamp and locks it into one fixed position, thereby preventing conformational oscillations that are critical for RNAP function as it progresses through the transcription cycle. TFS4 engages with RNAP in a similar manner to transcript cleavage factors such as TFS/TFIIS through the NTP-entry channel; TFS4 interferes with the trigger loop and bridge helix within the active site by occlusion and allosteric mechanisms, respectively. The conformational changes in RNAP described above are universally conserved and are also seen in inactive dimers of eukaryotic RNAPI and several inhibited RNAP complexes of both bacterial and eukaryotic RNA polymerases, including inactive states that precede transcription termination. A comparison of target sites and inhibitory mechanisms reveals that proteinaceous repressors and RNAP-specific antibiotics use surprisingly common ways to inhibit RNAP function. Archaea originally rose to fame due the unorthodox environmental niches they dominated, including extremes of temperature, pressure, salinity and pH. Over the last decade, however, it has transpired that archaea are highly abundant organisms in the biosphere, and many species thrive under moderate conditions on a par with mesophilic bacteria. As an integral part of the biosphere in the Earth’s crust and oceans, archaea play an important role in the elemental cycles including the recycling of sulphur and nitrogen . While aThe archaeal genomes are organised in polycistronic operons, such as in bacteria, and compacted using a range of nucleoid proteins that are either specific for archaea like Cren7 and Alba) or eukaryotes (histones) ,7. The l and AlbaFollowing the escape of RNAP from the promoter, the elongation factors Spt4/5 and Elf1 are recruited to the transcription elongation complex (TEC) and improve the processivity of elongation by sealing the DNA-binding channel and forming a tunnel through which the downstream DNA is directed to the catalytic centre of RNAP . TerminaViruses and their hosts are engaged in an endless battle, whose outcome is called by the arms race between host and viral measures and countermeasures . Yet, incoliphage T7 encodes Gp2, a small protein that binds the RNAP-σ70 holoenzyme inside the DNA-binding channel, which inhibits the open complex formation ..46].The clamp is a flexible domain, which opens and closes over the channel upon DNA binding and in response to transcription factors as RNAP progresses through the transcription cycle ,50. SingRIP expression is induced during the late stages of infection prior to cell lysis ,46. The The transcript cleavage factors (TFS) are a large family of transcription regulators that interact with RNAP through its NTP-entry channel. This channel consists of a pore at the bottom of a funnel that enables the access of nucleotides to the RNAP active site, and the egress of the RNA 3′-end in the backtracked TEC ,52. TFS N) binding between the lobe and the upper jaw, and, through the linker, positioning the C-terminal zinc ribbon (ZRC) inside the NTP-entry channel  bindiC of TFS4 physically clashes with and displaces the trigger loop. As the bridge helix and trigger loop are essential for the catalytic addition and translocation of NTPs during RNA synthesis, TFS4 effectively inhibits the enzymatic activity [However, as the resulting binding surface is not solvent-exposed, TFS4 displaces the jaw, which causes a large conformational change with concomitant widening of the DNA-binding channel and melting of the bridge helix c. Finallactivity . Moreoveactivity .Saccharolobus solfataricus TFS4 paralogue is not expressed during standard cell growth conditions, either during the exponential or stationary phase, but 12 h after infection with the Sulfolobus turreted icosahedral virus (STIV), the protein becomes detectable and shortly after the cell growth stagnates. The expression of a TFS4 variant from a plasmid induces growth arrest of Sulfolobus acidocaldarius, making it tempting to speculate that TFS4 is the causative agent of the growth phenotype caused by STIV infection. A dormant state or quiescence is often associated with a persister state in bacteria [RNAPs are large and dynamic molecular machines; the engagement with transcription factors, nucleic acids and the enzymatic reaction itself all involve conformational changes in RNAP, most of which are intrinsic to its structure and, therefore, fundamentally evolutionary conserved ,50. The bacteria , allowinThe wide-spread emergence of multidrug resistant bacterial pathogens calls for urgent efforts to discover and develop novel classes of antibiotics . RNAPs aAntibiotics need to inhibit pathogen RNAPs with sufficient affinity and high selectivity to circumvent off-target effects, such as inhibiting the host RNAP and/or other macromolecules. In addition, when considering specific RNAP motifs to be drugged, it is important to consider the likely emergence of resistance . One of The structurally mobile motifs of RNAP include the active site bridge helix and trigger loop, and the RNAP clamp that moves on the so-called switches. While the continuous conformational changes in the bridge helix and trigger loop enable the catalytic activity and nucleotide translocation ,82, the These compelling similarities demonstrate, that while many roads lead to Rome, travelers often share the same path.RNA polymerase inhibition plays an important role for the global regulation of gene expression in response to environmental changes, such as oxidative stress and nutrient deficiency, and in response to virus infection. Surprisingly, it appears that the global repression of transcription can be of both selective advantage for the host and the virus, but for entirely different reasons. These include improved survival for the host, and aiding virus particle assembly and release for the virus. As repressors often use a range of measures to inhibit RNAPs that are reminiscent of antibiotics, characterising the underlying inhibitory mechanisms is not only of academic interest but has the potential to benefit the development of more potent and specific antibiotics that are less prone to resistance.RNAPs and the molecular mechanisms of RNA synthesis are universally conserved in all domains of life.Both the virus and host cell can encode repressors that tightly bind to RNAPs and efficiently inhibit their functions, leading to transcriptome repression or attenuation.modus operandi of these factors can unravel the underlying molecular mechanisms of RNAP activity and identify critical pressure points of enzyme functionThe ○steric occlusion of DNA, NTPs and transcription factor binding sites;○allosteric regulation by inducing conformational changes that perturb the active site (bridge helix and trigger loop) and widening of the DNA-binding channel.Inhibitory mechanisms include:Allosteric mechanisms of inhibition can reveal movements inherent to RNAP function. As these are evolutionary conserved, it is possible to draw intriguing parallels between the regulation of different transcription systems.Antibiotics that target and inhibit RNAPs and proteinaceous repressors act via functionally closely related molecular mechanisms.A thorough understanding of RNAP inhibition in all domains of life, including archaea, could be beneficial for the development of novel antibiotics.


