Saccharomyces cerevisiae. Nitrogen conditions influence general nucleosome profiles and the expression of nitrogen catabolite repression (NCR) sensitive genes. The nucleosome occupancy of TATA-containing genes was higher compared to TATA-less genes. TATA-less genes in high or low nucleosome occupancy, showed a significant change in gene coding regions when shifting cells from glutamine to proline as the sole nitrogen resource. Furthermore, a correlation between the expression of nucleosome occupancy induced NCR sensitive genes or TATA containing genes in NCR sensitive genes, and nucleosome prediction were found when cells were cultured in proline or shifting from glutamine to proline as the sole nitrogen source compared to glutamine. These results also showed that variation of nucleosome occupancy accompany with chromatin-dependent transcription factor could influence the expression of a series of genes involved in the specific regulation of nitrogen utilization.Well-organized chromatin is involved in a number of various transcriptional regulation and gene expression. We used genome-wide mapping of nucleosomes in response to different nitrogen conditions to determine both nucleosome profiles and gene expression events in  Nucleosomes form the basic repeating unit of eukaryotic chromatin, consisting of ~147 bp DNA wrapped around a histone octamer core124Saccharomyces cerevisiae is able to use the nitrogen source that enables best growth by a mechanism called nitrogen catabolite repression (NCR)101113914In response to different nutritional conditions or perturbation of the environment, yeast cells tend to fine tune their transcriptional, translational and metabolic processes in order to maximize survival abilityS. cerevisiae. To map the location of individual nucleosomes on a genomic scale in response to different nitrogen conditions, we used a MNase-seq method to sequence the ends of nucleosome-associated DNA. This study focused firstly on the regions around TSSs and stop codon sites, which are well-defined nucleosome distributions23Recent advances in high-throughput sequencing technology have dramatically expanded our understanding of genome-wide nucleosome organization61618621Knowledge of the detailed remodeling of nucleosome positioning across genomes and the mechanism underlying nucleosome profile changes is crucial for understanding gene regulation and expression. The nucleosome profiles of the 5′ and 3′ ends of a gene-coding region have been considered as a marker for gene transcription initiation and termination. Here, the whole genome data of nucleosome occupancy profiles were exhibited at the 5′ and 3′ ends of all genes . There iApproximately 20% of yeast genes contain a TATA box. Based upon the presence or absence of a TATA box in the promoter region, the genes can be classified as TATA-containing or TATA-less genesR2 < 0.009) between the nucleosome occupancy amount of gene coding regions and NFRs  between the nucleosome occupancy and nitrogen metabolic gene expression under different nitrogen conditions, although the correlation in M.Gln-Pro medium  and q-value < 1.0E-30 under nitrogen source change were analyzed  and then the ends of the DNA fragments were sequenced. After aligning sequence reads to the reference genome, the genome positions of individual nucleosomes in the genome were estimated. The precise positions calculated by analysis corresponded to the known positions of these nucleosomes and NUCwave software was used for the mapped sequence reads35We used a MNase-seq method to isolate mononucleosome-associated DNA from 600) of 0.6–0.8. Yeast cells were treated with 2% (v/v) formaldehyde for 30 min (with vigorous shaking every 10 min) and stopped with 125 mM glycine. Cells were collected, washed twice with sterile water and then suspended in 40 mL of buffer Z , 10 mM β-mercaptoethanol) with Zymolyase®-20T  at 0.25 mg/mL. Cells were spheroplasted at 28–30 °C for about 40 min, depending on how much time was necessary to remove the cell wall of each species completely. Spheroplasts were pelleted by centrifugation (3000g for 10 min) then suspended in 3.5 mL of NP buffer , 5 mM MgCl2, 1 mM CaCl2 and 0.075% nonylphenoxypolyethoxylethanol (NP-40), with 1 mM β-mercaptoethanol and 500 μM spermidine freshly added). Samples (600 μL) of spheroplasts were added to six Eppendorf tubes containing a range of concentrations  of micrococcal nuclease (MNase) and incubated at 37 °C for 20 min. Reactions were stopped by the addition of 150 μL of stop buffer (5% (w/v) SDS, 50 mM EDTA), 5 μL of proteinase K (20 mg/mL) was added and reactions were incubated at 65 °C overnight. Samples were extracted with phenol/chloroform/isoamyl alcohol (25:24:1 by vol.), precipitated, with ethanol and treated with DNase-free RNase. Samples were examined by gel electrophoresis with ~90% mononucleosomal DNA, and mononucleosomal DNA was further purified by gel isolation and used for high-throughput sequencing with Illumina HiSeq 2500 Yeast cultures (400 mL of strain S288C) were grown in YNB medium in 2 L flasks to optical density at 600 nm (ODSaccharomyces cerevisiae Genome Assembly. The raw MNase-seq data and processed data files were deposited at the NCBI Gene Expression Omnibus (GEO) (http://www.ncbi.nlm.nih.gov/geo) under accession numbers GSE75705.MNase-seq data were aligned to S288C p-value between Gln_NFR/ Pro_NFR and Gln_NFR/Gln-Pro_NFR. There is an option to generate the statistically significant genes, which calculates an adjusted p-value using the Benjamini-Hochberg method, the adjusted p-value is q-value. While for TATA genes, TATA 0 represent TATA less genes; TATA 1 represent TATA containing genes.The genome wide nucleosome occupancy maps from sequence reads under different nitrogen conditions were generated, annotated and normalized by using a bioinformatics tool, NUCwave35S. cerevisiae S288C was grown overnight at 30 °C in YPD medium. The cells were recovered, washed and diluted to OD600 = 0.1 then transferred to and cultured in M.Gln until OD600 = 0.6–0.8. Cells were pelleted by centrifugation, washed and transferred to M.Pro for 2 h, pelleted again, washed and stored at −80 °C. The procedures for RNA extraction and cDNA synthesis are described fully elsewhere® Premix Ex TaqTM kit . The PCR protocol was: incubation at 95 °C for 30 s then 40 cycles of amplification at 95 °C for 5 s, 55 °C for 20 s and, finally, cooling at 50 °C for 30 s. Reactions (in triplicate) took place in a LightCycler 480 II Real-time PCR instrument  and mean values were used for further calculations. The fold change was determined by the 2CT–ΔΔ method normalized to the ACT1 genePrimers used for qRT-PCR were designed by Beacon Designer 7.0 software . The qRTHow to cite this article: Zhang, P. et al. Genome-wide mapping of nucleosome positions in Saccharomyces cerevisiae in response to different nitrogen conditions. Sci. Rep.6, 33970; doi: 10.1038/srep33970 (2016).


