Ephb4 and Coup-TFII (Nr2f2) vein-specific enhancers to demonstrate that ETS factors are equally essential for vein, arterial and angiogenic-specific enhancer activity patterns. Further, we show that ETS factor binding at these vein-specific enhancers is enriched by VEGFA signalling, similar to that seen at arterial and angiogenic enhancers. However, while arterial and angiogenic enhancers can be activated by VEGFA in vivo, the Ephb4 and Coup-TFII venous enhancers are not, suggesting that the specificity of VEGFA-induced arterial and angiogenic enhancer activity occurs via non-ETS transcription factors. These results support a model in which ETS factors are not the primary regulators of specific patterns of gene expression in different endothelial subtypes.Correct vascular differentiation requires distinct patterns of gene expression in different subtypes of endothelial cells. Members of the ETS transcription factor family are essential for the transcriptional activation of arterial and angiogenesis-specific gene regulatory elements, leading to the hypothesis that they play lineage-defining roles in arterial and angiogenic differentiation directly downstream of VEGFA signalling. However, an alternative explanation is that ETS binding at enhancers and promoters is a general requirement for activation of many endothelial genes regardless of expression pattern, with subtype-specificity provided by additional factors. Here we use analysis of  •Vein-specific enhancers can contain essential ETS motifs.•VEGFA induced an increase in ETS binding at vein, arterial and angiogenic enhancers.•VEGFA stimulation cannot induce vein-specific enhancer activity. The vascular system is subdivided into arteries, veins, lymphatics and capillaries, each comprised of genetically distinct ECs expressing specific fate-determining genes . The essE-26 transformation-specific) transcription factor family  identified a group of ETS binding motifs required for arterial and angiogenic activity  signalling may act via ETS transcription factors to enable specific activation of arterial and angiogenic genes. VEGFA signalling influences many processes during early vascular growth, and plays essential roles in vasculogenesis, arterial specification and angiogenesis . ETS traactivity . ERG binactivity . A similactivity , whilst activity .Despite their hypothesised role in arterial and angiogenic-specific patterns of gene expression, binding motifs for ETS factors are also a common feature of many pan-endothelial expressed gene promoters and enhancers . ETS1, Ein vivo. These results indicate that within the endothelium, VEGFA-stimulated ETS factor binding is a shared feature at enhancers associated with multiple different patterns of gene expression, and suggests that additional transcription factors may be primarily responsible for directing arterial, angiogenic and venous-specific gene expression patterns downstream of different growth factor signalling inputs.In this paper, we undertake a detailed analysis of two recently characterized vein-enriched gene enhancers. We demonstrate that, similar to arterial and angiogenic enhancers, ETS factor binding at these venous enhancers is necessary for enhancer activation and vein-specific patterns of reporter gene expression, and that this binding is also enriched by VEGFA signalling. However, unlike arterial and angiogenic enhancers, these venous enhancers cannot be directly activated by over-expression of VEGFA 22.1Ephb4 and Coup-TFII (Nr2f2) gene loci (Coup-TFII. Neither enhancer was active in the mature microvasculature (We have recently identified enhancers within the venous-enriched ene loci . Both enA/T)  were able to compete for binding of either ETS1-DBD or ERG, or both proteins   and Fig.A/T) A. Of theCGGAAG/A . We perfCGGAAG/A B–C and fCGGAAG/A D–E, bothproteins C while Eproteins E. The spproteins C and E. EPHB4 and COUP-TFII/NR2F2 were significantly enriched in these HUVECs . Similar to the venous enhancers investigated, ERG binding around these arterial-, angiogenic- and pan-endothelial-expressed enhancers was seen in both HUVEC and HUAEC cells, suggesting that binding of ERG to specific enhancer regions was not routinely restricted to the EC subtypes in which the associated genes are preferentially active . Taken together, these results demonstrate that functional ETS binding motifs can be present within vein EC-specific enhancers, and show that the ability to bind ERG and other ETS factors is not restricted to enhancers that are active in arterial and angiogenic ECs.Since the binding of ETS factors to vascular enhancers has been previously associated with arterial-specific and angiogenic-specific enhancers, we next investigate whether ETS factor binding was also a feature at venous-specific enhancers . We found significant binding for ERG, FLI1 and ETS1 at both Ephb4-2 and CoupTFII-965 venous enhancers . EMSA analysis confirmed that the mutated ETS binding motifs could not bind ETS proteins   exclusively to the venous endothelium .In agreement with previous  studies , our resproteins C and E. on (hpf)  and Fig.on (hpf) A–B. Howeon (hpf) A–B. We sothelium  and Fig.othelium C–D, the The loss of Ephb4-2 and CoupTFII-965 vein enhancer activity after ETS motif mutation observed here is similar to the loss of activity seen when ETS motifs are mutated in the arterial and angiogenic Dll4in3 enhancer , in othe2.3erg and fli1. There are multiple ETS factors expressed in the developing zebrafish vasculature  zebrafish was significantly reduced after morpholino knockdown of erg and fli1, with the strength of Ephb4-2:GFP transgene activity inversely correlated with the levels of erg/fli1 MO . Conversely, we saw a reduction in the expression of endogenous ephb4 and stab1l, a zebrafish venous marker, when assessed by whole mount in situ hybridization analysis . Similar reductions in the expression of some venous-enriched genes was also observed after ERG depletion in HUVECs  transgenic zebrafish, although this was not as marked . As previously reported, erg/fli1 knockdown also resulted in reduced activity of the arterial/angiogenic Dll4in3 enhancer in transgenic zebrafish  and tg(CoupTFII-965:GFP) zebrafish lines compared to the arterial (tg(Dll4in3:GFP) zebrafish line. At the lowest concentration of inhibitor, Dll4in3:GFP activity was more notably reduced than either venous enhancer, while higher SU5416 doses significantly reduced activity of all enhancers  results in EC apoptosis, lower levels of VEGF inhibition result in reduced arterial and venous marker gene expression, although the reduction in venous genes was less pronounced and sometimes compensated by expansion of vein gene activity into the dorsal aorta . We therAlthough Dll4in3:GFP was more sensitive to inhibition of VEGFA signalling than either the venous Ephb4-2:GFP or the CoupTFII-965:GFP, our previous results show that changes in ETS factor binding after VEGFA stimulation can be seen at both venous and arterial enhancers. Consequently, it is unlikely that changes in ETS factor occupancy at enhancers can explain the differences between venous and arterial enhancer responses to VEGFA inhibition. An alternative explanation may be that additional transcription factors  may be instead responsible for allowing arterial and angiogenic enhancers a greater sensitivity to VEGFA signalling.2.6in vivo. We first used an established model of VEGFA-stimulated blood vessel growth in mice, in which an adenovirus expressing VEGFA164 (Ad-VEGFA164) is injected intradermally into the ears of adult mice . In both enhancers, angiogenic expression was lost by 60 days after injection, a time-point known to be independent of VEGFA signalling.We next examined whether VEGFA over-expression, and subsequent increased ERG occupancy, is alone sufficient to initiate activity of venous-, arterial- and angiogenic-specific enhancers ult mice . This reult mice . Vessel ult mice . To deteenic ECs A. Ad-VEG164 injection can specifically re-activate the Dll4in3 and HLX-3 enhancers in their native EC sub-types. However, they cannot determine whether this occurs via a VEGFA-mediated increase in ERG binding, VEGFA-mediated increase in other transcription factors binding to these enhancers or VEGFA-mediated removal of repressive factor binding. If VEGFA-mediated activation of these enhancers occurs primarily via changes to ETS factors, we would expect a similar reactivation of venous enhancers. We therefore next determined if Ad-VEGFA164 injection was able to directly activate the Ephb4-2:lacZ and CoupTFII-965:lacZ transgenes. Unlike with the arterial and angiogenic enhancers, we observed no endothelial transgene activity at any time point in either Ephb4-2:lacZ or the CoupTFII-965:lacZ Ad-VEGFA164 injected ears, although occasional ectopic expression could be detected  embryos (following protocol and concentration from tg(Ephb4-2:GFP) embryos . This did not change significantly when we increased the amount of vegfaa injected . This result therefore further indicates that increased VEGFA signalling does not increase Ephb4-2 enhancer activity. Additionally, at 50 pg vegfaa121 and vegfaa165 we saw no clear expansion of Ephb4-2:GFP expression beyond the venous endothelial expression pattern observed in control embryos (vegfaa121 and vegfaa165 mRNA into 1-cell stage tg(Dll4in3:GFP) embryos resulted in slightly increased GFP intensity and expansion into the caudal vein plexus, as previously described by vegfaa levels . Taken together, these results suggest that the failure of Ad-VEGFA164 to activate venous gene enhancers in the mouse ear is unlikely to be a result simply of developmental context. Furthermore, the Ephb4-2:lacZ transgenes can be reactivated in injured neonatal hearts , suggesting that absence of normal activity does not affect enhancer reactivation. Taken together, these results indicate that VEGFA stimulation is not sufficient to activate transcription from the Ephb4-2 venous EC-specific enhancer, despite its reliance on VEGFA-augmented ETS transcription factors.Although developmental arterial and angiogenic enhancers were reactivated in the adult mouse ear by Ad-VEGFA164 A and B,  reduced . Furthern plexus . To dete3The role of ETS transcription factors in the regulatory hierarchy of endothelial cells has been unclear. The specification and maintenance of the endothelial cell lineage requires the shared activation and repression of many lineage-defining genes. However, differential gene expression within specific sub-populations of endothelial cells is also essential for vascular function. Consequently, spatial and temporal control of endothelial gene