The tumor microenvironment (TME) is an important place with regard to the growth and sustenance of tumor cells. It comprises an area within close proximity of tumor cells and is marked by the presence of several different cell types, such as fibroblasts, extracellular matrix, immune cells, etc., that regularly communicate with the tumor cells . Within The precursors of macrophages are monocytes, which remain in the bloodstream for a short duration before they migrate into tissues and differentiate into macrophages. In addition, it is worth noting that there are indications for the non-monocyte origin of macrophages . ActivatThis article begins with a discussion on the role of several transcription factors that have been documented to play a role in macrophage polarization. Specific transcription factors that are discussed in considerable detail include PU.1, the STAT family, NF-κB, c-Myc, interferon regulator factors (IRFs), the Snail family, and the Maf family. As is well known, transcription factors influence the transcription of genes by binding to target DNA sequences, and, interestingly, more than half of the known transcription factors in the human genome are expressed in macrophages under different states of polarization ,11. ThesThe discussion then moves on to epigenetic changes that help reprogram macrophages, including DNA methylation, histone modifications, and regulation involving miRNAs. Based on the studies on DNA methyltransferases (DNMTs), specifically DNMT1, DNMT3A, and DNMT3B, the authors discuss how these DNMTs have been linked to differential macrophage activation and polarization, particularly under different stimuli. It is concluded that DNA methylation potentially plays a more prominent role in the inflammatory behavior of macrophages compared to the formation of TAM phenotypes, or at least there is a lack of data on the subject. DNA methylation, or the transfer of a methyl group to DNA, is linked to reduced transcription and, therefore, reduced eventual expression of gene products. Histone modifications involve various events, such as methylation, acetylation, phosphorylation, ubiquitination, sumoylation, etc., on the histone proteins that affect chromatin packing  and geneSubsequent to the discussion on transcription factors and epigenetic events, this article focuses on the metabolic regulation of macrophage plasticity. Based on the available information, the M1 macrophages seem to be more ‘normoxic’ as compared to the M2 macrophages that reside in the comparatively more inner cores of the TME marked by ‘hypoxia’. As expected, M1 macrophages favor metabolic changes that support increased inflammation, including high glycolytic metabolism through the pentose phosphate pathway, fatty acid synthesis required for inflammatory signaling and impaired mitochondrial oxidative phosphorylation, and the tricarboxylic acid cycle . M2 macr


Cre2 Prdm6fl/fl ROSA26LacZ mice revealed normal neural crest–derived SMC investment of the outflow tract. In contrast, myography measurements on DA segments isolated from E18.5 embryos indicated that Prdm6 depletion significantly reduced DA tone and contractility. RNA-Seq analyses on DA and ascending aorta samples at E18.5 identified a DA-enriched gene program that included many SMC-selective contractile associated proteins that was downregulated by Prdm6 depletion. Chromatin immunoprecipitation–sequencing experiments in outflow tract SMCs demonstrated that 50% of the genes Prdm6 depletion altered contained Prdm6 binding sites. Finally, using several genome-wide data sets, we identified an SMC-selective enhancer within the Prdm6 third intron that exhibited allele-specific activity, providing evidence that rs17149944 may be the causal SNP for a cardiovascular disease GWAS locus identified within the human PRDM6 gene.Based upon our demonstration that the smooth muscle cell–selective (SMC-selective) putative methyltransferase, Prdm6, interacts with myocardin-related transcription factor-A, we examined Prdm6’s role in SMCs in vivo using cell type–specific knockout mouse models. Although SMC-specific depletion of Prdm6 in adult mice was well tolerated, Prdm6 depletion in Wnt1-expressing cells during development resulted in perinatal lethality and a completely penetrant patent ductus arteriosus (DA) phenotype. Lineage tracing experiments in Wnt1  However, the absence of SMC marker gene expression in the many cell types that express high myocardin and/or MRTF levels strongly suggests that additional mechanisms are critical for the overall pattern of SMC-specific gene expression observed in vivo. One mechanism that contributes to SMC-specific gene expression is the facilitation of MRTF nuclear localization by RhoA-dependent actin polymerization. Based upon seminal studies from the Treisman lab (It is well established that the regulation of smooth muscle cell (SMC) phenotype plays an important role in cardiovascular development and disease and that serum response factor (SRF) and the myocardin family of SRF cofactors  Although the lack of a suitable PRDM6 Ab prevented us from examining the interaction between endogenous proteins, both flag-MRTF-A  lysates. The SMC-selective PR/SET domain-containing protein, Prdm6, was identified in washed MRTF-A immunoprecipitates, as were several known MRTF-A interacting proteins including p300 , 30 and g-MRTF-A  and endog-MRTF-A  were shog-MRTF-A .PRDM6 mRNA levels in humans were highest in blood vessels and other SMC-containing organs (see data from the Genotype-Tissue Expression [GTEx] consortium presented in Cre Prdm6fl/fl mouse embryos (P < 0.05). SMC differentiation marker protein levels were also reduced when primary mouse outflow tract SMCs isolated from Prdm6fl/fl animals were treated with Cre-expressing adenovirus  by visual scoring of blood within the DA, by outflow tract casting, and by microscopic analysis of paraffin-embedded and frozen sections. As depicted in Cre2 Prdm6fl/fl mice, but not in littermate control animals, which included the following genotypes: Wnt1Cre2 PRDM6wt/fl, Wnt1Cre2 PRDM6wt/wt, PRDM6fl/fl, PRDM6wt/fl, and PRDM6wt/wt. Taken together these data indicated that PRDM6 expression was absolutely required for DA closure, that Cre expression alone or deletion of 1 copy of PRDM6 had no effect on ductus closure, and that the floxed PRDM6 allele behaved in a manner similar to the WT allele.To directly test whether Prdm6 was required for normal neural crest-derived SMC development and function, we bred Prdm6ly E10.5 . Wnt1Crefl model . We careCre2 Prdm6fl/fl mice, we hypothesized that Prdm6 expression in SMC was required for the contractile pathways that mediate DA vasoconstriction. To begin to test this idea, we stimulated ductus closure in E18.5 embryos by treating pregnant dams for 4 hours with indomethacin (20 mg/kg), a nonsteroidal antiinflammatory drug used to treat patent DA in humans because it inhibits the production of prostaglandin E2 (PGE2), which helps maintain ductus patency by stimulating ductus SMC relaxation. While this procedure resulted in premature DA closure in all littermate controls, it had no effect on DA closure in Wnt1Cre2 Prdm6fl/fl fetuses . Importantly, we observed functional DA closure of WT mice within 30 minutes of the C-section procedure even under ambient air conditions.Functional DA closure in mice is initiated within minutes after birth and is mediated by severe vessel constriction. Full anatomic closure takes place over a longer period and involves intimal thickening and eventual fibrotic remodeling of the DA into a remnant structure known as the ligamentum arteriosum . Based u fetuses . We alsoCre2 Prdm6fl/fl mice to the ROSA26LacZ reporter strain, which allowed us to perform lineage tracing of WT and Prdm6-deficient neural crest cells during development. As shown in Cre2 Prdm6fl/fl ROSA26LacZ pups at P1 was consistent with normal neural crest cell migration and was not detectably different from littermate controls (Wnt1Cre2 ROSA26LacZ). To further examine the effects of Prdm6 depletion on outflow tract morphology and neural crest cell identity, we stained frozen sections from P1 pups with anti-LacZ and anti–SM α-actin Abs. As shown in Cre2 Prdm6fl/fl ROSA26LacZ mice was similar to that of genetic littermate controls. Importantly, virtually all cells within the medial layers of the outflow tract vessels including the DA costained for LacZ and SM α-actin, and we observed no visible immunofluorescence in any sections with secondary Ab alone  and working pressures (20 mmHg) and failed to exhibit a myogenic  contractile response . Of interest, Prdm6 depletion had no effect on U46619-induced contraction in ascending aorta segments or on the relatively modest constriction induced in aortic segments by KCl and oxygen exposure .To test whether Prdm6 deficiency inhibited depolarization-dependent contractility, vessel segments were exposed to increasing concentrations of KCl that are known to activate voltage-dependent calcium channels. While 50 mM KCl resulted in an 80% decrease in the diameter of DA segments isolated from littermate controls, it