A single nucleotide substitution, ‐13910 C>T, the first causal variant to be identified, accounts for lactase persistence over most of Europe. Located in a region shown to have enhancer function in vitro, it causes increased activity of the LCT promoter in Caco‐2 cells, and altered transcription factor binding. Three other variants in close proximity, ‐13907 C>G, ‐13915 T>C and ‐14010 G>C, were later shown to behave in a similar manner. Here, we study four further candidate functional variants. Two, ‐14009 T>G and ‐14011 C>T, adjacent to the well‐studied ‐14010 G>C variant, also have a clear effect on promoter activity upregulation as assessed by transfection assays, but notably are involved in different molecular interactions. The results for the two other variants  were suggestive of function, ‐14028*C showing a clear change in transcription factor binding, but no obvious effect in transfections, while ‐13779*G showed greater effect in transfections but less on transcription factor binding. Each of the four variants arose on independent haplotypic backgrounds with different geographic distribution.The genetic trait that allows intestinal lactase to persist into adulthood in some 35% of humans worldwide operates at the level of transcription, the effect being caused by  The expression of the intestinal enzyme lactase, which is critical for the majority of Eutherian mammals during the suckling period, is generally downregulated after weaning, but persists into adult life in some humans. This trait, known as lactase persistence , is carried by some 35% of the world's population and is notably more frequent in populations with a long history of pastoralism and dairy farming  , about 14 kb upstream of the LCT transcriptional start site, in intron 13 of the neighbouring Minichromosome Maintenance 6 (MCM6) gene was the first causal variant to be identified  in adults is regulated at the level of transcription and the effect is LCT enhancer sequence for novel variation, we and other investigators found a number of other variants clustering in close proximity to each other. In the first studies, three further functional alleles were identified in Middle Eastern and African populations: ‐13915*G (rs41380347), ‐13907*G (rs41525747) and ‐14010*C (rs145946881). These also showed increase reporter gene expression compared to the ancestral variants and/or differences in protein‐DNA binding affinity  was selected because it was strongly associated with digester status   was of interest because it was found to be relatively common in some groups in India, in the milk drinking Toda, for example, though it was also found in hunter‐gatherers  had previously been found as the only LCT enhancer allele identified in the second highest expressing transcript of a homozygous lactase persistent person , and left to differentiate for 13 days after seeding, before preparation of nuclear extracts, as described elsewhere . The probes were purified using MicroSpin G‐25 Columns .For gel shift assays, sense and antisense oligonucleotides (Table S1) were annealed and radioactively 5′ labelled using T4 Polynucleotide Kinase  and 25–35 μCi . Constr14009*G and ‐14011*T show increased luciferase expression in both undifferentiated and differentiated Caco‐2 cells compared to the ancestral construct that carries T at ‐14009 and C at ‐14011 . The ‐13779*C construct also showed a consistent increase in reporter gene expression in both undifferentiated and differentiated cells and, despite a high intraexperimental variance, was highly significant across all experiments, as tested by two‐way ANOVA. In contrast to the other variant, alleles ‐14028*C did not show any evidence of influence on enhancer activity.The constructs containing the alleles ‐LCT enhancer, electrophoretic mobility shift essays (EMSAs) were performed to evaluate differences in DNA binding properties to nuclear proteins extracted from Caco‐2 cells.To further investigate the influence of the four new variant alleles on the To examine the effect of the ‐14009 T>G and ‐14011 C>T substitutions flanking the previously reported ‐14010 G>C functional variant, oligonucleotides were designed to cover these positions, and were derived from the same length of sequence used previously to study ‐14010 G>C , which is more intense for the ‐14011T and ‐14010C variant probes than for ‐14009G or the ancestral probe, as observed previously for ‐14010C. The sequence overlaps an Oct‐1 and HNF‐1α binding site . Binding of GATA‐3 and 4 specifically to ‐14011T was predicted bioinformatically. However, preliminary experimental results with different GATA competitor oligonucleotides point to a competition of both the ancestral as well as the derived ‐14011 probes (data not shown).The ‐14009G probe showed a pattern that differed more dramatically from the three other probes tested at that locus, in that a prominent additional DNA–protein complex was formed  showed a very slight competition of the binding for NF‐kappaB, Pax, Pax4‐8 and Pbx. However, ‐14009G complex 1 could be fully competed with the ancestral sequence. The prominent lower band . Competitor oligonucleotides were designed taking into account specific transcription factor binding motifs found in TRANSFAC, or using information about matching competitors available from the Affymetrix EMSA kit (Table S1) and Figure The reciprocal experiments, using c‐Ets‐1 and Aff_ELK‐1 as probes showed that the c‐Ets‐1 probe was more effectively competed with ‐14009G than with the ancestral sequence, whereas Aff‐ELK‐1 was competed with both competitor sequences (not shown).Further gel shifts, examining the ‐13779 G>C SNP, revealed a strong DNA–protein complex for both the ancestral and derived variant probes that could be competed in a similar way with the unlabelled probe containing either the ancestral or derived allele Fig. . An HNF‐Probes corresponding to both alleles of the ‐14028T>C SNP bound strongly to proteins of the nuclear extract Fig. . This bi‐14011*T, which occurred most frequently on A (like ‐13910*T and 13907*G) but twice on B  and resides close to an HNF‐1α binding site  and the C substitution alters gene transcription in vitro in Caco‐2 cells  factors are known to interact with other proteins to regulate DNA binding or transcription and to play an important role in the differentiation, survival and proliferation of cells, including those of the intestine  the lack of effect in transfection experiments, at least under the conditions tested, was surprising.The variant at ‐14028 T>C is located within a proposed Cdx‐2 binding site . Early experiments showed that ‐958*T disrupts binding to an unknown transcription factor  carries the 13779*C had been not very strong since it had in fact previously been reported in one Somali individual who was diagnosed as a lactose mal‐digester , failing to show any noticeable impact of selection. The two occurrences of ‐14028*C both from the United Kingdom appear to be on the same fairly extended B haplotype background as each other (B), and interestingly this variant has recently been reported in two other instances (rs759157971), also both in the United Kingdom. The ‐13779*C allele is located on a C haplotype background and is most prevalent in India, where it is clustered geographically (Table S2).The results presented here provide further strong support for the functional role of in vivo since the Caco‐2 cell line expresses only low levels of lactase and in some cases shows effects that are not important in vivo  but shows how a combination of experimental and bioinformatic approaches will be needed to identify causal rSNPs in complex disease.Disclaimer: Supplementary materials have been peer‐reviewed but not copyedited.Table S1‐2, Figures S1‐4.Click here for additional data file.


ESR1 and ESR2. These bind to DNA and modulate the expression of target genes. Identification of estrogen target genes is greatly facilitated by the use of transcriptomic methods, such as RNA-seq and expression microarrays, and chromatin immunoprecipitation with massively parallel sequencing (ChIP-seq). Combining transcriptomic and ChIP-seq data enables a distinction to be drawn between direct and indirect estrogen target genes. This chapter discusses some methods of identifying estrogen target genes that do not require any expertise in programming languages or complex bioinformatics.Estrogen is a steroid hormone that plays critical roles in a myriad of intracellular pathways. The expression of many genes is regulated through the steroid hormone receptors  ESR1 and ESR2, the nuclear receptors through which estrogen exerts much of its effect . It. ItConveConsiderations for experimental design: Replicates are essential for ChIP-seq and transcriptomic analysis when attempting to distinguish biologically meaningful variation from noise. As mentioned in the introduction, if using stimulation with estrogen or an estrogen receptor agonist, it is vital to decide on the time scale of stimulation to ensure that the correct cross section of binding and transcriptomic changes are sampled.Limitations of current bioinformatic methods: There are several important limitations to consider when interpreting lists of direct gene targets. The output is only as good as the data input into the model in the first place. This can be an issue particularly for ChIP-seq datasets, which are noisy and frequently irreproducible in the main between different studies nominally using the same material and methods [ methods . However methods . Another methods . Methods methods .Further functional annotation of direct gene targets: As mentioned above, many of the thresholds used are rather arbitrary and so it can be informative to include other forms of functional annotation to hone down a list of potential gene targets to ones of higher confidence. ESR1 binding sites are more likely to be consistent between different ChIP datasets if they possess a classical ESR1 recognition motif or are located in a region of open chromatin (as assessed by DNase-seq) [ase-seq) . BETA wiase-seq) . There iase-seq) .


