Plasmodium falciparum. In addition, most of the genes in Plasmodium falciparum are transcriptionally poised and only a handful of cis-regulatory elements are known to operate in transcriptional regulation. Here, we employed an epigenetic signature based approach to identify significance of previously uncharacterised intergenic regions enriched with histone modification marks leading to discovery of enhancer-like elements.The molecular mechanisms of transcriptional regulation are poorly understood in Plasmodium falciparum. Our study also suggests that the Plasmodium genome segregates functionally related genes into discrete housekeeping and pathogenicity/virulence clusters, presumably for robust transcriptional control of virulence/pathogenicity genes.We found that enhancer-like elements are significantly enriched with H3K4me1, generate unique non-coding bi-directional RNAs and majority of them can function as cis-regulators. Furthermore, functional enhancer reporter assay demonstrates that the enhancer-like elements regulate transcription of target genes in Plasmodium falciparum.This report contributes to the understanding of parasite regulatory genomics by identification of enhancer-like elements, defining their epigenetic and transcriptional features and provides a resource of functional cis-regulatory elements that may give insights into the virulence/pathogenicity of The online version of this article (doi:10.1186/s12864-017-4052-4) contains supplementary material, which is available to authorized users. Plasmodium species from apicomplexan genera, with an estimated 200 million cases every year . Mo. MoP. faPlasmodium enhancer-like elements are also co-enriched with H3K4me3, H3K9ac and H4ac  were extracted from PlasmoDB (http://plasmodb.org). Coding Potential Calculator (CPC) was found to predict 100% known non-coding RNAs from Plasmodium  was reported in lciparum . We obseIn higher eukaryotic systems enhancers are shown to regulate the transcription of nearest neighbourhood gene and their RNA (eRNA) level correlates with expression of the nearest gene . We wondP. falciparum genome at three different stages [Plasmodium genome [Plasmodium pathogenesis like cytoadherence and rosetting while genes with 5 or less contacts are involved in housekeeping functions  family of proteins and are the major known transcription factors in genome [Plasmodium enhanceosome will aid in our understanding of the concerted action of sequence- and/or structure-specific transcription factors, chromatin modifiers and remodelers that participate in transcriptional regulation mediated by enhancer-like elements.n genome . Genome-n genome –60. The n genome , 61. Ourn genome . Future P. falciparum that function as transcriptional enhancers. As observed in higher eukaryotes such as human and mouse, intergenic regulatory elements produce non-coding RNAs, which positively correlate with the gene expression. Even though the function of these elements remains to be established individually in P. falciparum, our study is the first report in any lower eukaryotic parasitic system where the enhancers are characterized in such greater details. Thus, this report contributes to the fundamental understanding of parasite regulatory genomics by first time identification of novel genome-wide enhancer-like elements, defining their epigenetic and transcriptional features and provides a resource of functional cis-regulatory elements that may give insights into the virulence/pathogenicity of Plasmodium falciparum.In conclusion, this study identified genome-wide intergenic regulatory elements in P. falciparum culture-adapted parasite lines were kindly provided by Dr. Vasudevan Seshadri  and were cultured as previously described [−1 gentamicin sulphate at 37 °C. Parasites were subcultured by replacing the entire spent medium and splitting the culture into two or more flasks to maintain parasitemia to 10% and quickly restoring the hematocrit to 1% in the required volume of culture medium.escribed . Brieflyhttp://www.ncbi.nlm.nih.gov/gds) under the accession number GSE63369 . ChIP-sequencing data for histone modifications  and RNA-sequencing (GSE23865) were downloaded from the public databank Gene Expression Omnibus for P. falciparum [http://r-project.org/). Sequences of primers used for quantitative ChIP-PCR validation are listed in Additional file ChIP-seq datasets were downloaded from the public data bank Gene Expression Omnibus  and transcription termination sites (TTSs) of coding gene in  earlier . Interge earlier  and +/− seqMINER .We extracted the tag density in a 1.5 kb window surrounding the intergenic peaks using the program seqMINER which generates heatmap as well as the profiles . The seqDistance from Transcription Start Sites (TSSs) for each intergenic peak was calculated from the centre of the intergenic peak to the nearest TSS. The distribution of peaks is plotted for every 500 bps around the TSSs.P. falciparum. Length of intergenic peaks is calculated by taking the mean tag density over the intergenic peaks in a window of +/− 1000 bps. Background tag density was used to put an arbitrary cut-off for determining the average intergenic peak length.P. falciparum3D7_Genome_v9.3 was used to calculate the length of all the genes in Total tag density of histone modifications was calculated as described above in average profile calculation over the strong intergenic peaks, 500 strongly and weakly expressed promoters. The profiles of histone modifications over the intergenic peaks, strongly and weakly expressed promoters are calculated using seqMINER .Plasmodium falciparum. pHC1 vector was first digested with the HindIII restriction enzyme to remove the extra set of promoter and 3′ UTR (CAM5’ promoter and HSP86–3′ UTR) and re-ligated using T4 DNA ligase. Later Tg-DHFR-TS gene was replaced with the Luciferase gene from pGL3 promoter vector (Promega) and resultant vector is called pHC1 enhancer vector. Predicted intergenic sites enriched with H3K4me1 and negative regions devoid of any regulatory marks (~1 kb) were amplified from Plasmodium genomic DNA and cloned into pHC1 enhancer vector in single HindIII restriction site upstream of luciferase gene and PcDT 5′ promoter. Confirmation of orientation was performed by restriction mapping and DNA sequencing. Co-ordinates of the cloned sites and primer information are provided in Additional file pHC1 vector (obtained from BEI resources) was used as a template to develop a enhancer luciferase system for 0 C. Parasite pellets obtained by centrifugation at 2800 rpm for 5 min were washed with 1X PBS to remove haemoglobin. Parasites were lysed in 30 uL of 1X Passive Lysis Buffer by incubating on ice for 10 min. Luciferase assay was performed with Promega Kit as per instructions. Lysates were spun at top speed for 3 min and 20 uL of supernatant is added in 100 uL of Luciferase Assay Reagent II (LAR II) and firefly luciferase activity measured luminometrically. Luminescence Units were expressed as fold change above the empty vector control and also normalised with parasitemia counts measured at the time of harvesting. Experiments were performed at least twice.Equal amount of parasites were cultured on RBCs loaded with indicated DNA constructs for 2 passages. Culture was spun at 1500 rpm for 5 min, and the pellet subjected to saponin lysis in 1X PBS for 10 min at 4Genes were categorized based on increasing expression levels (based on reads assigned per kilobase of target per million mapped reads (RPKM)) using the RNA-seq data (GSE23865). Datasets for different stages were normalized for the total number of uniquely mapped tags. Stage specific RNA-Seq data was corrected for each stage by a correction factor (based on the amount of RNA per parasite nucleus) present in each stage as described earlier. Distribution of RNA-seq reads over the intergenic peaks, and 500 strongly and weakly expressed promoter is calculated using the seqMINER . Correlahttp://plasmodb.org/common/downloads/release-9.3/Pfalciparum3D7/).Circular plot was generated in R v3.2.2 using circlize v0.3.5 package of CRAN. The genomic interactions observed in Hi-C data  were filwww.plasmodb.org) and the 462 enhancer sequences were 22.867 and 20.949, respectively. GC% of a sequence was calculated by dividing the number of G or C nucleotides in the sequence by the sequence length, and multiplying with 100. Similarly, the GC% at a base position in the enhancer sequences was calculated by dividing the number of G or C nucleotides at the position by the number of enhancer sequences accounted for that position, and multiplying with 100.Nucleotide sequence of the 462 enhancer-like elements were prepared by fetching 250 bases up and down of the peak centre using P. falciparum3D7_Genome_v9.3, and per base GC content and per sequence GC content was calculated. The average GC% in 5398 protein coding genes (downloaded from meme-suite.org/tools/meme-chip). It is a comprehensive motif analysis tool for large nucleotide sequences. Input DNA was MALARIA  DNA and database was Malaria . The prhttp://cpc.cbi.pku.edu.cn/), PLEK , CNCI  and CPAT .Tools tested for predicting coding potential of given sequence are CPC  and ChIP-qPCR are performed as described previously , 67. InfParasites were treated with 10 mM sodium butyrate for 0, 5, 10, 30 min, 1, 2, 4, 8 and 16 h. Parasites were cross-linked and ChIP was carried out as described above using H3K9ac antibody . Levels http://madiba.bi.up.ac.za/).Gene ontology analysis was performed using MADIBA ; an onliAdditional file 1:Supplementary Figures (S1-S9). (DOCX 2070 kb)Additional file 2: Table S4.Plasmodium falciparum. (XLS 71 kb)List of the enhancer-like elements identified in Additional file 3: Table S5.Overlap between New TSS List from Adjalleyet. al., 2016and the enhancer-like elements identified in this study. (XLS 19 kb)Additional file 4: Table S1.Table S2. Co-ordinates and primers used for the cloning of Intergenic regions for luciferase reporter assay are provided as a separate excel-file. (XLSX 12 kb)Primers used for ChIP-qPCR are provided as a separate excel-file. Additional file 5: Table S3.Contact information for ring, trophozoite and schizont stages is provided as a separate excel-file. (XLS 11820 kb)