had virtually no effect on DA segments isolated from Wnt1 embryos . Based u embryos , but thiexposure . Taken tCre2 Prdm6fl/fl and littermate control mice at E18.5 (before DA closure). We also isolated mRNA from the ascending aorta to better define the differences between these 2 neural crest–derived SMC populations and to test whether Prdm6 had vessel-specific effects. Importantly, we sequenced at least 5 samples for each genotype and tissue, which increased the statistical power of subsequent comparisons. As summarized in the principal component analysis and hierarchical clustering in To help deduce the mechanisms by which Prdm6 affected DA contractility, we performed bulk RNA-Seq analysis on DA tissue isolated from Wnt1P < 0.01) downregulated by Prdm6 depletion. Similarly, of the 399 genes that exhibited lower expression in the DA (less than 70% versus aorta), 228 of those were significantly upregulated by Prdm6 depletion. Gene Ontology analysis of the most significant differentially expressed genes between control and Wnt1Cre2 Prdm6fl/fl mice (Padj < 0.01 and logFC > 0.5 or logFC < –0.5) revealed that the SMC differentiation, SMC proliferation, and muscle contraction gene programs were downregulated in the Prdm6-deficient DA while those related to nerve function and extracellular matrix expression and organization were upregulated  or is recruited as part of a larger DNA binding complex. To further identify the mechanisms by which Prdm6 controls gene expression, we performed ChIP-Seq experiments in outflow tract SMCs isolated from E17.5 mouse embryos to identify Prdm6 binding sites within the SMC genome. Since suitable Abs for Prdm6 are not available, we used lentivirus to express flag-Prdm6 in our cells. Our final data set included only ChIP peaks detected in 2 separate immunoprecipitations from the same sample. We observed nearly 14,000 Prdm6 ChIP peaks that were associated with approximately 5,200 genes. Of the approximately 1,500 genes that were differentially expressed in Prdm6-deficient ductus tissue, about half were associated with Prdm6 binding . AlthougA breakdown of Prdm6 binding by gene component  demonstrThe fact that noncoding polymorphisms within the Prdm6 gene were associated with both cardiovascular disease and Prdm6 expression in arteries see  stronglyThe regulatory regions depicted in green at the bottom of r2 > 0.78), and the risk allele at each variant has been associated with a similar decrease in Prdm6 expression in human arteries. Thus, any one or more of these could be the causal variant at this important locus. Closer examination of the chromatin environment surrounding these SNPs revealed that only 2 were within regions that might exhibit regulatory activity. Since rs17149944 (but not rs2287696) was within a region that exhibited positive transcriptional activity in luciferase assays (Int3.1), we hypothesized that it was the causal variant and that the minor allele at this locus reduced Prdm6 expression by inhibiting the activity of the Int3.1 enhancer. In support of this idea, mutation of the major rs17149944 allele (G to A) inhibited the activity of the Int3.1 enhancer by over 50% (As shown in over 50% .fl/fl mice with a well-characterized tamoxifen-inducible Cre line driven by the SM MHC promoter (SMMHCCreERT2) , to induce hypertension. Although l-NAME treatment resulted in an 18 mmHg rise in blood pressure, we did not observe significant differences between the tamoxifen- and vehicle-treated groups.The early lethality observed upon deletion of Prdm6 globally or in SM22- or Wnt1-expressing cells has made it difficult to determine whether Prdm6 expression in vascular SMCs plays a role in blood pressure regulation in adult animals. To directly test this, we crossed the Prdm6CreERT2) . In brieCre2 Prdm6fl/fl mice to demonstrate that Prdm6 depletion in neural crest cells results in a patent DA phenotype (LacZ), or differences in the sensitivity of reporter gene detection methods used . It is important to note that in the same study, these authors reported a patent DA phenotype in SM22Cre2 Prdm6fl/fl mice. Since Prdm6 depletion in this model occurs only after the migration and SMC differentiation of neural crest cells, it seems unlikely that the patent DA phenotype observed in Wnt1Cre2 Prdm6fl/fl mice was due primarily to a failure of neural crest migration.Patent DA is a common congenital condition that frequently afflicts preterm infants. Although human genetic analyses and the reported patent DA phenotypes in many genetically modified mouse models have increased our knowledge of DA function, we do not fully understand the mechanisms that control DA closure. The current studies originally designed to characterize SMC-selective gene expression have led to several interesting insights in this regard. Our data provide strong evidence that Prdm6 expression in DA SMC was required for DA closure, adding support for human genetic data implicating Prdm6 in this process . Importahenotype . Based ohenotype , PRDM6 dAlthough our RNA-Seq studies support some previous results on DA-selective gene expression , 46, theThe dramatic effect of Prdm6 depletion on myogenic tone and contractility of isolated DA segments provides convincing evidence that Prdm6 is required for normal DA constriction. However, since Prdm6 depletion in DA SMCs resulted in many significant gene expression changes, it is difficult to ascertain which mediate the contractile deficiencies observed. The downregulation of the SMC gene program may be important, and the fact that patent DA phenotypes have been observed in neural crest cell–specific myocardin-KO  and globAlthough many of the genes altered by Prdm6 depletion were also shown to bind Prdm6 by ChIP-Seq, the mechanisms by which Prdm6 alters gene expression are still unclear. The present studies were initiated by our demonstration that Prdm6 could bind MRTF-A, and the observation that Prdm6 depletion decreased SMC marker gene expression supports the idea that these factors cooperate to regulate SMC identity. However, with a few exceptions, we did not detect Prdm6 binding at the SMC-specific promoters, perhaps suggesting that the effects of Prdm6 on SMC identify may be secondary to effects on other transcription mechanisms. We did detect Prdm6 binding to a region shown to drive SMC-selective expression of myocardin , and it Many GWAS have identified genetic loci that contribute to variations in blood pressure and other cardiovascular disease endpoints. However, most of these are within or near genes that have no known connection to cardiovascular function and are defined by multiple noncoding variations in high linkage disequilibrium. Thus, critical next steps are to identify the causal variants at these loci and to determine the mechanism by which they exert their effects. The current studies are the first to our knowledge to identify the transcription mechanisms that control Prdm6 expression in SMCs, and our characterization of an SMC-selective enhancer within the Prdm6 third intron is an important advance. The observation that Int3.1 activity is mediated by SRF, RBPJ, and TEAD, 3 transcription factors known to be important for SMC-specific gene expression , 53, 54,Somewhat surprisingly, we did not observe effects on blood pressure when Prdm6 expression was depleted in adult SMCs. Compensation by one or more of the many diverse mechanisms that maintain blood pressure homeostasis may have masked an effect of Prdm6 in our model, but we cannot rule out that the effects of Prdm6 on blood pressure are mediated by as-yet-uncharacterized expression in a non-SMC cell type. The association of Prdm6 with blood pressure could also be due to effects of Prdm6 during development. Given our demonstration that Prdm6 affected DA contractility, it is possible that similar effects in the vascular tree could lead to subtle structural alterations or compensatory remodeling that have downstream effects on blood pressure regulation in adults. Of interest in this regard, the third intron locus that contains rs17149944 has also been associated with pulse pressure , an endpCre2 Prdm6fl/fl mice and in humans that have Prdm6-coding mutations. Our characterization of the regulatory elements that drive the SMC-selective expression of Prdm6 should add to our understanding of DA development and the mechanisms by which cardiovascular disease–associated variations within the Prdm6 affect its expression. Finally, our studies provide a strong foundation for future examination of mechanisms that control DA contractility and closure.In summary, our studies provide potentially novel evidence for the importance of Prdm6 in DA function and closure. Our data suggest that Prdm6 is critical for a DA-selective gene program characterized in part by strong expression of the SMC differentiation marker genes. We show for the first time to our knowledge that Prdm6 depletion in neural crest–derived SMCs results in decreased DA tone and contractility, likely explaining the patent DA phenotype observed in Wnt1fl/fl mice  (Cre2 transgenic line (The Jackson Laboratory)  (fl/fl mice