expression must involve multiple layers of regulation. While analysis of arterial and angiogenic-specific enhancers has supported a proposal that ETS factors play a specific role in the activation of arterial and angiogenic genes downstream of VEGFA signalling, ETS factors have also been implicated in the more general activation of genes, and their cognate regulatory elements, involved in endothelial identity and maintenance. However, the analysis of the precise roles played by ETS factors in the vasculature has been complicated by the abundance of different ETS factors in the endothelium coupled with extensive redundancy between different ETS family members. Parsing their two potential functions is further challenged by the multiple roles played by VEGFA signalling in the vasculature. Recent analysis in zebrafish concluded that low levels of VEGFA signalling promotes general endothelial identity and survival, while higher levels of VEGF signalling primarily promotes arterial specification . ConsequIn this paper, we have clearly demonstrated that vein-specific gene enhancers can be reliant on ETS factors for activation in venous ECs. This is similar to that previously reported in arterial-specific and angiogenic-specific enhancers, even though expression of venous genes is not induced to the same extent by high VEGFA signalling. Further, we have shown that VEGFA signalling can also increases ETS factor binding at venous enhancers, indicating that selective arterial and angiogenic gene activation is unlikely to be achieved through this mechanism. Because our analysis was predominantly restricted to two venous enhancers, the conclusions may not equally apply to all venous-expressed genes. Of note, Notch1 expression in arterial ECs , Tg(CoupTFII-965:lacZ) Tg(Dll4in3:lacZ) and Tg(HLX-3:lacZ) were generated as previously described  containing 5 mM potassium ferrocyanide, 5 mM ferricyanide, 0.1% sodium deoxycholate, 0.2% Nonidet P-40, 2 mM MgCl2 and 1 X PBS. After staining, embryos were rinsed through a series of 1 X PBS washes, then fixed overnight in 4% paraformaldehyde at 4 °C. All embryos were imaged using a Leica M165C stereo microscope equipped with a ProGres CF Scan camera and CapturePro software (Jenoptik). In instances that images have been altered to improve quality and colour balance, each image within a set have been altered using the same parameters. This occasionally included to selective depletion of the yellow or red colour channel, in order to counteract issues from the X-gal stain solution (which is orange). All samples are stored in 4% PFA indefinitely and slowly become less orange. Consequently, samples stained more recently have a greater yellow/orange hue. An example of this alteration can be seen in  and tg(CoupTFII-965:GFP) zebrafish lines were generated in  at 28.5 °C. To image, all embryos were dechorionated and anesthetized with 0.01% tricaine mesylate. For analysis of F0 transgenic zebrafish, single embryos were transferred into a flat bottom 96-well plate, and GFP reporter gene expression screened with a Zeiss LSM 710 confocal microscope at 46–50 hpf. Whole zebrafish were imaged using the tile scan command, combined with Z-stack collection under a confocal microscope Zeiss LSM 710 MP (Carl Zeiss) at 488 nm excitation and 509 nm emission (EGFP).All animal procedures comply with all relevant ethical regulations, were approved by Clinical Medicine Local Ethical Review Committee, University of Oxford and licensed by the UK Home Office. Stable rated in . F0 mosarated in . EmbryosFor pharmacological inhibition of VEGF signalling, embryos were manually dechorionated and 0.625 μM, 1.25 μM, 2.5 μM and 5 μM of SU5416 (Stratech Scientific Ltd.) added at approximately 5ss as described in . Control4.3Ephb4-2mutETS and CoupTFIImutETS enhancer sequences were initially generated as custom-made, double-stranded linear DNA fragments . DNA fragments were cloned into the pCR8 vector using the pCR8/GW/TOPO TA Cloning Kit  following manufacturer’s instructions. Once cloning was confirmed, each enhancer was transferred from the pCR8/GW/enhancer entry vector to a suitable destination vector using Gateway LR Clonase II Enzyme mix  following manufacturer’s instructions. For mouse transgenesis, the enhancer was cloned into the hsp68-LacZ-Gateway vector (provided by N. Ahituv). For zebrafish transgenesis, the enhancer was cloned into the E1b-GFP-Tol2 vector (provided by N. Ahituv).4.3.1AATCAGTGCGTGCTCGTTAAGTCCTGGAGATCCACTGAGCGCGCAGCCTAACGCTGGAGAAAGTGGTTTGAAACCCAAAGTATAGAAAATGTAAATAAAAGGCAGGCGTGTCAGAGAGGGTGAGGGATCTCCGTAACACCTCATTTCATTTTTTTAAAGGAGGGGGACACTTCCCCGCCGCCTGCAGCCTTGACCTCCAAGGCGGGGGTAGGGACCGTTGTGGCTCTTTCCTGAGGCTGTTTCCTGTCTGGCTCCTGGGGGCCCTCGGGATGGCTGGGAGGGCCCTTCCTCTCATTTGCTAGCACCCCCTCTCATCCATCAGTTTGAGGGGAGGGTCCAGGAAAGACGGCCTCCTATCTACATCAGGGCACTGTGAGTGTGGGGCACGGGATGGTTGGATGAGAGAGGTGCTGTTCCCGAAGTCGGTCCTTTAAGGGCTGCGGTAAGGAGACTTTAATTTAAGGTAATTAGTACAGGGTCTGGAAACTCTGAGGTAGGAGTCTGGGGCACCTGGGAGTCTGCCAAATACCCTAAGGGCGCACACACACACCCCAGCGGGCGACCGGTGATGACCTCTTGTCCGCCTGCGCGCACACACACACCAGCGGGCGCGGGAGACCCGTGATGGCCTTTTGTCCCCGTGCACTTATCTTCCTGGCGCAAGTAGTGCTCCCCACCCCCTGCCCTTCCTCACAGCCCTGCCTGGGTCCCGCTCCGGGGTGGGTCAGCCAGGGCAGGAAACAGCCGGCTTGGCTGGAGCCAGGCTGACCGGCTAGATCTGGGAGTCCCCTCCTCCTTCCCCACGCAGACTCAGGCTCCCCTTCTCTTATCCACAGACACCCCCTTTTTTGCAGCTATCATTCTGCATCCGGGTCCCCCTGAATTTCTGAGTCGTGGCTTGTTCTCAC.4.3.2agCTGAGGCTGTTagCTGTCTGGCTCCTGGGGGCCCTCGGGATGGCTGGGAGGGCCCTagCTCTCATTTGCTAGCACCCCCTCTCATCCATCAGTTTGAGGGGAGGGTCCAGGAAAGACGGCCTCCTATCTACATCAGGGCACTGTGAGTGTGGGGCACGGGATGGTTGGATGAGAGAGGTGCTGTTCCCGAAGTCGGTCCTTTAAGGGCTGCGGTAAGGAGACTTTAATTTAAGGTAATTAGTACAGGGTCTGGAAACTCTGAGGTAGGAGTCTGGGGCACCTGGGAGTCTGCCAAATACCCTAAGGGCGCACACACACACCCCAGCGGGCGACCGGTGATGACCTCTTGTCCGCCTGCGCGCACACACACACCAGCGGGCGCGGGAGACCCGTGATGGCCTTTTGTCCCCGTGCACTTATCTagCTGGCGCAAGTAGTGCTCCCCACCCCCTGCCCTagCTCACAGCCCTGCCTGGGTCCCGCTCCGGGGTGGGTCAGCCAGGGCAGAGAACAGCCGGCTTGGCTGGAGCCAGGCTGACCGGCTAGATCTGGGAGTCCCCTCCTCCTTCCCCACGCAGACTCAGGCTCCCCTTCTCTTATCCACAGACACCCCCTTTTTTGCAGCTATCATTCTGCATCCGGGTCCCCCTGAATTTCTGAGTCGTGGCTTGTTCTCAC.AATCAGTGCGTGCTCGTTAAGTCCTGGAGATCCACTGAGCGCGCAGCCTAACGCTGGAGAAAGTGGTTTGAAACCCAAAGTATAGAAAATGTAAATAAAAGGCAGGCGTGTCAGAGAGGGTGAGGGATCTCCGTAACACCTCATTTCATTTTTTTAAAGGAGGGGGACACTTCCCCGCCGCCTGCAGCCTTGACCTCCAAGGCGGGGGTAGGGACCGTTGTGGCTCTT4.3.3GCTGAGACAAATGGAAAGCTGAAGATAAGGATCCTCTGAGGTGCGAACATACAGCTGTTGGGAATTGCCAGAGAATCGGACCAATAAAGGAAGTCACTATTTTTCCAGGCCTGAAGTGAGTTATAGGGCGAGACGGGTGTTGTATATTTATGTAAGGCAACAGCAGGGAGTTTAAGCGGCTGGATATTGCTGAAAGAGCATCATTCACATTCAGGCGGAGACAAAAGGTGGAAATGAAGCAACATCCTGGCCAAAGAAGGCCTCAAGACAGAATAATAACAGTTCAGAGAGGGGGGCTGTGTGCACGGCCGAGGGTCGGCCTCAAAACCAGGAAATGATCGAGATGCCTTGTCAGATCTTC.4.3.4GCTGAGACAAATGGAAAGCTGAAGATAAGGATCCTCTGAGGTGCGAACATACAGCTGTTGGGAATTGCCAGAGAATCGGACCAATAAAtcAAGTCACTATTTTTCCAGGCCTGAAGTGAGTTATAGGGCGAGACGGGTGTTGTATATTTATGTAAGGCAACAGCAGGGAGTTTAAGCGGCTGGATATTGCTGAAAGAGCATCATTCACATTCAGGCGGAGACAAAAGGTGGAAATGAAGCAACagCTTGGCCAAAGAAGGCCTCAAGACAGAATAATAACAGTTCAGAGAGGGGGGCTGTGTGCACGGCCGAGGGTCGGCCTCAAAACCAtcAAATGATCGAGATGCCTTGTCAGATCTTC.4.4Mouse and human sequences of putative enhancers were aligned using ClustalW . Binding4.5italic underlined nucleotides modified (GGA to TCA or TCC to TGA) in mutant version:Proteins were made using the TNT Quick Coupled Transcription/Translation system as described in the manufacturer’s directions. The truncated ETS1 DNA binding domain (ETS-DBD) and full length ERG were in the pCITE2 plasmid, and transcribed using T7 polymerase. To make each probe, double stranded oligonucleotides with CTAG 5′ overhangs were labelled with 32P-dCTP using Klenow (Promega) to fill in overhanging 5′ ends, and purified on a non-denaturing polyacrylamide-TBE gel. 20 μl binding reactions consisted of 2–3 μl protein or lysate control, 2 μl 10X binding buffer  and 0.5 μg poly dI-dC. For competitor lanes, a 100-fold excess of competitor DNA was added in a volume of 1 μl. Binding reactions were incubated at room temperature for 10 min before the addition of radiolabelled probe, after which they were incubated an additional 20–30 min. Gels were electrophoresed on a 10% non-denaturing polyacrylamide gel. Sequences of the probes and competitor regions are listed below, with tccccgccg;ETS control probe  CTAGtaaatcctgaggctg.Ephb4-2 ETS-b CTAGgttgtggctctttcctgtctggc.Ephb4-2 ETS-c CTAGgaggctgttggatggctggga.Ephb4-2 ETS-d CTAGggccctcgtcctctcatttg.Ephb4-2 ETS-e CTAGagggccctggatggttgg.Ephb4-2 ETS-f CTAGgtggggcacgggatgagagaggtgc.Ephb4-2 ETS-g CTAGgatggtttcctggcgcaagta.Ephb4-2 ETS-h CTAGcacttatcttcctcacagcccEphb4-2 ETS-I CTAGcctgccctggaaacagcc.Ephb4-2 ETS-j CTAGagccagggcaggaaagctgaagataa.Coup-965 ETS-a CTAGacaaatggatcctctgag.Coup-965 ETS-b CTAGgctgaagataaggaattgccagaga.Coup-965 ETS-c CTAGagctgttgggaagtcactat.Coup-965 ETS-d CTAGcggaccaataaaggaaatgaagcaacatc.Coup-965 ETS-e CTAGaaggttcctggccaaag.Coup-965 ETS-f CTAGaagcaacaggaaatgatcgagatc.Coup-965 ETS-g CTAGcaaaaccatccaggcctg.Coup-965 ETS-h CTAGgtcactatttt4.6165 (PeproTech) at 25 ng/ml for 1.5 h. Cells were then trypsinised and the cell pellet collected.For VEGF stimulation experiments in cells, human umbilical vein pooled endothelial cells  were grown in Endothelial Cell Growth Medium 2 with BulletKit (PromoCell). Media was changed every 48 h. Four 80% confluent 15 cm dishes per condition were serum starved in 0.5% Foetal Bovine Serum (Gibco) overnight before being stimulated with VEGFAChromatin immunoprecipitation was carried out as previously described . BrieflyImmunoprecipitated DNA was analyzed by qPCR using TaqMan Custom Gene Expression Assay Probes (ThermoFischer) designed against 100bp regions of the Ephb4-2 enhancer, the CoupTFII-965 enhancer or a gene dessert region of Chromosome 7 as a negative control.4.6.1ACCCCTGCCCTTCCTTGCTGTTCTGCCTGGGTCCTGCGCCCGGGTTGGGGGGGGTGGGCCGGTCACCGAGGGCAGGAAACAGCCGGCTTCACTGGAGCCAGGCAGACCAG.4.6.2AGCGGCTGTATATTGCTGAAAGAGCATCATTCACATTCAGGCAGAGACAAAAGGTGGAAATGAAGTAACATCCTGGCTGAAGAAGGCCTCACGACAGAATA.4.6.3CCTCAGCCTCCCAAGTAGCTGGGATTACAGGTGTGTGCTACCATGCCTGGCTAATTTTTGTATTTTTAGTAGAGACAGGGTTTCACCATGTTGGCCAGGCTGGTCTCGAACTCCTGAACTCAGG TGATCTA.Each ChIP was performed on at least three biological replicates, with three technical replicates for each. Statistical analysis was performed in StepOne plus software, Microsoft Excel. Input was taken as the supernatant from the non-antibody control condition. Results are expressed as the mean of the % input defined as 100∗(2^(adjusted Input ct – ct IP)) across all replicates. Significant differences were calculated using ANOVA f test with p values subsequently derived from Tukey HSD test, accounting for multiple comparison correction. Graphs were produced using R statistical package.4.7https://ncbi.nlm.nih.gov/geo/) under accession