Red blood cell production (erythropoiesis) is the single largest consumer of iron in the body; this need is satisfied by maintaining a sensitive regulation of iron levels. The level of erythropoietic demand regulates the expression of the iron hormone hepcidin and thus iron absorption. Erythropoiesis-mediated regulation of hepcidin is an area of increasing importance and recent studies have identified a number of potential regulatory proteins. This review summarizes our current knowledge about these candidate erythroid regulators of hepcidin and the relation between transferrin receptors and erythropoiesis. Red blood cells (RBCs) perform one of the most vital functions in the human body; the transport of oxygen to every tissue and organ. Oxygen binds to the iron atoms in the heme part of hemoglobin, the protein that makes up more than 90% of the dry content of RBCs . The humThe other requirement for iron in erythropoiesis relates to its functional requirement in metabolically active and dividing cells. Iron acts as a cofactor for a number of enzymes including the DNA replicases, the three DNA polymerases  and DNA helicases (FANCJ) (reviewed in ) which aKassebaum et al.  estimateRBCs develop from hematopoietic stem cells (HSCs); this process involves a number of steps and is regulated by the interplay between a number of cytokines, growth factors, and environmental cues . The firIn the body, iron levels are regulated by modulating the levels of hepcidin, the iron regulatory hormone . HepcidiErythropoiesis is a known negative regulator of hepcidin ,18. AlthHypoxia links iron homeostasis to erythropoiesis. Oxygen transport utilizes the iron present in heme of the RBCs; in conditions of low oxygen availability, signals are sent to the bone marrow and hematopoietic compartment to increase RBC production. These signals are primarily mediated by HIFs, which regulate the transcription of a number of genes including EPO. HIF has an oxygen dependent α-subunit, either HIF1α ,23, HIF2In normoxic conditions the oxygen-dependent HIFα subunits are degraded by prolyl hydroxylase domain-containing enzymes (PHD). These enzymes need both iron and oxygen in order to be activated, forming an important link between iron and oxygen concentrations. The active PHDs are responsible for hydroxylating the HIFα’s at their proline residues, which makes them susceptible for ubiquitination. In hypoxic conditions, PHDs become inactive thus increasing the stability of HIFα. These subunits then bind to ARNT and other transcription factors to regulate the transcription of genes in response to hypoxia.Mice with a hepatocyte-specific deletion of HIF1α do not respond to iron deficiency or hypoxia by decreasing hepcidin expression as is seen in wild-type (WT) mice . HIF1α aTMPRSS6 [furin mRNA levels through HIF1α in HepG2 and Hepa1-6 (mouse hepatoma) cells [Hypoxia may also be acting indirectly in iron homeostasis by interrupting the expression of molecules involved in hepcidin regulation. Hypoxia and iron deficiency were shown to increase the mRNA levels of transmembrane serine protease 6 (TMPRSS6) . TMPRSS6TMPRSS6  develop a) cells . Furin ca) cells , sHJV acHypoxia also induces the expression of EPO in the kidney and EPO was also thought to be one of the candidates for erythropoiesis-mediated regulation of hepcidin. It appears that this hypoxia-mediated repression of hepcidin requires EPO; mice where EPO was deleted were unable to reduce hepcidin expression in response to hypoxia . A separAn increase in erythropoietic demand also leads to an increase in the production of EPO, and it was shown that an increase in erythropoietic activity was a negative regulator of hepcidin ,18,36. THAMP in primary hepatocytes, but this reduction was not observed in the absence of GDF15 [Gdf15 knockout mice which were subjected to two consecutive phlebotomies and did not exhibit any differences in hepcidin levels as compared with WT mice; these results suggested that either GDF15 was not the putative erythroid factor or its effects could be overcome by other pathways involved in hepcidin regulation [Two ‘erythroid regulator’ candidate genes were identified by performing a microarray analysis on RNA isolated from primary erythroblasts donated by normal subjects . This reof GDF15 . This waof GDF15 , multiplof GDF15 , and aneof GDF15 . This inof GDF15 . Gdf15 kgulation .Gdf15 mRNA levels increased in the bone marrow and spleens of five different models of anemia whereas Twsg1 showed no change as compared with controls [Unlike GDF15, TWSG1 has not received as much attention and the only study which suggested that it could be a factor secreted by early erythroblasts capable of regulating hepcidin was published in 2009 [controls . These sFam132b gene, known as erythroferrone (ERFE) [ERFE in the erythropoietic organs (bone marrow and spleen) [−/−Fam132b mice do not suppress hepcidin in response to phlebotomy as strongly as WT mice [Fam132b [−/−Fam132bmice injected with Plasmodium berghei K173 were not able to suppress hepcidin in response to the anemia induced by malaria [−/−Fam132b mice injected with Brucella were able to recover faster from anemia as they did not suppress hepcidin as efficiently as WT mice [Recently, there has been another addition to this list of potential erythroid regulators of hepcidin: the product of the e (ERFE) . EPO inj spleen) . Fam132b WT mice . Phenylh[Fam132b . As expe[Fam132b . ERFE al malaria . Similar WT mice . These r−Tfr1in mice leads to embryonic lethality [Transferrin receptor 1 is a ubiquitously expressed protein that is responsible for the uptake of transferrin (TF)-bound iron. This mechanism of iron uptake is the most common among all cells of the body; it is also essential during development as the loss of ethality . The homethality . These cTfr1; mice lacking Stat5 had less Tfr1 on the cell surface of erythroid cells. Tfr1 also has three Stat5 binding sites in its first intron [The binding of EPO to the erythropoietin receptor (EPOR) results in the activation of Janus activated kinase 2 (JAK2) which in turn phosphorylates the cytoplasmic tail of EPOR . The actt intron .Tfr1, body iron levels can also regulate Tfr1 mRNA levels through the iron responsive element (IRE)–IRE binding proteins (IRP). The IRE–IRP system is mediated by the binding of the RNA-binding proteins (IRPs) to a conserved sequence in the 5′- or the 3′-untranslated region (UTR) of the mRNA, thus either inhibiting translation or stabilizing the mRNA [In addition to erythropoiesis-mediated regulation of the mRNA . This cothe mRNA .Tfr1 regulation in erythroid cells may also be heme-dependent and iron-independent, suggesting an erythroid-specific regulation of iron uptake [n uptake . When din uptake . On the n uptake ,53, indin uptake . These sTFR2 was mapped to chromosomal position 7q22 in the human genome and 5qG2 in the mouse genome, and is close to EPO (on the opposite strand). An upstream analysis of the promoter region of TFR2 revealed binding sites for several erythroid-specific transcription factors [TfR2 promoter, these are potential erythroid Kruppel-like factor (EKLF) binding sites (another erythroid-specific transcription factor) [TFR2 promoter, although in one case where a shorter promoter was used EKLF decreased the activity suggesting it has an inhibitory effect [TfR2 could be regulated in an erythroid-specific fashion.The homolog of TFR1, TFR2 is thought to be involved more in the regulation of iron than its uptake. Patients  and mice factors . There a factors . Two put factors . In addi factor) . It was y effect . These oTFR2–EPO locus and various hematological traits including RBC number and hematocrit [TFR2 expression is lower in patients with high risk myelodysplastic syndromes (MDS), such as refractory anemia with excess blasts type 2 (RAEB2) [TFR2 are associated with higher survival rates in patients with acute myeloid anemia [Genome wide association studies have identified associations between the matocrit ,59. TFR2 (RAEB2) . Increasd anemia .Tfr2-deficient mice had a reduced colony forming ability even in the presence of physiological concentrations of EPO [The erythroid function of TFR2 was further supported by the demonstration that it may be required for the proper localization of EPOR . Erythros of EPO . Furthers of EPO ,64 and os of EPO –67. Usins of EPO , or incrIn summary, the mechanisms and molecules discussed above point to a very tightly regulated and sensitive erythroid control over iron homeostasis. This is mediated through regulating hepcidin and molecules involved in systemic iron homeostasis e.g. TFR1. Additional studies on the role of ERFE are no doubt underway and will contribute to defining its role as the erythroid regulator. Further studies should concentrate on the role of ERFE in human erythropoietic disorders and its role in hepcidin regulation.