In fact, many human cancers and diseases are associated with the malfunction of enhancers. Recent studies have shown that enhancers can produce enhancer RNAs (eRNAs) by RNA polymerase II. In this review, we discuss eRNA production, characteristics, functions and mechanics. eRNAs can determine chromatin accessibility, histone modification and gene expression by constructing a ‘chromatin loop’, thereby bringing enhancers to their target gene. eRNA can also be involved in the phase separation with enhancers and other proteins. eRNAs are abundant, and importantly, tissue‐specific in tumours, various diseases and stem cells; thus, eRNAs can be a potential target for disease diagnosis and treatment. As eRNA is produced from the active transcription of enhancers and is involved in the regulation of cell fate, its manipulation will influence cell function, and therefore, it can be a new target for biological therapy.Enhancers are important  We introduce the production and characteristics of enhancer RNA (eRNA) as well as its functions to regulate gene expression and potential as a marker for the diagnosis and target for the treatment of cancer and other diseases. These cis‐acting elements can bind to trans‐acting factors that regulate transcription. Previous studies on cis‐acting elements have focussed on promoters; these elements initiate transcription in a position‐ and direction‐dependent manner. Meanwhile, enhancers are able to ignore these conditions as they can form a loop over long genomic ranges to approach distant promoters, thereby achieving specific gene expression.cis‐elements. Although TFs can bind to both promoters and enhancers to form multi‐protein complexes that regulate transcription in a spatial and temporal fashion,Gene transcription is a very accurate regulatory process influenced by many factors, among which enhancers and promoters are important 2eRNA is mainly actively transcribed by enhancers that have the following features: (1) low levels of DNA methylation (low‐methylated regions)eRNA transcription generally occurs before the transcription of the code gene.3The RNAs produced by enhancers and that produced by promoters are different, although they have many similarities. The mRNAs produced by promoters are long, spliced, polyadenylated and stable transcripts that can encode proteins in the cytoplasm. Long non‐coding RNAs (lncRNAs) are long and stable, usually spliced and polyadenylated, transcribed by their own promoters.4Enhancers were thought to regulate gene expression in an orientation‐independent manner, completely different from that of promoters. Interestingly, Mikhaylichenko et al. revealed that bidirectionally transcribed enhancers act as promoters in both directions.Moreover, promoters can interact with enhancers and with other promoters to increase gene expression.Research has shown that promoters and enhancers may have similar characteristics, such as sequence motifs, transcription machinery, chromatin environment, changes in activity upon binding of activators or repressors and H3K27ac occupancy.5eRNA transcription has a strong correlation with enhancer activity, but the function of eRNA is controversial. Some researchers believe that eRNA is just a ‘noise’ product that is created when RNAP II complexes are recruited to the open chromatin environment of enhancers. Others have suggested that the complicated process of eRNA transcription is important for enhancer activation rather than the formation of eRNA itself. However, most researchers believe that the presence of eRNA itself acts as a transcription activator of the enhancer to promote the expression of the target gene.eRNA is produced by active enhancers; thus, they are responsible for tissue and lineage specificity and can be used to evaluate the cell type and state‐specific gene expression.SERPINB2, a strongly expressed gene in inflammatory states, can be upregulated by upstream eRNA, which is transcribed prior to SERPINB2 mRNA. Knockdown of eRNAs inhibited the transcription of SERPINB2 mRNAs, while eRNA overexpression was associated with upregulated SERPINB2 mRNA. This provides direct evidence that control of eRNA expression can influence mRNA levels.In macrophages, Rev‐Erbs nuclear receptors regulate the expression of Mmp9 and Cx3cr1 by reducing the transcription of distal enhancers selected by macrophage lineage determinants.The transcription of eRNA is closely associated with the regulation of nearby target genes, and knockdown of eRNA decreases the expression of relative target genes.6cis, ie intrachromosomally.trans, ie interchromosomally.trans.trans in human prostate cancer.As the majority of eRNAs are non‐polyadenylated, short‐lived and unstable, they act in 6.1Enhancers can span the space to regulate the expression of one