GSE93030. The four ChIP-seq datasets used have accession numbers GSM2442775 to GSM2442778. Data consisted of ChIP-seq Model-based Analysis of ChIP-Seq (MACS) (https://CRAN.R-project.org/package = data.table and https://CRAN.R-project.org/package=ggpubr). GRCh37 ROIs: HLX-3 chr1:221,049,659–221,050,776, Dll4in3 chr15:41,222,807–41,223,778, Ephb4-2 chr7:100,426,194–100,427,393, and CoupTFII-965 chr15:95,908,708–95,909,301.ChIP-seq analysis was conducted on the published and publicly available data from q (MACS)  regions q (MACS) . MACS pe4.8Antisense MOs were ordered from GebeTools LLC and dissolved in water before injected into 1–2 cell stage zebrafish embryos as previously described . Sequencfli1 MO (3 – 6 ng) TTTCCGCAATTTTCAGTGGAGCCCG (GGAGCCCG .erg MO (3 – 6 ng) CAGACGCCGTCATCTGCACGCTCAG .4.9in situ hybridization ephb4, efnb2 and stab1l probes were generated as custom-made, double-stranded linear DNA fragments , cloned into the pCR2 vector using the TOPO/TA cloning kit (Invitrogen 450641) and transcribed using SP6 and T7. The sequences are provided below. dll4 probe was kindly provided by R. Patient, University of Oxford, Oxford. Whole-mount in situ hybridization was conducted as previously described . Embryos were washed through a dilution series of 2 x SSC followed by 0.2 x SSC at 65 °C and thereafter taken through room temperature dilution washes of 100% MABT . Nonspecific sites were blocked with MAB block (MABT with 2% Boehringer block reagent) and the embryos incubated for 15 h with 1:4000 antiDIG antibody (Roche) at 4 °C, before washing in MABT. Prior to staining, embryos were washed in AP buffer and the in situ signal developed at room temperature with BM Purple (Sigma-Aldrich). Staining was stopped as appropriate, and embryos were bleached in 3% H2O2/0.5% KOH until pigmentation disappeared, then re-fixed in 4% PFA for 20 min and washed 4 times with PBST. Embryos were transferred to 80% glycerol for imaging.For zebrafish whole-mount escribed . Brieflyin situ/condition. Experimental blinding was not used as phenotypes of control and treated were easily detectable due to morphological defects.Analysis was qualitative not quantitative, therefore no statistical analysis was applied to the observations of staining intensity and pattern. Numbers of zebrafish embryos was no less than 30/4.9.1TCTCAGCTCTGGACAAGCTGATCCGCAACCCGGCCTCACTCAAAATCACAGCGCAGGAGGGGGCGGGCCCCTCTCACCCTCTGCTGGACCAGCGGTCTCCACTCACGCCCTCATCCTGCGGGACAGTGGGTGACTGGCTGCGGGCCATCAAGATGGAGCGCTACGAGGAGACATTTCTGCAGGCGGGATACACGTCCATGCAGCTCGTCACCCACATCAACACGGAGGATCTGCTGCGTTTGGGAATAACTTTAGCAGGTCACCAGAAGAAGATTCTCTCCAGCATTGAGGCTCTCGGGATTCAAAACAAAGCACCAGGGAATGTGCTGTACTGA.4.9.2AAAACCAAGTCGATGAAAATCATCATGAAGGTTGGACAAAACCCCTCTGATCCCATTTCCCCCAAAGACTACCCTACCAGTTACCCTCCCAAACACCCTGACTTAGGGGGCAAGGACAGCAAATCGAATGAAGTACTTAAGCCAGATGCATCTCCTCATGGGGAAGATAAGGGAGATGGAAATAAATCCTCATCAGTCATTGGCTCAGAGGTGGCCCTGTTTGCCTGCATCGCCTCAGCAAGCGTCATCGTCATCATCATAATCATCATGCTAGTTTTCCTTCTCCTGAAGTATCGACGA.4.9.3GGATTCAGCAGCTACAGACACACCCAACCTCATCGACTAGCACAGACAGCAGCGTTAAACTCTCCCTTCATTTACCTGCAATCAGCTGACCGCTCTTAAAATAAAGGTTCTGTATTGGCATTGATGGTTCCGCGAAGAATCTTTATAAGCCATAACATCTTTCCATTTCCATGAGGTGTAAAAAGACTCTTTAGAATATTAAAATGTTACTTCATAAACATTTGATGTGTTTGATTGCAGATACTTCAGAGTGTTTAACTTCCACCCATTTATTTCTGCGTTTCACACATATTTTTGACTAAAAATGTTCTTTACATTAAGAAAAAATGGTGTACTCACCCTCAAGTAGGTCCAAACCTTCACGAGTTTCTTTTTTTGCCTTCTGTTGAACACAAACAAGAAAATATTTTGATAATGCGTTAAGCAAGGGGCCATTAGCTGTTTTGATCCAACTTTTTTCATGCGCATTTTAAATTATCGCATGTAAAAAAGCTTAATGGAAACCCAAGATGTGCTTAATTTGTCAAAATCTGCAT.4.10164 injections were performed on nude mice as described in (164 (provided by Lilly) diluted 1:30 in sterile 3% glycerol/PBS. At the required time-point after injection, ears were harvested and skin removed from the dorsal side. Ears were fixed in 2% paraformaldehyde and 0.2% glutaraldehyde in PBS for 20 min at 4 °C, washed twice in PBS then X-gal stained overnight at room temperature. Ears were then placed in 4% paraformaldehyde for storage.Intradermal Ad-VEGFAribed in . Briefly164 injected ears, ears were harvested and X-gal stained as described above, then dehydrated through a series of ethanol washes, cleared by xylene and paraffin wax-embedded. 5 or 6-μm sections were prepared and de-waxed. For imaging of X-gal staining, slides were counterstained with nuclear fast red (Electron Microscopy Sciences).For histological analysis of Ad-VEGFA4.11vegfaa121 and pCS2+vegfaa165 plasmids were kindly provided by S. Sumanas, Cincinnati Children’s Hospital Medical Center, Ohio, USA. vegfaa121 and vegfaa165 mRNA was synthesised in vitro using the mMessage mMachine SP6 transcription kit (ThermoFisher Scientific) and injected into 1 cell stage zebrafish embryos at a final concentration of 50 pg of mRNA per embryo. Analysis was qualitative not quantitative, therefore no statistical analysis was applied to the observations of staining intensity and pattern.pCS2+No competing interests declared.


Stem cells represent a key resource in regenerative medicine, however, there is a critical need for pharmacological modulators to promote efficient conversion of stem cells into a myogenic lineage. We have previously shown that bexarotene, an agonist of retinoid X receptor (RXR) approved for cancer therapy, promotes the specification and differentiation of skeletal muscle progenitors. To decipher the molecular regulation of rexinoid signaling in myogenic differentiation, we have integrated RNA-seq transcription profiles with ChIP-seq of H4K8, H3K9, H3K18, H3K27 acetylation, and H3K27 methylation in addition to that of histone acetyl-transferase p300 in rexinoid-promoted myoblast differentiation. Here, we provide details regarding data collection, validation and omics integration analyses to offer strategies for future data application and replication. Our analyses also reveal molecular pathways underlying different patterns of gene expression and p300-associated histone acetylation at distinct chromatin states in rexinoid-enhanced myoblast differentiation. These datasets can be repurposed for future studies to examine the relationship between signaling molecules, chromatin modifiers and histone acetylation in myogenic regulation, providing a framework for discovery and functional characterization of muscle-specific loci. Epigenetic mechanisms control the patterns of gene expression in stem cell differentiation and play an essential role in regulating the balance between expression patterns for self-renewal of stem cells and activation of lineage-specific genes . A thoroProper formation of skeletal muscle is orchestrated by sequential expression of myogenic regulatory factors (MRFs). Amongst the MRFs, Myf5 and MyoD initiate the transcription of muscle-specific genes, directing cells into the skeletal muscle lineage, whereas myogenin mainly regulates late myoblast differentiation and the fusion of myoblasts into myotubes . We haveEpigenetic mechanisms, including post-translational modification of histones, regulate chromatin structure and accessibility of genes to the transcriptional machinery. Histone modifications may result in disruption of histone-DNA interactions, leading to an open chromatin structure that is accessible to binding of transcriptional machinery or may strengthen histone-DNA interactions to create a tightly packed chromatin structure which favors gene silencing . Based oAs global analyses of the combinations of histone marks can be exploited to delineate distinct lineage specific chromatin states , we have.Here, we provide technical validation of 16 genomic data sets that profile and integrate global gene expression, histone acetylation, and p300 association with chromatin state dynamics in an effort to obtain an unbiased, genome-wide view of rexinoid-enhanced myoblast differentiation. To facilitate interpretation of this data, we provide detailed information on quality control , validatRNA samples from two biological repeats of differentiating myoblasts in the absence or presence of bexarotene were processed by McGill University Genome Quebec Innovation Centre for transcriptome library construction and sequenced using an Illumina HiSeq 2000 sequencer as single-end 50 base pair reads. To assess the quality of reads in Illumina RNA-seq datasets, the mean per sequence quality scores, per base quality scores, and GC content were analyzed using the FastQC program . The quavia fingerprint plot analysis displayed intersection of the x-axis at approximately 0.1 by the fingerprint plot traces of all samples indicating an absence of read coverage across 10% of the genome  transformation which minimizes differences between samples with small counts and normalizes with respect to library size  of key muscle-specific proteins in Cluster I including myogenin, and the troponin and myosin families which all displayed increased expression in response to bexarotene (p = 3.00 × 10–49). E2F4 is a critical molecule in the RB/E2F pathway and is known to play an important role in control of the cell cycle and action of tumor suppressor proteins  software . Based oxarotene . In contproteins . As a trproteins . Taken tvia integration of RNA-seq and ChIP-seq datasets, to depict epigenetic mechanisms of early myogenic gene transcription in rexinoid signaling. We utilized our chromatin state model to characterize p300 localization in myoblasts differentiated for 24 h in the absence or presence of bexarotene (de novo motif discovery tool as part of the Hypergeometric Optimization of Motif EnRichment (HOMER) software  associated with p300 loci unique to bexarotene. The k-means machine learning algorithm partitioned motifs into a set of four groups such that motifs within the same cluster displayed the most similar enrichment in each condition. As seen in As the p300 peaks in Cluster I and II exhibited an increasing association with poised enhancers , we examTaken together, the integration of p300 ChIP-seq with chromatin state classification and chromatin modification coverage can be used to delineate the distinct functions of HATs in the regulation of myogenic expression in specific biological contexts such as rexinoid responsive gene expression. Additionally, the model of rexinoid-enhanced myogenesis offers a system to identify new genetic targets and molecular interactions which may have pharmacological significance related specifically to muscle-related disease. This report represents biologically valid and high-quality next generation sequencing data that will be useful for future studies examining the role of novel and known myogenic regulators including p300, histone acetylation and their relation to chromatin state dynamics in early myogenic differentiation.


