Herein we identify three putative G4-forming sequences (G4FS) within the KRAS gene, explore their G4 formation, and develop oligonucleotides targeting these three regions and the G4mid forming sequence. We tested Polypurine Reverse Hoogsteen hairpins (PPRHs) for their effects on KRAS regulation via enhancing G4 formation or displacing G-rich DNA strands, downregulating KRAS transcription and mediating an anti-proliferative effect. Five PPRH were designed, two against the KRAS promoter G4mid and three others against putative G4FS in the distal promoter, intron 1 and exon 5. PPRH binding was confirmed by gel electrophoresis. The effect on KRAS transcription was examined by luciferase, FRET Melt2, qRT-PCR. Cytotoxicity was evaluated in pancreatic and ovarian cancer cells. PPRHs decreased activity of a luciferase construct driven by the KRAS promoter. PPRH selectively suppressed proliferation in KRAS dependent cancer cells. PPRH demonstrated synergistic activity with a KRAS promoter selective G4-stabilizing compound, NSC 317605, in KRAS-dependent pancreatic cells. PPRHs selectively stabilize G4 formation within the KRAS mid promoter region and represent an innovative approach to both G4-stabilization and to KRAS modulation with potential for development into novel therapeutics.KRAS is a GTPase involved in the proliferation signaling of several growth factors. The KRAS gene is GC-rich, containing regions with known and putative G-quadruplex (G4) forming regions. Within the middle of the G-rich proximal promoter, stabilization of the physiologically active G4 KRAS gene abolish inherent GTP hydrolysis; these mutations render the protein constitutively active. The highest incidence of mutational activation occurs in lung, colorectal, and >95% of pancreatic cancers . As PPRH 1 and PPRH 2 both target the same G4-forming region stabilized by 317605, combinations of SCR, PPRH 1 or PPRH 2 and the small molecule were examined in the AsPc-1 cell line  significantly decreased KRAS promoter activity within 48 h of transfection and treatment B, middleell line C. Both PKRAS gene.In the present study, we sought to develop a novel means of modulating KRAS expression and/or function by targeting gene regulation with PolyPurine Reverse Hoogsteen hairpins (PPRH) targeting putative G4-forming DNA sequences within the KRAS gene. PPRHs are novel therapeutic tools that can be used for gene silencing , gene reKRAS–one distal promoter, one intronic and one exonic. Of those, we identified one region capable of forming interstrand G4 structures which have the potential to form in the pre-transcriptionally modified mRNA. PPRHs were further designed to target these three novel G-rich DNA regions independent of their G4-forming capabilities, in addition to the previously identified and described G4-forming region within the proximal promoter, G4mid , cells were plated at 3–4 × 105 cells/well in a 6 well plate and allowed to attach overnight. Cells were treated with 21 mM NSC 317605 for 72 h, lysed and RNA was harvested with the Roche GeneJet RNA isolation kit (Thermo Fisher). 500 ng of RNA was reversed transcribed into cDNA, and 1/5 of the resultant cDNA was mixed with 1× TaqMan PerfeCTa qPCR SuperMix , 1 μL of FAM-labeled KRAS probe . PCR cycling conditions were 3 min denaturation at 95 °C, followed by 40 cycles of 10 s at 95 °C and 30 s at 60 °C. Relative expression of KRAS was determined using the DDCT method from regression modeled CT values; experiments were performed in triplicate with minimally duplicate qPCR samples. One-way ANOVA with post-hoc Tukey analysis was used to determine statistical significance.To determine Statistical analyses were carried out using GraphPad Prism 9  using the tests described in the corresponding text. All data, with a minimum of three independent experiments, are shown as the mean ± SEM.


Discovering that heredity is encoded in DNA sparked relentless efforts to reveal the processes perpetuating the genetic code and deciphering how it is read and executed. Such efforts have shed light on the intricate interactions of multiprotein complexes with diverse regulatory elements in the DNA that dictate where and when genes are activated or shut down. DNA methylation, histone modification, chromatin accessibility, repetitive elements, non-coding RNAs, chromatin looping mediating interaction of distal regulatory elements, and structuring of chromatin into higher-order structures, add complexity layers to the molecular mechanisms behind transcriptional gene regulation. Genome-wide approaches applied to an ever-growing diversity of organisms, tissues, and cell types at multiple developmental stages and under different conditions are helping to progressively integrate these elements in a multilayered model of genome control. This integrative and evolving concept is now at the core of our understanding of the fundamentals of cellular identity and function, embryogenesis, homeostasis, and disease.The present Research Topic includes 14 reports on key processes and regulators of chromatin modification and three-dimensional organization of the genome in the nucleus, and their link to gene control.Penagos-Puig and Furlan-Magaril, who walk us through discovery of the processes that control chromatin folding into heterochromatin and how its repressive activities are key for transcriptional dynamics. The authors then focus on recent findings suggesting that tethering heterochromatin domains toward the nuclear lamina instructs large-scale genome organization. Finally, they discuss evidence that altering such tethering is associated with aging disorders like progeria. This highlights functions of heterochromatin in multiple cellular processes  (Lee et al., Konkova et al. They found that the length of the repeat in the 1Q12 locus determines its repositioning relative to the nucleolus toward the center of the nuclei in response to ionizing radiation. Interestingly, the authors suggest that susceptibility to oxidative stress in response to radiation favors cells with shorter repeats and higher expression of satellite DNA in lymphocytes of patients affected by schizophrenia.Repeat elements were also considered “junk DNA,” but they are important regulators of the spatial configuration of the genome (Slotkin and Martienssen, Hernández-Hernández et al. discuss a multilayered regulatory network in which myogenic transcription factors interact with histone modifiers and genome organizers like CTCF to modify chromatin accessibility and mediate the interaction of distal elements, e.g., promoter-enhancer to turn on skeletal muscle gene expression. Yuan et al. provide an overview of cardiogenic transcription factors and how their activity is coordinated via interaction with enhancers to establish the logic of cardiac cell differentiation. The authors then engage in lively discussion of the evolutionary origin of enhancers and the use of genome-wide chromatin features coupled to multi-species alignment as tools for their identification. This collection of expert contributions highlights the outstanding progress being made toward our understanding of genome regulation. Constant technological and conceptual leaps in single-cell genomics, proteomics, and mutagenesis predict a more integrative and in-depth insight into the fundamentals of life.The function of chromatin modifiers and genome architecture in developmental processes or in physiology are the subject of active research (Van Der Weide and De Wit, MF-M, the guest editor, and the co-editors KO and PD invited expert contributions and handled peer review. PD wrote the Editorial with input from MF-M and KO. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Four-stranded DNA (guanine quadruplex) can regulate many cellular processes such as replication, recombination and also gene expression. This study shows that four-stranded DNA can also affect the activity of mobile genetic elements (transposable elements). We inserted a quadruplex-forming motif into a non-coding region of the Ty1 transposable element and tested its activity in yeast cells. We found that the quadruplex inhibited Ty1 jumping. Especially when the quadruplex was formed on the transcribed strand, the amplification of the element was hindered. Our study shows that DNA conformation can tune the activity of mobile genetic elements that in turn shape eukaryotic genomes during evolution.Ty1his3-AI retrotransposon and measured the frequency of retrotransposition in yeast strains BY4741, Y00509 (without Pif1 helicase) and with G4-stabilization by N-methyl mesoporphyrin IX (NMM) treatment. We evaluated the impact of G4s on mRNA levels by RT-qPCR and products of reverse transcription by Southern blot analysis. We found that the presence of G4 inhibited Ty1his3-AI retrotransposition. The effect was stronger when G4s were on a transcription template strand which leads to reverse transcription interruption. Both NMM and Pif1p deficiency reduced the retrotransposition irrespective of the presence of a G4 motif in the Ty1his3-AI element. Quantity of mRNA and products of reverse transcription did not fully explain the impact of G4s on Ty1his3-AI retrotransposition indicating that G4s probably affect some other steps of the retrotransposon life-cycle . Our results suggest that G4 DNA conformation can tune the activity of mobile genetic elements that in turn contribute to shaping the eukaryotic genomes.Guanine quadruplexes (G4s) serve as regulators of replication, recombination and gene expression. G4 motifs have been recently identified in LTR retrotransposons, but their role in the retrotransposon life-cycle is yet to be understood. Therefore, we inserted G4s into the 3′UTR of  Guanine-rich sequences  are abundant in eukaryotic genomes, e.g., almost 150 thousand putative non-telomeric QSs were found in the maize genome . In the QSs are often present in regulatory regions of genes but we have recently found accumulation of QSs within human and plant transposable elements TEs; ,10,11). ,11. 