were crossed with a tamoxifen-inducible Cre line driven by the SM MHC promoter (SMMHCCreERT2)  (All mouse lines examined were on a C57/Black6 background (at least 6 backcrosses). To knock out Prdm6 in neural crest cells, Prdm6Germany)  were crooratory)  in whichoratory) . To knocGermany) . Cre actCre2 Prdm6fl/fl ROSA26LacZ and Wnt1Cre2 ROSA26LacZ mice were euthanized at P1, and chest cavities were opened to expose the heart and outflow tract. After a brief rinse in phosphate-buffered saline (PBS), mice were fixed in 4% paraformaldehyde (PFA) for 20 minutes. After three 10-minute incubations in wash buffer , pups were incubated overnight at room temperature in X-gal staining solution  as previously described  and stored at –80°C or fixed in 4% PFA for 24 hours and processed for paraffin-embedding. Both frozen and paraffin-embedded embryos were cut into 10- to 15-micron sections, and those that contained the outflow tract arteries were collected onto charged slides. For histological observations, sections were stained with H&E using standard protocols. For immunostaining, frozen sections were thawed and then fixed in ice-cold acetone for 10 minutes while paraffin-embedded sections were deparaffinized, rehydrated, and subjected to antigen retrieval by incubating in 10 mM sodium citrate buffer (pH 6.0) at 95°C for 10 minutes. All sections were then washed 3 times in 1× PBS and blocked for 1 hour at room temperature with 5% goat serum in PBS. Abs against the following proteins were applied overnight at 4°C; β-galactosidase , CNN1 , CD31 , and SM α-actin (MilliporeSigma A5228). Sections were washed with PBS 3 times for 10 minutes, then incubated with secondary Abs (A21202 and A21422 from Invitrogen) (1:500) and DAPI  for 1 hour at room temperature before being mounted with Fluoromount-G mounting medium (SouthernBiotech 0100-01). For RNAscope-based in situ hybridization, slides were processed and stained using the RNAscope Multiplex Fluorescent V2 Assay (ACDBio 323110) according to manufacturer instructions. Target retrieval was performed at 99°C for 15 minutes, followed by Protease Plus (ACDBio 322331) treatment at 40°C for 30 minutes. Probes used include Prdm6 (ACDBio 456891), Tfap2b (ACDBio 536371-C2), and EP4 (ACDBio 441461-C3). Images were taken with a confocal microscope  and were analyzed using ImageJ/Fiji software.To better visualize the status of the DA in mice, intracardiac perfusion was performed using Microfil silicone rubber injection compound  per manufacturer’s instructions.A single dose of indomethacin (20 mg/kg) was administered to pregnant dams at E18.5 by oral gavage. Four hours after dosing, fetuses were delivered by C-section and processed for histological analysis as described above.Cre2 Prdm6fl/fl (n = 5) and littermate control mice (n = 7). Ascending aorta and DA samples were isolated by microdissection, and total RNA from each sample was isolated using the RNeasy UCP Micro Kit (QIAGEN 73934) per manufacturer’s instructions. Low-input RNA-Seq  was performed by Novogene Corporation Inc., with an average of 25.8 million paired reads generated per sample. All RNA samples sequenced had RNA integrity number values greater than 9.0. For each sample, sequences were filtered using fastq quality filter (FASTX-Toolkit) (RRID:SCR_005534) requiring at least 90% of bases (-p 90) to have a minimum quality score of 20 (-q 20). Adapter sequences were removed using tagdust  to determine vessel viability and peak contractility. Chambers were then changed from a flow-through system to a recirculating system  and allowed to re-equilibrate for 20 minutes. Lumen diameter was initially recorded as the resting diameter at 5 mmHg baseline for deoxygenated conditions. Pressure-induced tone (myogenic response) was monitored as the change in lumen diameter (from peak to trough after 10 minutes at each new pressure) during the pressure ramps. In some studies, progressive changes in lumen diameter in response to increased concentrations of KCl  were recorded and compared. For oxygen treatment studies, vessels were changed from a recirculating system that was continuously aerated with deoxygenated gas (pO2 ~38–42 Torr) to one aerated with increasing concentrations of oxygen   to evaluate maximal contractile response. At the end of each study, vessels were exposed to 50 mM KCl to verify vessel responsiveness and integrity.Ductus and ascending aorta segments from 4 to 8 fetuses representing at least 4 litters were used for each myography study. Wnt1escribed –41, 61. gas pO2 ~–42 Torr CreERT2 Prdm6fl/fl mice aged 12–16 weeks as previously described  every 7 days.Radiotelemetry  was used to measure blood pressure in tamoxifen- and vehicle-treated SMMHCescribed . In somefl/fl mouse embryos at E18.5 as previously described (4 cells/well and transfected with 250 ng of luciferase plasmid per well. Luciferase activity was measured 48 hours after transfection using the Steady-Glo Luciferase Kit (Promega) according to the manufacturer’s instructions. Raw luciferase values were normalized to the activity of the empty pGL3 basic or pGL3 promoter vectors. For transactivation experiments, 125 ng of expression plasmids (empty vector or myocardin) was cotransfected along with 125 ng of luciferase plasmid.HuBrSMCs were purchased from Lonza and maintained in Clonetics Smooth Muscle Growth Medium-2. Primary rat aortic SMCs were isolated as previously described  and mainescribed . After 3escribed . COS-7 cescribed  were maiCells were transfected with 30 nM siRNA targeting Prdm6 or GFP using Dharmafect transfection reagent (Dharmacon). Quantitative PCR for Prdm6 and several SMC markers was performed 72 hours after transfection. siRNA sequences used were Prdm6 5′GGUGCGCUCCUGGACGUAGCC3′ and GFP 5′GGUGCGCUCCUGGACGUAGCC3′.P < 0.001). Overrepresented sequences in the ChIP-Seq data set were identified using HOMER , anti-myc , or nonimmune mouse IgG  overnight at 4°C with rotation. Immunoprecipitants were washed 3 times in wash buffer , eluted in sample buffer, resolved by SDS-PAGE, transferred to nitrocellulose, and probed with anti-flag , anti-myc , or anti-MRTF-A  Abs. For Western blots used to measure SMC differentiation marker gene expression, cleared RIPA lysates were boiled in sample buffer, run on an 10% SDS-PAGE gel, transferred to nitrocellulose, and probed with the following Abs: SM α-actin , CNN1 , SM22 , and GAPDH .Flag-tagged MRTF-A variants were immunoprecipitated from COS-7 cells as above, resolved by SDS-PAGE, transferred to nitrocellulose, and then renatured according to previously established protocols . Blots wAn anti–MRTF-A Ab  was used as above to immunoprecipitate endogenous MRTF-A from mouse AoSMCs. Washed immunoprecipitates were submitted directly for analysis by mass spectroscopy on a nanoACQUITY-Orbitrap Velos system. Samples were eluted over a 150-minute gradient from 1% to 40%, where mobile phase A was 0.1% formic acid and mobile phase B was acetonitrile with 0.1% formic acid. The top 8 most intense ions were chosen for higher energy collisional dissociation. Peptide coverage of immunoprecipitated MRTF-A was 26.8%.t test, and statistical significance was considered as a P value less than 0.05. For myography studies, change in lumen diameter was plotted as percentage change compared with baseline diameter at 20 mmHg resting tone . One-way ANOVA with repeated measures was used to detect differences in vessel caliber in response to increasing concentrations of KCl or oxygen. Knockout and control vessel response curves were compared by 2-way ANOVA. Bonferroni’s post hoc analysis was performed when significant differences were found. An unpaired 2-tailed Student’s t test was used to compare the effects of the thromboxane agonist, U46619, on DA and aorta contractility.All data represent at least 3 independent experiments presented as means ± SEM. Means were compared by Student’s 2-tailed All animal procedures were approved by the University of North Carolina and/or Vanderbilt University Institutional Animal Care and Use Committees. All animals were housed in facilities accredited by the American Association for Accreditation of Laboratory Animal Care.MZ designed research studies, conducted experiments, collected data, analyzed data, and wrote the manuscript. KDM designed research studies, conducted experiments, collected data, analyzed data, and wrote the manuscript. JCM designed research studies, conducted experiments, collected data, and analyzed data. HHC conducted experiments, collected data, and analyzed data. MTY conducted experiments, collected data, and analyzed data. ELS designed research studies, conducted experiments, collected data, analyzed data, and provided reagents. JMT designed research studies, analyzed data, wrote the manuscript, and provided reagents. JR designed research studies, conducted experiments, collected data, analyzed data, and provided reagents. TSF designed research studies, analyzed data, wrote the manuscript, and provided reagents. CPM designed research studies, conducted experiments, collected data, analyzed data, wrote the manuscript, and provided reagents.