DMPK (dystrophia myotonica protein kinase) gene on chromosome 19, while DM2 is caused by CCTG repeat expansions in intron 1 of the CNBP (cellular nucleic acid-binding protein) gene on chromosome 3. Recent advances in genetics and molecular biology, especially in the field of RNA biology, have allowed better understanding of the potential pathomechanisms involved in DM. In this review article, core clinical features and genetics of DM are presented followed by a discussion on the current postulated pathomechanisms and therapeutic approaches used in DM, including the ones currently in human clinical trial phase.Myotonic dystrophies (DM) are the most common muscular dystrophies in adults, which can affect other non-skeletal muscle organs such as the heart, brain and gastrointestinal system. There are two genetically distinct types of myotonic dystrophy: myotonic dystrophy type 1 (DM1) and myotonic dystrophy type 2 (DM2), both dominantly inherited with significant overlap in clinical manifestations. DM1 results from CTG repeat expansions in the 3′-untranslated region (3′UTR) of the  DMPK (dystrophia myotonica protein kinase) gene on chromosome 19 [CNBP (cellular nucleic acid-binding protein), previously known as ZNF9 (zinc finger protein 9), on chromosome 3 [Myotonic dystrophies (DM) are the most common muscular dystrophies in adults, with an estimated prevalence of 1/8000 . DM1 andosome 19 ,7 while Clinical presentations of myotonic dystrophies have a wide spectrum, from asymptomatic or pauci-symptomatic DM2 patients to severe neuromuscular and cognitive deficits in congenital DM1. Myotonic dystrophies are systemic diseases and their clinical manifestations are not limited to skeletal or cardiac muscles . SometimClinical features of classic DM1 (Steinert’s disease) include muscle weakness in the distal extremities, leading to the inability to perform fine motor tasks with the hands, and foot drop, leading to falls. In more severe cases, proximal muscles may be involved, causing difficulty with climbing stairs and reaching up above the head ,19. WeakCardiac complications are a major cause of unexpected morbidity and mortality and can include various dysrhythmias and heart blocks. DM1 patients need regular cardiac surveillance and if needed, pacemaker placement or other interventions. Cardiac pump malfunction can also be seen in DM1 .Central nervous system involvement leads to cognitive/intellectual and/or behavioral impairment as well as central sleep disorders . Eye invPatients with congenital DM1 show the most severe form of DM. Congenital DM1 presents with reduced fetal movements, polyhydramnios, congenital hypotonia and contractures, difficulty with sucking or swallowing during the first month of life and congenital/infantile neuromuscular respiratory insufficiency ,11,23. SChildhood-onset DM1 is often defined with an onset of symptoms from 1 to 10 years of age and with an intermediate phenotype between congenital DM1 and classic DM1. These children can have gastrointestinal symptoms, learning disabilities, facial weakness, myotonia and cardiac conduction defects ,23.DM2, which was originally called proximal myotonic myopathy (PROMM) has significant clinical overlap with DM1 but tends to have a milder phenotype and can be harder to recognize compared to DM1 . The maiDMPK (dystrophia myotonica protein kinase) gene on chromosome 19 [CNBP (cellular nucleic acid-binding protein), previously known as ZNF9 (zinc finger protein 9), on chromosome 3 [DM1 and DM2 share many clinical features but are caused by two different genes. DM1 results from CTG repeat expansions in the 3′-untranslated region (3′UTR) of the osome 19 ,7, whilemosome 3 ,9  is an extreme example of the parent-of-origin effect since most CDM1 children receive the CTG repeat expansion from their mothers . CTG len progeny .In DM2, the smallest reported mutations in CNBP vary from 55 to 75 CCTG repeats and the largest are about 11,000. Similar to DM1, there is significant somatic instability with increase of the CCTG repeat length over the lifespan of the individual . DM2 repSimilar to DM1, a bidirectionally labelled PCR method is recommended to avoid false negative results caused by CCG, CTG, and GGC sequence interruptions; however, due to large CCTG repeat size range in DM2  and somatic instability, genomic southern analyses fail to detect expansions in 20% of known carriers. Quadruplet-repeat primed (QP)-PCR has been used with an additional primer within the elongated CCTG repeat to detect expansions independent of their lengths. Although expansions in all size ranges can in theory be detected by QP-PCR, no reliable information about the length of the expanded repeat will be obtained because of extinction of the signal in the higher size region ,37. Due Expansions of DNA repeat sequences cause numerous neurological and neuromuscular diseases including Huntington’s disease, fragile X syndrome type A (FRAXA), Friedreich’s ataxia (FRDA), spinocerebellar ataxias (SCAs) and myotonic dystrophies (DM). In unaffected people, these DNA repeats are short and stable, showing no significant length changes; however, in affected individuals, the tracts are longer and unstable. These length changes are dynamic across generations, over the life span of an individual, and also between and within tissues from the same individual. Formation of unusual DNA, DNA-RNA, and RNA structures and their interference with nucleic acid replication, repair, recombination and transcription have been suggested as processes whereby DNA is damaged, repeats expand or contract, mis-splicing occurs, proteins are sequestered and, ultimately, translation is disrupted ,39. TranDifferent mechanisms have been suggested to explain the various pathologies in DM1 and DM2 . There iMBNL1 knockout mice show similar features characteristic of DM1 disease [MBNL2 knockout mice exhibit a number of DM-related central nervous system abnormalities [The most prominent pathomechanisms in DM1 and DM2 are thought to be abnormalities in alternative splicing. Alternative splicing (AS) is a regulatory mechanism of gene expression allowing the production of more than one unique mRNA from a single gene. Splicing regulation in skeletal muscle is crucial for normal myogenesis and adaptation to changes in post-natal metabolic and functional requirements. Splicing occurs in spliceosomes, which are large nuclear complexes made of smaller nuclear ribonucleoprotein particles and many regulatory factors including RNA-binding proteins (RBPs). RBPs are involved in inclusion or exclusion of different exons in each transcript depending on the specific cell context, cell type and tissue-specific developmental processes. Muscle-blind-like (MBNL) family, CUG-BP and ETR-3-like factors (CELF) family and the RNA-binding Fox (RBFOX) are the most important splicing regulators in skeletal muscle ,53. MBNL disease . MBNL2 smalities . In addimalities .CELF1 , a member of the family of CELF  proteins, is another RBP that seems to play a role in DM1 pathogenesis. CELF1 binds to single-stranded UG-rich RNA sequences but not to double-stranded sequences, unlike MBNL1, suggesting a key difference in their splicing regulatory mechanisms. CELF1 binds to CUG-repeat containing RNA with a lower affinity than MBNL1 and loss of functional MBNL1 induces CELF1 upregulation. CELF1 overexpression in MBNL1-depleted DM1 tissues leads to expression of embryonic variants of transcripts, the main characteristic of spliceopathy in DM1 ,59. MBNLIntranuclear accumulation of splicing and cleavage factors, such as heterogeneous nuclear ribonucleoproteins (hnRNPs) and small nuclear ribonucleoproteins (snRNPs) has been shown in both DM1 and DM2 patient biopsies . These fDysregulation of stress granule (SG) dynamics has been suggested as an important pathogenic factor in certain neurodegenerative diseases. SGs are transient cytoplasmic cell compartments that are formed in response to different stress stimuli and are implicated in the regulation of translation, mRNA storage and stabilization, and cell signaling during stress. They are formed by coalescence and storage of translation factors, mRNAs, RNA-binding proteins (RBPs) and other proteins that facilitate cell survival during stress. Upon resolution of stress, SGs should disperse; however, in age-related and neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), rigid complexes are formed in SGs potentially leading to accumulation of irreversible toxic aggregates . AlteredRAN translation has been described in several different types of repeat expansion mutations  and results in the production and accumulation of potentially harmful proteins. It has been shown that expansion mutations can be bidirectionally transcribed . Repeat microRNAs (miRNAs) are small non-coding RNAs that modulate gene expression at the post-transcriptional level and their expression and intracellular distribution is dysregulated in many human diseases, including muscular dystrophies. miRNAs promote messenger RNA (mRNA) degradation, destabilization or translation blocking with participation of additional protein factors, forming the RNA-induced silencing complex (RISC). It is estimated that about 2000 miRNAs identified in humans can modulate up to 60% of protein-coding genes . Some miCircular RNAs (circRNAs) are a large class of endogenous non-coding and very stable RNAs produced by a non-canonical splicing event called backsplicing. They have tissue-specific and cell-specific expression patterns and their biogenesis is regulated by specific cis-acting elements and trans-acting factors . It is nDMPK gene was correlated with the phenotypic variability in several muscular and respiratory parameters; this association was not linked to the CTG repeat length [Epigenetic modifications may impact the development or severity of phenotype in DM1. This has been well-characterized in congenital DM1 with the role of DNA hypermethylation at the CTCF1 binding site, which was discussed above in the genetics section of this review article. A recent review article summarizes available evidence on the potential role of epigenetics in DM1 and DM2 . DNA mett length . Anothert length . Genetict length . PatientCNBP knock-out (Cnbp −/+) mouse model showed some of the multi-organ phenotypic features of myotonic dystrophy, including muscle histopathology, myotonic discharges and heart conduction abnormalities. The crossing of these mice with transgenic mice overexpressing CNBP increased CNBP and CLCN1 levels and rescued the phenotype, suggesting that, in addition to RNA gain of function, a loss of CNBP may contribute to DM2 pathology [Cellular nucleic acid binding protein (CNBP) is a small human single-stranded nucleic acid-binding protein and is involved in transcription regulation by binding to both single-stranded DNA and RNA molecules and by activating or repressing the expression of many genes . A heterathology . Howeverathology . This stCLCN1 and SCN4A are associated with non-dystrophic myotonic myopathies ; however, studies of these genes in case series or case reports of patients with DM2 from Germany and Italy have suggested their possible role in the development or severity of myotonia and/or muscle pain in some patients with DM2 [CLCN1 and SCN4A seem to enhance the myotonia and/or muscle pain in DM2 patients.Mutations in with DM2 ,89,90. CBased on the evolving knowledge of possible pathogenetic mechanisms involved in DM1 and DM2, various therapeutic approaches may be considered. DM is a slowly progressing disease and the pattern of gene dysregulation, including spliceopathy, could be different in any specific tissue and also vary over the lifetime of the individual. These limitations impair development of accurate molecular outcome measures, which are crucial for initial drug development. Similar to other genetic diseases, limitations of the preclinical animal models and applicability of their biological mechanisms to humans pose a challenge to drug discovery.A number of therapeutic strategies have been suggested for DM  and someReducing the transcription of expanded CUG RNA by inhibition of RNA polymerase co-factors or by small molecules that bind to GC-rich repeats is one strategy. Pentamidine and related compounds may work through binding the CTG-CAG repeat DNA to inhibit RNA transcription in cell and mouse models of DM1 . The newSeveral nucleic acid technologies, primarily using antisense oligonucleotides (ASOs) have been used to achieve post-transcriptional silencing of DMPK . In a trIn view of the role of MBNL in the pathogenesis of DM, inhibiting interactions between MBNL and toxic RNA, reduction of CELF1 concentration, and dispersion of RNA foci have been used as therapeutic strategies in various in vitro and in vivo models. Nakamori et al. tested 20 FDA-approved small molecules in a DM1 cell model and found erythromycin to have the highest affinity to expanded CUG and a capacity to inhibit its binding to MBNL1. They then used erythromycin in a DM1 mouse model and showed splicing reversal and improvement of myotonia . MBNL ovAs discussed above, dysregulation of microRNA (miRNA) has been implicated in DM pathogenesis. miRNA-targeting approaches have been suggested as a treatment strategy in various models of DM1. There are many technical challenges in implementing such treatments in terms of identifying appropriate targets and biomarkers as well as stability and delivery issues. Various approaches for miRNA intervention have been used or suggested: one is to inhibit miRNA by using synthetic antimiR  products, such as ASOs that are perfectly complementary to the specific miRNA target; a second approach is to use blockmiRs, which contain a sequence complementary to one of the mRNA (messenger RNA) sequences that serve as a binding site for a microRNA and sterically block the miRNA from binding to the same site, hence preventing degradation or transcription inhibition of the target; the third approach is to use miRNA sponges that contain several tandemly arranged miRNA target sequences (same or different sequences); and the last one is using synthetic double strand miRNA mimics as replacement therapy. These proof-of-concept approaches have been used in in vitro models as well as in drosophila and mouse models of DM1 and further discussed in a review by Lopez Castel . In a moSignaling pathways downstream of CUG/CCUG expanded repeat expansions have also been used in therapeutic experiments. These include targeting glycogen synthase kinase 3 beta (GSK3beta), protein kinase C (PKC) and AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTORC1) pathways ,110,112.With recent advances in molecular genetic knowledge and techniques, our understanding of genetic diseases such as myotonic dystrophies has significantly improved; however, there is still much more to learn about these diseases and, as more details are revealed, new questions arise about the pathogenesis of DM1 and especially DM2. Disease-modifying therapies such as siRNA are just entering human clinical trials, but efforts to better understand nucleotide repeat diseases and to identify the best therapeutic targets should continue. Short- and long-term efficacy and safety of current therapeutic approaches are yet to be determined and this knowledge gap is even more noticeable for DM2, where animal models and therapeutic trials are lagging behind DM1. Since various potential pathomechanisms are involved in the development or progression of DM1 and DM2, future disease-modifying therapies may need to be used synergistically to target more than one disease mechanism.