10,1Ty1 elements, LTR retrotransposons of the Ty1/copia superfamily, belong to the most studied transposable elements in yeast. They represent a useful model system for studies of retrotransposition regulation [Ty1 element containing the HIS3 gene with an artificial intron (AI), that is spliced before reverse transcription, enables the detection of new Ty1 retrotransposition events in His3+ colonies [Ty1his3-AI has the potential for inserting additional sequences  into Ty1 and studying their effect on retrotransposition.gulation . The engcolonies . Such a Ty1his3-AI retrotransposon and studied its effect on retrotransposition in yeast as well as the impact of the G4-stabilizing drug N-methyl mesoporphyrin (NMM) and an absence of Pif1 helicase, an enzyme which unwinds quadruplexes [Ty1his3-AI retrotransposition are affected by G4 formation.Previously, we have shown the effect of LTR located G4s on a downstream reporter gene . Here, wruplexes ,20,21. WTy1his3-AI plasmid ; RRID:Addgene_62228). In order to clone the sequences of interest into the plasmid, a SacII recognition site was introduced into the Ty1his3-AI 3′UTR using a single mutagenic primer GAGGATCCCGCGGGGAGCTC and the QuikChange Multi Site-Directed Mutagenesis Kit . The mutant product was electroporated into XL-1 blue competent cells (Agilent) and the mutation was verified by SacII digestion and sequencing. The resulting plasmid pVTy1 was then digested by SacII, blunted, dephosphorylated and used for cloning annealed oligonucleotides (All plasmids were based on the pGleotides . To anneCircular dichroism and polyacrylamide gel electrophoresis (PAGE) were performed as described in . The CD UV absorption spectra were collected on a UV/VIS spectrometer Varian Cary 4000  in the range of 230–330 nm in 1 cm cells. The temperature was increased/decreased in 1 °C steps and the samples were equilibrated for 2 min before each measurement. The melting curves were expressed as the change in molar absorption MATa; ura3Δ0; leu2Δ0; his3Δ1; met15Δ0; pif1::kanMX4; EUROSCARF) were transformed by pVTy1-based plasmids  and shaken at 250 rpm at 22 °C for several days until saturation. The 8 µM NMM concentration was chosen based on literature [p ≤ 0.05) and Two-way ANOVA (for relative retrotransposition frequencies) followed by Dunnett’s multiple comparisons test (p ≤ 0.05) were performed using GraphPad Prism version 8.0 for Windows ) at a 0.05 significance level.Yeast strains BY4741 and Y00509  with 4-bromoanisole . This protocol produced RNA with only a very small amount of DNA contamination that was then completely removed by TURBO DNA-free™ Kit . Reverse transcription was done using High-Capacity RNA-to-cDNA™ Kit . RT-qPCR was performed in triplicate with Rotor-Gene Q  and SensiFAST™ 2X HRM kit , 10 ng of cDNA and 5 µM of each primer (p ≤ 0.05) to carry out multiple comparisons.For RNA isolation, 10 transformants were grown in the same way as for the transposition assay. RNA isolation was then performed in a pool containing 1 mL of each culture. Hot acidic phenol extraction protocol was used for RNA isolation , but with primer . Data anTy1 without galactose induction (glucose only) and cells without plasmid but with galactose induction . Spheroplasts were prepared by incubation of cells with 1 mg/mL of Zymolyase 20T  at 37 °C for 60 min in 0.9 sorbitol, 0.1 M EDTA containing 1 µL of ß-mercaptoethanol per mL (Sigma Aldrich) and 0.1 mg/mL RNase A (Qiagen). DNA from spheroplasts was extracted by incubation with CTAB extraction buffer for 45 min at 62 °C  followed by two chloroform extractions and isopropanol precipitation. About 3 µg of total DNA were digested by AflII and subjected to electrophoresis on 1% agarose and transferred to Hybond-N+ . The membrane was hybridized with HIS3 PCR product . Two controls were included: cells with pV product  labeled Ty1his3-AI retrotransposon system  as well as control sequences into the element. The QS originated from the LTR retrotransposon Machiavelli family found in Zea mays B73 [Following the confirmation that quadruplexes were not formed by motifs present within mays B73 , and theTy1his3-AI element . All tested oligonucleotides represented the G-rich strand and also contained an additional G-track present on the plasmid carrying  element . Both th element . M2 prod+. The CD spectra showed that NMM sped up the G4 formation however the effect on the final structures was negligible  supplemented with only 15 mM Kgligible . UV absogligible B. Absorpgligible C. The me+ which produced similar results as in lower potassium concentration , we measured the retrotransposition frequency in Saccharomyces cerevisiae strain BY4741 .As mentioned above, we cloned the QS and control sequences into the modified ne tract . Tested ne tract A. Similan BY4741 B. We obspif1::kanMX4 mutation). The inhibitory effect of both G4 stabilizing conditions on Ty1his3-AI retrotransposition is scaled in the heatmap of aligned and clustered data normalized to pVTy1 control  suggesting that the presence of G4 was critical and that G-quadruplex orientation was more important than its stability.In order to further enhance the effect of G4s, we treated the BY4741 cells with G4 stabilizing ligand NMM and and also used an isogenic strain Y00509 lacking the G4 unwinding helicase Pif1  since all other fragments originating from genomic DNA also contain a junction with surrounding chromosomal DNA and hence are longer (HIS3 gene. Such probe design ensures that the cDNAs produced from plasmid borne elements will be distinguished from endogenous (genomic) Ty1 elements.Consequently, we used Southern blot analysis to assess the effect of G4 on products of reverse transcription B–F. We ue longer B. We useTy1his3-AI elements with motif adopting G4s exhibited similar amounts of cDNA as elements not forming quadruplexes  gave rise to an additional band around 1.8 kb  where quadruplexes are close to promoters and the functional importance of G4s is strengthened by their higher number in evolutionary younger elements ,10. Nevetellites . MoreoveTy1his3-AI retrotransposon impeded retrotransposition frequency. Notably, the effect of G-rich strand orientation was more important than the stability of the G4s. Although the inhibitory effect was more pronounced when G4s were formed on the template strand for transcription and potentially could block RNA polymerase, we did not observe any correlation between Ty1his3-AI mRNA levels and the presence of G4s. It should be noted that transcription from Gal1 promoter leads to strong overexpression compared to LTR promoter [Ty1his3-AI retrotransposition since it is missing in control samples where there is neither galactose induction nor plasmid carrying Ty1his3-AI element . This could be explained by the fact that NMM treatment and pif1::kanMX4 mutation exhibited lower amounts of Ty1his3-AI mRNA compared to untreated BY4741. However, the reduction of Ty1his3-AI transcript due to the absence of Pif1 helicase was not proportional to the overall retrotransposition reduction  which indicates that the lower mRNA levels only partially explain the reduction of retrotransposition frequency. Another reason is that both pif1::kanMX4 and NMM also decreased the retrotransposition frequency of pM2+/− relative to pVTy1 , has been studied with respect to Ty1his3-AI transposition [rrm3Δ mutation leads to increased Ty1his3-AI retrotransposition and cDNA multimer insertion. These opposite effects of Rrm3p and Pif1p deficiency on Ty1his3-AI retrotransposition are puzzling, but several studies showed opposite effects on other processes that could be G4 dependent. Antagonistic roles have been shown in maintaining unidirectional replication fork barriers in rDNA genes, telomere erosion/lengthening, and rDNA recombination [Ty1his3-AI retrotransposition [Ty1his3-AI retrotransposition. However, Pif1 is a multifuncional helicase with distinct roles in replication through non-histone proteins, lagging strand replication, replication fork convergence and double strand break repair [Ty1his3-AI retrotransposition and therefore more evidence is needed.To our knowledge, there is no evidence of Pif1p regulating the position . Unlike bination . rDNA gebination  and telobination . Moreoveposition , and alsposition  and Xrn1position . It is tk repair . InhibitThere are a few studies that show that G4 can have both stimulatory and inhibitory effects on retrotransposition. For example, small-ligand stabilization of G-quadruplexes within the 3′UTR of human LINE-1 elements stimulated retrotransposition . On the Even though we used an artificial retrotransposon system, our results suggest that from the mechanistic point of view DNA conformation, namely four-stranded structures, may represent a challenge for the enzymes to go through. Since G4 formation is a cell cycle dependent and controlled process ,43, thes