or more genes, forming the ‘chromatin loop’ structure to take the enhancer to the vicinity of the target‐gene promoters, thereby fully approaching and interacting with the target gene in space by activating or inhibiting the target gene. TFs, transcriptional coactivators, steroid hormone receptor, CTCF, cohesion and other protein complexes play an important role in the construction of the ‘chromatin loop’.TFs can bind to the specific recognition sequences of enhancers, even when the enhancer is located far from where the transcription initiation complex is bound.Cohesin has been proved to occupy sites bound by CTCF and participate in the formation of the ‘chromatin loop’ by connecting two DNA segments.Enhancers involved in the ‘chromatin loop’ with core promoters have high expression of eRNA, suggesting that eRNAs may act in the formation of the ‘chromatin loop’.6.2An active enhancer has an accessible chromatin architecture accompanied by enhanced DNase I hypersensitivity.6.3In accordance with the central role of RNAP II in the ‘chromatin loop’‐mediated transcription,In addition, eRNAs promote the transition from paused RNAP II into active elongation by acting as decoys for the negative elongation factor (NELF) complex upon induction of immediate early genes (IEGs) in neurons. Knockdown of eRNAs impairs the transient release of NELF from the target promoters during transcription, resulting in a reduction in the elongating form of RNAP II, thus leading to a decrease in target mRNA expression.7Using the Cancer Genome Atlas (TCGA) RNA‐seq database, we found higher concentrations of eRNAs in a broad range of cancers relative to those in normal tissues.7.1Androgen receptors (ARs) can influence the development of prostate cancer by binding at different gene loci to initiate transcripts, leading to important cellular activities.cis activity) or interchromosomally (trans activity) , a gene encoding prostate‐specific antigen (PSA), can be regulated by AR, and its enhancer is known as androgen response element III (ARE III) marked by AR, H3K27ac and H3K4me1.) Figure . Moreove7.2MYC is an important proto‐oncogene that influences tumour development in many tumour types.7.3P53 is a key tumour‐suppressor gene that can bind to the enhancer region of target genes to determine cell fate.Based on the cancer‐specific expression of eRNAs, we suggest that eRNAs are valid diagnostic or prognostic biomarkers in cancer therapy. Moreover, some eRNAs can be utilized to determine cancer‐related clinical features, such as subtype, targets for cancer therapy, stages and grades.8Peblr20 binds to the Oct4 enhancers and recruits the TET2 demethylase to promote enhancer transcription, thereby controlling the fate of stem cells.Embryonic stem cells (ESCs) have the properties of self‐renewal and pluripotency, and their transcription can be affected by specific TFs such as Oct4, Sox2, Nanog and Sall4.9We have mentioned that SEs bind with many TFs to control gene transcription.10Although the existence of eRNAs is not debatable, there are still many questions about their mechanism and specific applications. The secondary structure of eRNA is unknown, and it is also unknown how the structure influences cell function. Proteomic methods such as ChIRP, RAP and iDRiP can be used to recognize tumour‐specific eRNA‐protein interactions, and this can be further used to explore what role they play in the progression of tumours.11Enhancers and promoters have many similar features. eRNA is produced from enhancers and is involved in the selective expression of specific genes. Most eRNAs are short, 5‐capped, non‐polyadenylated, and only a small portion of eRNAs are long and polyadenylated. As most eRNAs are not stable, global run‐on sequencing (GRO‐seq), precision nuclear run‐on sequencing (PRO‐seq), chromatin immunoprecipitation (ChIP)‐seq or cap analysis of gene expression (CAGE) sequencing is used to recognize eRNAs instead of the common RNA‐seq.An increasing number of studies have shown that eRNAs are involved in tumour oncogenic signalling pathways and manipulation of tumour genesis. Most eRNAs have specific expression patterns with associated TFs, and they are related to cancer types. KLK3e, NETIe and CCAT1 are eRNAs that can be found in specific tumours, and their suppression will influence tumour progression. eRNA can be used for cancer prevention, early diagnosis and therapy. Many studies have shown that most disease‐associated loci represent sequence variations in enhancers.Previous studies have shown that many disease‐associated genetic variants occur in putative enhancer elements.The authors declare no conflict of interest.All authors contributed to the review conception and design. Zhenzhen Han wrote the manuscript; Wei Li revised and approved the manuscript.