Zea mays) pollen-predominant gene Zm908, a novel small-peptide gene, was reported to play critical roles in pollen germination and pollen tube growth in our previous work. In this study, we aimed to explore the regulatory mechanism of Zm908. The putative promoter of Zm908 was cloned and analyzed. The activity analysis of a series of promoter truncations in different tissues of transgenic tobacco plants indicated that the Zm908 promoter is pollen-specific and that the –126 to –68 region is crucial for pollen expression. The 5′ deletion analysis of the –126 to –68 region revealed that the –126 to –102 region functions as a transcriptional suppression element. ZmDof30, which is predominantly expressed in pollen and whole anthers, was cloned and characterized. ZmDof30-GFP localized to the nuclei of maize protoplasts and possessed no transcriptional activation activity in a yeast system. ZmDof30 could bind to the AAAG elements in p184 sequence containing the –126 to +58 region of the Zm908 promoter in vitro and in vivo, and negatively regulated p184 activity in tobacco leaves. Collectively, ZmDof30 may function as a Zm908 transcriptional repressor in pollen, and these results may provide a better understanding of the regulation of the Zm908 gene. Additionally, the pollen-specific Zm908 promoter may be valuable for genetically engineering male sterility.The maize ( Arabidopsis thaliana, the INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) gene encodes a small peptide protein that controls floral organ abscission (Zm401 (AY911609), an anther-specific sORF gene in maize, leads to pollen sterility in transgenic tobacco and maize  which contains tandem Dof common recognition elements , encoding small peptides with less than 100 amino acid residues . Many plscission . The ovend maize . Zm401p1elopment . Furtherelements . Dong ete growth .Arabidopsis . Through an activity analysis of a series of promoter truncations in different tissues of transformed tobacco, the expression of pZm908 was investigated. The full-length of pZm908 was pollen-specific and the –126 to –68 region was crucial for promoter activity. The 5′ deletion analysis of the –126 to –68 region showed that the –126 to –102 region of pZm908 acted as a negative element. Deletion of the –126 to –102 region resulted in lacking one Dof core element, destruction of two tandemly repeated Dof core elements and lacking one lat52 pollen-specific transcription activation cis-regulatory elements indicating that the Dof core elements were likely negative regulatory elements and a Dof transcription factor might participate in suppressing the promoter activity. ZmDof30, a pollen-predominant Dof transcription factor, interacted directly with the AAAG motif in –126 to –100 region of the Zm908 promoter in vitro and in vivo, and negatively regulated the activity of p184 containing the –126 to +58 region of Zm908 promoter in tobacco leaves. Collectively, ZmDof30 may function as a Zm908 transcriptional repressor in pollen, and these results provide a better understanding of the regulation of Zm908 in pollen germination and pollen tube growth.In this study, we cloned and analyzed the putative promoter of TM19-T simple vector (TaKaRa) for sequencing. The inserts were digested from the recombinant pMDTM19-T simple vector and cloned into the corresponding restriction sites of pBI121. Each recombinant vector was confirmed by digestion and sequencing.pZm908 was cloned from the key maize inbred line, Zong31, which was cultivated by the Chinese scientist Jingrui Dai , and fused with the GUS gene in the pBI121 plasmid to generate p908::GUS. For the generation of the truncated constructs, p908::GUS was used as a template for PCR using 2× Taq Platinum PCR Master Mix . Primers designed with restriction endonuclease sites are listed in Supplementary Table Nicotiana tabacum cv. Xanthi-nc) grown in bottles with Murashige and Skoog (MS) medium at 26°C under a 16 h light/8 h dark photoperiod were used to obtain stably transformed lines in this study. Each truncated construct was introduced into tobacco by Agrobacterium tumefaciens (LBA4404)-mediated leaf disk transformation (Sterile tobacco seedlings (ormation . After s3(CN)6, 5 mM K4Fe(CN)6, 100 mM sodium phosphate buffer [pH 7.0] and 0.1% Triton X-100) for 12 h at 37°C. Following alcohol decolorization, the samples were photographed under a microscope .The leaves and roots of tobacco seedlings, immature seeds, indehiscent anthers and mature pollen shed from intraday dehiscent anthers were used in the GUS histochemical staining assay. GUS staining was performed using the chromogenic substrate 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid . Each sample was incubated with GUS staining buffer  as the substrate and employing a F-4500 fluorescence spectrophotometer  to measure fluorescence. GUS activity, expressed as nanomoles of 4-MU produced per gram protein per minute, was normalized to total protein concentration.Total protein was extracted from mature pollen grains using extraction buffer (50 mM sodium phosphate (pH 7.0), 10 mM EDTA, 0.1% SDS, 10 mM β-mercaptoethanol, and 0.1% Triton X-100). After vortexing, the samples were incubated on ice for 15 min and then centrifuged at 13,500 Bradford . The fluZmDof30 coding region, cloned from maize anther cDNA, was fused to GFP in the pUC-GFP plasmid to construct the ZmDof30-GFP vector. The AT-hook motif nuclear-localized protein 22 gene (AHL22) was used as a positive control. For subcellular localization assays, maize protoplasts were isolated according to a previously reported protocol . The ZmDof30 coding region fused with GAL4 was inserted into the pBD-GAL4 plasmid using the EcoRI and SalI sites to generate the pBD-ZmDof30 vector. The pBD-ZmDof30, pBD-GAL4 (negative control), and pGAL4 (positive control) plasmids were transfected into yeast. The transfected yeast cells were cultured on YPDA or SD/-Trp/-His medium (lacking threonine and histidine) at 30°C for 3 days. β-galactosidase activity assays of the transfected yeast cells were performed as described in the Yeast Protocols Handbook .The yeast strain YRG-2, containing Figure 4A), which was transformed into yeast strain Y1HGold according to the instructions of the YeastmakerTM Yeast Transformation System 2 . As a negative control, the E (p184)-M sequence was mutated from AAAG to AATC. Bait strains were selected from colonies that grew well on a synthetic defined (SD) medium lacking uracil. The coding sequence of ZmDof30 was cloned into the EcoRI and BamHI sites of pGADT7 to generate the prey vector. After screening for the minimal inhibitory concentration of aureobasidin A (AbA), the bait strains were transformed with the prey vector and cultured on SD medium lacking leucine and containing the minimal inhibitory concentration of AbA at 30°C for 3 days.The bait sequence double E (p184), synthesized in tandem, was cloned into pAbAi to generate the bait plasmid  according to the manufacturer’s instructions.Full-length ZmDof30 could not be expressed in the prokaryotic expression system. Therefore, the truncated fragment encoding the 26–127 amino acids, which contains the DNA binding domain of ZmDof30, was cloned into pMAL-C2x to construct the MBP-ZmDof30Nicotiana benthamiana plants at the six-leaf stage, grown in a phytotron at 23°C with a 16 h light/8 h dark cycle, were used for transient transformation. The reporter plasmid (p184::GUS), the effector plasmid (pSuper::ZmDof30) and the internal control (35S::LUC) were transformed into Agrobacterium (GV3101) individually and then co-infiltrated into the leaves of N. benthamiana as previously described . AGAAA is a pollen-specific transcription activation cis-regulatory element of the tomato lat52 gene . p1670 and p1126 showed strong GUS staining in seedling leaves. GUS staining was detected in mature pollen with all constructs except p126. GUS staining was found in both mature pollen grains and leaves of seedlings for p1670 and p1126, consistent with the Zm908 gene expression pattern in maize  was prepared as outlined in in maize . The fulcis-acting elements revealed that there were two tandem repeated Dof core elements (AAAG), two lat52 pollen-specific cis-regulatory elements (AGAAA), one pollen Q element and one CAAT box in the –126 to –68 region. One of the AGAAA motifs was between and overlapped with the two AAAG motifs, and the other motif was downstream and overlapped with the AAAG motif close to the 3′ end of the promoter . Moreover, sequence alignment showed that the cis-regulatory elements of the p184 promoters from Zong31, B73, and Mo17 were identical . To elucidate the element here, as shown in Figure 2A, stable transformation of tobacco was performed with five promoter deletion constructs . GUS activity in the mature pollen grains of the transformed tobacco lines was subsequently measured by histochemical staining assays and fluorometric quantitative analysis. The results showed that p151, lacking one Q-element, had much lower GUS activity in pollen grains than p155, and p155, lacking one AGAAA element, which is a pollen-specific transcription activation cis-regulatory element in the tomato lat52 gene, had much lower GUS activity than p160. Deletion of the region from –126 to –102 resulted in lacking one Dof core element, destruction of two tandemly repeated Dof core elements and lacking one lat52 pollen-specific transcription activation cis-regulatory elements (AGAAA). The promoter activity of p160 was much higher than that of p184 indicating that the one Dof core element, especially two tandemly repeated Dof core elements were likely negative regulatory elements . These results implied that a Dof transcription factor might participate in suppressing promoter activity by binding to the AAAG motifs.An analysis of ZmDof1 (GRMZM2G162749) and ZmDof30 . This result confirmed that ZmDof30 localized to the nucleus. A yeast assay system was