Heart failure is a complex disease process with underlying aberrations in neurohormonal systems that promote dysregulated cellular signaling and gene transcription. Over the past 10 years, the advent of small-molecule inhibitors that target transcriptional machinery has demonstrated the importance of the bromodomain and extraterminal (BET) family of epigenetic reader proteins in regulating gene transcription in multiple mouse models of cardiomyopathy. BETs bind to acetylated histone tails and transcription factors to integrate disparate stress signaling networks into a defined gene expression program. Under myocardial stress, BRD4, a BET family member, is recruited to superenhancers and promoter regions of inflammatory and profibrotic genes to promote transcription elongation. Whole-transcriptome analysis of BET-dependent gene networks suggests a major role of nuclear-factor kappa b and transforming growth factor-beta in the development of cardiac fibrosis and systolic dysfunction. Recent investigations also suggest a prominent role of BRD4 in maintaining cardiomyocyte mitochondrial respiration under basal conditions. In this review, we summarize the data from preclinical heart failure studies that explore the role of BET-regulated transcriptional mechanisms and delve into landmark studies that define BET bromodomain-independent processes involved in cardiac homeostasis. Alt. Alt2+-cGene expression is controlled by DNA binding TFs and chromatin changes that allow the transcriptional machinery to be recruited to target genes. The binding of TFs to DNA elements known as enhancers stimulates the recruitment of other cofactors and RNA polymerase II (pol II) to the transcription start site . UsuallyHistone acetylation, in particular, is an important component of cardiac remodeling in the progression to HF. ChIP-Seq analysis of cardiomyocytes from hypertrophied hearts has indicated that nearly 10% of the genome undergoes changes in the distribution of histone marks during stress, with all histone marks being redistributed to transcription start site . The difEp300 prevented mice from developing hypertrophy in response to pressure-overload, whereas overexpression of p300 led to pathological remodeling associated with hyperacetylation of chromatin and pro-hypertrophic cardiac transcription factors [Further evidence of the involvement of histone acetylation in HF comes from studies investigating histone acetyl transferases (HATs) and histone deacetylases (HDACs), which add or remove acetyl-lysine groups from histone side chains. The HAT p300 is a transcriptional coactivator that increases during cardiac hypertrophy . Haploin factors . Similar factors ,18,19. D factors . CollectAcetylated lysine residues are specifically recognized by bromodomains (BDs), which are highly conserved protein motifs found in many chromatin-associated proteins . BDs areBET proteins function as epigenetic readers of acetylated histone marks and a scaffold to localize the transcriptional machinery at the transcription start site . The intBETs also regulate gene expression by associating with the Mediator complex, a multimeric protein complex that links TFs and pol II . It remaThe presence of BRD4 at SEs, in combination with the Mediator complex, helps to compartmentalize the transcription machinery. BRD4 and MED1 (a component of the Mediator complex) localize in nuclear bodies and occupy SEs in embryonic stem cells . These nGene transcription is also facilitated by the switching/sucrose non-fermentable (SWI/SNF) complex that rearranges nucleosome positions thereby permitting the access of transcription machinery to target genes. BRD4 pairs with BRG1, a component of SWI/SNF, to increase transcription at pluripotency genes in embryonic stem cells . NucleosUnderstanding the role of BET proteins in the regulation of gene transcription has been aided by the development of small molecule-inhibitor that actively compete with acetyl-lysine for BD binding. The first-in-class BD inhibitor, JQ1, was found to disrupt assembled transcriptional machinery and block gene expression . The advAnf, Bnp, and Serca2a [Anf further clarified that JQ1 is effective at disrupting the transcriptional machinery at specific loci in cardiomyocytes. Further, BRD4 is upregulated in NRVM without changes in Brd4 mRNA levels under hypertrophic stress [Brd4 identified multiple microRNA (miR) binding sites. With phenylephrine treatment (a pro-hypertrophic stimulus), miR-9 expression levels decreased suggesting that this particular miR is involved in regulating Brd4 at the post-transcriptional level. Indeed, a miR-9 mimic suppressed phenylephrine-induced hypertrophy and BRD4 expression with transcriptomic changes that were largely similar to those observed with JQ1. Genome-wide binding of BRD4 identified 459 SEs, with increased BRD4 binding to 65 SEs and a concomitant decrease observed at 19 SEs under hypertrophic stimuli. ChIP-seq targeting RNA pol II also elucidated three subsets of promoter regions that had either increased, decreased, or unchanged the binding of BRD4. The miR-9 mimic strongly suppressed BRD4 accumulation on SEs and promoters confirming its key role in BRD4 expression and BRD4 dependent transcriptional regulation. Finally, the AP-1 transcription factor complex was identified as a key TF that binds to BRD4-enriched SEs to promote gene transcription. Collectively, these landmark studies in cardiomyocytes demonstrated that (1) BETs are involved in cardiomyocyte hypertrophic gene expression; (2) hypertrophic signaling promotes a shift in BRD4 loading on SEs, which facilitates gene transcription; and (3) miR-9 is an important regulator of BRD4 expression.In neonatal rat ventricular myocytes (NRVMs), BET inhibition with JQ1 prevented cardiomyocyte hypertrophy, and blunted the differential expression of hypertrophy-associated genes such as  Serca2a ,38. Chroc stress . EvaluatIn addition to JQ1, other BET inhibitors including I-BET, I-BET-151, RVX-208, and PFI-1 were successful at inhibiting cardiomyocyte hypertrophy, suggesting that this is a class effect of BET inhibitors . RNA-seqTransverse aortic constriction (TAC) leads to LV pressure overload and serves as a model of acute HF. Daily JQ1 administration immediately following TAC surgery successfully inhibited cardiac hypertrophy and LV systolic dysfunction for up to 4 weeks ,38. LikeGene set enrichment analysis (GSEA) was performed to compare JQ1-induced gene changes with previously established clusters of genes known to be regulated by specific TFs including calcineurin-A, NF-κB, and GATA4 . This reIn a follow-up study to examine whether BET inhibition could effectively treat established cardiomyopathy, a cohort of mice that underwent TAC were treated with JQ1 2.5-weeks after surgery . JQ1 sucIn a model of MI caused by proximal LAD occlusion, BET inhibition with JQ1 suppressed the development of cardiomyocyte hypertrophy, LV systolic dysfunction and cavity dilation . FurtherIn contrast to the importance of BETs in pathological stress-induced cardiomyopathy, BET inhibition by JQ1 had no effect on physiological hypertrophy. Adult mice subjected to a high-intensity endurance swimming protocol for 3 h/day for 4 weeks displayed mild cardiomyocyte hypertrophy with a concomitant increase in LV systolic function irrespective of treatment with JQ1 . A key d2+-regulatory gene phspholamban (PLN) lead to the development of dilated cardiomyopathy (DCM) characterized by LV chamber dilation and HF. Our lab previously characterized transgenic mice overexpressing a specific human PLN mutation , that causes HF and early death within 5–6 months [R9C mice with DCM exhibit significant myocardial fibrosis associated with increased fibroblast proliferation. To discern cell-type specific changes, RNA-seq was performed after the separation