Saccharomyces cerevisiae, we identified 37 genes with a G4 motif in the promoters including 20 genes that contain stress response element (STRE) overlapping a G4 motif. STRE is the binding site of stress response regulators Msn2 and Msn4, transcription factors belonging to the C2H2 zinc-finger protein family. We show here that Msn2 binds directly to the G4 DNA structure through its zinc-finger domain with a dissociation constant similar to that of STRE-binding and that, in a stress condition, Msn2 is enriched at G4 DNA-forming loci in the yeast genome. For a large fraction of genes with G4/STRE-containing promoters, treating with G4-ligands led to significant elevations in transcription levels. Such transcriptional elevation was greatly diminished in a msn2Δ msn4Δ background and was partly muted when the G4 motif was disrupted. Taken together, our data suggest that G4 DNA could be an alternative binding site of Msn2 in addition to STRE, and that G4 DNA formation could be an important element of transcriptional regulation in yeast.Sequences capable of forming quadruplex or G4 DNA are prevalent in the promoter regions. The transformation from canonical to non-canonical secondary structure apparently regulates transcription of a number of human genes. In the budding yeast   Four guanines interact together through Hoogsteen hydrogen bonds to form guanine tetrads which are stacked on top of each other and held by intervening loops to form the four-stranded G4 DNA , which is the binding site of a master regulator of stress response, Msn2. We show that Msn2 protein, a C2H2 zinc-finger transcription factor, binds to G4 DNAs MATa, ura3-52 ade2-101 trp1Δ1)  and MscI-digested pRS306-TSL1pG4Mut-TSL1(1–300) plasmids, respectively. These plasmids were constructed by introducing the G4 mutation into promoter sequence by PCR site directed mutagenesis method and then cloned into the integration plasmid pRS306 using NEB Builder HiFi DNA assembly master mix . The sequence encoding for Msn2(596–704) zinc-finger domain (BamHI and NotI sites for expressing GST-Msn2(596–704) in Escherichia coli. The plasmids are listed in Yeast strains used in this study were derived from YPH45 strain ( trp1Δ1)  and are ribed in . The SUBribed in . The ATGr domain  was ampl600 ∼ 0.1 in YEP-GE medium and grown to mid-log phase (OD600 ∼ 0.5), and then further diluted to OD600 ∼ 0.1 in YEP-GE medium before adding 50 μM TMPyP4  or 25 μM PhenDC3 . Cultures were grown until OD600 reached ∼0.5 and harvested by centrifugation. RNA was extracted using hot acid phenol method as described in . The probe set included 37 genes (34 target genes and 3 house-keeping genes). The data were analyzed using nSolver 4.0 Analysis Software  and normalized to 3 house-keeping genes ACT1, ALG9 and TFC1  and the cDNA copy numbers were determined by Bio-rad QX200 Droplet Digital PCR (ddPCR) system and QX200 ddPCR EvaGreen Supermix . The ddPCR primers are listed in ALG9  and then grown overnight. Cells were diluted to ODribed in . The extIn vitro DNA pull-down assays were performed as previously described in  were carried out as described below.ribed in ,47 with E. coli BL21(DE3) cells harboring pGEX4T1-GST-Msn2(596–704) plasmid. Bacterial culture was grown in 500 ml LB medium supplemented with 100 μg/mL ampicillin and was incubated at 30°C with shaking at 150 rpm. Protein expression was induced with 0.05 mM IPTG  after the culture reached OD600 of ∼0.4 and then incubated with shaking at 150 rpm at 20°C for additional 1 hour before being harvested by centrifugation . Cell pellet was washed with cold PBS and stored at −80°C until further use. Frozen cell pellets were dissolved in lysis buffer ) and disrupted by sonication (QSONICA sonicator). The crude extract (45 ml) was clarified by centrifugation  before mixing with 3 mL glutathione agarose gel  for batch purification. The unbound proteins were washed away using wash buffer ) and the target protein was eluted using elution buffer , 150 mM NaCl, 0.05% Triton X-100). The eluted protein was then exchanged to storage buffer  using Zeba desalting spin column  and then flash-frozen in liquid N2 and stored at −80°C for further use. The protein purity was analyzed by SDS-PAGE followed by Coomassie staining and protein concentration was determined by Total Protein Assay Kit . For EMSA, 5′ Cy5-labeled oligos (Sigma) (4) and incubated at room temperature for 30 min before running on 6% nondenaturing TBE-polyacrylamide gel  with 0.5× TBE buffer. Gel images were captured using Bio-Rad ChemiDoc™ MP imaging system and band intensity was measured by Bio-Rad Image Lab software. The signal intensities of bound and unbound fractions were determined and used for Kd calculation.The recombinant zinc-finger domain GST-Msn2(596–704) was expressed in  (Sigma)  were res600 ∼ 0.1 were grown in synthetic complete (SC) media supplemented with 2% glucose until OD600 reached ∼0.5 and then shifted to SC media supplemented with 3% glycerol for 20 min to activate Msn2-13Myc protein  of target loci and ΔCt value (IP – Input) of UBC13 promoter which is not a target of Msn2 protein and does not contain G4 DNA motif. The relative DNA enrichments were calculated by dividing the relative DNA fold changes of tagged samples (containing Msn2-13Myc) to those of un-tagged samples. P-values were determined by Student's t-test and value that is less than 0.05 was considered significant.ChIP of Msn2-13Myc protein was performed as described in ,51 with  protein . The chrProtein samples were prepared as previously described in . Whole cSaccharomyces cerevisiae. We utilized QGRS mapper, a web-based G4 mapping tool (https://bioinformatics.ramapo.edu/QGRS/index.php) developed by the bioinformatics team of Ramapo College of New Jersey (≥3N≤10G≥3N≤10G≥3N≤10G≥3 and the maximum length of the motif was 45 nt. Although it has been reported that the loop lengths of no longer than 7 nt is necessary to form very stable G4 structures (Saccharomyces Genome Database (SGD), we surveyed 6275 yeast genes to identify 37 genes containing G4 motifs in their promoters, with the G-scores ranging from 62 to 100 , genes encoding for cellular enzymes , stress response genes , or trehalose synthesis genes . The colocation of G4 motifs and STREs in these gene promoters suggests that G4 DNA might regulate expression of these stress response genes.Since G4 motifs contain guanine runs, we searched for transcription factors that have binding sites also containing guanine runs with at least three guanines. Amongst >150 transcription factors in budding yeast ,60, we fproteins . We founproteins . This coproteins ,62–65. Aand TSL1 ,61,64,66TPS1pG4 oligos. TPS1 is a well characterized target of Msn2  + Figure . This dao Figure . Msn2-13o Figure . Circulaructures  whereas lel form .TPS1pG4 sequence did not significantly disrupt the binding of Msn2-13Myc to the oligos. And Msn2-13Myc bound to the non-STRE-containing rPEX5 G4 oligos with sequences from rat PEX5 gene  did not bind when the G4 motifs were mutated as in ATG39pG4Mut and TPS1pG4Mut  was significantly more robust in the presence of K+ than that in the presence of Li+  of 23 ± 5.2 nM  protein concentrations were used for the EMSA experiment with the BRCA1-G3 oligo folded in K+ buffer. The EMSA data showed that the binding of GST-Msn2(596–704) to BRCA1-G3 G4 DNAs increased with the increasing protein concentration and reached binding saturation at ∼100 nM   . These rin vitro led us to investigate the binding of Msn2 to the G4 motifs in vivo by using the ChIP-qPCR approach. The promoter region of CTT1 gene, which is a well-characterized target of Msn2/4  containing 20 copies of the repeat (GAGCT)nGGGGT, which has been shown to form G4 DNAs in vivo .The surprising finding that Msn2 binds to the G4 DNA structures f Msn2/4  and doess Figure . This res Figure ,73. Thers Figure . After t in vivo ,47,72,74ACT1, ALG9 and TFC1, the data showed that stabilizing G4 structures by TMPyP4 upregulated the transcription of 19 genes, downregulated the transcription of 5 genes, and 10 genes had no change in the transcript levels  in the transcript level, while ATP10 encoding for a mitochondrial protein was most reduced by TMPyP4 treatment  and trehalose synthesis genes  were upregulated and three genes, ADO1, SUT1 and TRM3 were downregulated when treated with TMPyP4  that contained conserved G4 motifs in the promoters , but also with the binding site of Mig1/2 transcription repressor factors . Repeating the binding assay with the c-KIT2 oligo with additional flanking nucleotides did not result in significant interaction with the purified Msn2(596–704) , as inferred from the dissociation constants, were comparable to that of STRE-binding  bound directly to a variety of G4-forming DNA oligos Figure . Similarr Figure  and C. T596–704) . For botg Figure . ImportaATG20, ATG39), trehalose synthesis genes , HSP150, HXK1, MSN4, RAD54, and SOD1 genes. Their transcription levels are significantly affected by G4 ligands (Figure ATG20, ATG39, TPS1 and TSL1 caused by the G4 ligands (Figure SμG4 locus only upon glucose down-shift-to-glycerol media condition (Figure A recent study using the yeast one-hybrid system placed a G4 DNA motif in the promoter of a reporter gene as a bait to identify G4 DNA-binding proteins in budding yeast and found 157 proteins . Howevers Figure . The loss Figure . Also, as Figure , and 67%s Figure . This ins Figure . We expen Figure . More exWe did a literature search and found 31 transcription factors of budding yeast that have the binding sites containing a guanine-run  and, notin vitro independent of a STRE with binding affinity similar to that of the STRE binding. We also confirmed that Msn2 protein is enriched at the G4-containing genomic loci under stress conditions and that Msn2 regulates the transcription of the promoter-G4 containing genes. These key findings suggest that G4 DNA may play a role in stress response through the interaction with the Msn2 transcription factor and that C2H2 zinc-finger proteins may be a new class of G4 binding protein.In summary, we have identified a new G4 binding protein, the C2H2 zinc-finger protein Msn2, and identified 37 yeast genes that have a PQS in the protomer with 20 of them having an overlapping STRE. We demonstrated that Msn2 binds to G4 DNA structures gkad684_Supplemental_FileClick here for additional data file.