subsequently used to determine the transcriptional activation activity of ZmDof30 . The coding region of ZmDof30 was cloned into the pBD-GAL4 vector for fusion with the GAL4 DNA-binding domain. We transformed the fusion plasmid (pBD-ZmDof30) and the control plasmids into the yeast strain YRG-2. The negative control plasmid was the pBD-GAL4 vector with only the GAL4 DNA-binding domain, and the positive control plasmid included both the GAL4 DNA-binding domain and the GAL4 activation domain. The growth of the transfected yeast cells was then examined on nutritionally deficient selection medium (SD/-His). Yeast cells transfected with the positive control construct grew well on histidine-free SD medium and exhibited high β-galactosidase activity. However, no growth was observed for yeast cells transfected with pBD-ZmDof30 or for the negative control, showing that ZmDof30 possessed no transcriptional activation activity in the yeast system and implying that ZmDof30 might act as a transcriptional suppressor.The investigation of spatiotemporal expression patterns is useful for understanding gene function. Therefore, we performed expression profiling of 46 maize Dof genes identified by Chen . Accordid pollen . Therefoin vitro. The MBP-ZmDof3026-127aa fusion protein, containing the DNA binding domain of ZmDof30, was expressed in a prokaryotic expression system. The three repeated E (p184) sequence was synthesized in tandem and labeled with biotin. The retardant band indicated that ZmDof3026-127aa could bind to the E (p184) sequence. The binding signal could be reduced by the 50× and 200× unlabeled probe, but not the mutant probes, demonstrating that ZmDof3026-127aa could specifically bind to the E (p184) sequence . We also investigated whether ZmDof30 could bind directly to the E (p184) sequence using a yeast one-hybrid system. The double E (p184) sequence was synthesized in tandem and cloned into pAbAi to construct the bait plasmid and the double E (p184)-M sequence was used to generate the negative bait plasmid . The two bait plasmids were independently transformed into yeast. Subsequently, the minimal concentration of AbA needed for growth inhibition was determined in the yeast cells transformed with bait plasmids. A concentration of 800 ng/mL AbA was chosen for the E (p184) bait yeast strains, and 200 ng/mL AbA was used for the E (p184)-M bait yeast strains . The prey plasmid pGADT7-ZmDof30 was transformed separately into the bait yeast strains. The transformed E (p184) bait yeast cells could grow on medium that lacked leucine but contained 800 ng/mL AbA, while the transformed E (p184)-M bait yeast cells could not grow on medium that lacked leucine and contained 200 ng/mL AbA , indicating that ZmDof30 protein could bind specifically to E (p184) in a yeast system. In conclusion, ZmDof30 could bind to the core recognition element (AAAG) of p184 in vitro and in vivo.An EMSA was performed to characterize the interaction between ZmDof30 and the Dof core recognition element (AAAG) in p184 N. benthamiana. In this system, the reporter plasmid (p184::GUS), the effector plasmid (pSuper::ZmDof30) and the internal control (35S::LUC) were transformed into Agrobacterium (GV3101) individually and then co-infiltrated into leaves of N. benthamiana. The negative control was empty pSuper vector. Three days after inoculation, the GUS and LUC activity were assayed in protein extracts prepared from infiltrated leaves. Compared to the control, co-expression of p184::GUS with pSuper::ZmDof30 resulted in a 0.4-fold decrease in relative GUS/LUC activity . The results indicated that ZmDof30 could bind to the E (p184) sequence to suppress promoter activity.For further analysis, a transcriptional activation experiment was carried out in the leaves of Zm908p11 in maize decreased pollen germination efficiency, whereas maize with Zm908p11 RNAi pollen could produce normal pollen tubes. The success of pollen germination depends on normal or low levels of the Zm908 protein, which can interact with profilin. The appropriate level of Zm908 protein might maintain the dynamic equilibrium between profilin-actin and Zm908p11-profilin-actin, resulting in the normal growth of pollen tubes  gene were also analyzed in tobacco , indicating that a Dof transcription factor might participate in suppressing the promoter activity.The function of Zm908 in pollen germination has been previously identified . The oveen tubes . In thiso system . The cis tobacco . Here, tDof gene family are clearly vital for plant development in a number of cases. PBF (prolamin-box binding factor) and ZmDof3, both of which are members of the Dof transcription factor family in maize, were reported to play critical roles in seed development . And ZmDof30 possessed no transcriptional activation activity in a yeast system . The EMSA and yeast one-hybrid results indicated that ZmDof30 interacted directly with p184 by binding to the AAAG element . Agroinfiltration of N. benthamiana leaves is a widely used transient system to analyze the transcriptional regulation of the target genes. For example, a transient transcription assay in leaves of N. benthamiana revealed that the 27-kD γ-zein promoter was transactivated by OHPs and PBF . All of these results indicate that ZmDof30 is a Dof transcription factor and functions as a Zm908 transcriptional repressor, providing evidence that a Dof transcription factor participates in pollen germination. The expression profile of a gene may shed light on its biological function. In addition to pollen, ZmDof30 was also predominantly expressed in whole anthers, implying that ZmDof30 may also be associated with the maize pollen development processes.Members of the elopment . PBF is ize seed . ZmDof3,ndosperm . Dof1, wnagisawa , is an eelopment . Accordid pollen . ZmDof30 and PBF . In our DAG1 (Dof affecting germination), affected seed germination when expressed in the maternal testa layer of the seed. The DAG1 mutant line showed reduced dormancy (DAG2 mutant (AtDOF4:2 was involved in phenylpropanoid metabolism in A. thaliana, negatively influenced flavonoid synthesis, and positively affected hydrocinnamic acid production. The variable terminal region of Dof had transcriptional regulation activities, mediating activation or repression of gene expression by interacting with other regulatory proteins, including the Dof domain itself (Zm401, thereby controlling pollen development and suggesting that there may be a suppressor that interacts with the Dof1 protein to abolish its transcription-activating activity (Zm908 in transient expression assays in N. benthamiana. These results imply that ZmDof30 might mediate the repression of Dof1 transcriptional activity by interacting with Dof1. However, further analyses are needed.The binding of Dof transcription factors mediates the activation or suppression of gene transcription, depending on the target gene. One Dof gene, dormancy . However2 mutant . It seemn itself , a bZIP n itself , a Myb tn itself  and othen itself . Furthern itself . Howeveractivity . In thisZm908 promoter was cloned and identified. pZm908 is pollen-specific, and the –126 to –102 region acts as a negative element. ZmDof30 could bind to the AAAG elements of p184 and negatively regulate pZm908 activity in tobacco leaves. ZmDof30 may function as a transcriptional repressor of Zm908 in pollen. These results provide a better understanding of the regulation of the Zm908 gene and the pollen-specific Zm908 promoter may have a potential for application in genetically engineering male sterility.The JY and JP conceived and designed the research. JP prepared materials, designed and performed the research, and wrote the original manuscript. XQ, XC, and NL participated in the preparing materials. JY revised thoroughly the manuscript and finalized the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


SET gene, and identify a novel multi-protein transcription complex, composed of MYC, SP1, RUNX1 and GATA2, which activates SET expression in AML. The role of MYC is crucial, since it increases the expression of the other three transcription factors of the complex, and supports their recruitment to the promoter of SET. These data shed light on a new regulatory mechanism in cancer, in addition to the already known PP2A-MYC and SET-PP2A. Besides, we show that there is a significant positive correlation between the expression of SET and MYC, RUNX1, and GATA2 in AML patients, which further endorses our results. Altogether, this study opens new directions for understanding the mechanisms that lead to SET overexpression, and demonstrates that MYC, SP1, RUNX1 and GATA2 are key transcriptional regulators of SET expression in AML.The SET (I2PP2A) oncoprotein is a potent inhibitor of protein phosphatase 2A (PP2A) that regulates many cell processes and important signaling pathways. Despite the importance of SET overexpression and its prognostic impact in both hematologic and solid tumors, little is known about the mechanisms involved in its transcriptional regulation. In this report, we define the minimal promoter region of the  Acute myeloid leukemia (AML) results from multiple genetic and epigenetic alterations in hematopoietic stem cells, and is characterized by a differentiation blockade of the myeloid hematopoietic progenitor cells accompanied by uncontrolled proliferation . AlthougSET gene  encodes a multifunctional protein that mediates essential functions in cell cycle control [PP2A, one of the main serine/threonine phosphatases in mammalian cells, is a tumor suppressor which regulates several essential functions and counteracts most of the kinase-driven intracellular signaling pathways . Our gro control , apoptos control –13, diff control , DNA rep control , DNA rep control , transcr control –20, as w control , 22. SET control , and soo control . SET bin control , 24–26.  