Streptomyces avermitilis. Eleven genes/operons were demonstrated to be directly regulated by Rex; these genes/operons are involved in aerobic metabolism, morphological differentiation, and secondary metabolism. Rex represses transcription of target genes/operons by binding to Rex operator (ROP) sequences in the promoter regions. NADH reduces DNA-binding activity of Rex to target promoters, while NAD+ competitively binds to Rex and modulates its DNA-binding activity. Rex plays an essential regulatory role in aerobic metabolism by controlling expression of the respiratory genes atpIBEFHAGDC, cydA1B1CD, nuoA1-N1, rex-hemAC1DB, hppA, and ndh2. Rex also regulates morphological differentiation by repressing expression of wblE, which encodes a putative WhiB-family transcriptional regulator. A rex-deletion mutant (Drex) showed higher avermectin production than the wild-type strain ATCC31267, and was more tolerant of oxygen limitation conditions in regard to avermectin production.The regulatory role of redox-sensing regulator Rex was investigated in  Streptomyces, a genus of filamentous Gram-positive soil-dwelling bacteria, are obligate aerobes1Streptomyces strains are widely used in industrial production of various antibiotics, and oxygen supply is a key parameter determining product yield during the antibiotic fermentation processStreptomyces strains sense and respond to oxygen limitation.+ is reduced to NADH by accepting electrons during substrate oxidation, and NADH is then reoxidized by the electron transport chain. Changes of oxygen status are reflected by the intracellular ratio of NADH to NAD+. When NADH/NAD+ ratio is low, Rex binds to target genes and represses transcription of genes involved in NAD+ regeneration. In contrast, high NADH/NAD+ ratio inhibits DNA-binding activity of Rex and derepresses transcription of its target genes467Streptomyces coelicolor and shown to regulate expression of cytochrome bd terminal oxidase (cydABCD operon) and heme biosynthesis (rex-hemACD operon)Bacillus subtilis, a facultative aerobe, Rex represses expression of cytochrome bd oxidase (cydABCD), NADH dehydrogenase (yjlC-ndh), NADH-linked fermentative lactate dehydrogenase (lctP-ldh), and a formate-nitrate transporter (ywcJ)69Staphylococcus aureus, Rex directly regulates at least 19 genes. It acts as a central regulator of anaerobic metabolism leading to anaerobic NAD+ regeneration, which includes lactate, formate, and ethanol fermentation  and nitrate respiration S. coelicolor (5′-TGTGAACNNNTTCACA-3′)B. subtilis S. aureus (5′-TTGTGAAWWWWTTCACAA-3′)S. coelicolor.Rex is a redox-sensing regulator widely distributed in Gram-positive bacteria4S. coelicolor and its regulatory mechanism has been extensively studied, few target operons/genes of Rex in Streptomyces have been confirmedS. avermitilis is an important species used for industrial production of avermectins, a group of anthelmintic antibiotics widely used in the medical, veterinary, and agricultural fieldsS. avermitilis. Our findings have potential application to novel genetic engineering strategies for high antibiotic-producing strains and hypoxia-tolerating strains of this genus.Even though Rex was first characterized in rex gene is conserved within the genus Streptomyces and is cotranscribed with the heme synthesis genes hemACDS. avermitilis, we constructed a rex-deletion mutant (termed Drex) by homologous recombination in wild-type strain ATCC31267. rex deletion had no effect on growth in liquid fermentation medium  encodes putative NADH dehydrogenase I (complex I), cydA1B1CD (SAV4260-4258) encodes putative cytochrome bd-I oxidase (cytochrome bd complex), atpIBEFHAGDC (SAV2888-2880) encodes putative F-type proton-transporting ATPase, and hemAC1DB (SAV4739-4742) is cotranscribed with rex and encodes heme synthesis enzymes . Transcription level of t 30 min . These fand Drex . Transcr6-tagged Rex fusion protein was overexpressed in E. coli and purified for DNA binding analysis. EMSAs were performed to evaluate interactions between Rex and the promoters in vitro. Rex-His6 bound to the promoter regions of atpIBEFHAGDC, cydA1B1CD, nuoA1-N1, and rex-hemAC1DB operons  of rex. A consecutive ROP site (5′-TTGTGCACGCGTTCACAA-3′) was found in the protected region; the site is located between −35 region and −10 region and encompasses −35 region (cydA1 promoter region. One ROP site (5′-ATGTGAACGCGTTCACAA-3′) was found in the protected region downstream from TSS. A half-site ROP (5′-TTGTGAA-3′) was also found in the protected region; it is located upstream from the ROP site and encompasses TSS (cydA1 promoter region (TTGTGATACGGTTCACGA-3′) was detected in the atpI promoter region   . ROP in S. coelicolor, NADH at concentrations <5 μM inhibits DNA-binding activity of Rex, whereas 1 mM NAD+ has no inhibitory effect. NAD+ competes with NADH for Rex bindingB. subtilis and S. aureus, NAD+ enhances binding of Rex to putative Rex-binding sites, while NADH competes with NAD+ for Rex binding and reduces Rex activity6+ and NADH on DNA-binding activity of Rex to upstream regions of cydA1 in S. avermitilis. DNA-binding activity of Rex was reduced by addition of NADH, but not by NAD+ concentrations up to 1 mM  and a half-site ROP (5′-TTCACAA-3′) located downstream of TSS. Rex may inhibit the progress of RNA polymerase by binding to ROP downstream of the wblE promoter, and thereby repress transcription.The promoter region of ng motif . wblE enTCC31267 . EMSAs sin vitro . In in v of Drex . These f of wblE . The prowblE in Drex results in delayed morphogenesis, we attempted to delete wblE in S. avermitilis. However, this attempt was unsuccessful. wblE is evidently an essential gene in Streptomyces; an attempt to delete it in S. coelicolor was also unsuccessfulwblE was overexpressed in ATCC31267, the resulting strain had a phenotype similar to that of Drex  resulted in a 20% reduction of avermectin production, and static culture for 2 h on day 5 resulted in a 35% reduction. In Drex, avermectin production was reduced by 3.8% and 25%, respectively, under the above two conditions  were found in the protected region and downstream region. Rex can evidently interact with the half-site ROP; however, because the interaction is weak and easily dissociated in vitro, EMSA did not reveal a clear shifted band.qRT-PCR analysis was performed to determine whether Rex regulates avermectin production at the transcriptional level. Drex showed significantly increased transcription levels of pathway-specific regulatory gene and aveD . EMSAs rc region . AlthougRex-His6 . No conswblE, cydA1B1CD, rex-hemAC1DB, atpIBEFHAGDC, and nuoA1-N1 are directly controlled by Rex. To investigate whether Rex binds to promoter regions of other putative target genes, we selected 16 genes with predicted gene function for EMSAs (hppA (encodes an inorganic H+ pyrophosphatase), ndh2 (encodes a NADH dehydrogenase [complex I]), echA7 (encodes an enoyl-CoA hydratase), ectABC (encodes ectoine biosynthesis enzymes), SAV828 (encodes a rhamnosidase), and SAV2652 (encodes a regulatory protein). Probes whose binding motif had one or two mismatches showed higher affinity than probes whose binding motif had three mismatches . Eleven wbl genes (including whiB and whiD) have been identified in S. coelicolorwblA, whiB, and whiD are essential for sporulation, and WblA also negatively regulates antibiotic biosynthesis in Streptomyces132526wbl genes are not involved in morphological development, with the exception of wblE. Fowler-Goldsworthy et al.wblE could not be deleted in various strains of S. coelicolor, and we made a similar observation in S. avermitilis. Thus, wblE appears to be essential in this genus. The homolog of wblE in Mycobacterium tuberculosis is whiB1, which encodes an essential transcription factor in response to nitric oxide exposurewblE is directly negatively regulated by Rex, and that wblE overexpression results in delayed morphogenesis similar to that of Drex. Expression of wblE, like that of atpIBEFHAGDC, is downregulated by oxygen limitation in both ATCC31267 and Drex, suggesting that (i) wblE is jointly regulated by Rex and some other regulator, or (ii) wblE itself responds to low oxygen concentration via its own redox-sensitive [4Fe-4S] cluster. Under oxygen limitation condition, wblE expression in Streptomyces is downregulated, with consequent stimulation of sporulation and production of a large number of spores to maintain viability under conditions of little or no oxygen. Another Rex target, ectABC, encodes enzymes for biosynthesis of ectoine (a compatible solute) that serves as an osmolyte and promotes survival under osmotic or temperature stressectABC transcription, Rex facilitates ectoine biosynthesis to enhance viability under these types of stress.WhiB-like family transcription factors are widely present in actinomycetes, but not found in other bacterial orders. WhiB was first identified as a small transcription factor-like protein essential for sporulation in aveR and biosynthetic genes, and avermectin production, were notably increased. Although EMSA showed no clearly retarded band between aveR promoter region probe and Rex-His6, DNase I footprinting analysis revealed one 15-nt protected region consisting of two adjacent half-site ROP on the coding strand of aveR by Rex-His6. Thus, Rex may directly regulate aveR expression by interacting with the half-site ROP in the aveR promoter region. Expression of electron transfer chain components was enhanced in Drex, thus promoting aerobic respiration rate, ATP production, and secondary metabolism. The notable increase of atpIBEFHAGDC, cydA1B1CD, nuoA1-N1, and rex-hemAC1DB expression in Drex enhanced the tolerance of cells to oxygen limitation. The findings described here provide a basis for construction of new Streptomyces strains with