ZMIZ1. Taken together, our results reveal important interactions between host genetic variation and EBNA2-driven disease mechanisms. Further, our study highlights a critical role for EBNA2 in rewiring human gene regulatory programs through rearrangement of the chromatin landscape and nominates these interactions as components of genetic mechanisms that influence the risk of multiple autoimmune diseases.The interplay between environmental and genetic factors plays a key role in the development of many autoimmune diseases. In particular, the Epstein–Barr virus (EBV) is an established contributor to multiple sclerosis, lupus, and other disorders. Previously, we showed that the EBV nuclear antigen 2 (EBNA2) transactivating protein occupies up to half of the risk loci for a set of seven autoimmune disorders. To further examine the mechanistic roles played by EBNA2 at these loci on a genome-wide scale, we globally examined gene expression, chromatin accessibility, chromatin looping, and EBNA2 binding in a B cell line that was (1) uninfected, (2) infected with a strain of EBV lacking EBNA2, or (3) infected with a strain that expresses EBNA2. We identified more than 400 EBNA2-dependent differentially expressed human genes and more than 5000 EBNA2 binding events in the human genome. ATAC-seq analysis revealed more than 2000 regions in the human genome with EBNA2-dependent chromatin accessibility, and HiChIP data revealed more than 1700 regions where EBNA2 altered chromatin looping interactions. Autoimmune genetic risk loci were highly enriched at the sites of these EBNA2-dependent chromatin-altering events. We present examples of autoimmune risk genotype–dependent EBNA2 events, nominating genetic risk mechanisms for autoimmune risk loci such as  Cross talk between genetic risk polymorphisms and environmental factors is thought to influence the onset and progression of many human diseases . Many di–Barr virus (EBV) in mononucleosis  protein and the human TF RBPJ , or (3) infected with an EBV strain (B95.8) that has EBNA2 (EBVEBNA2+). Using this approach, we identify human genes whose expression level changes coincide with the presence of EBNA2 (RNA-seq), and we resolve the effects of EBNA2 on chromatin accessibility (ATAC-seq) and chromatin looping (HiChIP). Further, we examine the enrichment of autoimmune disease risk genetic variants within these EBNA2-dependent regulatory mechanisms and identify allele-dependent EBNA2 behavior at autoimmune-associated variants.Understanding the molecular mechanisms mediating interactions between EBNA2 and the human genome is important for achieving an understanding of the development and progression of autoimmune diseases. In our previous study, analysis of public data sets revealed the presence of EBNA2 ChIP-seq peaks at up to half of the risk loci for particular autoimmune diseases. In this study, we explore the role of EBNA2 within the human B cell gene regulatory network by examining EBNA2-dependent alterations to the human chromatin landscape and investigating the impact of autoimmune disease–associated genetic polymorphisms on these mechanisms. To identify EBNA2-dependent effects, we use an experimental design comparing human B cells that are (1) uninfected, (2) infected with an EBV strain (P3HR-1) that lacks EBNA2  across the three cell types. As expected, we detected transcripts for EBERs only in the EBV-infected Ramos cells (both EBVEBNA2+ and EBVEBNA2−), and we detected EBNA2 transcripts and protein only in EBVEBNA2+ cells . Further, no RNA-seq reads mapped to the EBV genome in the uninfected data set, whereas 7535/7764 and 4249/4423 reads mapped for EBVEBNA2+ replicate 1/replicate 2 and EBVEBNA2− replicate 1/replicate 2, respectively.To globally measure the effect of EBNA2 on human gene expression patterns, we performed RNA-seq in Ramos B cells in three experimental conditions: uninfected, EBVBVEBNA2+ . We usedEBNA2+ and uninfected, which captures the effect of EBV infection on human gene expression in B cells. In total, 493 human genes were differentially expressed upon EBVEBNA2+ infection , with 290 genes up-regulated and 203 down-regulated . Among these EBV-dependent differentially expressed genes, 67 of the 290 up-regulated genes and 18 of the 203 down-regulated genes were consistent with a previous study examining EBV infection in primary B cells at day 28 post-infection  .To investigate the effect of EBNA2 on host gene expression, we identified differentially expressed genes (DEGs) using these three experimental conditions. First, we compared gene expression changes between EBVP-value < 0.05) between EBVEBNA2+ and uninfected cells, and between EBVEBNA2− and uninfected cells . This procedure identified 421 genes that are differentially expressed in the EBVEBNA2+ condition but not in the EBVEBNA2− condition (243 up-regulated genes and 178 down-regulated genes) , which we designate the EBNA2 DEGs. As expected, GO Biological Process enrichment analysis for EBNA2 DEGs revealed processes involved in the immune response, including response to virus, lymphocyte activation, and cytokine production . Further, a significant proportion of the EBNA2 DEGs, including CD80, MAP3K8, SLAMF1, and ZMIZ1, are involved in leukocyte cell–cell adhesion  . Collectively, these results indicate that the expression levels of hundreds of human genes are affected by the presence of EBNA2.Next, we identified the EBNA2-specific effects of these gene expression changes. We identified significant changes in gene expression . We also performed ChIP-seq for EBNA2 in GM12878 cells, an EBV-transformed lymphoblastoid cell line. Quality control analyses indicated high data quality  and strong agreement between experimental replicates . We note that the FRiP score of our EBNA2 GM12878 data set is almost double the FRiP score of a publicly available EBNA2 GM12878 data set (0.036 vs. 0.0185). Our EBNA2 Ramos ChIP-seq experiment has a lower FRiP score (0.010), but this still compares favorably to the majority of publicly available virus ChIP-seq data sets (rank is 16 of 54) . Likewise, our EBVEBNA2+ and GM12878 ChIP-seq peaks aligned significantly with published ChIP-seq peaks of established EBNA2 partners and coregulators performed in EBV-infected B cells . As expected, we observed enrichment within our EBNA2 peaks for the DNA binding motifs of established EBNA2 partners, such as RBPJ, EBF1, and SPI1 . EBNA2 peaks are strongly enriched within 100 kb, 10 kb, and 5 kb of EBNA2-dependent gene transcription start sites (TSSs) . Collectively, these results indicate that our EBNA2 ChIP-seq experiments are of high quality.We performed ChIP-seq for EBNA2 in EBV6 of 54) . Through6 of 54)  to compa B cells , includiEBNA2+ Ramos ChIP-seq peaks were enriched within both proximal  and distal  regions of EBNA2 DEGs. EBNA2 ChIP-seq peaks were more enriched near up-regulated than down-regulated genes within proximal regions (likely promoters)  and distal regions  . For example, EBNA2 ChIP-seq peaks are located in the promoter region of the EBNA2 DEG LY9 , in agreement with a recently published finding that LY9 gene expression is induced by EBV infection . We identified 45,207, 35,894, and 64,679 ATAC-seq peaks in these three conditions, respectively. Quality control analyses indicated high data quality , strong agreement between experimental replicates , and enrichment at transcription start sites . These genomic regions corresponded strongly with previously published data sets performed in relevant cell types, including DNase-seq and ChIP-seq peaks for H3K27ac histone marks . We examined the impact of EBNA2 on chromatin accessibility by performing differential analysis using MAnorm . As expected, EBNA2 ChIP-seq peaks in Ramos EBVEBNA2+ cells were highly enriched within both EBNA2-dependent open chromatin accessibility gains  and EBNA2-dependent chromatin accessibility losses  .We examined the impact of EBNA2 on genome-wide chromatin accessibility by performing ATAC-seq in uninfected, EBVg MAnorm  and the g MAnorm  to discoP-value: 4.30 × 10−68), but not proximal to down-regulated EBNA2 DEGs . Likewise, EBNA2-dependent closed chromatin showed the opposite effect, with enrichment for down-regulated EBNA2 DEGs  but not up-regulated EBNA2 DEGs  . Similar results were obtained for distal (100 kb window) regions of EBNA2 DEGs. Particular TF binding motifs are preferentially enriched within EBNA2-dependent open versus closed chromatin regions. For example, Ets-like (including SPI1), TCF, and E-box motifs are much more strongly enriched in open regions, whereas ZBED, SOX, and several C2H2 zinc finger motifs are much more strongly enriched in closed regions . These results collectively reveal an important role for EBNA2 in genome-wide alteration of human chromatin accessibility.We next tested the hypothesis that EBNA2 up-regulates genes by opening chromatin and down-regulates genes by closing chromatin. We used RELI to examine the significance of the intersection of EBNA2-dependent ATAC-seq peaks and genomic loci harboring EBNA2 DEGs. As expected, these analyses revealed that EBNA2-dependent open chromatin regions tend to fall proximal (within 5 kb of the TSS) to up-regulated EBNA2 DEGs , further supporting a possible role for EBNA2 in chromatin looping alteration. To further elucidate the impact of EBNA2 on chromatin looping across the human genome, we next performed HiChIP-seq with an antibody against H3K27ac in the uninfected, EBVEBNA2−, and EBVEBNA2+ conditions (Methods).Previous reports support a role for EBNA2 in regulating the three-dimensional structure of chromatin looping . Yet, EBtrans interactions were between 8.8% and 9.9% of the sequenced pairs , similar to the results obtained in the original HiChIP study . The full quantification of each chromatin looping event and comparisons of these events between conditions are provided in Supplemental Table S9. As expected, EBVEBNA2+ looping interactions significantly align  (Methods) with data from a previously published Hi-C experiment performed in GM12878 cells  . ChIP-seq peaks obtained from relevant cell types for POL2RA and chromatin looping factors such as CTCF, RAD21, and YY1 significantly intersected with HiChIP loop anchors, along with active chromatin marks and DNase peaks . Collectively, these results indicate that our HiChIP-seq data are of high quality.Analysis of the HiChIP data revealed 93,354, 131,296, and 136,689 chromatin looping interactions in the three conditions, respectively. QC analyses using HiC-Pro  indicateIP study . Likewis78 cells . FurtherSupplemental Table S9). As expected, EBNA2-dependent “loop gains” intersect much more significantly with the promoters of up-regulated EBNA2 DEGs than with the promoters of down-regulated EBNA2 DEGs. Likewise, EBNA2-dependent “loop losses” significantly intersect with down-regulated EBNA2 DEGs, but not up-regulated EBNA2 DEGs . These findings support a model in which EBNA2-induced promoter interactions increase gene expression levels, whereas EBNA2-induced loss of promoter interactions decreases gene expression levels. In total, 102 newly formed EBNA2-dependent loops fall within the promoters of 70 EBNA2 DEGs (45 up-regulated and 25 down-regulated genes) . EBNA2-dependent gains and losses in chromatin looping were highly enriched for EBNA2 ChIP-seq peaks  . EBNA2-dependent gains in chromatin looping were enriched for EBNA2-dependent chromatin accessibility gains  but not losses . Likewise, EBNA2-dependent losses in chromatin looping were enriched for chromatin accessibility losses , but not gains  . Fifty-eight EBNA2-dependent ATAC-seq peaks are in the promoters of EBNA2 DEGs (TSS ± 5kb). Of those, 43 EBNA2-dependent ATAC-seq peaks are within Ramos EBVEBNA2+ loop anchors that loop to within 20 kb of an EBNA2 DEG. Collectively, these results indicate strong agreement between EBNA2 binding and EBNA2-dependent changes in chromatin accessibility, chromatin looping events, and gene expression. For example, we identified strong EBNA2 ChIP-seq peaks and highly EBNA2-dependent open chromatin in the promoter region of NAALADL2-AS2, an uncharacterized antisense long noncoding RNA that is the most highly up-regulated EBNA2 DEG , identifying 1432 and 311 looping events that are significantly stronger or weaker in the presence of EBNA2, respectively . Among these, 20 and 19 EBNA2 DEGs are