control , 27. In  control . In divi control . The nuc control , and by  control , 31, 32. control –35.SET in order to investigate the mechanisms that lead to SET overexpression in AML. We determine its minimal functional promoter region, and demonstrate that MYC, SP1, RUNX1 and GATA2 form a multi-protein transcriptional complex that is involved in the transcriptional activation of SET in AML.Nevertheless, despite the importance of SET, and the prognostic impact of SET overexpression in both solid and hematologic tumors , 36–39, In vivo studies with mouse xenografts injected subcutaneously with SET shRNA-infected HL-60 cells produced tumors that grew at a slower rate  for putative binding sites for transcription factors (TFs). This analysis revealed that RUNX1, GATA2, MYC and SP1, four TFs with essential roles in hematopoiesis [SET. The SET distal promoter region (-932/-699bp) contains DNA motifs for RUNX1 and GATA2, and the proximal promoter region (-318bp/TSS) for SP1 and MYC   and re-ChIP assays confirmed the binding of MYC, together with SP1, RUNX1 and GATA2 to the -318/-144bp region of ) Figure . IntriguSET transcription, we silenced the expression of these two TFs with siRNAs. Luciferase assays showed a significant decrease in SET promoter activity, along with reduced SET expression at mRNA and protein levels  decreased markedly after treatment with 10058F4, mirroring the results obtained with the siRNA of MYC; furthermore, the PP2A function was re-established in HL-60 and HEL cells , and mRNA and protein expression , suggesting that RUNX1 and GATA2 co-operation is necessary to activate SET. These data, together with the ChIP results, suggest the presence of a transcription complex that comprises at least MYC, SP1, RUNX1 and GATA2, which binds to the SET promoter and enhances its expression, along with the expression of each members of the aforementioned complex that may be the cause of PP2A inactivation in AML.Since the ChIP and re-ChIP assays showed that RUNX1 and GATA2 bind to a region of the s Figure . Next, wn Figure . Moreoven Figure . Single n Figure , signifitivation . No signSET promoter with maximum activity contains MYC binding sites, and these TFs interact among them, we studied whether depletion or inhibition of MYC could affect the formation and localization of the transcriptional complex of MYC, SP1, RUNX1 and GATA2. Co-IP and ChIP experiments in cells transfected with a siRNA for MYC show that the decrease of MYC, and consequentially reduction of RUNX1, GATA2 and SP1, did not totally prevent the formation of the multi-protein complex in the cells  or GATA2 , though the strongest significance was obtained when comparing SET and MYC expression .To evaluate the clinical relevance of our results, we analyzed the data from a series of patients with reported , 52. We me Atlas . There w; Figure . There wSET in both solid and hematologic tumors, little is known about the mechanisms involved in the transcriptional regulation of this oncogene. Here, we have studied the promoter region of SET in order to investigate the mechanisms that lead to SET overexpression in AML. We have characterized for the first time a minimal promoter region in the human SET gene, and we have demonstrated that MYC, SP1, RUNX1 and GATA2 form a multi-protein transcriptional complex that is involved in the transcriptional activation of this gene.Despite the importance of in vivo model should be used to address the role of SET in leukemogenesis, these results are consistent with published data in chronic myeloid leukemia [SET deregulation may play an important role in the pathogenesis of leukemia in AML patients.In this report, we have first confirmed that SET is an important regulator of proliferation and apoptosis in AML. Our experiments with shRNA and siRNAs showed the function of SET as an inducer of cell proliferation (by activating AKT and ERK pathways), and as an inhibitor of apoptosis in AML cells, through its binding and inhibition of PP2A. Although other leukemia , 54 and leukemia , 39, 55,SET overexpression is associated with poor prognosis in AML and other malignancies [SET expression, little is known about the causes of its overexpression in cancer. This prompted us to analyze the transcriptional regulation of SET. Here, we have defined the SET minimal promoter region (-318bp before the TSS), and found that this region is bound and activated by MYC and SP1, together with RUNX1 and GATA2. In silico analysis found no DNA motifs for RUNX1 and GATA2 in this region of SET promoter, and we hypothesized and confirmed that these TFs could form a complex with MYC and SP1. Furthermore, our results indicate that MYC is interacting with and recruiting SP1, RUNX1, and GATA2 on the chromatin; nevertheless, we cannot say whether they bind to the DNA directly or through MYC. Moreover, our results show that the depletion of any of these four TFs results in a significant reduction of SET expression. Previous separate studies have reported collaboration either between SP1 and MYC [SET transcription in AML cells. Additionally, our results prompt us to speculate about the presence of an intricate network of regulations among these TFs, in which MYC activates the expression of SP1, RUNX1 and GATA2; RUNX1 and GATA2 increase the expression of MYC, and finally, SP1 increases the expression of GATA2. Likely, the expression of RUNX1 and GATA2 is affected by other proteins, or by a general lessening of cell proliferation, and not exclusively by MYC. Still, our data show that MYC is crucial for the recruitment of RUNX1, GATA2 and SP1 to the proximal promoter of SET. MYC is a well-known target of PP2A, since its protein stability is reduced by PP2A-dependent dephosphorylation of Serine 62 [SET transcription, and possibly CIP2A and SETBP1, defining a new layer of regulation that exists in the connection PP2A-MYC and SET-PP2A in cancer.We and others have previously reported that gnancies , 8, 37.  and MYC , 49, 50  and MYC , 57, or  and MYC . Here, werine 62 , 60. Conerine 62 . Our stuSET expression associates with MYC, RUNX1 and GATA2 expression in AML patients, corroborating our data and highlighting their clinical importance. However, the expression of SP1 does not correlate with SET expression. The reason for this finding remains unclear. It has been reported that SP1 plays an important role in regulating cell proliferation by modulating the expression of several cell cycle regulatory proteins through specific sequences in G/C-rich promoter regions [SET promoter. It can be hypothesized that SP1 acts as a housekeeping factor in AML cells, being constitutively present, and exerting different functions depending on the partners available to form multi-TF complexes. Supporting this hypothesis, the variability of SP1 expression in the data series of The Cancer Genome Atlas that we have analyzed is quite low (CV= 22.2 %) compared to that of MYC (66.9%), GATA2 (67.5%) and RUNX1 (42.3%).Finally, correlation analyses show that  regions , and thi regions , such asSET transcriptional regulation is controlled by at least four TFs. Interestingly, these are essential TFs that control different stages of hematopoiesis [SET, which would trigger PP2A inactivation in AML, contributing to the leukemogenic phenotype. In this regard, treatments combining inhibitors for MYC, GATA2 and/or RUNX1 could be more effective and could minimize the development of resistance by targeting two or more proteins in the same complex. Further preclinical investigation of these possible combinatorial therapies in AML is still needed.In summary, we have found that opoiesis –45 and aopoiesis , 64–66. -/-γc-/- mice were bred in house and treated according to the guidelines established by the University of Navarra Animal Experimentation Ethics Committee (No.:138-11). Stably transfected SET shRNA or control shRNA AML cells were harvested and re-suspended at 2 × 107 cells per ml in sterile phosphate-buffered saline. Two groups  of 6 week-old female Rag2-/-γc-/- mice were subcutaneously injected at the shoulder with 0.2 ml of the cell suspensions, one group for SET shRNA expressing cells and another for the control shRNA cells. Tumor growth was first measured 4 days after injection and then every day until the end of the experiment. Tumor volume (V) was monitored by measuring the length (L) and width (W) with calipers and calculated according to the formula (LxW2) x 0.5. After 10 days, tumor-bearing mice and controls were sacrificed and the tumors were excised and measured. Animals received a standard diet and water ad libitum at the Animal Core Facilities of the Center for Applied Medical Research (University of Navarra). All efforts were made to minimize the suffering of the animals.Rag2http://www-bimas.cit.nih.gov/molbio/proscan/), and NNPP (http://www.fruitfly.org/sequence/human-datasets.html) softwares. Then, the analysis was performed with MotifScanner program [For prediction of SET promoter and putative TF binding sites within the 2000 bp upstream the TSS of the SET transcript (NM_00320X), we used Promoter 2.0 , ElDorad program . The pro program  and the  program  and Tran program  database6 HL-60 or HEL cells with 5 μg of the promoter-luciferase construct and 1 μg of pRL-SV40. For the TF silencing experiments, the corresponding siRNAs for SP1, MYC, RUNX1, GATA2 or control were added to the electroporation mix. Firefly luciferase activity was normalized to Renilla luciferase signals to adjust the variability in transfection efficiencies. To study the effect of RUNX1 and GATA2 on the SET promoter, 100.000 HEK293t cells were transfected using the phosphate calcium method with 100 ng of pGL3-construct and 10 ng of pRL-SV40, together with 445 ng each of RUNX1 and GATA2 expression plasmids or 890 ng of empty vector.Promoter-luciferase reporter constructs were obtained by amplifying several regions of the SET promoter from BAC-RP11-216B9 . The amplicons were digested and cloned into NheI–XhoI sites in the