of cardiomyocytes from non-myocytes ; this identified the upregulation of profibrotic cytokines including TGFβ2, TGFβ3, and CTGF in cardiomyocytes, whereas periostin and components of NF-κB signaling were upregulated in the non-myocyte population [Genetic mutations in the Ca6 months . PLNR9C pulation . GSEA ofR9C mice with JQ1 inhibited systolic HF, cardiac fibrosis, and fibroblast proliferation and increased the lifespan by 10%, thereby verifying the role of BETs in genetic DCM [R9C nonmyocytes with DCM. In contrast, the cellular metabolic changes observed in cardiomyocytes remained unperturbed by JQ1 treatment. Mechanistically, acetylated RelA (aK310 –RelA) was markedly increased in PLN hearts and was identified to be bound to BRD4. JQ1 blocked this RelA-BRD4 interaction while having no effect on aK310-RelA levels, thus suggesting that NF-κB is still activated, but BRD4 is necessary for the expression of at least a subset of NF-κB-activated genes in PLNR9C hearts. Treatment of PLNetic DCM . RNA-seqLMNA account for approximately 6% of familial DCM cases [R9C, RNA-seq analysis of LMNA-DCM hearts identified the upregulation of genes involved in inflammation, epithelial-to-mesenchymal transition (EMT), and apoptosis, with TGFβ1, RelA/NF-κB, and STAT1 pathways being highly activated. ChIP-seq analysis of BRD4 revealed that BRD4 peaks were redistributed in cardiomyocytes from LMNA-DCM mice and localized with genes that have increased transcript levels compared to WT cardiomyocytes. BET inhibition with JQ1 increased median survival from 23 to 32 days, limited negative LV remodeling, decreased cardiac arrhythmias, and markedly inhibited fibrosis and cell death. BET inhibition was associated with suppression of the innate immune response, EMT, and fibrosis-associated gene expression.Lamin A/C (LMNA) is a component of the nuclear envelope and genetic mutations in CM cases . In an aCM cases . SimilarAlthough numerous studies utilized JQ1 to demonstrate the vital role of BETs in inflammatory and fibrotic gene expression during HF development, it remains to be defined whether BRD4 was the dominant BET protein mediating transcriptional changes. To assess this, two separate groups created cardiomyocyte-specific BRD4-null mice and made similar observations. Cardiomyocyte-specific deletion of BRD4 in early gestation was embryonically lethal by postnatal Day 5 . In contRNA-seq analysis of whole LV between JQ1-treated and BRD4-null hearts identified enrichment in genes associated with mitochondrial metabolism and the electron transport chain (ETC) pathway . MitochoRNA-seq analysis of whole LV between JQ1-treated and BRD4-null hearts indicated that there was only a 25% overlap of differentially expressed genes . There aAnother important consideration is that the non-BD functions of BETs have a wide range of effects, some of which are vital to maintaining normal cell functions . Hence, BET proteins are epigenetic coactivators that are recruited during cellular stress to mediate dynamic changes in gene transcription. They integrate the vast array of pathologic signaling cascades activated in HF of varying etiologies to produce a coordinated set of gene networks that result in negative cardiac remodeling. JQ1 is a powerful inhibitor of inflammatory and fibrotic gene transcription, which helps preserve cardiac function. While JQ1 has shown therapeutic efficacy in limiting HF in preclinical models, deletion of BRD4 from cardiomyocytes is detrimental, leading to spontaneous DCM . FurtherThere is significant ongoing effort to translate our current knowledge of the molecular function of BETs into clinical application. Numerous clinical trials are studying BETs as primary and adjunctive chemotherapeutics for a variety of cancers . In card


Epigenetic memory plays a critical role in the establishment and maintenance of cell identities in multicellular organisms. Polycomb and trithorax group (PcG and TrxG) proteins are responsible for epigenetic memory, and in flies, they are recruited to specialized DNA regulatory elements termed polycomb response elements (PREs). Previous transgene studies have shown that PREs can silence reporter genes outside of their normal context, often by pairing sensitive (PSS) mechanism; however, their silencing activity is non-autonomous and depends upon the surrounding chromatin context. It is not known why PRE activity depends on the local environment or what outside factors can induce silencing.Drosophila, we find that the so-called neutral chromatin environments vary substantially in their ability to support the silencing activity of the well-characterized bxdPRE. In refractory chromosomal contexts, factors required for PcG-silencing are unable to gain access to the PRE. Silencing activity can be rescued by linking the bxdPRE to a boundary element (insulator). When placed next to the PRE, the boundaries induce an alteration in chromatin structure enabling factors critical for PcG silencing to gain access to the bxdPRE. When placed at a distance from the bxdPRE, boundaries induce PSS by bringing the bxdPREs on each homolog in close proximity.Using an attP system in This proof-of-concept study demonstrates that the repressing activity of PREs can be induced or enhanced by nearby boundary elements.The online version contains supplementary material available at 10.1186/s12915-021-01047-8. Drosophila embryo by the three bithorax complex (BX-C) homeotic genes: Ultrabithorax (Ubx), abdominal-A (abd-A), and Abdominal-B (Abd-B) [Ubx determines segment identity in two parasegments, PS5 and PS6, and its expression in these parasegments is controlled by the abx/bx and bxd/pbx regulatory domains, respectively. abx/bx is activated at the blastoderm stage in PS5, while bxd/pbx is silenced. In PS6, both domains are active, but Ubx expression is controlled by bxd/pbx.In many developmental systems, the mechanisms involved in the choice of cell fate are distinct from those deployed for maintaining or remembering cell fate decisions. One classic example of choice is the establishment of parasegment identity in the posterior 2/3rds of  (Abd-B) –3. Theseon or off during the rest of development. The memory mechanism is epigenetic. The off or silenced state is remembered by Polycomb group proteins (PcG), while the on or active state is maintained by trithorax group proteins (TrxG) [The maternal and early zygotic gene products that initially determine the appropriate activity state of the BX-C regulatory domains for each parasegment disappear after the onset of gastrulation. Consequently, a different mechanism is required to remember whether the domain is s (TrxG) –10. PcG s (TrxG) –16.Drosophila, most of the known PcG proteins are components of three complexes: polycomb repressive complexes 1 and 2 (PRC1 and PRC2) and Pho repressive complex (PhoRC). The canonical PRC1 core subunits are sex combs extra , posterior sex combs (Psc), polycomb (Pc), and polyhomeotic (Ph) proteins [In proteins –19. The proteins , 21. Finproteins  and DNA-proteins . The Trxproteins , 25.abx/bx and bxd/pbx, that control Ubx expression [engrailed and even-skipped [. The binding of PcG/TrxG proteins to PREs is mediated by sequence-specific DNA-binding proteins, including Pho [The PcG and TrxG proteins are recruited to their regulatory targets by specialized DNA elements called PREs (polycomb response elements) –29. PREspression , 31. The-skipped , 33. Theding Pho , 34 and ding Pho .white reporter and a PRE were compared in hemizygotes and homozygotes. In classical PSS, there is little or no evidence of silencing in hemizygous flies, while strong silencing is evident when the transgene is homozygous. Since PSS is typically observed for transgenes inserted at the same chromosomal site, but not at different sites, the enhancement of repression in homozygotes is thought to be due to trans interactions between the two copies of the PRE, one on each homolog [One characteristic activity of PREs that can be readily assayed is their ability to induce pairing-sensitive silencing (PSS) , 36. Thi homolog , 36. The homolog .Abd-B region of BX-C, the PREs for the iab-5, iab-6, iab-7, and iab-8 regulatory domains are located in relatively close proximity to the chromatin boundary elements (insulators) that flank these domains [Mcp for iab-5; Fab-6 for iab-6; Fab-7 for iab-7, and Fab-8 for iab-8 [Fab-7 and Mcp, the PRE and boundary are integral parts of the same regulatory element [Mcp boundaries were shown to facilitate PSS between transgene inserts megabases from each other and even between transgenes inserted on different chromosomes [The activity of PRE elements in these transgene experiments is influenced by the surrounding chromatin environment. Depending on the integration site, PREs can repress and have a neutral or even a positive impact on gene transcription , 38–46.  