Ecotropic viral integration site 1 (EVI1), encoded at the MECOM locus, is an oncogenic zinc finger transcription factor with diverse roles in normal and malignant cells, most extensively studied in the context of hematopoiesis. EVI1 interacts with other transcription factors in a context-dependent manner and regulates transcription and chromatin remodeling, thereby influencing the proliferation, differentiation, and survival of cells. Interestingly, it can act both as a transcriptional activator as well as a transcriptional repressor. EVI1 is expressed, and fulfills important functions, during the development of different tissues, including the nervous system and hematopoiesis, demonstrating a rigid spatial and temporal expression pattern. However, EVI1 is regularly overexpressed in a variety of cancer entities, including epithelial cancers such as ovarian and pancreatic cancer, as well as in hematologic malignancies like myeloid leukemias. Importantly, EVI1 overexpression is generally associated with a very poor clinical outcome and therapy-resistance. Thus, EVI1 is an interesting candidate to study to improve the prognosis and treatment of high-risk patients with “EVI1high” hematopoietic malignancies. This could potentially be explained by context or expression level-dependent activities of EVI1 that are different when EVI1 is lentivirally overexpressed in vitro versus a leukemia setting in patients.Evi1 gain of function models similarly illustrates an important role for Evi1 in HSC biology, with lentiviral overexpression of EVI1 expression is tightly restricted to the most primitive HSC compartment in normal hematopoiesis. However, approximately 10% of acute myeloid leukemia (AML) patients display abnormally high expression of the EVI1 transcript, but not the MDS1-EVI1 mRNA.27 Elevated EVI1 expression can be a result of 3q rearrangements, which comprise a high-risk AML entity as defined by the World Health Organization (WHO),28 but can also occur outside the context of such translocations, often in co-occurrence with mixed-lineage leukemia (MLL) rearrangements or chromosome 7 deletions.26 In Fanconi anemia patients suffering from MDS or AML, approximately every second patient shows EVI1 overexpression. Importantly, regardless of the presence of a 3q rearrangement and the exact mechanism of aberrant expression, all EVI1 overexpressing (EVI1high) leukemias are associated with a low rate of disease remission, high rates of relapse and chemotherapy resistance and very poor overall survival.31 A significant proportion of the EVI1high AML patients remains refractory, and of those who achieve complete remission,25 many relapse after a short time. Indeed, the median survival of 3q-rearranged AML patients is only 10 months.32EVI1 is overexpressed in around 10%–28% of pediatric AML cases, where it predicts a poor prognosis and is associated with MLL rearrangements in around one-third of the cases, while 3q rearrangements are very rare.33As noted above, EVI1 is overexpressed in 10% of patients with myelodysplastic syndrome (MDS),36 and in 30% of patients with chronic myeloid leukemia (CML) in blast crisis.39 Moreover, EVI1 is elevated in around 10% of chronic phase CML patients who demonstrate therapy resistance to the thymidine kinase inhibitor imatinib, with these patients not surprisingly exhibiting a particularly poor prognosis.38 In mouse models of both chronic phase CML, as well as blast crisis, Evi1 overexpression enhanced the proliferation and leukemogenic potential of these cells and conferred resistance to tyrosine kinase inhibitors.40In addition to AML, EVI1 apparently plays an important causal role in the evolution of other myeloid malignancies. 41 As in AML, CML, and MDS, high EVI1 expression in pediatric ALL may serve as a predictive factor for adverse clinical outcome. 3q rearrangements are rare in ALL and an association of high EVI1 expression with cytogenetic subgroups was not found, but elevated levels of EVI1 were again linked with poor prognosis in patients and enhanced cell survival, increased growth, and elevated leukemia initiation capacity in an experimental setting.42{Stevens, 2014 #151}While EVI1 is most frequently associated with myeloid malignancies, it is also dysregulated in some cases of adult and pediatric acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia, most commonly in B-ALL.44 breast cancer,45 pancreatic cancer,46 colorectal cancer,47 and prostate cancer.48 While transcription factors like EVI1 might have cell type-specific targets and functions resulting from a context-dependent remodeling of the epigenetic landscape and gene expression, it would be of interest to investigate the commonalities in the mechanism of action of EVI1 across these diverse cancer entities.Interestingly, aberrantly high levels of EVI1 have also been observed across a range of non-hematologic malignancies including ovarian cancer,Although EVI1 is overexpressed in a variety of cancers and is associated with therapy resistance and poor clinical outcome, the exact oncogenic mechanism of action downstream of EVI1 overexpression is still the subject of extensive investigation. Broadly speaking, EVI1 is canonically thought of as a classical transcription factor that can both positively and negatively modulate the expression of target genes via the recruitment of the transcriptional machinery to specific genomic loci, or by the alteration of local chromatin structure into an active or repressive state as a result of interactions with proteins with epigenetic remodeling activity. However, some literature also suggests that EVI1 may elicit effects that are not directly related to the regulation of transcription.13 Indeed, the proliferation of definitive HSCs in mouse embryos was heavily impaired in Evi1-/- mice, presumably due to the decrease in Gata2 expression.13 EVI1 also binds to an upstream regulatory element of Spi1, which encodes the pioneer transcription factor PU.1.49 Binding to this site, approximately 14kb upstream of Spi1, requires both zinc finger domains and increases Spi1 transcription. As a consequence, erythropoiesis and lymphopoiesis are suppressed while myelopoiesis is enhanced. Aberrant overexpression of EVI1 can thus result in aberrant myeloid expansion and, ultimately, in leukemic transformation.49 A recent study found the ETS transcription factor ERG to be a direct transcriptional target of EVI1.50 EVI1 can drive ERG expression by binding to an intragenic +85 enhancer that is conserved between humans and mice. This region is occupied by EVI1 in AML cell lines and also in primary AML patient samples, leading to aberrant ERG expression that maintains cells in an immature differentiation stage and constitutes a dependency in EVI1-driven AML.51 Furthermore, EVI1 can also directly bind to the promoter of the proto-oncogenic transcription factor PBX1 and activate its transcription52  gene  signaling. Treatment of murine Evi1-driven leukemias with the mTOR inhibitor rapamycin significantly prolonged the survival of recipient mice.54 This sensitivity towards inhibition of mTOR using rapamycin might also be an attractive therapeutic strategy for human EVI1high leukemia. EVI1 also recruits other transcriptional co-repressors to chromatin. Evi1 can form a co-repressor complex with C-terminal binding protein (CtBP)55 and phosphorylation at S436 of the EVI1 protein seems to influence the association with CtBP1.10 Moreover, it was shown that Evi1 can bind directly to the MH2 domain of Smad3, an intracellular mediator of TGFβ signaling,56 via its first zinc finger domain.57 In this context, through the interaction with the EVI1/CtBP co-repressor complex, binding of the Smad3 complex to DNA is prevented  inhibitor trichostatin A, the transcriptional repression of TGFβ signaling could be prevented, suggesting an involvement of HDAC in this EVI1-mediated process.55 In addition to Smad3, Evi1 has also been shown to interact with, and thereby repress the transcription of, other proteins of the Smad protein family, including Smad1 and Smad2 which are targets of bone morphogenetic protein (BMP) and activin signaling. Moreover, following stimulation with TGFβ, Evi1 binds to the promoter of Smad7 in a complex with CtBP to inhibit Smad7 transcription.57 In this context, the deletion of the CtBP1 binding site in EVI1 prevented an increase in proliferation observed in 32Dcl3 cells upon EVI1 overexpression, demonstrating the functional relevance of this interaction.58 Taken together, EVI1 mediates signaling of several TGFβ family ligands, including TGFβ, BMP, and activin, that are all implicated in embryogenesis and are often deregulated during malignant transformation, resulting in de-regulation of cellular functions including proliferation, differentiation, and