promoterless luciferase reporter vector (pGL3-Basic) . E-box mutants were obtained by triple ligation by using the primers listed in the Chromatin from crosslinked HL-60 and HEL cells was sonicated, pre-cleared and incubated overnight with 3 mg of the corresponding antibody in RIPA buffer, and precipitated with protein G/A-Sepharose. For the re-ChIP assay, three fourth of the sample were incubated at 37°C for 30min in 10mM DTT. Supernatants were then incubated with the second antibody overnight, and protein G/A-Sepharose beads were added for two hour at 4°C the following day. The DNA-protein-antibody complexes were then washed three times with RIPA, three times with RIPA and NaCl, twice with Litium Buffer, and twice with 1X TE. Cross-linkage of the co-precipitated DNA–protein complexes was reversed and DNA was used as a template for quantitative Real Time PCR. Primers are listed in the Cell pellets were lysed in 3 ml of co-IP buffer . After centrifugation, protein samples were quantified by the BCA method. Overnight pre-clearing with non-specific IgG plus protein A/G beads was performed; then 1 mg protein was incubated with 5 μg of antibody crosslinked to protein A/G-Dynabeads following manufacturer's instructions (Invitrogen). The immunocomplexes were extensively washed with co-IP buffer and subsequently eluted with 50 μL of 0.1M Citrate pH 2.5 and boiled at 100°C in Laemmli buffer (2X) for Western blot analysis. IP control samples were incubated with irrelevant crosslinked IgG.For protein expression studies, 30 μg total protein lysates were separated in a 10-12% SDS-PAGE gradient gel and transferred onto a PVDF membrane. Overnight incubation at 4°C with primary antibodies was performed followed by the incubation with the appropriate secondary antibodies conjugated to horseradish peroxidase for 45′ at RT. The protein levels were detected by chemiluminescence . The list of the primary antibodies used is described in the PP2A activity was determined using the PP2A IP phosphatase assay kit . Briefly, protein lysates were prepared in TBS 1X, 1% Triton and phosphatase inhibitors tablets (Roche). Fifty micrograms of protein were immunoprecipitated with 2 μg of PP2A antibody and 25 μL of protein-A-agarose beads for 2 hours at 4°C. After extensive washing with TBS first and with Ser/Thr assay buffer last, the beads were then used in the phosphatase reaction for measuring dephosphorylation of the phosphopeptide (K-R-pT-I-R-R) with malachite green phosphate detection solution, following the manufacturer's instructions. The level of free phosphate is then normalized to total amount of immunoprecipitated PP2Ac, assessed by densitometry analysis of the western blots.t-test, Mann-Whitney U test, Oneway or Twoway-ANOVA and Bonferroni test, as appropriate. To evaluate the correlation analysis between SET and TFs in AML patients, Spearman's rank correlation coefficient was used, because the values did not fit the normal distribution. For contingency studies on the distribution of frequencies of high or low expression of SET and GATA2 in AML patients, the Chi square test was applied. Statistical analysis was performed using the SPSS 17.0 statistical package  and GraphPad software . A P-value of 0.05 was considered to be significant (*), 0.01 or 0.001 very significant (** or ***).Statistical significance of differences between means was estimated using Student's


Rhodobacter capsulatus photosystem is CrtJ, which functions as an aerobic repressor of photosystem genes. Recently, we reported that a vitamin B12 binding antirepressor of CrtJ called AerR is required for anaerobic expression of the photosystem. However, the mechanism whereby AerR regulates CrtJ activity is unclear. In this study, we used a combination of next-generation sequencing and biochemical methods to globally identify genes under control of CrtJ and the role of AerR in controlling this regulation. Our results indicate that CrtJ has a much larger regulon than previously known, with a surprising regulatory function under both aerobic and anaerobic photosynthetic growth conditions. A combination of in vivo chromatin immunoprecipitation-DNA sequencing (ChIP-seq) and ChIP-seq and exonuclease digestion (ChIP-exo) studies and in vitro biochemical studies demonstrate that AerR forms a 1:2 complex with CrtJ (AerR-CrtJ2) and that this complex binds to many promoters under photosynthetic conditions. The results of in vitro and in vivo DNA binding studies indicate that AerR-CrtJ2 anaerobically forms an extended interaction with the bacteriochlorophyll bchC promoter to relieve repression by CrtJ. This is contrasted by aerobic growth conditions where CrtJ alone functions as an aerobic repressor of bchC expression. These results indicate that the DNA binding activity of CrtJ is modified by interacting with AerR in a redox-regulated manner and that this interaction alters CrtJ’s function.Purple nonsulfur bacteria adapt their physiology to a wide variety of environmental conditions often through the control of transcription. One of the main transcription factors involved in controlling expression of the  12  as a blue-light-absorbing chromophore have been described. In this study, we analyzed the mechanism by which the vitamin B12 binding photoreceptor AerR controls the DNA binding activity of the photosystem regulator CrtJ. This study shows that a direct interaction between the vitamin B12 binding photoreceptor AerR with CrtJ modulates CrtJ binding to DNA and importantly, the regulatory outcome of gene expression, as shown here with photosystem promoters.Photoreceptors control a wide range of physiology often by regulating downstream gene expression in response to light absorption via a bound chromophore. Different photoreceptors are known to utilize a number of different compounds for light absorption, including the use of such compounds as flavins, linearized tetrapyrroles (bilins), and carotenoids. Recently, a novel class of photoreceptors that use vitamin B Rhodobacter capsulatus is capable of growth utilizing aerobic or anaerobic respiration, fermentation, or anoxygenic photosynthesis , crt (carotenoid), and puc (light-harvesting complex II)  . CrtJ isplex II) . CrtJ isplex II) , 11. Thein vitro using the bchC promoter that contains two closely linked copies of a recognition palindrome sequence TGTN12ACA. One palindrome spans the −35 promoter recognition sequence, and the other palindrome spans the −10 promoter recognition sequences  via oxidation/reduction of redox-active cysteines. For example, cysteine 420 (C420) located in the DNA binding domain has been shown to form a disulfide bond with C249 , chromatin immunoprecipitation-DNA sequencing (ChIP-seq), and ChIP-exo to dissect the in vivo roles of CrtJ and AerR. Our results indicate that the number and variety of CrtJ target promoters are much more extensive than previously thought and that CrtJ regulates gene expression under both aerobic and anaerobic photosynthetic growth conditions. We further show that AerR has a more nuanced role than previously appreciated, as its main role is to function as a switch that alters CrtJ binding at target sequences to relieve repressor activity.Even though current models for redox and light regulation of CrtJ explain both the aerobic repressor role of CrtJ and the light-regulated antirepressor role of AerR, they are based primarily on in vivo  shows that there are a large number of genes that exhibit reproducible changes in expression with a P value of ≤0.01  and aerobic repression of a phosphotransferase system (PTS) fructose-specific transporter (rcc02541 and rcc02543) and an aldehyde dehydrogenase (rcc02015).Analysis of differentially expressed genes (DEGs)  that codes for an anaerobic FeS assembly protein. The bacteriochlorophyll a biosynthetic pathway codes for several enzymes such as dark protochlorphyllide reductase and chlorophyllide reductase that contain essential 4Fe4S centers, so anaerobic activation of sufB expression may be a consequence of the increased need to assemble 4Fe4S centers for these enzymes (12 binding methionine synthase (rcc03352) which is notable, as an early step in the bacteriochlorophyll biosynthetic pathway, Mg-protoporphyrin IX methyltransferase, utilizes S-adenosylmethionine as a cofactor , flgA coding for a flagellar basal body P-ring assembly protein (rcc03514), and a cheW homolog (rcc01356) are also anaerobically repressed by CrtJ. Other notable anaerobically regulated genes include three transcription factors  and an RNA polymerase sigma-32 (rcc02811) each of which is anaerobically repressed by CrtJ. There are also three diguanylate cyclase/phosphodiesterases  that are photosynthetically activated by CrtJ -induced plasmid gene-encoded promoter and a 3×V5-CrtJ epitope that was recombined into the native chromosomal location with expression driven by the native CrtJ promoter. Functional complementation of these constructs, as well as analysis of CrtJ expression levels, is provided in model-based analysis of ChIP-Seq program (MACS) in at least three of five independent biological replicates and also present in both the 3×FLAG-CrtJ and 3×V5-CrtJ data sets.We analyzed the location of CrtJ binding to the genome crtJ as assayed by RNA-seq  that is 8 nucleotides upstream of the upper TGTN12ACA palindrome that spans the −35 promoter region . However, the lower strand right-hand borders are distinctly different between aerobically and photosynthetically grown cells. In aerobically grown cells, there is a prominent right border located between the upper and lower TGTN12ACA palindromes and a second slightly less prominent right border 8 nucleotides downstream of the lower TGTN12ACA palindrome   . Howeverctively) . This recrtJ is colocalized in the genome with its coregulator AerR   . It has  of CrtJ , 21. In n strain , 37. Aerin vitro . Becausenditions  and S2. nditions . A previomophore , so it i2).We next addressed