E75B and Hr4, ecdysone-responsive genes, enhancer activity, and transcription required the histone acetyltransferase activity of the CBP. EcR binding was critical in inducing enhancer activity and H3K27 acetylation. The CREB-binding protein (CBP) HAT domain catalyzed H3K27 acetylation and CBP coactivation with EcR, independent of the presence of ecdysone. Increased H3K27 acetylation promoted chromatin accessibility, with the EcR and CBP mediating a local chromatin opening in response to ecdysone. Hence, epigenetic mechanisms, including the modification of acetylation and chromatin accessibility, controlled ecdysone-dependent gene transcription.The steroid hormone ecdysone regulates insect development via its nuclear receptor (the EcR protein), which functions as a ligand-dependent transcription factor. The EcR regulates target gene expression by binding to ecdysone response elements (EcREs) in their promoter or enhancer regions. Its role in epigenetic regulation and, particularly, in histone acetylation remains to be clarified. Here, we analyzed the dynamics of histone acetylation and demonstrated that the acetylation of histone H3 on lysine 27 (H3K27) at enhancers was required for the transcriptional activation of ecdysone-responsive genes. Western blotting and ChIP-qPCR revealed that ecdysone altered the acetylation of H3K27. For  In insects, the steroid hormone 20-hydroxyecdysone (20E) functions as a generalized systemic signaling molecule that coordinates the critical developmental events of embryogenesis, larval molting, metamorphosis, and reproduction ,2. The sDrosophila melanogaster , the ecdysone-induced expression of Eip74EF and E75B was associated with the acetylation of histone H3 on lysine 23 [Hormone-triggered transcriptional responses are highly cell-specific, and the chromatin environment surrounding regulatory regions is an important determinant of target-gene transcriptional responses ,11,12. Tysine 23 . AlthougSox14, an ecdysone-induced gene crucial for regulating dendrite pruning during Drosophila metamorphosis [sox14 transcription. Current research on this topic is based mainly on D. melanogaster. These studies have mainly focused on gene promoters; few have addressed regulatory regions such as enhancers, or the whole genome [Histone acetylation is catalyzed by histone acetyltransferase (HAT), and deacetylation is influenced by histone deacetylase (HDAC) . The dynorphosis . The CBPe genome ,30,31. TE75B and Hr4, after treatment with 20E. This treatment rapidly and significantly increased H3K27 acetylation in the regulatory elements of E75B and Hr4 loci. This increase was necessary to activate the regulatory elements. Our results showed that the EcR plays a central role in H3K27 acetylation and chromatin accessibility by recruiting the histone acetyltransferase CBP at enhancers. Thus, we demonstrated that epigenetic regulation plays critical roles in controlling the expression of ecdysone-responsive genes.Using ChIP-seq experiments in silkworm BmE cells, it has previously been shown that ecdysone triggers genomic changes in histone modification, and that histone acetylation is important in regulating ecdysone-responsive genes . To bettWe classified ecdysone-responsive genes in BmE cells as up- or downregulated using published RNA-seq and ChIP-seq data, and analyzed changes in H3K27 acetylation in these differentially expressed genes. The results showed that the 20E treatment significantly increased H3K27 acetylation in upregulated genes, and reduced it in downregulated genes A. This rE75B, an early response gene, and Hr4, an early–late gene, play key roles in ecdysone-triggered genetic cascades [E75B and Hr4 loci  to examine H3K27 acetylation levels in BmE cells. Consistent with the ChIP-seq results, H3K27 acetylation levels were significantly higher in regions a, b, c, and d of  of E75B D,F.E75B and Hr4 loci and tested their activity using a dual luciferase reporter assay; these regions significantly increased reporter gene expression, indicating their function as enhancers  exhibited at least a 1.5-fold increase in H3K27ac in response to 20E. Some enhancers exhibited a high level of H3K27 acetylation before and after the 20E stimulation. We also identified enhancers  that underwent a decrease in H3K27 acetylation in response to 20E. We then classified these responses as “increasing”, “constant”, or “decreasing” -induced EcR knockdown in ecdysone-treated BmE cells C. LikewiHr4 loci D. Hence,D. melanogaster, and to the CBP orthologs in other insect species [E75B and Hr4 loci after the CBP knockdown via ChIP-qPCR; these levels were substantially lower after the CBP knockdown  treatment with C646, which was sufficient to suppress H3K27 acetylation D, fully pression E. This snditions D. These nditions D,E. ThesCBP mRNA expression remained unaffected after the EcR knockdown  signal changed dynamically across the genome following the 20E treatment. For example, at the and tylation . We thentylation A. This sE75B and Hr4 loci increased significantly after the 20E treatment  enhancers containing H3K4me1 alone . In the Based on the H3K27 acetylation signal, we categorized the previously identified enhancers . The