cell survival.In its role as both a transcriptional activator as well as a transcriptional repressor, EVI1 also interacts with a range of proteins with chromatin remodeling activities. In bone marrow hematopoietic cells, Evi1 directly binds to several members of the polycomb repressive complex (PRC), including PRC1 and PRC2/3/4, facilitating their recruitment to the promoter of the 59 These HMTs methylate histone H3 at lysine 9 (H3K9) which is generally associated with the silencing of genes. Inactivation of SUV39H1 prevented the transcriptional repression of TGFβ-responsive elements by Evi1 and knockdown of either of these 2 HMTs decreased the colony-forming potential of Evi1-overexpressing hematopoietic stem and progenitor cells in a CFU assay and thus restricted their self-renewal capacity.59EVI1 can also directly interact with the histone methyltransferases (HMTs) G9a and SUV39H1  DNMT3A and DNMT3B via its proximal zinc finger domain and mediates de novo DNA methylation of target regions.58 ; and p300/CBP-associated factor (P/CAF), which are both known to exhibit acetyltransferase activity, acetylating both transcription factors and histone lysine residues62 EVI1 also binds to GATA1 via 2 zinc fingers in the proximal zinc finger domain, preventing DNA binding and repressing GATA1 function, resulting in a block in erythroid differentiation.63 The distal zinc finger domain of EVI1 is able to mediate interaction with AP1, modulating its transcriptional regulatory activity.64 Indeed, the ChIPseq experiment in human ovarian carcinoma cells revealed an approximate 25% overlap in EVI1 and AP1 binding sites, suggesting a synergistic interaction of these transcription factors. The eighth zinc finger within the distal zinc finger domain of EVI1 mediates interaction with the DNA-binding Runt domain of RUNX1, thereby reducing the binding of RUNX1 to DNA.65 Consequences of this interaction include a block in differentiation and an increase in cell death.65 Thus, overexpression of EVI1 effectively leads to a functional repression of RUNX1. One consequence of this inhibition is the upregulation of the creatine kinase CKMT1, which in turn modulates WNT and GSK3 signaling.66In addition to its association with transcriptional co-activators, co-repressors, and chromatin remodeling enzymes that cannot directly bind DNA, EVI1 forms complexes with other transcription factors, resulting in a range of functional consequences. EVI1 binds to PU.1 and therefore interferes with its function by specifically blocking its interaction with the coactivator c-Jun , which plays a crucial role in signal transduction related to stress-induced cell death7 Figure D. As a c8 Figure C, which In summary, the EVI1 protein is able to directly interact with a wide range of additional proteins, including transcription factors, transcriptional co-activators, transcriptional co-repressors, chromatin remodelers, and other signaling proteins such as protein kinases. These diverse interactions may in part explain the apparent “master regulator” effect that EVI1 appears to play in both normal and malignant HSC biology and how it is able to coordinately impact upon several cellular processes that are directly implicated in malignant disease, including differentiation, cell proliferation, and cell death.The downstream functional activity of EVI1 is frequently deregulated in a variety of different cancer entities and is often associated with a particularly poor prognosis. This oncogenic deregulation of EVI1 frequently relates to a simple alteration in mRNA expression which mediates elevated protein levels but can additionally be a consequence of changes in protein function.MECOM locus exert their oncogenicity via aberrant overexpression of EVI1 while leaving the protein structure intact, the translocation t results in a fusion of the EVI1 protein with another transcription factor required for normal hematopoiesis: RUNX1  patients is only 14%.32 In a chimeric knock-in mouse model, Runx1-Evi1 expression alone was sufficient to induce a megakaryoblastic leukemia in adult mice,71 while Runx1-Evi1 expression early during hematopoietic development was embryonically lethal around day E13.5. There are several molecular consequences resulting from expression of this transcription factor fusion: transcriptional activation and repression mediated by wild-type RUNX1 and EVI1 are disrupted; and the fusion protein gains new oncogenic functions and novel transcriptional targets,72 resulting in extensive epigenetic reprogramming of hematopoietic progenitor cells undergoing lineage fate decisions.73 In the t SKH-1 cell line, knockdown of the RUNX1-EVI1 fusion gene resulted in myeloid differentiation, indicating a role of the oncogenic protein in blocking the normal differentiation process.72 The effect of the RUNX1-EVI1 fusion was also studied in an in vitro differentiation model for hematopoietic specification using mouse embryonic stem cells. Combined analysis of transcription factor binding and gene expression in these cells revealed that the cell fate decision of multipotent hematopoietic progenitor cells was disrupted by RUNX1-EVI1, and a multi-lineage gene expression pattern was induced. Both the RUNX1- and the EVI1-mediated gene expression programs were disturbed by RUNX1-EVI1 expression. In addition to the observed changes in the differentiation and lineage specification of the cells, the fusion also led to a block in the cell cycle, decreased colony-forming potential and increased apoptosis.73 In summary, the RUNX1-EVI1 fusion both disrupts the normal RUNX1 and EVI1-mediated gene expression programs and regulates its own unique targets. This results in alterations of the cell cycle and the failure of hematopoietic progenitor cells to undergo cell fate commitment.73While many translocations involving the ocation t;21 translocation have been reported in poor prognosis AML, MDS, and blast crisis MDS.;12 and inv(3), occurring in approximately 2.5% of all AML cases.32 Indeed, these rearrangements are defining features of a distinct AML entity recognized in the WHO classification system28 and the more recently established International Consensus Classification (ICC) of myeloid neoplasms and acute leukemias,75 and can also be observed in CML in blast crisis.76 In AML with t or inv(3), an enhancer of GATA2 at 3q21, also called the GATA2 distal hematopoietic enhancer (G2DHE), is relocated proximal to the MECOM locus at 3q26 /inv(3) cases.81 The mutated SF3B1 causes alternative splicing of the EVI1 transcript, leading to a novel oncogenic isoform with an addition of 18 base pairs/6 amino acids in the distal zinc finger domain.82 This EVI1+18 isoform exhibits altered DNA binding and increased oncogenicity.Mutations in the splicing factor EVI1 overexpression, with enhancer hijacking operating as a recurrent mechanism of dysregulation. In AML with t, an MYC “super-enhancer” comprised of several enhancer modules is translocated to the MECOM locus /inv(3), other translocations can also lead to ;3/inv(3)3q26 translocations that involve super-enhancers of genes that play an important role in myeloid development, including CD133 (PROM1), CD164, and CDK6 ).86 The mechanism of EVI1 overexpression as well as the pathophysiology of these leukemias resembles that of the classical inv(3)/t(q21q26) AML entity in that EVI1 expression is high, the expression of MDS-EVI1 is low or absent and in half of the cases, levels of GATA2 are also reduced.85 Since the translocation in these leukemias does not involve the GATA2 enhancer, the mechanism behind the low GATA2 expression in these cases is unclear. Interestingly, some of the transcription factors known to bind to the GATA2 enhancer at 3q21, for example, ERG, GATA2, LMO2, and RUNX1, are reported to also interact with the loci involved in these atypical 3q26 translocations.87 All in all, it is possible that atypical 3q26 rearrangements and the classical inv(3)/t constitute a single AML entity that is characterized by elevated EVI1 levels through enhancer hijacking, absent MDS-EVI1 and a reduction in GATA2, while clinically sharing a poor prognosis and the occurrence of resistance to chemotherapy.There are also other atypical EVI1 overexpression was accompanied by an insufficiency of GATA2 as a result of the translocation events described above, other translocations leading to high levels of EVI1 in the absence of altered GATA2 expression have also been described. Cases of t, as reported by Haferlach et al88 and D’Angió et al,89 relocate the EVI1 gene proximal to regulatory elements of the Nuclear Receptor Interacting Protein 1 (NRIP1) gene. As is the case for the t RUNX1-EVI1 translocation, there is no evidence that t results in the expression of a novel fusion protein.90 