high antibiotic production and hypoxia tolerance.In Drex, expression of regulatory gene S. avermitilis strains used were ATCC31267 (wild-type), Drex (rex-deletion strain), Drex-C (rex-deletion complementary strain harboring plasmid pSET-rex), and Orex (ATCC31267 harboring rex overexpressing plasmid pKC-rex). E. coli strains JM109 and BL21 (DE3) were used for routine cloning and protein expression, respectively. YMS medium and SFM medium were used for sporulation and phenotype studies30S. avermitilis were as described previouslyThe rex (SAV4738) gene deletion mutant was generated through targeted gene deletion mediated by homologous recombination. A 566-bp fragment upstream of rex (position −460 to + 87 from start codon) was amplified by primers rex-up-Fw and rex-up-Rev, and a 579-bp fragment downstream of rex (position +539 to +1098) was amplified by primers rex-dw-Fw and rex-dw-Rev, using ATCC31267 genomic DNA as template  was confirmed by PCR using one pair of external primers (rex-V-Fw/rex-V-Rev) and one pair of internal primers (rex-V2-Fw/rex-V2-Rev)  ; Fig. 1.rex ORF and its putative promoter was amplified by PCR using primers rex-E-Fw and rex-E-Rev  was used as internal control.RNA was isolated using Trizol reagent  from S. avermitilis cultures grown in FM-II for 2 or 6 days were fixed in cross-linking buffer  containing 1% formaldehyde for 20 min at 28 °C. ChIP was performed using anti-Rex antibody. After DNA extraction, pellets were washed with 70% ethanol and resuspended in 50 μl Tris·EDTA buffer. 1 μl DNA solution was subjected to PCR using the primer sets listed in ChIP assay was performed as described previouslyrex coding region was amplified by PCR using primers His-rex-Fw and His-rex-Rev. The purified fragment was cut with NcoI/HindIII and cloned into NcoI/HindIII-digested pET28a (+) to generate expression plasmid pET-rex. pET-rex was introduced into E. coli BL21 (DE3) for overexpression of C-terminal His6-tagged Rex. Rex-His6 was induced by 0.2 mM IPTG at 37 °C and purified from whole-cell lysate by Ni-NTA agarose chromatography  according to the manufacturer’s instructions.The nd Generation, Roche) as described previously6 for 30 min at 25 °C in a total volume of 20 μl containing 1 μg poly[d(I-C)]. Electrophoresis  was performed to separate protein-bound probes from free probes. DNA was electroblotted onto a positively charged nylon membrane, and retarded and unbound bands were detected by chemiluminescence and recorded on X-ray film.EMSAs were performed according to the manufacturer’s instructions . Samples were subjected to phenol/chloroform extraction, ethanol precipitation, and capillary electrophoresis. Electrophoregrams were analyzed using GeneMarker software v2.2.0.A fluorescent labeling procedure was used for DNase I footprinting assaysS. avermitilis strains and estimation of avermectins yields by HPLC analysis were performed as described previouslyFermentation of rex and wblE were mapped by 5′-RACE using a 5′/3′ RACE Kit . Total RNA was extracted from ATCC31267 grown in FM-I for 2 days. A gene-specific primer (sp1) was used to synthesize cDNA, and template RNA was degraded with RNase H. A homopolymeric A-tail was purified and added to the 3′-end of cDNA using terminal transferase. Tailed cDNA was PCR amplified through 35 cycles with a specific nested primer (sp2) and an oligo (dT)-anchor primer  of r primer . PCR proS. avermitilis genome using Virtual Footprint softwareTo search for putative Rex target genes, Rex consensus motif 5′-TTGTGAANNNNTTCACAA-3′ was used to scan the intergenic regions of the How to cite this article: Liu, X. et al. Redox-sensing regulator Rex regulates aerobic metabolism, morphological differentiation, and avermectin production in Streptomyces avermitilis. Sci. Rep.7, 44567; doi: 10.1038/srep44567 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Long noncoding RNAs (lncRNAs) are a large class of regulatory RNAs with diverse roles in cellular processes. Thousands of lncRNAs have been discovered; however, their roles in the regulation of muscle differentiation are unclear because no comprehensive analysis of lncRNAs during this process has been performed. In the present study, by combining diverse RNA sequencing datasets obtained from public database, we discovered lncRNAs that could behave as regulators in the differentiation of smooth or skeletal muscle cells. These analyses confirmed the roles of previously reported lncRNAs in this process. Moreover, we discovered dozens of novel lncRNAs whose expression patterns suggested their possible involvement in the phenotypic switch of vascular smooth muscle cells. The comparison of lncRNA expression change suggested that many lncRNAs have common roles during the differentiation of smooth and skeletal muscles, while some lncRNAs may have opposite roles in this process. The expression change of lncRNAs was highly correlated with that of their neighboring genes, suggesting that they may function as cis-acting lncRNAs. Furthermore, within the lncRNA sequences, there were binding sites for miRNAs with expression levels inversely correlated with the expression of corresponding lncRNAs during differentiation, suggesting a possible role of these lncRNAs as competing endogenous RNAs. The lncRNAs identified in this study will be a useful resource for future studies of gene regulation during muscle differentiation. The major type of smooth muscle cells, vascular smooth muscle cells (VSMCs), are capable of converting between synthetic and contractile phenotypes , 2. In tIn contrast to the reversible change in phenotype of smooth muscle cells, terminally differentiated skeletal muscle, like many other cell types, cannot be dedifferentiated. Upon external signals such as muscle injury, skeletal muscle is regenerated from satellite cells, which are main myogenic progenitor cells. The satellite cells are divided asymmetrically, and expanded into myoblasts, which are further differentiated into myocytes. This differentiation process is influenced by diverse factors, and for the treatment of diseases such as skeletal muscle degeneration, the elucidation of signaling networks involved in this process is critical , 6.SENCR) [SMILR) [HAS2), an enzyme that synthesizes hyaluronic acid. The function of only a small number of lncRNAs has been elucidated in the phenotypic change of VSMCs; therefore, it is hypothesized that additional lncRNAs involved in this process await discovery. To date, various reagents have been used to model the differentiation process of VSMCs, such as platelet-derived growth factor (PDGF), myocardin (MYOCD), transforming growth factor beta (TGFβ), activin A, retinoids, and angiotensin II. The transcriptome responds quite differentially to each of these factors; therefore, to identify common lncRNAs, diverse experimental models should be combined.Many studies have shown that various types of noncoding RNAs have regulatory roles in most cellular processes. MicroRNAs (miRNAs), a class of small noncoding RNAs, have been studied extensively for a long time . The regSENCR) . Inhibit [SMILR) . SMILR rIn the present study, we aimed to comprehensively discover lncRNAs involved in muscle differentiation by combining diverse RNA sequencing (RNA-seq) datasets. By integrating the data from VSMCs where different extracellular cues were used for differentiation, we identified several lncRNAs with potential as regulators in VSMCs differentiation. We analyzed the correlation between the expression of lncRNAs and that of their neighboring protein-coding genes, and compared the expression change of lncRNAs between smooth and skeletal muscle cells.http://www.gencodegenes.org/) [http://genome.ucsc.edu/) [We obtained RNA-seq data from the Gene Expression Omnibus (GEO) database . These des.org/) , and thesc.edu/) . The GENp values were less than 0.1 in more than two datasets and less than 0.3 in all datasets.To select the lncRNAs with differential expression during differentiation of VSMCs, we selected only those lncRNAs with a log2 value of expression changes greater than 0.5 or lower than -0.5 between before and after treatment with external factors. To select the lncRNAs that increased in the synthetic phenotype, those lncRNAs with increased expression in the PDGF-treated sample, but with decreased expression in the MYOCD- and TGFβ-treated samples, were chosen. In the case of the lncRNAs that increased in the contractile phenotype, we chose those lncRNAs with decreased expression in the PDGF-treated sample, but with increased expression in the MYOCD- and TGFβ-treated samples. We removed the lncRNAs from the list if the FPKM values of ≥ 50% of the samples belonging to the denominator were zero. To select statistically significant lncRNAs, a two-tailed T-test was applied to each dataset, and labeled as ‘significant’ if the p value criteria in the T-test as 0.2.To select lncRNAs that are differentially expressed during skeletal muscle differentiation (GSE70389), we used the same criteria that were used for lncRNA selection in VSMCs, except that the To predict transcription factors that might regulate the expression of lncRNAs, we used the same approach as that detailed in our previous report for the analysis of miRNA promoters . We usedTo measure the expression level of lncRNAs and their neighboring protein-coding genes during differentiation of smooth muscle cells, we cultured HCASM cells (Gibco) according to the manufacturer’s protocol. These cells were maintained in Medium 231 (Gibco) supplemented with Smooth Muscle Growth Supplement (Gibco), and harvested for the sample of synthetic phenotype. To obtain the sample of contractile or differentiated phenotype, HCASM cells stabilized in the growth media were cultured in Medium 231 supplemented with Smooth Muscle Differentiation Supplement (Gibco) for three days, and then collected. We used TRIzol Reagent (Invitrogen) to extract total RNA, performed reverse transcription using RevertAid Reverse Transcriptase (Thermo Scientific) with Random Hexamers (Invitrogen). Quantitative real time polymerase chain reaction (PCR) was performed using SYBR Green PCR Master Mix (Applied biosystems) in Rotor-Gene Q (Qiagen). The primer sequences for PCR were included in the supplementary table. The differentiation of these cells was confirmed by measuring the level of Calponin 1 (CNN1) and Smooth muscle protein 22-alpha (SM22α). For normalization, the expression level of Glyceraldehyde 3-phosphate dehydrogenase (GADPH) was used.In the track data hubs of UCSC Genome Browser , we utilVSMCs can be switched between synthetic and contractile phenotypes in response to diverse extracellular cues , 2. To dMIR3142HG is the host gene of miR-3142 and miR-146a. miR-146a regulates the differentiation of VSMCs by targeting nuclear factor-κBp65 (NF-κBp65) and the proliferative cell nuclear antigen (PCNA) [MIR3142HG gene results in the change in miR-146a level, which regulates the physiology of VSMCs, although we could not exclude a miRNA-independent role of this lncRNA. For the other lncRNAs, their roles in muscle physiology have not been reported yet. Thus, the lncRNAs identified in this analysis represent good candidates for future studies to elucidate their roles during muscle differentiation.To identify those lncRNAs that are likely to play a role in phenotypic change of VSMCs, we statistically tested for the selected lncRNAs shown in n (PCNA) . Thus, iTo identify any transcription factors with enriched binding into the promoter regions of differentially expressed lncRNAs, we used the data released by the ENCODE project, where ChIP-seq was performed for 161 major transcription factors . Among tSMILR, has a high probability to function in skeletal muscle, although its role was only reported in smooth muscle [Although smooth and skeletal muscles have different morphologies and exert dissimilar roles in the body, they share several factors during their differentiation, such as serum response factor (SRF) . To idenh muscle . In contTo confirm the expression change of lncRNAs selected above, and to test whether the expression change of those lncRNAs is reproducible in another condition of VSMCs differentiation, we cultured HCASM cells with growth media including growth factors, and induced their differentiation with low concentration of serum and without growth factors see . Using tWe utilized Coding Potential Assessment Tool (CPAT) and Coding Potential Calculator , which has essential roles in vascular development [Previous studies that elucidated the working mechanisms of lncRNAs suggested that many lncRNAs regulate the expression of their neighboring genes in the genomic context . These ‘elopment , 33. ThuIn addition to the role of lncRNAs as cis-acting transcriptional regulators, lncRNAs can work as competing endogenous RNAs by binding miRNAs in a sequence-specific manner and suppressing their function . In thisFinally, we searched NPInter database where the experimentally verified association between lncRNAs and proteins are documented . SeveralAlthough many lncRNAs have been discovered in diverse species and experimental models, their roles and pathological effects in muscles have not been studied as extensively as those related to other diseases, such as cancer. One of the issues in discovering lncRNAs or other types of noncoding RNAs in an experimental model is their inconsistency when analyzed using different approaches. Thus, lncRNAs identified in one approach may not be replicated in another approach, although both approaches are aimed at the same experimental model. The comparison of the expression profile of lncRNAs in the differentiation model of VSMCs confirmed this issue . AlthougS1 FigThe cluster indicated in (PDF)Click here for additional data file.S2 FigThe cluster indicated in (PDF)Click here for additional data file.S1 Table2 (fold change), and the p values from two-sided T test for each dataset, are shown. For those lncRNAs whose promoters were analyzed, the position of the transcription start sites and the strand of transcription are included. For the lncRNAs with incomplete annotation (red letters), transcription start sites were identified by manual curation using the information from RefSeq annotation. Only the significant lncRNAs from For each lncRNA, their genomic locus (hg19), the fragments per kilobase of exon model per million mapped fragments (FPKM) values in each sample, log(XLSX)Click here for additional data file.S2 Tablep values lower than 0.1. There was no transcription factor enriched in the lncRNA promoters of the contractile phenotype against those of the synthetic phenotype.Among the transcription factors included in the ChIP-seq data from ENCODE, we identified those that were significantly enriched in the promoters of lncRNAs that increased in the synthetic phenotype compared with those increased in the contractile phenotype (one-sided Fisher’s exact test). This analysis resulted in three transcription factors with (XLSX)Click here for additional data file.S3 TableThe coding potential of selected lncRNAs in Figs (XLSX)Click here for additional data file.S4 TableWe used NPInter database to search the experimentally verified association between lncRNAs and proteins , and lis(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.


Regions of chromatin identified by FAIRE-seq, as having altered accessibility during differentiation, were found to be enriched with sequence-specific binding motifs for transcription factors predicted to be involved in driving basal and differentiated urothelial cell phenotypes, including forkhead box A1 (FOXA1), P63, GRHL2, CTCF and GATA-binding protein 3 (GATA3). In addition, co-occurrence of GATA3 motifs was observed within subsets of differentiation-specific peaks containing P63 or FOXA1. Changes in abundance of GRHL2, GATA3 and P63 were observed in immunoblots of chromatin-enriched extracts. Transient siRNA knockdown of P63 revealed that P63 favoured a basal-like phenotype by inhibiting differentiation and promoting expression of basal marker genes. GATA3 siRNA prevented differentiation-associated downregulation of P63 protein and transcript, and demonstrated positive feedback of GATA3 on PPARG transcript, but showed no effect on FOXA1 transcript or protein expression. This approach indicates that as a transcriptionally regulated programme, urothelial differentiation operates as a heterarchy, wherein GATA3 is able to co-operate with FOXA1 to drive expression of luminal marker genes, but that P63 has potential to transrepress expression of the same genes.Cell differentiation is affected by complex networks of transcription factors that co-ordinate re-organisation of the chromatin landscape. The hierarchies of these relationships can be difficult to dissect. During  Isolating this sheared DNA from nucleosomal DNA by phase separation enables characterisation of the relative extent of chromatin accessibility in a genome-wide manner. As transcription factors bind dynamically to nucleosome-depleted regions, motif matching within open chromatin, as identified by FAIRE, can be used to classify transcription factors that actively associate with chromatin and define cell phenotype.23 FAIRE identifies a complementary but partially distinct set of putative enhancer regions outside of gene promoters, as compared to DNase-seq,19 which uses DNase I enzyme to cleave regions of open chromatin. FAIRE-seq DNA has been shown to be enriched relative to DNase-seq for potential FOXA1-binding sites, which are known to contribute to urothelial differentiation,9 and chromatin-associated histone H3 monomethylated on lysine 4 (H3K4me1), which is associated with genomic enhancers specific to cell type.Formaldehyde-assisted isolation of regulatory elements coupled with next-generation sequencing (FAIRE-seq)4 alongside time-matched non-differentiated vehicle controls. Open chromatin regions unique to treated and control libraries were searched for matches to known sequence-specific transcription factor-binding motifs, both on a genome-wide basis and proximal to differentially expressed genes. Selected candidate transcriptional regulators were validated as modulators of urothelial differentiation using immunoblots of chromatin-enriched extracts and siRNA knockdown to investigate effects on urothelial phenotype.To obtain a genome-wide picture of the transcriptional drivers of different urothelial cell phenotypes, RNA-seq and FAIRE-seq were performed on serially propagated NHU cell cultures from three independent donors at 24 and 144 h time points after concurrent EGFR blockade and PPARγ activation to induce differentiation,UPK1A) and UPK2.26 Gene ontology analysis, performed using the GOrilla tool,27 showed that the 122 genes upregulated at both time points included genes involved in lipid metabolism (P=1.16 × 10−5) and water homeostasis <0.1), respectively . Transcr20 when genes were split into quartiles based on normalised RNA-seq read counts  and overlapping peaks at each time point . Consistd counts , a greatd counts . In addid counts .28 Motif searching was conducted using control-specific peak sets as the background for the differentiation-specific peak set and vice versa.To uncover transcription factors driving cell phenotype in differentiated and non-differentiated urothelial cells, sequence-specific transcription factor-binding motifs enriched in non-overlapping FAIRE peaks at each time point were identified using the motif discovery tool HOMER.29 and that within promoter regions, transcription start sites (TSSs) have fewer differences in transcription factor motifs than the rest of the genome.20 As such, FAIRE peaks in TSS promoter regions (−1 kb to +100 bp) were excluded from all analyses. To highlight any differences between motifs enriched proximal to genes and those found across the genome, control-specific and differentiation-specific FAIRE peaks were compared as either genome-wide groups, or analysis was restricted to those located within ±25 kb of the TSS of differentially regulated genes. Motifs matched