de Furthermore, enhancer activity repression after the 20E treatment appeared to be independent of the EcR binding, but seemed to involve transcription factors such as Eip74 and Ftz-F1. Given that the Eip74-motif-mutant enhancer also showed weaker activity in the absence of ecdysone , Eip74 mC/EBPg, which binds to the CBP promoter, activates CBP expression, and leads to histone H3K27 acetylation [CBP is a coactivator that forms a protein complex with the EcR in an ecdysone-independent manner. Studies in humans, mice, and other mammals have shown that the CBP and its family member p300 function as key transcriptional coactivators, interacting with hundreds of different factors, including hormonal nuclear receptors, to activate gene expression [The CBP is the primary histone acetyltransferase in insects ,44,45. Ttylation . Our respression . The CBPpression . At its pression ,48. In vpression ,50,51,52The organization of accessible chromatin across the genome reflects a network of possible physical interactions, in which enhancers, promoters, insulators, and chromatin-binding factors cooperatively regulate gene expression. Here, the local chromatin of enhancers opened after the 20E treatment, and chromatin accessibility was consistent with increased H3K27 acetylation. Prior to hormone treatment, chromatin remains closed, preventing access to transcription factors, and thereby preventing abnormal gene activation. Histone acetylation has been proposed to facilitate transcription by directly modulating histone–DNA contacts, or by targeting chromatin remodelers to disrupt nucleosomes . More imIn conclusion, these findings showed that the EcR plays a central role in H3K27 acetylation. Like other nuclear hormone receptors, ligand binding to the EcR induces structural changes that result in differential association with coregulator complexes. In the presence of 20E, the EcR binding at the target enhancer elements of ecdysone-responsive genes would increase H3K27 acetylation by recruiting the coactivator CBP. The CBP, via its intrinsic HAT activity, directly catalyzes the local H3K27 acetylation of enhancers. Histone acetylation neutralizes the positive charge of the lysine residue and results in a loose structure of chromatin accessible for transcriptional machinery, thereby activating gene expression .The BmE silkworm embryo cell line was maintained in Grace’s insect cell culture medium  supplemented with 10% fetal bovine serum  at 27 °C. Cells were incubated with 2.5 μM ecdysone  or 2 μM trichostatin A  in culture medium for 12 h, unless otherwise stated. BmE cells were treated with 30 μM C646  or 25 μM SGC-CBP30  for 30 min before harvesting. As a vehicle control, the cells were treated with DMSO in parallel.RP49 transcription as a normalization reference. The primers used for RT-qPCR analyses are listed in Total RNA from BmE cells was extracted using an RNeasy Mini Kit . cDNA was synthesized from 500 ng of total RNA using the GoScript Reverse Transcription System , as per the manufacturer’s instructions. RT-qPCR was performed using TB Green Premix Ex Taq II  on a 7500 Fast Real-Time PCR System . Amplifications were performed in triplicate. Triplicate mean values were calculated using delta–delta Ct quantification, with BmE cells in the logarithmic growth phase were inoculated in 12- or 24-well culture plates and cultured for 18 h. Cell transfection was conducted when the cells reached approximately 80% density. Regions of ca. 1 kb around the center of regulatory elements were PCR-amplified from silkworm genomic DNA and cloned into the pGL3-derived reporter vector. The primer sequences are provided in For BmEcR or BmCBP RNAi, double-stranded RNAs (dsRNAs) were synthesized using the T7 RiboMAX Express RNAi System , following the manufacturer’s instructions, with EGFP dsRNA as the control. dsRNA was transfected or cotransfected with pGL3-derived reporter plasmids to determine gene expression or regulatory element activity. Specific PCR primers for dsRNA synthesis were designed for this study .For immunoblotting, protein extracts were prepared by lysing BmE cells in RIPA buffer  and boiling in SDS sample buffer for 5 min. Protein samples were resolved on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, transferred onto activated polyvinylidene difluoride membranes, and blocked for 1 h in 5% skim milk. Membranes were incubated with primary antibodies against the EcR , created by the NICHD of the NIH, maintained at The University of Iowa, Department of Biology, Iowa City, IA, USA), CBP , FLAG , HA , H3K27ac , and α-tubulin  overnight at 4 °C, then further incubated with HRP conjugated secondary antibody for 1 h. Signals were detected using the SuperSignal West Pico Chemiluminescent Substrate .v/v) glycerol, and 0.5% NP40) with protease inhibitors  in the presence or absence of 20E. The supernatants were used for immunoprecipitation with either anti-HA  or anti-IgG  antibodies overnight at 4 °C, followed by incubation with protein A/G Dynabeads  for 2 h. The protein A/G Dynabeads were washed four times with cold PBS. Bound proteins were separated via SDS-PAGE and analyzed via Western blotting using anti-FLAG, anti-HA, and anti-CBP