located within SLE and MS-associated loci, respectively. In contrast, EBNA2-independent EBV DEGs  do not coincide with autoimmune disease risk loci , suggesting an EBNA2-specific, rather than an EBV-specific, role in autoimmune gene regulatory mechanisms.Previous studies  have nomSupplemental Table S12). Rather, we observed enrichment for autoimmune-associated variants within EBNA2-dependent chromatin closing events  . These results prompted us to further examine the relationship between chromatin accessibility and autoimmune risk loci. To this end, we examined the significance of the intersection between autoimmune-associated variants and several types of ATAC-seq peaks: (1) constitutively open chromatin regions ; (2) EBV-dependent peaks (gains and losses); (3) EBVEBNA2−-dependent peaks (gains and losses); and (4) EBVEBNA2+-dependent peaks (gains and losses). These analyses revealed significant intersection between autoimmune-associated variants and regions of the genome that are constitutively open in B cells . These results indicate that autoimmune risk variants also concentrate in regions of the genome that are already accessible for the binding of EBNA2 and other proteins before infection. Collectively, these analyses suggest that accessible chromatin before EBV infection and EBNA2-dependent chromatin closing are significant components of autoimmune risk loci but EBNA2-dependent opening of chromatin is not.Next, we inspected EBNA2-dependent chromatin accessibility regions for disease enrichment. Contrary to expectation, despite the vast changes in chromatin accessibility orchestrated by EBNA2, we did not identify significant intersection between EBNA2-dependent chromatin opening events and autoimmune-associated variants  . EBNA2-dependent loss of chromatin looping also shows significant intersection with autoimmune variants, albeit to a much lesser degree . Collectively, these data indicate that EBNA2-dependent alteration of long-range chromatin interactions is highly associated with autoimmune-associated genetic variants, revealing a key role for EBNA2-altered chromatin interactions in autoimmune disease etiology.Finally, we investigated the relationship between EBNA2-altered chromatin looping interactions and autoimmune disease risk loci. This analysis revealed highly significant intersection between EBNA2-induced changes to chromatin looping and autoimmune disease risk loci. In particular, 125 newly established EBNA2-dependent chromatin loop anchors intersect autoimmune-associated variants  , including validation in Ramos cells of the allele-dependent EBNA2 binding we previously observed for rs3794102 at the CD44 locus in Mutu cells (EBNA2+ Ramos cells to an MS-associated variant (rs1250567) located in the ZMIZ1 locus  and whole blood  . ZMIZ1 expression levels are threefold lower in EBVEBNA2+ Ramos cells compared to uninfected Ramos cells, consistent with a previous report by ZMIZ1 locus reveal EBNA2 and autoimmune risk allele-dependent mechanisms possibly underlying the established roles played by genetics and the environment in autoimmune diseases.Using this approach, we discovered 32 instances of allele-dependent EBNA2 binding at autoimmune risk variants . Among these, 24 are involved in allele-dependent EBNA2 binding, 41 are located within the promoters (TSS ± 500 bp) of EBNA2 DEGs, 46 are located within EBNA2-dependent chromatin accessibility regions, and 539 are located within EBNA2-dependent chromatin looping events. Fourteen of these variants involve multiple EBNA2-dependent mechanisms. For example, the inflammatory bowel disease–associated rs630923 variant is located within the promoter of the CXCR5 gene, which has EBNA2-dependent lower gene expression. This promoter also shows EBNA2-dependent loss of chromatin looping, providing a possible mechanism underlying the lowered expression of CXCR5 . Collectively, these data implicate EBNA2 in multiple types of autoimmune disease gene regulatory mechanisms.In total, 633 unique autoimmune-associated genetic variants can be implicated in at least one EBNA2-dependent mechanism  interacting with promoters and enhancers; (2) altering chromatin accessibility; and (3) forming new chromatin 3D interactions. We show that many of these mechanisms significantly intersect autoimmune-associated genetic variants, and we identify multiple EBNA2 interactions that are autoimmune risk allele-dependent.EBNA2− and EBVEBNA2+ cells compared to uninfected cells. These genes and elements are altered by EBV infection independently of EBNA2 and hence might involve other EBV-based regulatory molecules, such as Zta, Rta, or the EBNA3 proteins , which is expressed across lymphocyte subsets and has been shown to interact with the EBNA2 coactivator EBNA-LP  family, including patterns , with SLet genes . SLAM faet genes , developet genes , and celet genes . Our newZMIZ1 locus, we present genotype-dependent binding of EBNA2 at a disease risk variant, looping of the genetic variant to the “short isoform” promoter of ZMIZ1, EBNA2-dependent expression of the ZMIZ1 “short isoform,” and allele-dependent expression of ZMIZ1 as a function of the disease risk variant genotype. Although these results are consistent with our conclusion of EBNA2-dependent allelic expression of ZMIZ1, it is possible that additional gene regulatory mechanisms might also mediate the expression of ZMIZ1 and contribute to disease.At the A limitation of our study design is that P3HR-1 and B95.8 strains are different genetic isolates of EBV. Therefore, there are further genetic differences between the strains in addition to the EBNA2 deletion. For example, the last two exons (45 amino acids) of EBNA-LP are also deleted in the P3HR-1 virus. However, EBV mutants with these two exons deleted can still transform primary B cells, albeit at lower frequencies . FurtherIn summary, our findings reveal an important role for the EBV-encoded EBNA2 regulatory protein in multiple mechanisms that ultimately affect human gene expression levels. Several of these mechanisms are allele-dependent at variants associated with autoimmune diseases such as MS and SLE. It is possible that other viral transcriptional regulators play similar mechanistic roles in other diseases. Future studies will deepen our knowledge of the mechanisms underlying virus–host interactions, and ultimately they will provide both a rationale and a foundation for therapeutic approaches targeting these interactions.EBNA2+) and P3HR-1 EBV lacking EBNA2 (EBVEBNA2−) were prepared from cell supernatants and cultured in 10% FBS supplemented RPMI-1640 medium for 2 wk. Viral suspension was filtered via 0.45 µm Millipore filters and cells were pelleted. The concentrated viral stocks were stored at −80°C. 2 × 106 Ramos cells  were infected with 1 mL viral stock based on infection optimization assays and incubated for 4 h for virus adsorption. After infection, cells were washed and cultured. After 10 passages, we confirmed the infection by morphological changes and the expression of EBNA2 viral protein levels as previously published .RNA was extracted, sequenced, and analyzed using standard methods (EBNA2+) and GM12878 cell lines, using standard experimental procedures . The resulting libraries were sequenced targeting 100,000,000 unique single-end reads. We performed quality control of raw sequencing reads using FastQC (version: 0.11.2) (http://www.bioinformatics.babraham.ac.uk/projects/fastqc). All data were confirmed to pass all quality control checks , except for adapter sequence contents, which were removed using cutadapt   for EBNA2 binding partner RBPJ. We observed strong agreement between experimental replicates . Motif enrichment for established EBNA2 partners RBPJ, EBF1, and SPI1 was slightly stronger in peaks called by pooling experimental replicate reads compared to peaks obtained using an ENCODE-like IDR-based method . We thus pooled the reads between the replicates and re-called peaks using MACS2 to capture as many EBNA2 binding events as possible. We also obtained publicly available EBNA2 ChIP-seq data sets  and called peaks using MACS2 with the following arguments: -g hs -q 0.01.ChIP-seq for EBNA2 was performed in duplicate in Ramos (EBV: 0.4.2) . Alignme: 0.4.2) . Peaks w: 0.4.2)  with theSupplemental Methods). FastQC was used to perform quality control (http://www.bioinformatics.babraham.ac.uk/projects/fastqc) as described above. ATAC-seq reads were aligned to the human genome (hg19) using Bowtie 2 . We thus pooled the reads between the replicates and re-called peaks using MACS2 to capture as many open chromatin regions as possible. Differential chromatin accessibility was calculated using the MAnorm program  using MAnorm. Next, to obtain EBNA2-dependent regions, we subtracted EBVEBNA2−- unique peaks compared to uninfected using the BEDTools subtract command. To identify EBNA2-dependent closed chromatin, we first determined EBV-dependent closed chromatin by identifying uninfected-unique peaks compared to EBVEBNA2+ peaks, then we subtracted the uninfected-unique peaks compared to EBVEBNA2− peaks. Finally, we used the IDR approach (IDR threshold of 5%)   to identEBNA2− and EBVEBNA2+ conditions. We used HiC-Pro (version: 2.11.0) to align reads and identify the Hi-C contact map . Background correction, restriction site bias modeling, and looping interaction identification were performed using hichipper  . We thus pooled the reads between the replicates and reanalyzed the data as described above. We identified differential looping events using the diffloop Bioconductor R package (1.6.0) . Likewise, we also compared EBVEBNA2− looping events to uninfected looping events using the same cutoffs. Then to identify EBNA2-dependent looping events, we applied diffloop again, comparing EBVEBNA2+-dependent looping events to EBVEBNA2−- dependent looping events, using the same cutoffs.HiChIP libraries were prepared following tact map . HiC-Prochipper) . We obse (1.6.0) . BrieflyEBNA2+ condition and looping data from GM12878 cells , 99% peak agreement (ATAC-seq), 97% anchor agreement (HiChIP), and 90% shared expressed genes (RNA-seq). Peak and loop calls are available on the study GEO page for both hg19 and hg38 coordinates.P-value < 0.05, Bonferroni correction), following our standard practices.We used the RELI method to calculate the significance of intersection between the genomic coordinates of two or more data sets. In brief, RELI calculates the overlap between the input genomic coordinates and a library of ChIP-seq-derived genomic coordinates. It then permutes the input coordinates to calculate the overlap between randomized coordinates and the library coordinates. After 2000 iterations, RELI then compares the randomized overlap to the actual overlap and calculates the significance of this overlap . We usedAllele-dependent behavior was identified in sequencing reads using the MARIO pipeline . BrieflyWe used the HOMER software package  to calcu−8 was used to establish the statistical significance of a variant and its association to a given disease or trait. After filtering for genome-wide association, variants were grouped based on the disease or trait reported in the publication as well as the reported ancestries. For each disease or trait, independent loci were identified using LD-based pruning in PLINK (r2 < 0.2).We obtained GWAS data from multiple studies from the NHGRI-EBI GWAS catalog  . A genomin PLINK  (window https://www.ncbi.nlm.nih.gov/geo/) under accession number GSE148396. A UCSC Genome Browser session for the hg19 genome build is available at http://genome.ucsc.edu/s/ledsall/2021_EBNA2. Source code for RELI and MARIO are available on GitHub  and as Supplemental Code.All raw and processed sequencing data generated in this study have been submitted to the NCBI Gene Expression Omnibus (GEO;