However, the NRIP1 promoter and associated enhancers contain multiple elements that are responsive to retinoic acid and NRIP1 transcription can be stimulated by treatment with all-trans-retinoic acid (ATRA). It is therefore likely, that ATRA treatment will also lead to the upregulation of EVI1 in cases with t.While EVI1 is translocated to the MSI2 locus.91 While no fusion protein can be detected, the translocation increases the expression of EVI1.91 Also cases of translocations involving the short arm of chromosome 2, t, have been reported in AML and CML in blast crisis92 with poor clinical outcome, leading to a >20-fold overexpression of EVI1.93In myeloid malignancies with t, EVI1 locus at 3q, for example spanning the region of 3q26q29, is a frequent early event in myeloid malignancies that occur in Fanconi anemia (FA) patients,95 with the EVI1 locus being contained in the minimally amplified region  or the growth-restrictive cell fate outcomes that result from activation of the DDR. It is tempting to speculate that such a relationship may be more broadly relevant to the poor outcome and therapy resistance observed in EVI1high leukemias in non-FA patients.Amplification of the 98 Indeed, these transcription factors directly bind to a 318bp region that has been characterized as the minimal promoter region of EVI1. Knockdown of ELK1 or RUNX1 results in decreased EVI1 levels, while their overexpression led to an increase. EVI1 and RUNX1 levels also seem to positively correlate in AML patient samples, indicating a causal relationship which appears to relate to RUNX1 exerting a regulatory role on EVI1 expression via mediating acetylation of histone H3 in the EVI1 promoter region.98 Whether translocations involving the RUNX1 gene influence its ability to bind to the EVI1 promoter has yet to be determined.The majority of cases of EVI1 overexpression in hematologic malignancies do not occur in the context of translocations involving the MECOM locus, suggesting that aberrant epigenetic programming of the EVI1 promoter/enhancer elements is a more frequent driver of this oncogenic event. Indeed, known upstream regulators of EVI1 transcription include GATA1, ELK1, and RUNX1, meaning that altered expression or activity of any of these transcription factors could modulate EVI1 activity  enhancers to the MECOM locus via translocations at the 3q locus. Thus, an obvious therapeutic strategy in 3q-rearranged AML could be to prevent the aberrant activation of the EVI1 promoter by inhibition of such super-enhancers. One way that this could be achieved by pharmacologic BET bromodomain inhibition.111 Indeed, in both 3q-rearranged cell lines as well as primary t AML patient samples, treatment with the BET bromodomain inhibitor JQ1 led to a decrease of EVI1 and growth arrest of the AML cells.77 Other studies have specifically investigated the binding of important myeloid transcription factors to the GATA2 distal hematopoietic enhancer that is often relocated to the EVI1 locus, including CEBPA and RUNX1. Proteome analyses have identified PARP1 and IKZF1 among the colocalizing proteins at this locus, thereby identifying novel therapeutic targets to interfere with the oncogenic super-enhancer.112 Inhibition of PARP1, both pharmacologically with olaparib or talazoparib, and shRNA-mediated, was able to reduce the interaction of the super-enhancer with the EVI1 promoter and decreased the expression of EVI1. As a consequence, differentiation and apoptosis in 3q-rearranged AML cell lines was increased.112As described previously, a recurrent mechanism of 55 Thus, selective inhibition of the enzymatic activity of proteins that act as co-regulators of the EVI1 transcription factors may well comprise a feasible targeted approach for the treatment of EVI1high malignancies.The EVI1 protein contains several different functional domains, allowing it to associate with a range of proteins that can impact on target gene expression, including chromatin remodelers like HMTs and HADCs. With regard to the latter, HDAC inhibitors, such as trichostatin A, already exist. It has already been shown in vitro that trichostatin A treatment is able to revert EVI1-mediated transcriptional repression of TGFβ signaling.EVI1 gene falling under the control of regulatory elements of the retinoid acid-responsive gene NRIP1.90 Upregulation of NRIP1 can be observed in AML cases with elevated EVI1 levels, presumably due to binding of EVI1 to a NRIP1 enhancer. Knockdown of NRIP1 in 3q-rearranged cell lines reduced the proliferation and viability of these AML cells and was also able to increase their sensitivity towards treatment with all-trans-retinoic acid (ATRA),90 making NRIP1 a potential therapeutic target in EVI1high AML cases. Whether EVI1high AML cases might profit from treatment with ATRA is still under continued investigation.As described previously, the t translocation leads to the 3q-rearranged/EVI1high AML samples. Verhagen et al observed in vitro induction of differentiation upon ATRA treatment in 9 out of 13 EVI1high AML cases, coupled with a reduced viability of the AML blasts in some cases.113 The pretreatment with ATRA also resulted in decreased survival of these cells after treatment with doxorubicin. In 2 out of 3 cases, ATRA treatment also resulted in reduced leukemic engraftment of primary AML blasts in NOD SCID-IL2g knockout (NSG) mice. Interestingly, a second study has shown a synergistic effect of combining the bromodomain inhibitor JQ1 with ATRA to induce differentiation and apoptosis in vitro in the non-APL AML cell lines HL-60 and MV4-11.114 In a clinical study by Paubelle et al, 13 high-risk EVI1high AML cases were treated with ATRA, of which 7 achieved complete remission.115 While these results are promising, it is also clear that the response of EVI1high AML cells to ATRA is very heterogenous and warrants further investigation with larger cohort/sample sizes to determine whether it is possible to identify criteria that robustly sub-classify patients with EVI1high leukemias who would respond at ATRA treatment as a combined or mono-therapy.More generally, following on from the clinical success of treating acute promyelocytic leukemia (APL) with ATRA, this so-called “differentiation therapy” has been broadly tested against a range of hematologic malignancies, regardless of whether they harbored the APL-specific translocation between the retinoic acid receptor-α (RARA) and APL genes that specifically confers sensitivity to ATRA. Some of these studies have encompassed 116 which has been reported to constitute a metabolic vulnerability of EVI1high leukemic cells. Both the shRNA-mediated knockdown of CKMT1, as well as pharmacological inhibition using the small molecule cyclocreatine, reduced the growth of EVI1high AML cells and induced cell cycle arrest and apoptosis in human cell lines, orthotopic xenograft models and mouse models of primary AML. At a mechanistic level, cyclocreatine treatment interfered with the cell’s mitochondrial respiration and ATP production and altered both WNT signaling and GSK3 signaling.116 Given the important role of these signaling pathways in normal HSCs, it has to be clarified whether a therapeutic window exists to facilitate the successful treatment of EVI1high leukemias.The EVI1-mediated inhibition of RUNX1 results in the upregulation of the mitochondrial creatine kinase CKMT1,high AMLs towards the inhibition of ERG signaling.51 In the EVI1high leukemic cells, knockdown of ERG led to a decrease in proliferation and an increase in apoptosis, and also induced differentiation.50 However, achieving a pharmacological inhibition of ERG in AML patients, as is the case for many transcription factors, remains a challenge and is currently still a subject of research and development.117Two independent studies have demonstrated a therapeutic vulnerability of EVI1EVI1 locus and corresponding transcript levels can be preserved after the malignant transformation of HSCs with the MLL-AF9 oncogenic fusion gene.68 In an MLL-AF9 AML mouse model, the GMP-derived EVI1low murine leukemic cells showed a higher degree of apoptotic priming and were more sensitive to treatment with doxorubicin and to treatment with the LSD1 inhibitors IMG-7289 and IMG-98 than the HSC/multipotent-progenitor (MPP)-derived EVI1high malignant cells. Knockdown of Evi1 in HSC/MPP-derived MLL-AF9 leukemic cells sensitized the cells to treatment with several chemotherapeutic agents, including doxorubicin, ionizing radiation, and combination treatment with venetoclax and LSD1 inhibitors.68 The authors could ascribe this EVI1-mediated reduction in apoptotic priming to decreased p53 protein abundance via a proteasome-independent mechanism that is as yet uncharacterized68  and the Dietmar Hopp Stiftung (MDM).