antibodies.After the cotransfection of the pSL1180-A4 vectors overexpressing HA-tagged N-terminal CBP (aa1–494) and FLAG-tagged EcR in BmE cells, cells were treated with 2.5 μM 20E or DMSO for 12 h. BmE cells were homogenized with lysis buffer  assays were performed as previously described. Briefly, treated BmE cells were fixed with formaldehyde and subjected to overnight immunoprecipitation using the corresponding antibodies. The following antibodies were used: anti-H3K27 acetylation , anti-H3K27me3 , anti-EcR , and anti-CBP . Purified DNA fragments from immunoprecipitated products were used for qPCR validation. The primers used to amplify the specific region covering the potential regulatory elements for 7 BmE cells) were lysed, and the chromatin was sonicated to a size range of 300–800 bp using a Covaris S220 focused ultrasonicator . FAIRE DNA was recovered from the cell lysate via phenol–chloroform extraction, in which open chromatin DNA was recovered from the aqueous phase. The recovered chromatin was treated with RNAse A and proteinase K, then purified using a Zymo spin column . Further, FAIRE DNA was quantified via qPCR, to generate a FAIRE–seq library. The primers used for FAIRE-qPCR were the same as those used for ChIP-qPCR. The FAIRE-seq library was built using a NEBNext Ultra II DNA Library Prep Kit  and sequenced on an Illumina HiSeq 2500  to generate 150 bp paired-end reads.To understand the role of chromatin accessibility in the transcription of ecdysone-responsive genes, we performed a FAIRE-seq in BmE cells after the 20E treatment. FAIRE was performed as previously described, with minor modifications . Brieflyhttps://kaikobase.dna.affrc.go.jp/ (accessed on 20 August 2021). Only uniquely aligned reads were used. Highly enriched peaks were obtained using MACS2 using standard settings [We used bowtie2 (version 2.3.4.1) to map the clean paired-end reads to the silkworm reference genome , which wsettings , allowinWe identified enhancers with H3K4me1 modifications located >1.5 kb beyond the TSS. The number of input-normalized H3K27 acetylation ChIP-seq reads within a 1.5 kb window centered on each enhancer was taken to be the ChIP-seq signal for the enhancer. The enhancers were categorized based on their H3K27 acetylation signals: “increasing” reflected an increase of ≥1.5-fold in H3K27 the acetylation signal upon the 20E stimulation, with the stimulated H3K27 acetylation signal being above the bottom quartile of the stimulated signals; “decreasing” reflected a reduction of ≥33% in the H3K27 acetylation signal upon the 20E stimulation, with the unstimulated signal being above the bottom quartile of unstimulated signals; “constant” referred to the H3K27 acetylation signals in the top quartile of signals in both the unstimulated and stimulated conditions, and with a signal change of ≤10% upon stimulation; and “no response” referred to the enhancers whose H3K27 acetylation peaks did not overlap with those within the H3K27 acetylation datasets. The FAIRE-seq read density for the increasing, decreasing, and constant categories was calculated as described earlier.To identify the transcription factors involved in the 20E-induced alteration of H3K27 acetylation in enhancers, we performed a de novo motif analysis using these three categories. De novo motifs were identified using HOMER motif tools (findMotifs.pl) . Motifs


Drosophila insulators were the first DNA elements found to regulate gene expression by delimiting chromatin contacts. We still do not know how many of them exist and what impact they have on the Drosophila genome folding. Contrary to vertebrates, there is no evidence that fly insulators block cohesin-mediated chromatin loop extrusion. Therefore, their mechanism of action remains uncertain. To bridge these gaps, we mapped chromatin contacts in Drosophila cells lacking the key insulator proteins CTCF and Cp190. With this approach, we found hundreds of insulator elements. Their study indicates that Drosophila insulators play a minor role in the overall genome folding but affect chromatin contacts locally at many loci. Our observations argue that Cp190 promotes cobinding of other insulator proteins and that the model, where Drosophila insulators block chromatin contacts by forming loops, needs revision. Our insulator catalog provides an important resource to study mechanisms of genome folding. Topological screen identifies hundreds of Drosophila chromatin insulator elements and questions looping model of insulator action. Eukaryotic chromosomes are extensively folded to fit inside micrometer-size cell nuclei. The degree of folding varies between different regions of the chromosome, and the specific folding patterns vary from cell to cell. Nevertheless, high-resolution imaging , Su(Hw), Ibf1, Ibf2, Pita, ZIPIC, BEAF-32, and M1BP] and two candidate bridging proteins [Cp190 or Mod(mdg4)] were implicated in Drosophila was the first multicellular organism where genomic distributions of multiple insulator proteins became available . The y+1CTCF allele is a 3.3-kb deletion that removes the entire open reading frame of the CTCF gene and produces no protein  genotyping (fig. S1B) and sequencing (fig. S1D) so we focused