Nuclear factor erythroid 2‐related factor (Nrf2) is an important regulator of cellular antioxidant defence. We previously showed that SFN prevented Ang II‐induced cardiac damage via activation of Nrf2. However, the underlying mechanism of SFN’s persistent cardiac protection remains unclear. This study aimed to explore the potential of SFN in activating cardiac Nrf2 through epigenetic mechanisms. Wild‐type mice were injected subcutaneously with Ang II, with or without SFN. Administration of chronic Ang II‐induced cardiac inflammatory factor expression, oxidative damage, fibrosis and cardiac remodelling and dysfunction, all of which were effectively improved by SFN treatment, coupled with an up‐regulation of Nrf2 and downstream genes. Bisulfite genome sequencing and chromatin immunoprecipitation (ChIP) were performed to detect the methylation level of the first 15 CpGs and histone H3 acetylation (Ac‐H3) status in the Nrf2 promoter region, respectively. The results showed that SFN reduced Ang II‐induced CpG hypermethylation and promoted Ac‐H3 accumulation in the Nrf2 promoter region, accompanied by the inhibition of global DNMT and HDAC activity, and a decreased protein expression of key DNMT and HDAC enzymes. Taken together, SFN exerts its cardioprotective effect through epigenetic modification of Nrf2, which may partially contribute to long‐term activation of cardiac Nrf2. The RAS is traditionally regarded as a circulating endocrine system. It is now demonstrated that local RAS is an autocrine‐paracrine system that exists in nearly every target tissue throughout the body, including kidney,Ang II has blood pressure‐dependent effects that induce hypertension, leading to left ventricular hypertrophy reflected by the accumulation of extracellular matrix and increased cardiomyocyte size.The nuclear factor erythroid 2‐related factor (Nrf2) is a major transactivator of hundreds of cytoprotective genes, which have cytoprotective functions through restoring redox balance and repairing cell damage caused by exposure to stress conditions (such as oxidative or electrophilic stress). These cytoprotective genes encoding detoxification enzymes, such as NAD(P)H:quinone reductase (NQO1), haem oxygenase 1 (HO‐1), catalase (CAT), glutamate‐cysteine ligase (GCL), glutathione S transferase (GST), UDP‐glucuronosyltransferases (UGT) and superoxide dismutase (SOD).2O2–induced cellular oxidative damage and reduces ROS production, accompanied by an up‐regulation of Nrf2.Sulforaphane (SFN) is a naturally occurring isothiocyanate compound extracted from broccoli sprouts. Reportedly SFN with multiple pharmacological activities exerts anticancer, anti‐inflammatory, antioxidant and neuroprotective effects.Accumulating studies suggest that SFN, as a potent activator of Nrf2, exhibits the antioxidant ability by up‐regulating Nrf2‐mediated cytoprotective genes expression through modification of Keap1 cysteine residues, activation of MAPK, phosphatidylinositol 3‐kinase (PI3K) and protein kinase C (PKC) pathways, which results in the phosphorylation, nuclear accumulation and increased transcription and stability of Nrf2.22.1Male C57/BL mice at the age of 8 weeks were purchased from Beijing Experimental Animal Technical Co. LTD. The mice were housed in the Animal Center of Jilin University . All animal procedures were approved by the Animal Care and Use Committee of the Chinese Academy of Medical Sciences . The mice were randomly divided into four groups (n = 5‐7 each group), namely control group, SFN group, Ang II group and Ang II plus SFN (Ang II/SFN) group according to our previous method.Dose of SFN (0.5 mg/kg) was selected based on our and other publications.2.2Echocardiography (Echo) was performed to measure heart function for mice anaesthetized with Avertin  by using a high‐resolution imaging system suitable for small animals , equipped with a high‐frequency ultrasound probe (RMV‐707B). Echo analyses include chamber dimensions and cardiac function.2.3A high‐speed tissue homogenizer was used to lyse heart tissues in ice‐cold 1× RIPA buffer  supplemented with protein inhibitors . Use 4% to 15% SDS‐polyacrylamide gel electrophoresis (SDS‐PAGE) to separate proteins and transfer them to PVDF membrane (Millipore). After blocking with 5% BSA, incubate the membrane with specific primary antibodies from Santa Cruz Biotechnology at 4°C overnight, including CAT (1:500), HO‐1 (1:500), CTGF (1:1000), 3‐NT (1:1000), 4‐HNE (1:1000), ICAM‐1 (1:500), IL‐1β (1:500), DNMT3a and 3b (1:1000 each), HDAC2, 3, and 5 (1:1000 each), and β‐actin (1:1000), then incubate with HRP‐conjugated secondary antibodies  for 1 hour at room temperature. The antibody‐bound proteins were detected with a Super Signal enhanced chemiluminescence (ECL) detection and Gel Documentation 2000 system (Bio‐Rad). Use Image J software to analyse the densitometry of the bands.2.4The total RNA was extracted from the hearts of mice using Trizol reagent (Invitrogen). According to the manufacturer's instructions, first‐strand cDNA was synthesized from total RNA using the SuperScript III First‐Strand Synthesis System (Invitrogen). The mRNA expression level was qualified using the first‐strand cDNA as a template by quantitative real‐time PCR (qPCR) with Power SYBR Green PCR Master Mix (Applied Biosystems), and GAPDH was used as an internal loading control. The following primer sequences for Nrf2, HO‐1, CAT, GAPDH were used: Nrf2, 5′‐TCACACGAGATGAGCTTAGGGCAA‐3′ (sense) and 5′‐TACAGTTCTGGGCGGCGACTTTAT‐3′ (antisense); HO‐1, 5′‐CCTCACTGGCAGGAAATCATC‐3′ (sense) and 5′‐CCTCGTGGAGACGCTTTACATA‐3′ (antisense); CAT, 5′‐GGAGGCGGGAACCCAATAG‐3′ (sense) and 5′‐GTGTGCCATCTCGTCAGTGAA‐3′ (antisense); GAPDH, 5′‐TCAACAGCAACTCCCACTCTTCCA‐3′ (sense) and 5′‐ACCCTGTTGCTGTAGCCGTATTCA‐3′ (antisense).2.5After the mice were weighed and sacrificed, the hearts were taken out and weighed. Then calculate the ratio of heart to body weight. The heart tissues were fixed in 10% formalin, embedded in paraffin and cut into 5 µm thickness. The heart sections were deparaffinized and rehydrated in a graded alcohol series, then subjected to Masson's Trichrome staining for collagen deposition.2.6Genomic DNA was extracted from the heart tissues of mice using a QIAamp DNA Mini kit (Qiagen). Then, bisulfite was used to convert DNA with an EZ DNA Methylation‐Gold Kit (Zymo Research Corp.). TA cloning was performed to amplify the converted DNA using Platinum Taq DNA Polymerase (Invitrogen) with primers that amplify the first 15 CpGs located between −1226 and −863 of the murine Nrf2 gene with the translation initiation site (TIS) defined as position 1.2.7Total DNMT/HDAC activity was assayed with a fluorometric EpiQuick™ DNMT/HDAC Activity/Inhibition Assay kit (Epigentek). The fluorescence density was read at 405 nm by using a Tecan microplate reader plate reader.2.8g. Using a dilution buffer to dilute the chromatin solutions, 10 μL of each sample was used as input control. Diluted chromatin solutions were then immunoprecipitated with protein A magnetic beads and anti‐acetyl‐Histone 3  antibody or non‐specific Rabbit IgG. The solutions were then slowly shaken overnight at 4°C. Then a magnetic separator was used to collect the immunoprecipitated complex‐magnetic beads. The purified DNA solution was used for regular PCR amplification using the primers: 5′‐TGAGATATTTTGCACATCCGATA‐3′ (sense) and 5′‐ACTCTCAGGGTTCCTTTACACG‐3′ (antisense), which covers the DNA sequence of the first 15 CpGs of murine Nrf2.Chromatin immunoprecipitation (ChIP) assay was performed following the manufacturer's protocol. In short, heart tissues were cross‐linked with formaldehyde to a final concentration of 1% at room temperature for 15 min, then add 2.5 mol/L glycine to formaldehyde and incubate for 10 min. After washing twice with PBS, resuspend the tissues in Lysis Buffer containing a mixture of protease inhibitors, lyse with a homogenizer and sonicate to produce the 200‐500 bp DNA fragments in ice‐cold water. The samples were centrifuged at 13 500 2.9P < .05.Data are represented as means ± SD values (n = 5‐7 per group). The data analyses were performed using two‐way analysis of variance (ANOVA) or Tukey's test with Origin 7.5 software (OriginLab Corporation). Differences were considered statistically significant when 33.1To confirm the protective effect of SFN for cardiac dysfunction caused by Ang II, Echo was performed to detect cardiac function in mice. We found that 2‐month Ang II administration caused cardiac remodelling and dysfunction, defined by enhanced LVID and LVPW, and reduced EF and FS Figure . The car3.2It has been confirmed that the inflammatory response and oxidative stress involve the pathogenesis of cardiac damage caused by Ang II.Reportedly cardiac fibrosis, remodelling and dysfunction is often the ultimate consequence of cardiac inflammation and oxidative damage caused by Ang II.3.3To confirm the indispensable role of Nrf2 in the SFN‐mediated prevention of cardiac damage induced by Ang II, we next detected the transcription capacity of Nrf2, demonstrated by CAT and HO‐1 expression. Compared to the control group, Ang II injection did not cause a obvious difference in the transcriptional and protein expression of Nrf2, CAT and HO‐1, but SFN treatment significantly increased the Nrf2 expression and its downstream antioxidant genes expression, which was much higher in the Ang II/SFN group than that in the Ang II group Figure . These r3.4Transcription activation of Nrf2 is inhibited due to the hypermethylation of the first 15 CpGs at the Nrf2 gene promoter in a TRAMP mouse model and JB6 P+ cells.DNA methylation is mediated by DNMTs, mainly including DNMT1 (maintenance methyltransferase) and DNMT3a and 3b (de novo methyltransferases). We next examined the expression and activity changes of DNMTs in the cardiac tissues of mice. Ang II treatment greatly increased the protein expression of DNMT3a and 3b and the global DNMT activity Figure , which w3.5Histone modification is also an important epigenetic modification, and numerous reports have shown that SFN is an inhibitor of HDACs.4Our previous study has demonstrated that SFN exhibits its preventive effect for Ang II‐induced cardiomyopathy via activating Nrf2.Oxidative stress is a key contributor to cardiomyopathy induced by Ang II.Nrf2, as a transcription factor, enhances the capacity of endogenous antioxidant defence against oxidative damage.Growing evidence suggests that natural phytochemicals such as SFN, curcumin, angelica and tocopherol can reactivate the silenced Nrf2 gene by epigenetic modification, including DNA methylation and histone modification.To elucidate the mechanism accounting for the lasting activation of Nrf2 by SFN in the hearts of mice, we detected the CpGs methylation status located in the Nrf2 promoter region by bisulfite sequencing. To a large extent, these CpGs determine the transcription of Nrf2.While DNA methylation and histone acetylation can independently regulate gene expression, several studies indicate that they may interact with each other to establish and maintain different chromatin states.Under basic conditions, Keap1, a negative regulator of Nrf2, mediates the ubiquitination and proteasome degradation of Nrf2.The modulation mechanism of Nrf2 signalling pathway can be roughly summarized as Keap1‐dependent and Keap1‐independent manners. The direct modification of Keap1 cysteine residues by SFN is considered as the Keap1‐dependent model, which inhibits Nrf2 polyubiquitination and degradation, translocating Nrf2 to nucleus.5Current research indicates that SFN can protect against cardiac damage induced Ang II through up‐regulation of Nrf2 and its downstream antioxidative genes CAT, HO‐1. Furthermore, SFN can activate the Nrf2 gene under Ang II treatment through epigenetic modification, including DNA methylation and histone acetylation, by reducing the methylation level and enhancing the Ac‐H3 accumulation in the Nrf2 promoter region through the inhibition of DNMTs and HDACs. The epigenetic mechanism can partially contribute to the persistent long‐acting activation of Nrf2 by SFN in the heart Figure .The authors report no conflicts of interest in this work.Xuling Su: Data curation ; Formal analysis ; Methodology ; Writing‐original draft (lead). Shudong Wang: Investigation ; Methodology ; Software . Haiying Zhang: Data curation ; Investigation ; Methodology ; Software . Ge Yang: Data curation ; Methodology ; Software . Yang Bai: Data curation ; Investigation ; Methodology ; Software ; Visualization . Pinyi Liu: Data curation ; Formal analysis ; Methodology ; Writing‐original draft . Lingbin Meng: Conceptualization ; Investigation ; Project administration ; Writing‐review & editing . Xin Jiang: Conceptualization ; Project administration ; Writing‐review & editing . Ying Xin: Funding acquisition ; Project administration ; Supervision ; Visualization ; Writing‐review & editing .