by HOMER were filtered for those which occurred in at least ≥1.25-fold of the total percentage of regions in the target set as compared to the background set, in order to focus on motifs significantly enriched in each experimental situation.30 This approach identified divergent groups of transcription factor motifs across the different regions, with each group containing matches to motifs from both previously described urothelium-associated factors and others not previously associated with urothelium .Previous transcription factor motif matching studies using open chromatin isolation techniques have observed that particular motifs tend to be enriched at sites distal to genes,othelium . De novo31OCT4 transcripts are expressed by NHU cells, but the pluripotency-associated isoform OCT4A is not.32 P63, a transcription factor associated with a non-differentiated ‘basal-like' urothelial cell phenotype in normal cells and carcinoma,37 was enriched both proximal to downregulated genes and across the genome at 144 h, whereas STAT6 and ETS motifs were specifically associated with peaks ±25 kb of downregulated genes at this time point.Motifs with the highest fold change in abundance in peaks specific to control libraries and around downregulated genes at 24 h were dominated by cell cycle-associated transcription factors such as ETS family factors, JUN-AP1, EGR1 and EGR2, and a motif associated with combined binding of the OCT4-SOX2-TCF-NANOG pluripotency factors in mouse embryonic stem cells.9 GATA312 and PPARγ4 were enriched in differentiation-specific FAIRE peaks within ±25 kb of the TSS of genes with expression upregulated during differentiation. PPARγ motifs were only enriched around genes upregulated at 24 h, in agreement with observations that it drives early events during in vitro urothelial differentiation upstream of FOXA1,9 motifs from which were matched at 144 h. GATA3, CEBPB and GRHL2 motifs were enriched around upregulated genes at both time points. GRHL2 has been implicated in regulation of tight junction complex genes, which are central to barrier formation in several epithelia,38 including urothelium,39 whereas the closely related GRHL3 has been associated with urothelial differentiation in the mouse.15 CEBPB plays a key role in orchestrating CEBPA and PPARG expression during adipogenesis.2 CEBPB has no known role in normal human urothelial biology, although other groups have shown the CEBPB motif to be enriched in promoters of urothelial carcinoma gene sets,40 and it has been associated with urothelial differentiation in mouse.41 ELF5 and ELF1 motifs were enriched in regions proximal to up- and downregulated genes at 144 h, respectively. Although neither of these has been previously associated with urothelial biology, the closely related ELF3, whose motif is not in the HOMER database used here, is a driver of differentiation.10Motifs from urothelial differentiation-associated transcription factors FOXA1,9 and the closely related motif for IRF2 were enriched at 24 h, as were those from CTCF at both time points. As none of these motifs were enriched proximal to differentially regulated genes, these observations agree with previous studies that showed CTCF and IRF1 preferentially bind to regions distal to expressed genes.29Across the genome, in differentiation-induced cells, motifs from the known urothelium-associated transcription factor IRF128 Pioneer factors such as FOXA1, which can open repressed regions of chromatin, often bind proximally to differentiation-inducing nuclear receptors.44 To determine whether there was co-occurrence of differentiation-associated transcription factor motifs within FAIRE-seq peaks, P63 and FOXA1 motif-containing open chromatin regions specific to control and differentiated cells at each time point were searched separately for enriched motifs using the same approach as for the genome-wide investigation. P63- and FOXA1-containing peaks were enriched with motifs that overlapped the genome-wide set of peaks, but with significant differences  in all donors in both non-differentiated and PPARγ-activated conditions, although statistical significance was not reached due to a large variation in the fold increase between different donor cell lines  phenotype. This involves a major shift in gene expression, implying a change in genomic organisation to reflect the transcriptional landscape of urothelium. We have previously identified a network of PPARγ-regulated intermediary transcription factors that mediate the differentiated urothelial programme, although inter-relationships within the network have yet to be established. In other tissues, such as breast, a role has been identified for the so-called pioneer factors FOXA and GATA in defining the tissue-specific genomic organisation. GATA3 and FOXA1 have been shown to act co-operatively in promoting ESR1-driven transcription in MCF7 cells, with GATA3 lying upstream of FOXA1.51 The results in this study add to the weight of evidence that CTCF binding, although widespread and well-conserved in many genomic regions,51 shows tissue-specific binding around genes associated with cell phenotype.The motif matching performed here identified transcription factors not previously associated with urothelial differentiation, including CTCF. CTCF was not enriched at the protein level in chromatin extracts after induction of differentiation, most probably because CTCF is a constitutive chromatin-associated protein that, among other functions, facilitates looping of chromatin.GRHL3 in differentiation of human urothelium. However, no differentiation-associated changes in GRHL3 protein abundance or localisation were seen during differentiation, whereas the constitutively expressed GRHL2 gene showed increased protein abundance and relocation onto the chromatin of differentiating cells. Taken with the nuclear localisation of GRHL2 in situ, these data implicate GRHL2 as the more important factor and further illustrate that not all differentiation-associated events are transcriptionally regulated. GRHL2 has been observed to be expressed by human urothelium in another recent study52 and is known to reside within a genomic region that is commonly amplified in aggressive urothelial carcinoma.53Our initial analysis of differentially expressed gene transcripts indicated a potential role for 16 however KLF5 was not significantly differentially expressed in urothelium as determined by RNA-seq, nor were KLF5 motifs enriched in FAIRE peaks analysed herein. This suggests if KLF5 has a role in human urothelial development, it may function in early urothelial specification and not be directly associated with regulating genes associated with mature differentiation stages. Gata4 has been associated with urothelial differentiation in mouse.17 However, GATA4 was not detected in RNA-seq data in the current study, where GATA3 transcript was the most highly expressed GATA gene family member detected and, in addition, was the only GATA member to be upregulated upon differentiation and associated with post-differentiation chromatin. These data implicate GATA3 rather than GATA4 in the differentiation of human urothelium.Klf5 is reported to be upstream of Pparg and Grhl3 in mouse urothelial development,54 Cultures were subcultured by trypsinisation and maintained in keratinocyte serum-free medium containing bovine pituitary extract and epidermal growth factor  and further supplemented with 30 ng/ml cholera toxin . Differentiation was induced in just-confluent cell cultures using 1 μM troglitazone as PPARγ ligand with concurrent 1 μM PD153035 to block epidermal growth factor receptor activation.4 Non-differentiated vehicle control (0.1% DMSO) cultures were maintained in parallel and used at the same time points (24 and 144 h).NHU cells were maintained as finite, serially passaged cell lines, as described previously.55 to the reference UCSC hg19 human genome. Differential gene expression was performed between control and differentiation-induced cells at 24 and 144 h time points using DESeq.56 The results obtained from three independent cell lines were treated as replicates and genes with a FDR cutoff <0.1 were called significant.Cell monolayers were solubilised in Trizol , using the manufacturer's protocol for chloroform and isopropanol extraction, and DNA was digested using RNAse-free DNase I . Library construction was performed using TruSeq RNA Sample Prep Kit v2 . Sequencing was performed using an Illumina HiSeq 2500 sequencer and reads aligned using RSEM6 cells were lysed and sheared, and open chromatin extracted as described in the FAIRE protocol.57Cell monolayers were fixed in 1% formaldehyde for 10 min before quenching by addition of glycine to 125 mM for 5 min and scrape-harvesting in ice-cold PBS with added protease inhibitors. Approximately 5 × 1058 was used to call FAIRE-enriched peaks. Non-overlapping peaks between control and differentiated samples at each time point were identified using bedtools. HOMER motif discovery software28 was used to discover motifs over-represented in each treatment condition, using peaks uniquely present in control cells as the background when searching the differentiation-induced specific peaks, and vice versa. Motifs identified by HOMER as enriched were further filtered by fold change as percentage enrichment above background of ≥1.25.MACS peak-calling algorithm59 with optional RNase digestion step included.Cells were fixed and scrape-harvested as for FAIRE, then pelleted cells were subjected to a chromatin enrichment protocolAnti-FOXA1  used at 1:250 for IHC and 1:400 for immunoblot. Anti-CTCF  used at 1:250 for IHC and 1:1000 for immunoblot. Anti-P63  used at 1:1000 for IHC and 1:500 for immunoblot. Anti-GRHL2  used at 1:150 for IHC and 1:400 for immunoblot. Anti-PPARγ  used at 1:2000 for IHC and 1:500 for immunoblot. Anti-GATA3  used at 1:800 for IHC and 1:1000 for immunoblot. Anti-GRHL3  used at 1:500 for immunoblot. Anti-KRT13  used at 1:1000 for immunoblot. Anti-BACT  used at 1:250 000 for immunoblot).