domains , 47–49. or iab-8 . Moreove element , 50–52. omosomes –56. Simiomosomes .cis and the other in trans appear to be in play. The first is observed when the boundary is placed in close proximity to the PRE. In this case, silencing is activated in fly hemizygous for the transgene insert and is enhanced when the transgene is homozygous. The induction of silencing activity in hemizygotes is not dependent on the identity of the linked boundary and is observed for all three boundaries. ChIP experiments indicate that the linked boundary facilitates alterations in the local chromatin structure. In the region spanning the PRE, there is a depletion of histone H3 levels and a concomitant recruitment of factors required for PRE function. Interestingly, in the region to either side of the boundary, PRE sequences histone H3 levels are increased. The second mechanism is observed when the boundary element is separated by 1 kb or more from the PRE. In this case, silencing is not or at most only weakly activated in hemizygotes. However, silencing activity is induced in homozygous flies. In this case, the mechanism is dependent upon boundary:boundary pairing interactions as it is not observed when the PREs are linked to heterologous instead of homologous boundaries.In the present study, we tested whether a close linkage between boundaries and PREs can impact the acquisition of silencing activity. We show that in chromosomal contexts that are refractory to PRE-induced silencing, the PREs fail to recruit PcG/TrxG proteins. However, both silencing and recruitment of PRE-associated proteins can be triggered by linking the PRE to an artificial boundary element consisting of multimerized binding sites for the Su(Hw), CTCF, or Pita polydactyl zinc finger DNA-binding proteins. Two different mechanisms, one that occurs in Drosophila transgenic lines. For this purpose, we selected five attP sites that had previously been shown to provide a context in which the expression of a white reporter is not subject to obvious repression or activation by the surrounding chromatin neighborhood . The bxdPRE was flanked by ~1 kb “neutral” spacers derived from coding regions of the eGFP and RFP genes and by terminators of transcription (SV40 terminator upstream and yellow gene terminator downstream) to reduce the influence of potential transcription from surrounding genomic sequences. To better assess the effects of the PRE, we also inserted a control construct at each site, which has all of these components except for bxdPRE  with antibodies against Ph, which is a core component of the PRC1 complex  that is known to be enriched in PcG/TrxG proteins. Figure bxdPRE in the four lines that show silencing of the white gene. Moreover, the extent of association correlates well with the level of repression observed in each line. Consistent with the lack of silencing of white in the 96E insertion, Ph is not found to be associated with its bxdPRE sequence.Silencing is expected to be accompanied by the association of PcG proteins with the Abd-B regulatory domains are positioned close to the boundary elements for each domain. Another example is one of the PREs for the even-skipped (eve) gene that is located next to the distal boundary of the eve locus homie [PREs are often located near other transcriptional regulatory elements. As noted above, the PREs in the four us homie . These obxdPRE is unable to silence white at this insertion site. As the test boundary, we used an artificial element consisting of multimerized binding sites for the polydactyl zinc finger protein Su(Hw) rather than an endogenous boundary. Endogenous boundaries contain binding sites for many different proteins, and some are known to be required for PRE activity. For example, the GAF protein is implicated not only in insulation but also in PcG-dependent silencing [To explore this possibility, we selected the 96E attP since the ilencing .gypsy transposon [gypsy transposon has 12 binding sites for the Su(Hw) protein [gypsy insulator is sufficient for boundary activity in transgene reporter assays [bxdPRE  multimer would not be able to insulate white from PRE-dependent silencing [bxdPRE has a dramatic effect on white expression. Silencing of white is evident in hemizygotes, while strong PSS is observed when the transgene is homozygous. In contrast, the 4xSu(Hw) multimer alone has no effect on eye pigmentation either as a hemizygote or a homozygote. Thus, the presence of the 4xSu(Hw) multimer can induce the establishment of silencing by the bxdPRE in a chromosomal location that is not conducive to PcG-dependent silencing.The Su(Hw) protein is responsible for the boundary activity of the insulator element associated with the ansposon , 67. The protein , 69; howr assays  and in tr assays . This 4xilencing , 57, 72.bxdPRE insertions that are able to repress white expression but is not found associated with the bxdPRE at 96E. If the addition of the 4xSu(Hw) multimer induces PcG-dependent repression, it should also facilitate the recruitment of Ph to the 96E bxdPRE. To test this prediction, we used ChIP to examine Ph association at 5 sites in the Su-bxd and bxd transgenes: (1) the distal end of the spacer sequence upstream of bxdPRE, (2) bxdPRE, (3) the distal end of the spacer sequence downstream of bxdPRE, (4) the white transcription unit, and (5) the white promoter  associates with the bxdPRE element in the transgene construct, but not with other sequences even though white expression is silenced [bxdPRE and the 4xSu(Hw) multimer as Ph is not associated with the control transgene carrying only the 4xSu(Hw) multimer  multimer is included in the transgene, but not when it is absent. The DNA-binding proteins known or thought to be important for bxdPRE silencing include Pho, which binds PREs together with its partner Sfmbt (the PhoRC complex) as well as the Combgap DNA-binding protein. An alternative model is that the bxdPRE at 96E is unable to silence because it is occupied by TrxG proteins and these factors block association of Ph and other PcG proteins and/or their function. In this case, the presence of the 4xSu(Hw) multimer could shift the balance to favor of the recruitment of PcG complexes.Biochemical and genetic studies have shown that, like other PREs, the silencing activity of the bxdPRE in the Su-bxd transgene. These results are consistent with the predictions of the first model. We infer from these finding that the presence of the 4xSu(Hw) multimer induces the association of key DNA-binding proteins with the bxdPRE.To test these two models, we used antibodies against Pho, Sfmbt, Combgap, and two TrxG proteins, Trx and CBP, for ChIP experiments. In the bxd transgene, we observe only background levels of Pho, Sfmbt, and Combgap in ChIPs for the PRE and other sequences in the transgene Fig. c–e. In cbxdPRE alone, but will be displaced by PcG proteins when the 4xSu(Hw) multimer is present. However, like the PcG proteins, the Trx and CBP are recruited to the bxdPRE only when the 4xSu(Hw) multimer is present  protein. For this reason, we asked whether the 4xSu(Hw) multimer is able to augment bxdPRE silencing at the four other attP sites which do not have peaks for Su(Hw) nearby  multimer  is included next to bxdPRE in the transgene  multimer is present  peak that overlaps the attP site at 96E, while there are two larger peaks on either side of the attP ~5 kb and ~10 kb away , both of these multimers have insulating activity in BX-C boundary replacement experiments [bxdPRE induces silencing activity in hemizygotes and PSS in homozygotes. Control experiments show that white expression is not affected when the multimers are included in the transgene alone. Thus, multimerized sites for three different chromosomal architectural proteins can induce the silencing activity of the bxdPRE.Next, we wondered whether induction of PRE repressing activity is unique to Su(Hw) or whether other polydactyl zinc finger proteins that have chromosome architectural functions are able to induce PRE silencing. To test this, we linked the eriments , 75–77. enPRE (PSE2) from the engrailed locus, in different chromosomal environments. In previous P-element transgene experiments, this element was shown to repress white expression as a hemizygote and/or homozygote and maintain the parasegmental expression pattern of a Ubx reporter [white by the enPRE in hemizygote flies is weak or nonexistent at all five attP insertion sites and there is no evidence of PSS when the inserts are homozygous   multimer to the Hw) Fig. c.bxdPRE is increased. In the latter model, relatively small changes in distance (<10 kb) should have little or no effect on the ability of the boundary to target the transgene to a PcG-silenced compartment.How do the architectural proteins help establish PRE activity? Two different mechanisms could be in play. The architectural proteins could facilitate the establishment of PcG silencing by locally displacing nucleosomes