The study shows that estrogen activates transcription within pre-established chromosomal 3D architectures, strengthening enhancer interactions through local accumulation of the estrogen receptor α. The genome is organized in functional compartments and structural domains at the sub-megabase scale. How within these domains interactions between numerous cis-acting enhancers and promoters regulate transcription remains an open question. Here, we determined chromatin folding and composition over several hundred kb around estrogen-responsive genes in human breast cancer cell lines after hormone stimulation. Modeling of 5C data at 1.8 kb resolution was combined with quantitative 3D analysis of multicolor FISH measurements at 100 nm resolution and integrated with ChIP-seq data on transcription factor binding and histone modifications. We found that rapid estradiol induction of the progesterone gene expression occurs in the context of preexisting, cell type-specific chromosomal architectures encompassing the 90 kb progesterone gene coding region and an enhancer-spiked 5′ 300 kb upstream genomic region. In response to estradiol, interactions between estrogen receptor α (ERα) bound regulatory elements are reinforced. Whereas initial enhancer–gene contacts coincide with RNA Pol 2 binding and transcription initiation, sustained hormone stimulation promotes ERα accumulation creating a regulatory hub stimulating transcript synthesis. In addition to implications for estrogen receptor signaling, we uncover that preestablished chromatin architectures efficiently regulate gene expression upon stimulation without the need for de novo extensive rewiring of long-range chromatin interactions. Rapid cellular responses to external stimuli rely on regulation of gene expression. Among numerous steps required for this response, the spatial organization of the genome is known to modulate DNA accessibility to the transcriptional machinery and to promote contacts between genes and distant regulatory DNA elements such as enhancers . In the PGR) and the ERα coding gene itself (ESR1). Estrogens and hormones, in general, control transcription of hundreds of genes in the eukaryotic nucleus. It was shown that changes in gene expression are accompanied by modifications in global genome structure in ER-expressing cells  and their growth is independent of hormones. We selected 0.6–1.3-Mb domains around four ER-regulated genes: the estrogen receptor gene (ESR1) located on chromosome 6, the growth regulation by estrogen in breast cancer 1 gene (GREB1) located on chromosome 2, the cyclin D1 gene (CCND1) and the PGR on chromosome 11, and determined their 3D conformation using 5C , revealed slightly stronger interaction frequencies at long distances in MCF7 compared with MDA-MB-231 cells (proteinatlas.org). In MDA-MB-231 cells, SYNE1 is expressed and it is possible that transcription may lead to loss of this boundary, allowing increased interactions between the 5′ part of SYNE1 with the rest of the locus including ESR1. 5C contact maps and interaction counts for the control gene PUM1 (We used Chromosome Conformation Capture Carbon Copy (5C) with an alternating primer design  to assesF7 cells . In MCF7ll lines   were lost 3 h after E2 stimulation  appeared as soon as 45 min of E2 stimulation and remained stable at 3 h E2  by 3D DNA FISH  defining a 2D survival zone (Sz) with one degree of freedom. Using the measured distances, inverse mathematical modeling predicts each locus’ distribution inside survival zones . Survival zone radii of loci correlate with relative freedom of movement of one locus with respect to the two others  surrounding the PGR gene were largely reduced compared with the zones these segments can explore in hormone-deprived cells. In contrast, the centrally located Fos3 locus which corresponds to the TSS region of the PGR seemed to become more dynamic after transcription activation by E2. Upon activation, the distal upstream region (Fos5) folds toward the PGR gene body. Its freedom of movement is reduced twofold  freedom of movement was increased twofold, from r = 85 nm to r = 172 nm, observed 3 h poststimulation  for 3 d. Genomic sites for which distances were shortened in at least 50% of E2-treated cells (+E2) are linked by red arches PGR domaid arches . This fid arches . Distancd arches  and S3A d arches . The resanalysis . Overallanalysis , PGR domstradiol .PGR gene can be induced within minutes by adding E2. The ensuing mRNA synthesis is known to increase over time , necessary for ERα-dependent gene activation were also recruited to the enhancers  and more generally with transcription-induced reduced chromatin dynamics (Preprint) but here as a mechanism to reversibly regulate acute activation.Here, we show that chromatin fiber folding reflects transcriptional activity at the level of genomic domains and single genes. We discovered that domains encompassing silent genes show disordered structures that lack domain boundaries in triple-negative human breast cancer cell lines reminiscent of silent domains in multiple cell lines . In ERα-ced gene Preprintdynamics   for MCF7 or DMEM (Gibco) for MDA-MB231 with Glutamax and complemented with 50 μg/ml gentamicin, 1 mM sodium pyruvate, and 10% FCS. Cells were grown at 37°C in a humidified atmosphere containing 5% COPGR, ESR1, CCND1, and GREB1) cells were grown for 3 d in phenol red-free media supplemented with 10% charcoal-stripped FCS (−E2) and subsequently treated with 100 nM E2 (Sigma-Aldrich) for the indicated times.To study the effects of 17β-estradiol (E2) on estrogen-regulated genes (3D DNA FISH experiments were performed as previously described in Fosmids were purchased at C.H.O.R.I. (see Table S1) and were labeled using the BioPrime DNA Labeling System from Invitrogen by incorporation of fluorochrome-conjugated nucleotides Atto647N-dUTP-NT (Jena Bioscience), atto550-dUTP-NT (Jean Biosciences) or ChromaTide AlexaFluor 488-5-dUTP (Molecular Probe). Labeled probes were then purified trough a G50 column from the Illustra MicroSpin G50 kit  and precipitated overnight with salmon sperm DNA (Sigma-Aldrich), human Cot-1 DNA (Invitrogen), 3 M NaAc, and 100% cold EtOH. After centrifugation, the pellet was resuspended in an appropriate volume of HP  to obtain equal concentration (100 ng/μl) for all fosmid probes. For DNA-FISH, 200 ng of labeled fosmids were added to the prepared coverslips. The coverslips were sealed with rubber cement (Electron Microscopy Sciences) and placed into a hybridizer (Dako). Denaturation of the probes and target DNA was performed simultaneously at 85°C for 2 min and samples were then incubated overnight at 37°C. Before microscopy, coverslips were then washed, with gentle shaking, four times for 3 min in 2xSSC at 45°C, and four times for 3 min in 0.1x SSC at 60°C before being mounted with 4 μl of Vectashield Antifade Mounting Medium with DAPI at 1.5 μg/ml and sealed with transparent nail polish.Fosmids used in this study.Table S1. P-value ≤ 0.05 was considered statistically significant.3D-DNA FISH observations were performed using an Olympus IX-81 wide-field fluorescence microscope, equipped with a CoolSNAP HQ CCD camera (Photometrics), a Polychrome V monochromator (Till Photonics) equipped with a 150 W xenon source used with a 15-nm bandwidth, an electric PIFOC piezo stepper (PI) with an accuracy of 10 nm, and imaged through an Olympus oil immersion objective 100x PLANAPO NA1.4. Acquisitions of DAPI, Alexa 488, ATTO550, and ATTO647 fluorophores were performed using multiband dichroic mirrors (Chroma), specific single-band emission filters mounted on a motorized wheel (PI), and emission filters ET450/40, ET520/40, ET580/40, and ET685/60. Image acquisitions were performed in ∼21 focal planes with a 200-nm step size for 3D DNA FISH. This configuration was driven by MetaMorph . Images were analyzed using a home-made script running on ImageJ . Inter-phttps://www.ncbi.nlm.nih.gov/geo/) and treated as subsequently described. Raw data were downloaded with fastq-dump from sratoolkit (2.8.2-1). (https://github.com/ncbi/sra-tools). The quality of the reads was estimated with FastQC (0.11.7). Sequencing reads were aligned to the reference human genome assembly (hg19/GRCh37) using Burrows–Wheeler aligner  aligner . ERBS we aligner . Paused  aligner . ER cofaDatasets used in this study .Table S2. GSE62789) (RNA-Seq time course in MCF7 before and after E2 treatment were downloaded from GEO (accession number: SE62789) . Raw dat5C primers were designed using My5C . 5C emplWe used My5C  to desigPGR, ESR1, CCND1, and GREB1) have been computed with hicPlotDistVsCounts tool from HiCExplorer (2.2.1.1) utilities. Correlation Heatmaps were computed with hicCorrelate tool from HiCExplorer. To study interaction counts distributions at ERBS/ERBS and ERBS/TSS for the PGR gene, we considered a window region from each ER peak summit ±10 kb (±15 kb for ERE7) within the PGR gene region matrices and PGR gene TSS ±10 kb. We then plotted the corresponding averaged interaction count values around the two interacting regions, and compared each of the two E2 induction time-point distributions. Violin plots have been generated using R home-made scripts.Interaction counts by genomic distance for estrogen-regulated gene regions . Conversely, a decrease of contacts in at least 20% of models with respect −E2 models indicated a decrease of an arch (blue color).x,y,z coordinates of the fosmid.Euclidean distances in Cartesian space between selected fosmids were calculated for the entire ensemble and represented as box plots. If a fosmid occupied more than one particle, the center of mass of the constituting particles was used as the molmap function in Chimera). Finally, the “centroid” model  was represented as a thick worm-like structure for easy visualization (obtained using the shape tube function in Chimera).The UCSF Chimera package , a highldrgmin, drbmin, dgbmin, the minimal distances) and drgmax, drbmax, dgbmax the maximal distances between R–G–B, respectively.Distances between the three loci R, G, and B were measured in 3D (see above).We can note thatdrbmin, drbmax) and .We have the same inequalities for couples , , ) were deduced as follows:For each triplet of measured distances, c is the color index R, G or B. N is the number of survival zones subdivision and M is the polar angle subdivision.To solve system https://github.com/FlavienRaynal/PGR_Kocanova for 5C and ChIP-seq analyses and at https://src.koda.cnrs.fr/alain.kamgoue.3/tripleloci for triple loci.Code and data can be accessed at