Here, we for the first time directly investigated the influence of LitR on gene expression in the luxI/R QS system of psychrophilic bacteria Aliivibrio logei. Investigated promoters were fused with Photorhabdus luminescens luxCDABE reporter genes cassette in a heterological system of Escherichia coli cells, litR A. logei was introduced into the cells under control of Plac promoter. LitR has been shown to upregulate genes of autoinducer synthase (luxI), luciferase and reductase (luxCDABE), and this effect doesn’t depend on presence of luxR gene. To a much lesser degree, LitR induces luxR1, but not the luxR2 — the main luxI/R regulator. Enhanced litR expression leads to an increase in a LuxI-autoinducer synthesis and a subsequent LuxR-mediated activation of the luxI/R QS system. Effect of LitR on luxI transcription depends on lux-box sequence in luxI promoter even in absence of luxR (lux-box is binding site of LuxR). The last finding indicates a direct interaction of LitR with the promoter in the lux-box region. Investigation of the effect of LitR A. logei on luxI/R QS systems of mesophilic Aliivibrio fischeri and psychrophilic Aliivibrio salmonicida showed direct luxR-independent upregulation of luxI and luxCDABE genes. To a lesser degree, it induces luxR A. fischeri and luxR1 A. salmonicida. Therefore, we assume that the main role of LitR in cross-interaction of these three QS systems is stimulating the expression of luxI.LitR is a master-regulator of transcription in the  StructuxR2-luxI  (FidopiaxR2-luxI .luxR and luxI genes constitute a quorum sensing (QS) system. luxI-luxR system provides regulation of the expression of lux regulon genes (cell luminescence control) in dependence on cell culture density  concentration decrease detected at late growth phase , which have other lux regulon structure. Previously it was supposed that A. salmonicida LitR could activate the luxR1, luxR2 and luxI genes promoters .litR gene expression (plasmid p15Tc-litR) was made at the early exponential growth phase (OD about 0.1) by addition of IPTG  to a final concentration of one mM.Bacteria were grown in Luria-Bertani (LB) broth. The liquid LB medium was composed of 1% tryptone, 0.5% yeast extract and 0.5% NaCl, for the preparation of solid medium, agar was added to a final concentration of 1.5% m/v. The medium was supplemented with appropriate antibiotics: 100 μg/ml ampicillin, 10 μg/ml tetracycline, or their composition. Overnight cultures were grown at 37 °C with continuous agitation and then were used to inoculate liquid LB. The resulting cultures were grown at 22–25 °C with continuous agitation (200 rpm). The optical density (OD) of cell suspensions was measured with a KFK-3 photometer . An induction of the Plasmid DNA was isolated by alkaline lysis. Cell transformation with hybrid plasmids followed standard protocols. Endonuclease restriction, DNA fragment ligation, agarose gel electrophoresis and isolation of DNA fragments from agarose gel were performed according to Isopropyl β-D-1-thiogalactopyranoside (IPTG) was from Anatrace (USA).Bioluminescence intensities were measured in volume of 200 μl in plastic microtubes placed in front of a photomultiplier photocathode at room temperature using Biotox-7MB  or in 96-well plates using SynergyHT . Luminescence values were expressed in relative light units (RLU).lux regulons of the Aliivibrio genus bacteria was investigated for three species: A. fischeri, A. logei and A. salmonicida. For this purpose we used the heterological system of E. coli cells transformed with different biosensor plasmids from a set , one can find description of VL-XT, VSL2, VL3 and Charge-Hydropathy algorithms in Amino acid sequences alignment was performed using the Vector NTI software . The intrinsic disorder of proteins was predicted by their aa sequences with “Predictor of Naturally Disordered Regions” , luciferase and reductase (luxCDABE) genes are given in Aliivibrio species were tested with LitR A. logei in the heterological system of E. coli cells.Results of investigation of LitR E. coli MG1655 cells carrying combinations of the plasmid p15Tc-litR (litR under Plac) with different biosensor plasmids containing P. luminescens luxCDABE genes under control of PluxI and PluxCDABEG promoters of A. logei  activation of expression from the A. fischeri PluxICDABEG promoter. This effect has not been previously described and may indicate that litR is able to induce LuxI/R QS system through stimulation of both luxI and luxR gene expressions in A. fischeri cells. Moreover, the effect on luxI expression was higher in magnitude. The data obtained contradicts the results published by . To assess the effect of the lux-box sequence on the transcription activation of A. logei PluxCDABEG by the LitR protein, we compared the LitR-dependent activation of two variants of the A. logei PluxCDABEG promoter, which differ only in the lux-box sequences . The observed induction of PluxI and PluxCDABEGA. logei and consequently luminescence of cells is completely determined by the litR gene: in its absence , the luminescence of cells does not change in response to the addition of IPTG. The data in lux-box sequence affects the activation of the promoter by LitR. This indicates that the LitR binding site in the promoter region of A. logei luxCDABEG genes coincides or at least intersects with the LuxR binding site.As can be seen from the data presented in luxR-luxI regulon could be used for development of expression systems with target protein expression regulation in dependence on bacterial population density  transcription from promoters of luxI and luxCDABEG genes and to a lesser degree from promoter of luxR1 gene. Thus, 3-OH-C10 and AI-2 in the medium induce expression of litR and could enhance luxI expression and 3-O-C6 synthesis (luxI/R system to be significant, the conditions are needed preventing this system from normal function. Our finding could be a clue to understanding the cross-interaction of the luxS/PQ and ainS/R QS systems with the luxI/R one in psychrophilic bacteria of A. logei and A. salmonicida species. Such effect was not previously described for the luxI/R QS system of well-known mesophilic bacteria A. fischeri. Main effect of this cross-interaction is LitR-dependent stimulation of autoinducer synthesis through PluxI induction and this pathway could be activated in conditions, when luxI/R QS system was not activated by its own AI until high culture density.The results of this study demonstrated that LitR of psychrophilic bacteria ynthesis . But for10.7717/peerj.12030/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.12030/supp-2Supplemental Information 2E. coli cells transformed promoter-reporter plasmids in combination with plasmid carrying litR A. logei gene under control of Plac promoter. Luminescence was used for calculation of induction coeffitients.Measurements of luminescence of Click here for additional data file.10.7717/peerj.12030/supp-3Supplemental Information 3Click here for additional data file.


During the last 30 years, our understanding of the development and diversification of postganglionic sympathetic neurons has dramatically increased. In parallel, the list of target structures has been critically extended from the cardiovascular system and selected glandular structures to metabolically relevant tissues such as white and brown adipose tissue, lymphoid tissues, bone, and bone marrow. A critical question now emerges for the integration of the diverse sympathetic neuron classes into neural circuits specific for these different target tissues to achieve the homeostatic regulation of the physiological ends affected. According to the Danish anatomist Winslow, the great sympathetic or intercostal nerve is the principal means of bringing about the sympathies of the body Winslow . In the Here, we review key data on the importance of the sympathetic nervous system in homeostatic regulation of a range of crucial processes ranging from heartbeat and organ perfusion to water and mineral balancing, regulation of body temperature and metabolic expenditure, and the balancing of the immune system and skeletal health. We discuss the importance of target-selective sympathetic pathways to selectively regulate the different tissues involved in these processes. And we consider the developmental mechanisms for the generation of this cellular system and its integration into target-directed neural circuits.From the “sympathies of the body” Winslow , the “coFor a long time, the sympathetic/parasympathetic antagonism on heart activity and the sympathetic control of the vascular system constituted the prime focus of this analysis. With the increased interest in metab olic control and glucose homeostasis, the growing problem of adiposity and its linkage to cardiovascular and renal disorders, as well as the tight link with immune homeostasis and the interaction between nervous and immune system, this focus has been considerably extended. A detailed knowledge of the sympathetic efferent innervation of a wide range of target tissues from blood vessels, endocrine, and adipose tissue to lymphoid tissue and bone marrow widens our view of sympathetically regulated systems. An ever-increasing body of experimental and clinical studies reveals how deeply the sympathetic nervous system (SNS) intervenes in the most diverse aspects of bodily functions Fig. ; Table 1During the late nineteenth century, the opposing action of the vagal and sympathetic nerve supply on heart activity  emerged as a promising tool for studying autonomic control and its central regulation  on renal blood flow and glomerular filtration rate, tubular sodium, and water reabsorption, as well as renin release in humans, are analyzed in great detail , mechanisms of RSNA on sodium reabsorption and renin secretion were integrated  network critically involving PHOX2b, HAND1 and HAND2, and GATA2 and GATA3, linked by cross regulatory capacity Rohrer . NotablyImportantly, in noradrenergic neuroblastoma cell lines, super enhancers that govern gene expression programs are associated with gene loci for the TFs PhOX2a and 2b, HAND2, and GATA2 and 3 . Since this molecular class has been involved in multiple steps of neural circuit formation based on homophilic interactions (Kim et al. In studies on genome-wide RNA profiles of mouse CHAT-positive spinal cord neurons including preganglionic sympathetic neurons, 16 classes of molecularly distinct preganglionic neuron types could be distinguished (Blum et al. The amazing advancements in understanding of the cellular structure and developmental generation of the sympathetic nervous system now allows to investigate the generation of target-specific efferent pathways. The identification of the molecular players regulating formation of the appropriate synaptic connections in sympathetic ganglia is one of the most exciting imminent questions regarding the development of the autonomic nervous system. At the same time, autonomic dysfunctions and alterations in the development of the sympathetic nervous system remain an immense or even increasing clinical challenge.The urgent need for new therapies for childhood tumors (Zafar et al. The diverse spectrum of disease-associated autonomic dysfunctions is the subject of autonomic medicine (Goldstein