and/or by recruiting chromatin remodeling complexes so that key DNA-binding factors and PcG complexes are able to assemble on the PRE. Alternatively, the architectural proteins could enhance PRE activity by helping target the transgene from an active chromosomal compartment to a PcG-silenced chromosomal compartment . Since tbxdPRE by 1 kb and 3 kb . A 1-kb distance would be sufficient for approximately five nucleosomes, while 3 kb would correspond to about fifteen nucleosomes. All of the constructs were then introduced into the 96E attP site. The results of this analysis are shown in Fig. bxdPRE adversely impacts silencing activity. For the 4xSu(Hw) multimer, a distance of 1 kb is sufficient to substantially reduce silencing . In the case of the 4xCTCF multimer, the disruption of silencing is greater when it is located 3 kb away from the bxdPRE than it is at a 1-kb distance; however, even at 1 kb, silencing is reduced compared to the control CTCF-bxd construct. These findings argue in favor of the first model, namely that the 4xSu(Hw) and 4xCTCF multimers act in cis to facilitate the assembly of silencing complexes on the PRE  or 4xCTCF multimers and the  kb Fig. a. For thbxdPRE, this is not true for PSS. As shown in Fig. bxdPRE represses white expression even when the boundary multimers are located 3 kb away. A plausible explanation for this result is that pairing of the 4xSu(Hw) or 4xCTCF multimers in trans would tend to stabilize pairing interactions between the PREs on each homolog, and this interaction facilitates the PSS-dependent assembly of functional silencing complexes.While silencing activity in hemizygotes is significantly compromised when the multimers are moved away from the Drosophila depends upon specific interactions between proteins associated with each element [trans to CTCF-1kb-bxd or Su-3kb-bxd trans to CTCF-3kb-bxd. Figure trans and is not observed in heterologous combinations. A possible model is shown in Fig. Boundary pairing in  element , 84–86.  element , 87, 88. element . On othe element . Thus, ibxdPRE for silencing in hemizygotes and enhanced PSS in homozygotes suggests that the boundary has a local effect on chromatin structure that enables PcG factors to gain access to the PRE and assemble functional silencing complexes. If this suggestion is correct, then the chromatin organization of the bxdPRE should be altered when it is closely linked to a boundary element. To test this prediction, we analyzed histone H3 association with six unique sequences (1n-6n) located at different distances upstream and downstream of the BamH1 site used to insert test DNAs  has no apparent effect on histone H3 occupancy and the profile across the six sequences is similar to that of the E-w control. A different result is obtained for Su, CTCF, or Pita transgenes containing the multimerized binding sites for the boundary proteins. In all three cases, there is a reduction in histone H3 occupancy in the sequences located immediately next to the multimers  and is equivalent to that observed for the control genomic sequence in the coding region of the Ras64B gene (ras) Fig. b. The iners Fig. b. This fbxdPRE combination is reduced. In each case, the reduction is approximately the same as that observed for the corresponding multimer alone. Importantly, this is true for 4n, which is separated from the multimers by the 650 bp bxdPRE. These results provide strong support for the idea that closely linked boundary elements can induce alterations in the association of nucleosomes with the PRE. Interestingly, this is not the only alteration in nucleosome association evident in these transgenes. In all three of the multimer-bxdPRE combinations, we found that histone H3 association with the more distant sequences  is enhanced relative to the various control transgenes . Since enhanced association is not observed with the inactive bxdPRE or with any of the multimers alone, it seems likely that this effect is due to the recruitment of functional PcG complexes to the bxdPRE. It remains to be determined whether this alteration in histone H3 association reflects an increase in nucleosome density in the flanking DNA regions or an increase in the extent of compaction.We next examined the Su-bxd, CTCF-bxd, and Pita-bxd transgenes Fig. b. As wascis and requires a close linkage of the boundary and PRE. This mechanism locally alters the pattern of histone association and facilitates the recruitment of factors critical for PcG repression. The other takes place in trans and is mediated by boundary:boundary pairing interactions. In this second mechanism, boundaries appear to provide a “spot weld” that holds the homologs in close proximity. This facilitates PRE:PRE interactions and results in PSS even when the boundary multimers are separated from the PREs. We undertook several additional experiments to further explore this “pairing” mechanism.The studies in the previous sections indicate that boundaries can enhance PRE silencing by two different mechanisms. One takes place in Boundary pairing can induce silencing in trans: When 4xSu(Hw) or 4xCTCF is placed next to the bxdPRE, the PRE can assemble a functional silencing complex and repress white in hemizygotes. However, the bxdPRE would not be expected to efficiently silence white in trans unless the homologs are tightly paired in the immediate neighborhood. To test this expectation, we generated trans-heterozygotes between the starting transgene E-w  and either Su-bxd or CTCF-bxd , CTCF, or Pita-binding sites. Figure white in the three trans-heterologous combinations of multimerized binding sites is close to that observed when both transgenes have multimerized binding sites for the same protein.An inactive PRE can be transactivated by an active PRE: The bxdPRE insertion in the 22A attP site is a classic example of PSS. Little or no silencing is observed in hemizygotes, while silencing is quite efficient in homozygotes. The PSS phenomenon suggests that PRE:PRE interactions in trans can synergizes, promoting the assembly of functional PcG silencing complexes on both PREs. To test this idea for 96E, we generated trans combinations between the inactive PRE in the bxd transgene and transgenes in which bxdPRE is activated by closely linked 4xSu(Hw), 4xCTCF, or 5xPita sites. Consistent with prediction, the white reporter in the homolog carrying the bxdPRE (only) transgene is repressed when the other homolog has the boundary-bxdPRE , strong silencing of white is observed in hemizygotes, while at the other extreme (96E) there is no evidence of silencing even in homozygotes. For the three remaining attP sites, there is a range of silencing in hemizygous and homozygotes. For 22A and 58A, there is little evidence of silencing in hemizygous flies, while PSS is observed in homozygotes. In the case of 51C, white expression is clearly repressed in hemizygotes, and repression increases in homozygotes. The strength of silencing at the five attP sites correlates well with the recruitment of the PcG protein Ph. High levels are found associated with bxdPRE when it is inserted at 68E, while only background levels are evident at 96E.PcG-dependent silencing is widely used during the development of multicellular organisms as a mechanism for maintaining the determined state –10. In fbxdPRE insert at 96E.These findings are consistent with previous P-element transgene-based experiments, which showed that the silencing activity of PREs is non-autonomous and depends upon the local chromosomal context , 44–46. bxdPRE. We found that all three of these artificial boundaries were able to induce silencing in hemizygotes and PSS in homozygotes. For the 4xSu(Hw)-bxdPRE combination, we found that the acquisition of silencing activity is accompanied by the recruitment of both components of the PhoRC DNA binding complex, Pho and Sfmbt, and other PcG silencing factors to the bxdPRE. Surprisingly, we found that two TrxG proteins, Trx and CBP, are also associated with the bxdPRE, and like the PcG factors, this association is only observed when the bxdPRE is closely linked to the 4xSu(Hw) multimer.These findings led us to wonder what features of the chromatin landscape in the neighborhood of the PRE might facilitate the recruitment of the required silencing factors. In this respect, it was of interest that PREs in BX-C are often closely linked to chromatin boundary elements . This cobxdPRE. Induction of silencing is distance dependent, and for the 4xSu(Hw) multimer, a distance of 1 kb is sufficient to largely eliminate silencing in hemizygotes, while it is reduced but not entirely eliminated for the 4xCTCF multimer. The importance of proximity taken together with the de novo recruitment of DNA binding and PcG proteins suggests that the boundary might alter the local chromatin structure so that factors important for PcG-dependent silencing can gain access to the PRE. Consistent