Yarrowia lipolytica is increasingly used as alternative cell factory for the production of recombinant proteins. At present, several promoters with different strengths have been developed based either on the constitutive pTEF promoter or on oleic acid inducible promoters such as pPOX2 and pLIP2. Although these promoters are highly efficient, there is still a lack of versatile inducible promoters for gene expression in Y. lipolytica.The oleaginous yeast EYK1 gene coding for an erythrulose kinase. pEYK1 induction was found to be impaired in media supplemented with glucose and glycerol, while the presence of erythritol and erythrulose strongly increased the promoter induction level. Promoter characterization and mutagenesis allowed the identification of the upstream activating sequence UAS1EYK1. New hybrid promoters containing tandem repeats of either UAS1XPR2 or UAS1EYK1 were developed showing higher expression levels than the native pEYK1 promoter. Furthermore, promoter strength was improved in a strain carrying a deletion in the EYK1 gene, allowing thus the utilization of erythritol and erythrulose as free inducer.We have isolated and characterized the promoter of the Y. lipolytica.Novel tunable and regulated promoters with applications in the field of heterologous protein production, metabolic engineering, and synthetic biology have been developed, thus filling the gap of the absence of versatile inducible promoter in the yeast The online version of this article (doi:10.1186/s12934-017-0755-0) contains supplementary material, which is available to authorized users. Pichia pastoris, Hansenula polymorpha (Pichia angusta), and Yarrowia lipolytica as cell factories for the production of recombinant proteins or biomolecules with biotechnological or pharmaceutical applications has increased over the years  was replaced by [GGAACGCGTCC] and named motif a. The motif B [CTTGCATAATCCGATGAC] was replaced by [CTTGTACGCGTAGATGAC] and named motif b. This yielded to pEYK300aB and pEYK300Ab, respectively  to give rise to representative strains JMY6369 and JMY6372, respectively (Table EYK1) required for the promoter induction by both erythritol and erythrulose.The comparison of YFP fluorescence under pEYK450 and pEYK300 indicates that the TATA box may be involved in the expression of gene YALIPF01628g rather than gene YALI0F01606g. Thus, to determine the role of Box A and Box B in pEYK regulation, two mutated promoters, namely pEYK300aB and pEYL300Ab, were constructed as described in material and method by exchange of the On the opposite, the mean relative YFP fluorescence measured for strain JMY6372 carrying the pEYK300Ab mutated promoter  upstream of the wild-type pEYK300 promoter , glycerol (YNBG), erythritol (YNBOL) and erythrulose (YNBOSE) and compared to that of pEYK300 (strain JMY6245). As shown in Fig. EYK1 would allow to fine tune the strength of promoter induction. Indeed, several strong synthetic hybrid promoters have been created by fusing tandem repeats of upstream activation sequence (UAS) upstream to a core promoter region. The first one (hp4d) was based on four tandem repeats of the 108 bp UAS1XPR2 of the XPR2 gene upstream on the minimal LEU2 core promoter [XPR2 of the XPR2 gene upstream on the minimal LEU2 core promoter and 16 copies of UAS1XPR2 of the XPR2 gene upstream of TEF core promoters of different length [TEF upstream of the pTEF promoter [xpr2 upstream of the 100 bp proximal core POX2 promoter. This UAS-core promoter chimera showed a 4.2-fold higher expression level in oleic acid media than in glucose in contrast to a twofold higher expression level for the 8 copies of UAS1xpr2 upstream of the 136 bp proximal core TEF promoter [EYK1 upstream of the EYK1 promoter results in a 3.3-fold stronger promoter, thus we could expect to be able to construct stronger erythritol/erythrulose inducible promoters by introducing additional tandem repeats of the UAS1EYK1.Since the insertion of several copies of the 48 bp region encompassing the Box A motif resulted in a stronger promoter induction level, it could be assumed that increasing the copy number of UAS1promoter . Later Bt length . Promotepromoter . Oleic apromoter . Here weXPR2 gene upstream of a minimal promoter of the LEU2 gene  allowed a significant transcriptional activity [XPR2 (UAS1B) with the pEYK300 promoter leading to promoter HU4EYK300 (JME3998)  in the medium—on pHU4EYK300 induction can be specifically assessed over time. The regulation of pHU4EYK300 was investigated in regard to the growth rate of strain JMY6380 and the composition of the culture medium, more specifically in the presence of a mixture of glycerol/erythritol or glycerol/erythrulose.−1). The fluorescence levels of YFP were monitored by flow cytometry to assess the induction level at the single cell level. No significant difference in the promoter induction levels could be observed for the two dilution rates tested (data not shown). Indeed, the mean relative fluorescence of the cell population was equal to 8.86 ± 0.62 × 104 RFU at D = 0.16 h−1, and to 9.47 ± 0.31 × 104 RFU at D = 0.08 h−1. Moreover, cytograms showed that the cell population is homogenously induced in presence of erythritol , respectively. Glycerol, erythritol, erythrulose and YFP fluorescence were monitored for 8 h after inducer addition. In all experimental conditions tested, glycerol concentration remained almost constant (i.e. 3 g/L) in the bioreactor, confirming that a steady state was maintained in those experimental conditions.To assess the influence of inducer concentration on the regulation of pHU4EYK300, chemostat cultures of JMY6380 were performed on YNBG medium at a dilution rate of 0.2 h3 and 1.1  × 103 RFU, respectively). Similar observations were made for erythrulose. The maximal YFP fluorescence obtained 8 h after erythrulose addition was higher for the 0.6 CSP than for the 0.2 CSP . It could also be deduced from Fig. As shown in Fig. Y. lipolytica as main carbon source . Since eyk1∆ could not grow on erythritol and erythrulose as sole carbon source, strain RIY180 was grown in the presence of glucose or glycerol, used as energy source. Therefore, JMY6245 (pEYK300-WT) and RIY180 (pEYK300-eyk1∆) were grown in YNBDOL , YNBGOL , YNBDOSE , or YNBGOSE . Induction of the promoters was followed during time in microplates with glucose or glycerol for growth (0.25%) and with erythritol or erythrulose for induction (0.25%).Erythritol and erythrulose could be used by rce Fig. . Althougner Fig. . Therefo76 Table  was conseyk1∆ strains in the presence of erythritol occurred during the growth phase in media containing 0.25% of glucose or 0.25% of glycerol . In contrast, in the presence of erythrulose, YFP expression in wild-type and eyk1∆ strains was somewhat delayed from the growth phase in media containing 0.25% of glucose or 0.25% of glycerol .As shown in Fig. rol Fig. a, b. YFPeyk1∆, the rates of increase of YFP fluorescence were 10.5-fold higher .For strain JMY6245 (pEYK300-WT), the rates of increase of YFP fluorescence in the presence of erythritol were 97 and 83 FU/h in glucose and glycerol, respectively. In comparison, in the mutant eyk1∆ mutant (pEYK300-eyk1∆) as compared to the non-disrupted mutant (pEYK300-EYK1). The rate of YFP production in the mutant strain was 6.1-fold higher in glucose as compared to the wild-type strain . In the presence of glycerol, this increase was 7.3-fold .Similarly, in the presence of erythrulose, higher induction levels were obtained for the EYK1 gene. In such a strain, erythritol or erythrulose could be used as a free inducer and independently from growth, for having induction either during the growth phase or delayed from this latter.These results demonstrate that expression levels could be further improved by using a strain deleted for the Y. lipolytica [EYK1) and a potential upstream regulatory sequence (URSEYK). This study has generated new hybrid promoters combining core EYK promoter with either UAS1EYK1 or UAS1XPR2 upstream activating sequences, allowing at least a tenfold higher expression than the pTEF promoter. This opens the path to the design of new synthetic promoters containing UASEYK and/or URSEYK with higher tandem repeats number or with various core promoters to further widen the expression range and the induction profiles.Several groups have constructed hybrid promoters based on combination of repeats of upstream activating sequence (UAS), TATA box and core promoter for gene expression in polytica , 34, 48.polytica , this lapolytica –25, 49. EYK1 copy number, the induction time depending on the inducer used (erythritol or by erythrulose), the induction level depending on the media and the inducer concentration. One will have also to choice using erythritol or erythrulose as inducer and source of carbon for growth or use only as inducer in a ∆eyk1 genetic background.These promoters are poorly expressed in glucose or glycerol and could be induced by erythritol or by erythrulose with a tremendous advantage of being dose dependant thus allowing fine tuning of induction which will permit to vary the degrees of expression that could be obtained. One would be easily able to regulate the expression level depending on UAS1EYK1 expand the parts available for protein expression [Saccharomyces cerevisiae [These inducible promoters and UAS1pression  and for pression , 41 and pression , 51. Therevisiae .Additional file 1. Sequence of the upstream region of the EYK1 genes in Yarrowia clade. Sequence in Y. lipolytica (YALI-pEYK1), Yarrowia phangngensis (YAPH-pEYK1), Yarrowia yakushimensis (YAYA-pEYK1), Yarrowia alimentaria (YAAL-pEYK1), and Yarrowia galli (YAGA-pEYK1). Underlined are the nucleotidic sequences of the end of YALI0F01628g with the stop codon (CAT) and at the beginning of YALI0F01606g with the start codon (ATG).Additional file 2. Influence of dilution rate on the induction of hybrid EYK1 promoter pHU4EYK300 in continuous culture by erythritol. FL1-A/FSC-A cytograms corresponding to the chemostat of JMY6380 (pHU4EYK300) on YNB-erythritol medium (1% erythritol). The horizontal line at 4x103 FU represents the limit between induced cells (quadrant Q1-UR of the cytogram) and non-induced cells (quadrant Q1-LR of the cytogram). Cytograms are representative of two independent cultures, and are the result of the analysis of 40,000 cells. a Cytogram of the equilibrium cell population cultivated at a dilution rate of D = 0.16 h−1. b Cytogram of the equilibrium cell population cultivated at a dilution rate of D = 0.08−1.