our analyses on Cp190-deficient line CP-R6 and CTCF-deficient line CTCF 19.7-1c, hereafter referred to as Cp190 knockout (Cp190-KO) and CTCF knockout (CTCF-KO) cells. Western blot analyses detected no Cp190 in the Cp190-KO or CTCF in the CTCF-KO cell lines  . These enhancers and PREs are clustered in genetically defined domains  domains control the expression of abd-A  and Abd-B   are required to ensure that the enhancers and PREs activate or repress the correct genes in correct body segments . The latter directly interacts with Cp190 and can tether Cp190 to Fub even when CTCF is absent , and they are no longer visible in the contact maps from the Cp190-KO and CTCF-KO cells.In addition, two distinct density clouds of chromatin contacts within Fub insulator is capable to limit chromatin contacts when the entire bithorax complex is transcriptionally inactive and repressed by Polycomb mechanisms. Last, our observations suggest that a systematic screen for Cp190- and CTCF-binding sites that limit chromatin contacts in the control cells but lose this ability in Cp190-KO and/or CTCF-KO cells may serve as a genome-wide approach to discover robust insulator elements.Three conclusions follow from the observations above. First, our experimental system is sufficiently sensitive and accurate to detect topological changes around robust enhancer-blocking insulators. However, it may miss those associated with weaker elements. Second, the Drosophila insulator elements from genome-wide mapping data alone , Cp190, and Mod(mdg4) but not CTCF .Insulator proteins bind the genome in distinct combinations Mod(mdg4), Cp190, Ibf1, CTCF, and Su(Hw) were designated as MCIFS.Encouraged by this observation, we mapped the genomic binding of Cp190, CTCF, and several other key insulator proteins: Su(Hw), Mod(mdg4), and Ibf1 in the control and CTCF-KO and Cp190-KO cells using ChIP coupled to sequencing of the precipitated DNA (ChIP-seq). We performed two ChIP-seq experiments for each genetic background using independently prepared chromatins and had the DNA from the corresponding chromatin input materials sequenced to control for possible sample processing biases. We then used Model-based Analysis of ChIP-seq (MACS) algorithm . As expected, the median Δγ for the control (random) regions is close to zero for both Cp190-KO and CTCF-KO  thresholds. Second, using these thresholds, we selected all regions with Δγ ≤ FDR from classes that have a significantly higher fraction of regions with increased chromatin contact crossing in the corresponding mutant cells , are hard to cross (have high γ) in the control cells . However element .Further inspection of the C class indicates that most of these regions reside close to transcription start sites (TSSs) with a median distance of just 226 base pairs (bp) (fig. S7B). Sequencing of the total RNA indicates that most of the corresponding TSS belong to transcriptionally active genes (fig. S7C). Conceivably, proteins associated with transcription or spatial interactions between transcriptionally active genes restrict chromatin contact crossing regardless of Cp190 binding. The Hi-C bins encompassing “highly transcriptionally active” TSS  have γ comparable to that of the C class . Many inTwo conclusions follow from the observations above. First, transcriptionally active genes hinder chromatin contacts regardless of their association with Cp190 or any other insulator protein tested here. This may be due to binding of other, possibly undiscovered, insulator proteins or spatial segregation of transcriptionally active genes. Second, a previously recognized link between Cp190 and TAD boundaries (yellow (y), the gene most “up-regulated” in CTCF-KO cells (fig. S8B), comes from the transgene inserted in the y+1CTCF allele  at insulator protein binding sites and the transcription from the closest TSSs . The box plots of contact numbers (How  numbers  indicate1w67c23y) embryos recently published by Batut and coauthors   locus. The transgenes containing these elements can participate in trans-regulatory interactions over distances greater than 1 Mb. Separated by ~17 kb in their endogenous location, the two form a chromatin loop detected by Micro-C  algorithm , 60 mM KCl, 15 mM NaCl, 4 mM MgCl2, 0.1% Triton X-100, 0.5 mM dithiothreitol, and protease inhibitor cocktail (Roche)] for a total of 10 min at 25°C and 750 rpm on a shaker. After quenching with 5 ml of 2 M glycine, permeabilized cells were collected by centrifugation for 5 min at 4500g and 4°C and then washed once with 5 ml of fix buffer  and once with 1.25× NEB3 buffer (New England Biolabs), with centrifugation for 5 min at 4500g and 4°C each time. Permeabilized cells were resuspended in 300 μl of 1.25× Dpn II buffer (New England Biolabs) and 0.3% SDS and incubated for 1 hour at 37°C and 1000 rpm on a shaker. Triton X-100 was added to a final concentration of 2%, and the permeabilized cells were incubated for a further 1 hour at 37°C and 1000 rpm before overnight treatment with 1500 U of Dpn II (New England Biolabs) at 37°C and 1000 rpm. The restriction enzyme was inactivated by incubation for 20 min at 65°C and 1000 rpm with SDS at a final concentration of 1.3% before dilution