with this hypothesis, we find that multimerized binding sites for the boundary factors Su(Hw), CTCF, and Pita can, on their own, generate a local reduction in histone H3 occupancy, indicating that the multimers likely form a nucleosome-free region of chromatin like that observed for many endogenous boundaries [bxdPRE is placed next to these multimers, the nucleosome free region extends across the 650-bp bxdPRE fragment. While we did not investigate the mechanisms of nucleosome displacement, Su(Hw) and CTCF have been shown to interact with chromatin remodeling complexes. Su(Hw) was found to recruit the Brahma chromatin remodeling protein [In hemizygotes, silencing activity is induced when the boundary multimers are placed next to the undaries –92. When protein , 94. Mor protein . It woulbxdPRE, the presence of multimers in the transgene even at a distance of 3 kb is sufficient to promote PSS when the transgene is homozygous. Critically, the induction of PSS in this configuration is dependent on the ability of the boundaries on each homolog to pair with each other as it is not evident when the 4xSu(Hw) multimer is trans to the 4xCTCF multimer. A plausible interpretation of this finding is that when inactive PREs on each homolog are brought into close proximity by the paired boundaries, there is a synergistic interaction, which is able to promote the acquisition of PcG-dependent silencing activity. This model predicts that an active PRE should be able to trans-activate an inactive PRE. Consistent with this prediction, we found that an active bxdPRE (linked to a multimer) on one homolog can partially trans-activate an inactive bxdPRE (unlinked to a multimer) on the other homolog.Consistent with idea that the boundary multimers facilitate the displacement of nucleosomes from the PRE, we find that silencing activity is compromised when the multimers are placed at a distance of 1 to 3 kb from the PRE. This distance effect would seem to argue against a mechanism in which the multimers target the PRE to a PcG compartment. On the other hand, while the boundary multimers are unable to fully augment silencing activity in hemizygotes unless they are next to the bxdPRE, the artificial boundaries we tested are able to autonomously establish regions of chromatin that have reduced nucleosome occupancy. When the bxdPRE is placed next to one of these multimers, the region of reduced nucleosome occupancy extends over the PRE. Accompany this reduction in occupancy, PcG and TrxG factors are found associated with the bxdPRE and silencing activity is observed. In addition to this local effect on PcG silencing activity, we found that boundaries can induce PSS when placed at a distance from the PRE. In this case, our genetic experiments indicate that boundary:boundary pairing forms a “spot weld” which enables the PREs on each homolog to interact with each other and induce PcG silencing.Here, we show that boundary elements can help induce the silencing activity of PREs by facilitating the recruitment of DNA-binding and PcG/TrxG proteins. Unlike the yacw1118 flies, and the transgenic progeny was identified by their eye pigmentation. Details of crosses used for genetic analysis are given in Additional file All flies were maintained at 25°C on the standard yeast medium. The constructs were injected into embryos of lines ZH-attP-22A BDSC #24481), ZH-attP-51C (BDSC #24482), ZH-attP-58A (BDSC #24484), ZH-attP-68E (BDSC #24485), and ZH-attP-96E (BDSC #24487) . The res481, ZH-awhite expression, we visually determined the degree of pigmentation in the eyes (white) of 3- to 5-day-old males developing at 25°C, with reference to standard color scales. Pigmentation of all flies was analyzed in hemi- (P/+) or homozygotes (P/P).For phenotype analysis of BamHI-EcoRI fragment containing the white gene (mini-white version) without Wari insulator was clones from pCaSpeR∆700 vector [white gene (attBdir-white-rev-pSK). The sequence corresponding to the eye enhancer (E) of the white gene (regulatory sequences from position –1180 to –1849 bp relative to the transcription start site) was cut out of the Ee− pBluescript SK+ plasmid [white gene (attBdir-white-rev-E-rev-pSK).The 0 vector  into the plasmid  without yellow gene terminators of transcription were cloned downstream eGFP and Cherry respectively in orientation to stop the transcription from flanking regions (SV40dir-eGFPrev-Cherrydir-Ytermrev).The coding regions of eGFP (PCR-amplified with primers 5′-atggtgagcaagggcgaggagct-3′ and 5′-cttgtacagctcgtccatgccga-3′) and Cherry (PCR-amplified with primers 5′-atggtgagcaagggcgaggag-3 and 5′-ttacttgtacagctcgtccat-3′) were cloned into pBluescript SK+ vector in a “head-to-head” orientation (eGFPrev-Cherry-pSK). The SV40 and white-rev-E-rev-pSK plasmid between attB and the white gene. The resulting E-w construct has BamHI cloning site between the eGFP and RFP coding regions that was used to create the following constructs. The 656bp bxdPRE (corresponding to the genome sequences between PstI-NdeI) was PCR amplified from the frt(PRE) plasmid [BglII, XhoI, and SpeI restriction sites) and reverse primer . The bxdPRE PCR fragment was then cleaved by BamHI-BglII and cloned into E-w cleaved by BamHI (bxd construct). The 4xSu(Hw) sites (4xSu) were cut from a plasmid used previously [I and SpeI (Su-bxd construct) or into the E-w construct cleaved by BamHI (Su construct). CTCF-bxd, CTCF, Pita-bxd, Pita, en, and Su-en constructs were generated by the same scheme using the following plasmids and fragments. The plasmids with 4xCTCF [I restriction site was inserted upstream of eGFP (SphI-bxd). The 4xSu fragment or 4xCTCF was inserted into SphI-bxd cleaved by SphI. To create Su-3kb-bxd, CTCF-3kb-bxd constructs, the 2087-bp fragment containing the LacZ coding region was inserted into Su-1kb-bxd, CTCF-1kb-bxd constructs between 4xSu and eGFP. The sequences corresponding to construct fragments  are given in Additional file To obtain E-w construct, the fragment corresponding to SV40dir-eGFPrev-Cherrydir-Ytermrev was cloned into attBdir- plasmid  with direviously  and clonh 4xCTCF  and 5xPih 4xCTCF  sites weN-lauroylsarcosine, EDTA-free protease inhibitor cocktail) and sonicated to break chromatin into fragments with an average length of 700 bp. The material was pelleted by centrifugation at 18 000 g for 5 min, and the supernatant fluid was transferred to a new tube. The pellet was treated with the second 0.5-ml portion of lysis buffer, and the preparation was centrifuged at 18 000g for 5 min. The two portions of the supernatant fluid were pooled, cleared by centrifuging twice at 18 000g for 10 min, and the resultant chromatin extract (1 ml) was used for ChIP experiments after preincubation with A-Sepharose or G-Sepharose. One aliquot (1/10 volume) of chromatin extract after preincubation with Sepharose was kept as a control sample (Input).For each experiment, 150–200 mg of heads from 2- to 5-day-old adults homozygous for the different constructs were collected. Experimental procedures for chromatin immunoprecipitations were performed as described previously , 63. BriChIP experiments involved incubation with specific rabbit/rat antibodies or with nonimmune IgG that was used as nonspecific antibody control. All ChIP experiments were made at least in triplicate. The enrichment of specific DNA fragments was analyzed by real-time qPCR, using a C1000™ Thermal Cycler with CFX96 real-time PCR detection module (Bio-Rad) or a StepOne Plus Thermal Cycler . Primers used in ChIP/real-time PCR analyses are listed in Additional file Escherichia coli, affinity purified on Ni Sepharose 6 Fast Flow  according to the manufacturer’s protocol and injected into rabbits following the standard immunization procedure. Antibodies were affinity-purified from serum on the same antigen as was used for immunization. The specificity of Combgap, CBP, and Pho antibodies was verified by RNAi/Western blotting , Sfmbt (1-348 aa of isoform PB) , 63, andAdditional file 1: Table S1. Eye phenotypes of hemizygote transgene flies with insertion of white marker gene without enhancer at selected attP lines. Figure S1. X-ChIP for the “Su” transgene with Ph antibody and X-ChIP for the “Su-bxd” and “Su” transgenes with Su(Hw) antibody. Table S2. Primers used for X-ChIP-qPCR analysis to test PcG/TrxG, DNA-binding proteins recruitment to transgenes. Table S3. Primers used for X-ChIP-qPCR analysis to test histone H3 binding to transgenes. Figure S2. Antibody specificity test.Additional file 2. This file contains the analysis of homologue chromosomes contact frequencies at the 22A, 51C, 58A, 68E and 96E attP insertion sites.Additional file 3. This file contains the ChIP-seq data for PcG and boundary proteins binding to 22A, 51C, 58A, 68E and 96E genome regions.Additional file 4 This file contains: (1) the DNA sequences of the construct elements. (2) the details of Drosophila genetic crosses.