Biological aging is a complex process dependent on the interplay of cell autonomous and tissue contextual changes which occur in response to cumulative molecular stress and manifest through adaptive transcriptional reprogramming. Here we describe a transcription factor (TF) meta-analysis of gene expression datasets accrued from 18 tissue sites collected at different biological ages and from 7 different in-vitro aging models. In-vitro aging platforms included replicative senescence and an energy restriction model in quiescence (ERiQ), in which ATP was transiently reduced. TF motifs in promoter regions of trimmed sets of target genes were scanned using JASPAR and TRANSFAC. TF signatures established a global mapping of agglomerating motifs with distinct clusters when ranked hierarchically. Remarkably, the ERiQ profile was shared with the majority of in-vivo aged tissues. Fitting motifs in a minimalistic protein-protein network allowed to probe for connectivity to distinct stress sensors. The DNA damage sensors ATM and ATR linked to the subnetwork associated with senescence. By contrast, the energy sensors PTEN and AMPK connected to the nodes in the ERiQ subnetwork. These data suggest that metabolic dysfunction may be linked to transcriptional patterns characteristic of many aged tissues and distinct from cumulative DNA damage associated with senescence. The analysis of transcriptomes has become an important tool to study aging-associated processes, but has yet to deliver consistent datasets across tissues and experimental platforms. Gene expression studies comparing tissues from flies, worms, mice and humans have revealed tissue- and organism-specific aging profiles , with coTo gain a better understanding of changes in the transcriptome associated with aging in these different settings, we performed a transcription factor (TF) meta-analysis across multiple tissue datasets derived from tissues aged in-vivo as compared to experimental in-vitro aging models. Prior TF analyses of the aging process have been limited to specific TFs including Forkhead box TFs (FOXOs), signal transducer and activators of transcription (STATs), E2 family TFs (E2F) or nuclear factor kappa-b (NF-κB) –20. ThesAlternatively, TF activities can be estimated from gene expression data –23. To iSince transcription factors can both activate and repress genes based on cell type, cellular context and a complex dynamic of TF interactions , we perfWe generated transcription factor signatures representing 18 tissues collected at different biological ages and 7 experimental aging models. Datasets comparing young and aged human tissues included adipose, artery, heart, lung, muscle, nerve, skin, thyroid and blood , brain , kidney A set of classification methods was carried out to group samples according to ranked motifs. Self-organizing maps (SOM) and K-means Nearest Neighbor clustering (KMC) revealed three distinct phenotypical groups, shown here in combination with a sample distance matrix . A PrincWe noticed specific motifs contributing to an anti-correlating pattern in the complete-linkage dendrograms , emphasiThe analysis of anti-correlated transcription factors seeded a minimalistic protein-protein interaction (PPI) network , with a We observed an association of the ATM and ATR hubs with the senescence group of nodes. In contrast, PTEN and AMPK hubs connected to nodes associated with the energy restriction subnetwork, suggesting the existence of two specific subnetworks. The DDR pattern is connected to p53 and retinoblastoma protein (p53-Rb) pathway activation in senescence , 47. SpeThe PPI network predicts a role of histone deacetylases  and histone acetyltransferase p300 (EP300 HAT) as additional modulators of transcriptional regulation in ERiQ. ATM can activate HDAC1, which suppresses E2F transcriptional activation, which are avoided motifs in senescence. HDACs are also recognized as important mediators of autophagy, requiring ubiquitination . ActivatAging may be viewed as a process requiring continuous adaptive responses to chronic cellular stress caused by cumulative molecular damage and energetic challenges. It is poorly understood how this process is reflected at the transcriptional level. The meta-analysis presented here provides pivotal insights into gene regulatory signatures in biologically aged tissues in comparison to experimental cell models of aging. To enable cross-comparison of heterogeneous datasets, we restricted our exploratory examination to trimmed sets of differentially expressed target genes derived from gene expression studies providing whole genome coverage. Inclusion of both up- and downregulated transcripts in combination with subsequent analyses of ranked motifs allowed inclusion of smaller experimental studies. Motif enrichments provide initial global maps of transcriptional regulation in human aging that are suitable to generate new hypotheses , S1 Fig.Senescence presents as a multi-stage, diversifying process rather than a static endpoint . This diThe second senescence cluster aggregated samples lacking enrichment of cell cycle related motifs. Two experimental samples compared senescence with quiescent, growth factor-starved fibroblasts. Although both datasets were obtained with different platforms and analyses, they correlated closely in signatures and classifications Figs , 2 and 3There is an apparent lack of suitable experimental models for aging in post-mitotic tissues. Here we show that a major cluster of samples was associated with the energy-restricted (ERiQ) model described by us earlier , 42, whiA sign-less transcription factor analysis as conducted here limits inferences to function. Sign-sensitive approaches, until now, can utilize only very small TF catalogs . HoweverThe mechanisms of transcription are complex and fine-tuned by cell specific transcriptional networks, epigenetics, methylation and ubiquitination –82, cellTo include experimental studies in our analysis, we consistently trimmed published lists from all 25 samples of our panel to the 150 most significant differentially expressed genes per sample (75 up- and 75 down-regulated), improving statistical significance, as provided in To further reduce potential influences of experimental designs, TF scores within each profile were ranked. The resulting signatures of motifs were clustered hierarchically using Spearman Rank Correlation with complete linkage aggregation, emphasizing dissimilarities. The resulting dendrograms provided a global map of transcription factor regulation in human aging. In addition, ranked motif signatures were classified by non-parametric K-means nearest neighbor clustering (KMC), self-organizing maps (SOM), sample-distance maps and principal component analysis (PCA) to identify motif loads and detect dominant phenotypical patterns of regulation between samples. Tools to execute these analyses were provided in TM4/MeV  and ClusSignificant motifs characterizing groups, along with PCA loads, were further filtered to identify a subset of switching TFs between replicative senescence compared to quiescence and the energy restriction (ERiQ) data set. We required an opposing, greater than 15-fold change in scores between senescence and ERiQ, with a score of at least p < 0.05 in one, and a change in rank. Predictions for transcription factor protein-protein-interactions were performed with NetworkAnalyst , referenS1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file.


The multi-subunit Mediator complex, which links DNA-bound transcription factors to RNA Pol II during transcription, is an essential regulator of gene expression in all eukaryotes. Individual subunits of the Mediator complex integrate numerous endogenous and exogenous signals. In this paper, diverse regulatory functions performed by MEDIATOR25 (MED25), one of the subunits of the plant Mediator complex are reviewed. MED25 was first identified as a regulator of flowering time and named PHYTOCHROME AND FLOWERING TIME1 (PFT1). Since then, MED25 has been implicated in a range of other plant functions that vary from hormone signaling  to biotic and abiotic stress tolerance and plant development. MED25 physically interacts with transcriptional activators , repressors , and other Mediator subunits (MED13 and MED16). In addition, various genetic and epigenetic interactions involving MED25 have been reported. These features make MED25 one of the most multifunctional Mediator subunits and provide new insights into the transcriptional control of gene expression in plants. TFs also recruit via their trans-activation domains (TADs), the Mediator multiprotein complex, which bridges TFs to the RNA Pol II during transcription. In addition to its role in activator-mediated transcription, Mediator stimulates basal transcription and is involved in transcriptional silencing  play essential roles in activating or repressing transcription in response to endogenous and exogenous cues. However, TFs do not directly interact with RNA Pol II, the multiprotein enzyme complex involved in transcription. Instead, TFs directly bind to specific ilencing .Saccharomyces cerevisiae), which contains 25 subunits, while approximately 31 and 34 subunits are found in mammals and plants, respectively (reviewed in Mediator has been first discovered in yeast (Mediator can modulate RNA Pol II transcription by influencing the assembly of the pre-initiation complex (PIC), Pol II pausing, elongation and re-initiation and chromatin architecture . It is nIn this review, diverse functions performed by MED25, one of the relatively well-conserved subunits of the Mediator complex, will be reviewed. Please see recent reviews for additional information on the plant Mediator complex .pft1 (phytochrome and flowering time 1) mutant that showed a late flowering phenotype under suboptimal light conditions. This suggested that the mutated gene regulates shade-avoidance (rapid extension of stem and accelerated flowering in plants grown under dense canopies)  . Subsequ animals . Howeveres MED25 . As expl screens  and a nuThe yeast does not contain a MED25 homolog while a single gene encoding MED25 is found in plants . The locFigure 1). The conserved von Willebrand Factor Type A (vWF-A) domain located at the amino terminus of MED25 is required for its interaction with the Mediator complex via the Tail subunit MED16  mutant, which shows sensitivity to root inhibition by bestatin, a strong chemical activator of the JA signaling pathway, was found to be allelic to med25  screening of 1589 Arabidopsis TFs from 62 different families using the conserved ACID region of MED25 as bait has identified eight MED25-interacting TFs, including four AP2/ERFs belonging to Group IX of the AP2/ERF family  implicatignaling .Similarly to MYC TFs, JAZ repressors such as JAZ9 interact with MED25 through their conserved JAS (JA Associated) domains . So, it Figure 2 and Table 1). Interestingly, similarly to MED25, CDK8 positively regulates JA-responsive defense gene expression  and resistance to A. brassicicola. However, in contrast to MED25, which positively regulates resistance to Botrytis cinerea . Under high auxin levels, Aux/IAA14 is degraded and this results in the dissociation of TPL and MED13 from the complex. ARF7 and ARF9, by binding to the auxin responsive element (AuxE) found in the LBD16 (LOB DOMAIN 16) promoter, interact with MED25 to recruit the core Mediator complex and RNA Pol II , which is part of the CDK8 kinase domain of the Mediator complex, regulates auxin signaling through the association with ARF7 and ARF19 and Aux/IAA14 . Under lA Pol II .Jasmonate and auxin signaling share many mechanistic similarities , suggests that MED25 performs mechanistically generic roles in both signaling pathways. Furthermore, as stated above, MED25 interacts with CDK8 during the regulation of JA signaling  and repressors (JAZs) of JA signaling . Therefoignaling , and witignaling .med25 mutant shows increased sensitivity to Fe deficiency, suggesting that MED25 is a positive regulator of these responses. MED25 interacts with both MED16, another Mediator subunit involved in regulating Fe deficiency responses, and EIN3 (ETHYLENE INSENSITIVE 3) and EIL1 (ETHYLENE INSENSITIVE 3 LIKE), known regulators of both ethylene signaling and Fe deficiency responses (IRT1 (IRON-REGULATED TRANSPORTER 1) and FRO2 (FERRIC REDUCTION OXIDASE 2)  .med25 mutant shows increased sensitivity to the inhibition of germination and seedling growth by ABA, suggesting a role for MED25 in regulating ABA signaling. In addition, Em6, and RAB-RELATED GENE18 (RAB18) show increased ABA responsiveness in the med25 mutant, suggesting that MED25 is