of the lysate in 10 ml of 1× T4 DNA ligase buffer (New England Biolabs) and 1% Triton X-100 and incubation for 1 hour at 37°C and 750 rpm. The released chromatin was ligated for 4 hours at 25°C and 750 rpm with 40,000 U of T4 DNA ligase (New England Biolabs). Then, cross-links were reversed overnight at 65°C and 750 rpm in the presence of proteinase K (150 μg/ml). The resulting 3C DNA was purified by 1 hour of treatment with ribonuclease A (40 μg/ml) at 37°C and 750 rpm, phenol extraction, phenol/chloroform extraction, and ethanol precipitation. The DNA was quantified with the Qubit dsDNA (double-stranded DNA) assay (Invitrogen). Five-microgram aliquots of 3C DNA were sonicated with a Bioruptor (Diagenode) in 50-μl volumes in sonication buffer  to obtain a fragment range between 500 and 1500 bp. The sonicated 3C DNA was then purified by phenol/chloroform extraction and ethanol precipitation and quantified with the Qubit dsDNA assay (Invitrogen). Libraries for paired-end sequencing were made from 500-ng aliquots of sonicated 3C DNA using Illumina reagents and protocols, with size selection for products of ~800 bp. The libraries were sequenced on a HiSeq 2000 instrument (Illumina), following the manufacturer’s protocol.Hi-C was performed as described in . Two hundred nanograms of total RNA was used for indexed library preparation using Ovation RNA-Seq Systems 1–16 for Model Organisms Kit . Briefly, total RNA was treated with DNase I and reverse-transcribed using random oligonucleotide primers. The resulting complementary DNA (cDNA) was fragmented to 200 bp with a Covaris E220 focused ultrasonicator using microtubes (AFA Fiber Crimp Cap), and the cDNA fragments were end-repaired, ligated to oligonucleotide adapters, strand-separated, and subjected to 18 cycles of PCR amplification. After purification, the libraries were pooled and sequenced at the Science for Life Laboratory  using one flow cell of HiSeq2500 (HiSeq Control Software 2.2.58/RTA 1.18.64) with a 2 × 126 setup and HiSeq SBS Kit v4 chemistry. The Bcl to FastQ conversion was performed using None from the CASAVA software suite. The sequence read quality was reported in Sanger/phred33/Illumina 1.8+ scale.D. melanogaster dm3 genome release (see statistics in table S1), filtering, and normalization were performed as previously described (Sequence read mapping to the r) coefficients were calculated using the cor function in R (www.R-project.org/). To avoid spurious experimental noise, the matrices were filtered to remove bins with observed contacts of less than two, and contacts within individual bins  and between bins separated by more than 1.6 Mb were not considered. The similarity between Hi-C contact matrices of individual experiments was evaluated by hierarchical clustering as implemented in hclust function with distances between experiments calculated as 1 − absolute value of correlation coefficient. The cluster stability was evaluated by performing four variations of the procedure, i.e., using ρ or r and two agglomeration methods: complete and average Unweighted Pair Group Method with Arithmetic mean (UPGMA). For bins equal or larger than 40 kb, the clustering was robust to variations  and Pearson moment correlation  was computed for each Dpn II restriction fragment as described in .b0) starting from the two bins immediately adjacent to b0 and followed by pairs at progressively larger distances of up to 60 bins (300 kb = 60 × 5 kb) (supplementary code file: gen_ContactCcrossingCurves.sh). The values for the corresponding bin pairs calculated for mutant and control cells were subtracted to yield the contact crossing difference curves.To account for sampling biases between Hi-C experiments, the contact matrices were further normalized using multiHiCcompare algorithm  located at a distance L was supplemented with three matching control pairs. The first control pair consisted of one of the insulators (i1) and the control region (c1) located at a distance of L/2 toward the second insulator (i2). The second control pair consisted of the insulator (i1) and the control region (c2) located at the distance L to the left (upstream) of i1. Last, the third control pair included control sites c2 and c3, with the latter located at distance L to the left of c2. The number of contacts between regions of each pair was calculated using HiCmapTools (supplementary code file: gen_InsulatorLoopingTest.sh) (Putative chromatin insulator sites were grouped by chromosome and sorted by their starting position to identify the closest pairs (i.e., D. melanogaster dm3 genome assembly using bowtie2 (Reads were aligned to the To calculate ChIP-seq signal scores, read count profiles of individual insulator protein bound regions or TSSs were extracted from the normalized genomic read count profiles of corresponding proteins using BEDTools (D. melanogaster dm3 genome assembly using STAR_v2.6.1a (2 fold change = 1 and Wald significance test with P = 0.05 as thresholds. PCA and identification of the top 20 most variable genes were performed after regularized log transformation of transcription values. Figure S12 provides an overview of the entire data analysis pipeline.Paired-end reads were mapped to the