NLRP3. We found that a distal, rather than a proximal, promoter of human NLRP3 was predominantly used in the human monocytic cell lines and macrophages. Reporter analysis showed that an Ets/IRF composite element (EICE) at -309/-300 and an Ets motif at +5/+8 were critical for transcriptional activity of the distal promoter. Electrophoretic mobility shift assays and chromatin immunoprecipitation assays demonstrated that two transcription factors, PU.1 and IRF8, both of which play essential roles in development and gene expression of the monocytic lineage, were bound to the EICE site, whereas PU.1 alone was bound to the Ets site. Knockdown of PU.1 and/or IRF8 mediated by small interfering RNA downregulated expression of NLRP3 and related molecules and markedly diminished the LPS-induced release of IL-1β in THP-1, suggesting that activity of the NLRP3 inflammasome was suppressed by knockdown of PU.1 and IRF8. Taken together, these results indicate that PU.1 and IRF8 are involved in the monocytic lineage-specific expression of NLRP3 by binding to regulatory elements within its promoter and that PU.1 and IRF8 are potential targets for regulating the activity of the NLRP3 inflammasome.NLRP3 inflammasomes play crucial roles in the initiation of host defense by converting pro-Caspase-1 to mature Caspase-1, which in turn processes immature IL-1β and IL-18 into their biologically active forms. Although NLRP3 expression is restricted to monocytic lineages such as monocytes, macrophages, and dendritic cells, the mechanisms determining the lineage-specific expression of NLRP3 remain largely unknown. In this study, we investigated the transcription factors involved in cell-type-specific transcription of  Nod-like receptor pyrin domain containing 3 (NLRP3) is a pattern-recognition receptor belonging to the NLR family and is mainly expressed in monocytes, macrophages, and dendritic cells (DCs). After interacting with pathogen-associated molecular patterns or damage-associated molecular patterns, NLRP3 forms the NLRP3 inflammasome complex with pro-Caspase-1 in the presence of the adapter protein apoptotic scaffold protein containing a caspase recruitment domain (ASC), and subsequently converts pro-Caspase-1 to Caspase-1 , 3. PropSpi1, is essential for the development of myeloid and lymphoid lineages from hematopoietic stem cells  positively regulate gene expression of mouse and human NLRP3, respectively, by directly binding to the promoter region  were generated as previously described (+ monocytes isolated from the authors’ (TY and MH) peripheral blood mononuclear cells in the presence of 50 ng/mL hM-CSF  for 5 or 6 days. Human and animal experiments were performed in accordance with the approved guidelines of the Institutional Review Board of Tokyo University of Science .THP-1 and U937 cells  were cultured in RPMI1640 supplemented with 10% fetal calf serum, 100 U/mL penicillin, and 100 mg/mL streptomycin at 37°C in a humidified atmosphere in the presence of 5% COescribed . Human mRNA was extracted and reverse transcription (RT) performed as previously described . RT-PCR NLRP3 were amplified from human genomic DNA by PCR and inserted into the multi-cloning site of pGL-4.10  to generate reporter plasmids. Mutant reporter plasmids were generated using a PrimeSTAR Mutagenesis basal kit . The nucleotide sequences of the primers are listed in The distal and proximal promoters of The electrophoretic mobility shift assay (EMSA) was performed based on a method described previously . FluoresChromatin immunoprecipitation (ChIP) assays were performed as previously described , 37. AntChIP-seq data of human macrophages with an anti-H3K27ac antibody (GSM2942925 and GSM2942926) were obtained from Gene Expression Omnibus . The datSPI1 siRNA (HSS186060), IRF8 siRNA (HSS105171), Spi1 siRNA (MSS247676), Irf8 siRNA (MSS236848), and a Stealth RNAi siRNA negative control set were purchased from Thermo Fisher Scientific . THP-1 cells (2 × 106 cells) suspended in R buffer were mixed with 200 pmol siRNA and then transfected with the Neon transfection system (Thermo Fisher Scientific) setting program No.5. Transfection into BMDMs and human macrophages was performed with Nucleofector 2b  using the Amaxa Mouse Dendritic Cell Nucleofector Kit (Lonza), as previously described  kits for IL-1β (BioLegend) following the manufacturer’s instructions.Dead cells were stained with 1 μg/mL DAPI  and analyzed with a MACSQuant flow cytometer .t-test. p < 0.05 was considered as statistically significant.Multiple groups were compared by one-way ANOVA and Tukey–Kramer test. The difference between any two groups was analyzed by unpaired student’s hNLRP3 are registered in the Ensembl Genome Browser (http://asia.ensembl.org/index.html). They are classified into two groups: one is transcribed from exon 1 , and the other is transcribed from exon 2  (https://www.ncbi.nlm.nih.gov/geo/)  . Histonecis-acting element(s) in the distal promoter of hNLRP3, we performed luciferase reporter assays with vectors containing various lengths of the hNLRP3 promoter. As shown in cis-acting element(s). We then explored cis-acting elements within these regions using JASPAR (http://jaspar.genereg.net/), which is a database of transcription factor binding profiles. Although no promising sites recognizable by transcription factors were detected in -95/-61, we found a PU.1- and IRF-binding site at -309/-300 and PU.1-binding site at +5/+8 -labeled DNA probes (lane 2 (lane 3). Addition of an anti-IRF4 antibody did not affect the band pattern but the intensity of the major band was reduced in the presence of an anti-IRF8 antibody . Furthermore, this band gradually disappeared upon addition of an excess amount of non-labeled WT probe A  but not upon addition of the non-labeled probe A mutant bearing a nucleotide replacement at the EICE . These results suggest that PU.1 and IRF8, but not IRF4, can bind to the -309/-300 EICE by forming a heterodimer.To identify transcription factors that bind to the lane 2), in which a mixture of nuclear proteins and the probe was loaded, the shifted band showing the highest mobility (marked with an arrow) disappeared in the presence of the anti-PU.1 antibody. In contrast, addition of either the anti-IRF4 or the anti-IRF8 antibody did not affect the band intensity (lanes 3–5). As shown in NLRP3.In an EMSA using probe B, among the several shifted bands observed in the lane  that binds to the region around -95/-61 of the hNLRP3 promoter to contributes to its transcription. Given that deletion of this element significantly reduced reporter activity, this unknown transcription factor(s) may also play an important role in monocyte lineage-specific expression of NLRP3. Further studies are needed to fully understand the mechanisms by which NLRP3 expression is regulated. Overall, we demonstrated that PU.1 and IRF8 contribute to monocyte/macrophage-specific expression of hNLRP3. They positively regulate hNLRP3 transcription by forming a heterodimer on the -309/-300 EICE, which does not exist in the mNlrp3 promoter. PU.1 regulates hNLRP3 transcription by binding to the +5/+8 Ets site as a monomer. This mechanism is well-conserved between human and mouse macrophages.Through reporter assays and EMSA, we demonstrated that three The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by The Institutional Review Board of Tokyo University of Science. The patients/participants provided their written informed consent to participate in this study.TY designed research, performed experiments, analyzed data, and wrote the paper. MY and SA performed experiments and analyzed data. MH and KY performed experiments. KU and KK provided experimental tools. CN designed research and wrote the paper. All authors contributed to the article and approved the submitted version.This work was supported by the Grant-in-Aid for Scientific Research (B) , Grant-in-Aid for Scientific Research (C) , Grant-in-Aid for Young Scientists (B) , the MEXT-Supported Program for the Strategic Research Foundation at Private Universities , Tokyo University of Science Grant for President’s Research Promotion (CN), the Tojuro Iijima Foundation for Food Science and Technology (CN and TY), and the Takeda Science Foundation (CN). TY was supported by Research Fellowships of the Japanese Society for the Promotion of Science for Young Scientists (JSPS Research Fellowships for Young Scientists #10792).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.