a negative regulator of ABA responses during seed germination and early seedling growth . Upon binding of DREB2A to MED25, conformational alterations occur in the overall Mediator structure . Interestingly, the ubiquitination of MED25 by MBR1 and MBR2 was linked to the activation of FT transcription in a process called “activation by destruction”  seems to have a role in modulating flowering time. This section contains encodes a poly-glutamine track and transgenic plants expressing a MED25 isoform lacking this domain show similar flowering phenotypes as the pft1 mutant , suggesting that MED25-mediated changes in redox status regulate root hair differentiation. MED25 positively regulates class III peroxidases involved in H2O2 production while negatively regulating NADPH oxidases involved in ROS generation. This changes the redox status of the cells, which then acts as a signal in the regulation of cell specification, initiation and elongation of root hairs (PIN1 and PIN2 involved in auxin transport and response required for meristematic cell proliferation at the root tips (A role for MED25 in negative regulation of auxin (IAA) signaling as well as primary and lateral root formation and development has been shown (see below) . The medot hairs . In addioot tips .med25 mutant has larger floral organs , suggesting that MED25 limits organ growth most likely by negative regulation of genes that encode positive regulators of cell expansion and proliferation -mediated induction of PDF1.2 . Although MED25 and other Mediator subunits play essential roles in plant signaling, abolishing the function of a single Mediator subunit often does not result in complete loss of transcription, suggesting that engineering of Mediator subunits can be a potential strategy to improve stress tolerance in plants. The findings reviewed here might imply that the engineering of MED25 can potentially lead to pleiotropic effects due to its involvement in multiple plant functions. However, the ability of MED25 to perform different roles through its different domains can offer a flexibility in this regard. For instance, the modification of the MED25 ACID region through technologies such as CRISPR/CAS9  and may allow its interaction only with certain TFs while abolishing or attenuating interactions with others. Alternatively, new specificities can be generated by adding the ACID binding ability to heterologously-expressed TFs. Given that VP16 is a strong transcriptional activator and binds to the MED25 ACID region (The studies reviewed here show that MED25 is multifunctional protein (D region  various D region . The ideD region . Better After the acceptance of this article, two papers published have revealed additional insights into PFT1/MED25 functions in flowering and root development. KK reviewed the literature and wrote the paper.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Erythroid Krüppel-like Factor (EKLF/KLF1) is an erythroid-enriched transcription factor that plays a global role in all aspects of erythropoiesis, including cell cycle control and differentiation. We queried whether its mutation might play a role in red cell malignancies by genomic sequencing of the KLF1 transcription unit in cell lines, erythroid neoplasms, dysplastic disorders, and leukemia. In addition, we queried published databases from a number of varied sources. In all cases we only found changes in commonly notated SNPs. Our results suggest that if there are mutations in KLF1 associated with erythroid malignancies, they are exceedingly rare. Its roles in ß-like globin gene regulation during terminal erythroid differentiation have been well-established using genetic, biochemical, and molecular approaches3. Specific functional properties and expression characteristics of EKLF, along with recognition of its surprisingly broad role prior to and during red cell differentiation (reviewed in8), provide the conceptual basis for the present study.Erythroid Krüppel-like Factor (EKLF/KLF1) is a red cell-enriched, zinc finger DNA binding protein that interacts with its cognate 5′CCMCRCCCN3′ element at target promoters and enhancers12, raising the possibility that mutations at these sites or within their consensus sequences could have dramatic functional effects on gene expression control.First, single amino acids and their modifications are critically important for EKLF protein-protein interactions and its function as an activator or repressor17. Induction of p21 is reminiscent of a similar up-regulation that has been observed with KLF418 and with KLF619, known tumor suppressors that function in a p53-independent manner and that are frequently inactivated or downregulated in human cancer20.Second, subtle altering of EKLF cellular levels can change the erythroid cell cycle status from proliferation to differentiation, an effect mediated, at least in part, by its direct activation of the p21, p18, p27, and E2f2 genes21. Although most abundant in the erythroid cell, EKLF is also highly expressed in the megakaryocyte/erythroid progenitor (MEP)22. Its level is downregulated as MEPs differentiate towards the megakaryocytic lineage yet remains high in the erythroid lineage22. By using both gain- and loss-of-function approaches, we22 and others25 have found that the expression levels of EKLF impacts the bipotential lineage decisions that are made by the MEP; specifically, EKLF inhibits the formation of megakaryocyte colony and cell numbers while at the same time stimulating erythroid differentiation.Third, EKLF mRNA is highly restricted in its expression pattern during development to erythropoietic organs such as the yolk sac, fetal liver, adult bone marrow, and red pulp of the spleen28. For example, the semi-dominant mouse mutation Nan , which presents with hereditary spherocytosis, was mapped to a single amino acid change (E339D) within the second zinc finger of EKLF31. The mutation alters the DNA binding specificity of EKLF such that it no longer binds promoters of a subset of its DNA targets29. In addition, recognition of a novel target DNA sequence by Nan-EKLF leads to ectopic expression of genes not normally expressed in the red cell, yielding a neomorphic phenotype with cellular and systemic consequences33.Fourth, there are now links between KLF1 mutation and altered mammalian hematology39, where the same amino acid is altered  in human EKLF/KLF1. These patients are severely anemic with highly elevated HbF and reticulocyte levels, membrane abnormalities, severe hemolytic anemia, erythroid hyperplasia with dyserythropoiesis, splenomegaly, and growth delay36. KLF1/E325K is recognized as a characteristic feature of CDA type IV40.Excitingly, this murine mutation has converged with human disease, particularly a subtype of congenital dyserythropoietic anemia (CDA)43.Additionally, the regulation of some genes are uniquely sensitive to haploinsufficient levels of KLF1 , leading to altered genetic expression patterns and hematologic parameters in humans, of which hereditary persistence of fetal hemoglobin (HPFH) is particularly relevant to clinical outcome45. On the other hand, unrestricted proliferation and impaired differentiation are characteristic of acute myeloid leukemia (AML)47. Our test hypothesis is that dysregulation of KLF1 function may contribute or lead to either of these human malignancies.Collectively these functional properties suggest that genetic mutation or altered levels of KLF1 may also be a causative factor for a specific subset of hematopoietic disease. We focused our attention on two types of disorders. Myeloproliferative neoplasms (MPN) are chronic hematological malignancies that yield an excessive proliferation of blood cells with normal differentiation48. Recent studies provide an extensive catalogue of SNP variants of consequence for red cell parameters49. Perusal of the data enable us to extract the ones most relevant to KLF1 as summarized in Table 19p13.13, the chromosomal locus of KLF1, has been associated with variation in blood cell traits in meta-analysis studies50, enabling us to interrogate its proximal promoter, 5′ UTR, introns, exons, and 3′ UTR by eight overlapping amplimers. The proximal promoter contains highly conserved transcription factor binding sequences that are critical for its expression in erythroid cells55. To begin our evaluation of human KLF1 genomic status, we focused first on sequencing human leukemia cell lines that retain erythroid and/or megakaryocytic features57. These lines are derived from CML, AML-M6 or -M7 patients , and include those with mixed erythroid/megakaryocytic features . We conclude from genomic sequence comparison of these lines to the 1000 Genomes project59 that the KLF1 genomic changes observed represent known single nucleotide polymorphisms (SNPs), but no novel mutations  and essential thrombocythemia (ET), which represent abnormalities in the proliferation of erythroid and megakaryocytic lineage respectively45. We therefore sequenced genomic DNA from a collection of individuals with PV (eighteen), ET (eleven), and MF (five) samples. No novel mutations were identified, only SNPs  as cells whose aberrant properties might result from expression of mutated KLF1. MPNs are a heterogeneous group of clonal hematological malignancies that are characterized by hypercellular bone marrow, panmyelosis, and a gradual evolution to myelofibrosis (MF) and acute leukemiaPs Table . Althoug62. We hypothesized that the mutational status of KLF1 may provide a mechanistic basis to explain the differential expression signature in these patients, and potentially predict whether they will respond to lenalidomide. We analyzed 26 samples each of responders and non-responders. All variants identified in these samples were known SNPs  patients that vary in their response to lenalidomide treatment63, including four from patients with Down syndrome (DS) and six from those without DS. AMKL is an aggressive form of leukemia where a majority of the expanding cells are abnormal megakaryoblasts64, and whose DNA methylation patterns are distinct between DS and non-DS cells65. Again, all samples contain known KLF1 SNPs (Table We next examined ten acute megakaryoblastic leukemia (AMKL) samplesPs Table .26. The ones that result in non-synonymous coding changes are predicted not to affect KLF1 function67 , some with an overall low frequency (~5%) are nonetheless highly enriched (~20%) prevalently and exclusively in selected genetic or geographic sub-populations while completely absent in all others  , and AML tabulations74, along with those derived from The Cancer Genome Atlas (TCGA) data sets76 do not reveal a role for KLF1 in any case in these blood cancers. KLF1 is not one of 142 driver genes identified from an analysis of 1699 pediatric leukemias and tumors77. An shRNA screen of AML cells lines also did not implicate a role for KLF178, and KLF1 does not appear in differential analyses related to predicting therapy resistance in AML79. Examination of COSMIC data (v83)80 indicates that, out of 3478 curated hematopoietic and lymphoid samples, only a single coding sequence variant was observed (F27V) in a CLL patient81, one predicted to be benign67.Given our hypothesis, we were surprised by the absence of mutations associated with our target samples/sources. To expand our analysis, we also considered an 26). Two, KLF1 levels vary considerably across various malignancies as judged by tabulation of RNA sequence datasets82 . This line was established decades ago91, and early on was noted to exhibit variability93. We have noted major differences in transfection efficiency upon comparing our lab K562 stock with the ATCC K562 line (unpublished observations) although we did not find any KLF1 genomic sequence differences. We suggest labs are working with dissimilar isolates, differing in levels of GATA1 and/or KLF194.K562 cells were from our original lab stockPatient samples were procured after informed consent and IRB approval within the individual institutions . All methods were performed in accordance with the relevant institutional guidelines and regulations. Mononuclear cells from MPN patients were provided by the Myeloproliferative Disorders Research Consortium Tissue Bank Core C.Genomic DNA was isolated from all samples using a Qiagen DNeasy Blood & Tissue Kit. PCR primers spanning the complete KLF1 transcription unit were used to amplify eight overlapping regions across the locus  followed alignment to the GRCh38 reference sequence using Vector NTI ContigExpress software (ThermoFisher Scientific). SNPs and any associated parameters were identified from the 1000 Genomes databasesSupplementary Table 1