the nature of the interaction of AerR with CrtJ using multiangle light scattering (MALS), which can accurately determine the molecular weight of an AerR-CrtJ complex. This analysis  showed t2 complex. For this analysis, we used a 325-bp bchC DNA probe that contained the two tandem CrtJ binding sites in the bchC promoter region as well as 137 bp of the bchC coding region. These assays were performed under oxidizing condition or reducing conditions (10 mM dithiothreitol [DTT]). A binding isotherm of CrtJ2 and of AerR-CrtJ2 binding to the DNA probe shows that CrtJ2 had higher affinity to the bchC promoter under oxidizing conditions than under reducing conditions, which confirms prior DNA binding studies (2 complex exhibited significantly higher binding affinity (~6-fold) than CrtJ alone under both oxidizing and reducing conditions.We next investigated the role of AerR in regulating the DNA binding activity of CrtJ by performing gel mobility shift assays with CrtJ alone as well as with the AerR-CrtJ studies ; Fig. S2 studies , 16. Int10.1128/mBio.00261-17.3FIG S1 2 and AerR-CrtJ2 binding to the bchC promoter. Download FIG S1, PDF file, 0.2 MB.Results of DNase I footprint analysis of CrtJCopyright © 2017 Fang and Bauer.2017Fang and BauerCreative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.00261-17.4FIG S2 bchC promoter region under both oxidizing and reducing conditions. Download FIG S2, PDF file, 0.5 MB.Results of gel mobility shift assays of CrtJ and AerR binding to the Copyright © 2017 Fang and Bauer.2017Fang and BauerCreative Commons Attribution 4.0 International license.This content is distributed under the terms of the 2 complex to the bchC promoter by undertaking DNase I footprint protection studies in the presence of CrtJ2 and in the presence of AerR-CrtJ2. The results of this in vitro analysis  of the upper recognition sequence became hypersensitive to DNase I digestion. Furthermore, there is additional downstream protection and several hypersensitive sites extending ~100 bp within the bchC coding region.We also addressed the nature of binding of the AerR-CrtJanalysis  show thain vitro DNase I protection patterns with the in vivo ChIP-exo results indicates that the CrtJ2 only footprint results correlates well with the aerobic ChIP-exo protection pattern, which shows borders flanking the two CrtJ recognition palindromes  promoters under aerobic conditions, which could also be found in our ChIP-seq result in R. capsulatus. The authors found that PpsR showed strong binding only under aerobic conditions, but not under anaerobic fermentative growth conditions. In our study, we found that CrtJ bound to the ccoN promoter under both aerobic and photosynthetic growth conditions. Bruscella et al. (− (RegA homolog) strain, further disruption of PpsR would lead to differential expression of MetH and MetF homologs, but not in the strain with a PpsR disruption only. In our ChIP-seq study, we could clearly identify a peak for the metH1 promoter, but no change in gene expression was seen, which suggests that RegA could be playing a role in this regulation. Imam et al.  function as coregulators under this condition.Clearly, our study indicates that CrtJ binding to the bchC promoter region under anaerobic photosynthetic repressing conditions than under aerobic activating conditions. These in vivo ChIP-exo results are mimicked by in vitro footprint assays which show that the AerR-CrtJ2 complex exhibits a similar highly extended footprint pattern relative to that observed with CrtJ alone. Gel mobility shift assays with DNA segments that extended into the bchC coding region further show that the AerR-CrtJ2 complex is able to bind to the bchC promoter region in vitro and that AerR actually increases the affinity of CrtJ to this region. Taken together, we can propose a revised model for CrtJ regulation of photosystem gene expression in which oxidized CrtJ is capable of binding to the bchC promoter alone under aerobic conditions leading to repression of expression   in R. sphaeroides has little effect on synthesis of the photosystem  that exhibits significant similarity with the vitamin B12 binding domain of AerR/PpaA (44–blue light using FAD {flavin adenine dinucleotide} domain)  . Specifipression , 42, 43.tosystem , 37. Thipsulatus , 37. WhaerR/PpaA , 44–46.  domain)  that reg domain) . Deletiopsulatus . This supsulatus . ConsequR. capsulatus. On the basis of results from RNA-seq and CrtJ ChIP-seq, we have expanded the CrtJ regulon. ChIP-exo analyses also show that CrtJ exhibits altered binding to the bchC promoter under aerobic and anaerobic photosynthetic growth conditions. Finally, ChIP-seq and DNase footprint assays show that AerR colocalized with CrtJ to the bchC promoter in vivo and alter its binding on the promoter in vitro. Elucidating the molecular mechanism of the AerR-CrtJ system provides not only a new mechanism of regulating bacterial physiology but also additional understanding of the light-sensing tools used to regulate transcription in this organism and in other systems.In conclusion, we have applied a combination of next-generation sequencing and biochemical method to study CrtJ regulation in For more-detailed methodologies, see 10.1128/mBio.00261-17.1TEXT S1 TEXT S1, PDF file, 9.2 MB.Supplemental Materials and Methods. Download Copyright © 2017 Fang and Bauer.2017Fang and BauerCreative Commons Attribution 4.0 International license.This content is distributed under the terms of the Rhodobacter capsulatus SB1003 was used as the wild-type parental strain with the ΔaerR and ΔcrtJ single deletion strains and a ΔaerR ΔcrtJ double deletion strain derived from SB1003 as described previously (Escherichia coli BL21(DE3)   was used7-1 λpir  used to  at 34°C .Rhodobacter capsulatus SB1003 as previously reported  were used in the experiments. In one set of experiments, FLAG-tagged CrtJ was expressed from the pSRKGm plasmid in three independent biological replicates by the addition of 100 μM IPTG added 30 min before formaldehyde cross-linking. In a second set of experiments, V5-tagged CrtJ was recombined into the chromosome at its native location from which it was expressed from its native promoter. Chromatin immunoprecipitation-DNA sequencing (ChIP-seq) analysis of AerR involved the use of a C-terminal 3×FLAG-tagged AerR construct expressed from pSRKGm with no IPTG induction. ChIP-seq and exonuclease digestion (ChIP-exo) procedures were performed using FLAG-tagged CrtJ as previously reported . DetaileRhodobacter capsulatus strains were grown to early exponential phase  before collection. Total RNA was extracted using Isolate II RNA minikit (Bioline) followed by Turbo DNase (Ambion) treatment. Library construction and transcriptome sequencing (RNA-seq) were performed by the University of Wisconsin Madison Biotechnology Center DNA Sequencing Facility using the TruSeq RNA sample preparation kit (Illumina) according to the manufacturer’s protocol.E. coli BL21(DE3) strains bearing plasmid pSUMO-AerR or pSUMO-CrtJ were used to overexpress SUMO-tagged AerR or CrtJ according to previous studies  (Wyatt Technology Corp). The light scattering results were analyzed using ASTRA 6 (Wyatt Technology) with bovine serum albumin (BSA) for calibration.SRP082587.Raw sequence read files were deposited in the Sequence Read Archive (SRA) with the GenBank accession number


These molecules also provide an alternative for oncogenes deemed “undruggable” at the protein level, where the absence of solvent-accessible pockets or structural crevices prevent the formation of protein-inhibitor ligands; nevertheless, the genome-wide effect of pyrrole-imidazole polyamide binding remain largely unclear to-date. Here we propose a next-generation sequencing-based workflow combined with whole genome expression arrays to address such issue using a candidate anti-cancer alkylating agent, KR12, against codon 12 mutant KRAS. Biotinylating KR12 enables the means to identify its genome-wide effects in living cells and possible biological implications via a coupled workflow of enrichment-based sequencing and expression microarrays. The subsequent computational pathway and expression analyses allow the identification of its genomic binding sites, as well as a route to explore a polyamide’s possible genome-wide effects. Among the 3,343 KR12 binding sites identified in the human LS180 colorectal cancer genome, the reduction of KR12-bound gene expressions was also observed. Additionally, the coupled microarray-sequencing analysis also revealed some insights about the effect of local chromatin structure on pyrrole-imidazole polyamide, which had not been fully understood to-date. A comparative analysis with KR12 in a different human colorectal cancer genome SW480 also showed agreeable agreements of KR12 binding affecting gene expressions. Combination of these analyses thus suggested the possibility of applying this approach to other pyrrole-imidazole polyamides to reveal further biological details about the effect of polyamide binding in a genome.Pyrrole-imidazole polyamides are versatile DNA minor groove binders and attractive therapeutic options against oncological targets, especially upon functionalization with an alkylating agent such as  KRAS 2+ 1071.95; found 1072.00, [M+3H]3+ 714.94; found 715.25.The backbone chain  and Dulbecco’s Modified Eagle’s Medium , respectively. Media were additionally supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin (Gibco). For sequencing experiments, cultures were prepared in replicates at 6 × 105 cells/dish prior to KR12 administration. For WST viability assays, cells were seeded at a final density of 103 cells per sample in a 96-well plate for overnight attachment prior to treatment with 0.1% DMSO, 300 and 1000 nM KR12 as well as non-biotinylated variant (KR12 N/B) in 0.1% DMSO for 48 h to assess compound toxicity. After compound administration, WST-8 reagent (Dojindo) was added to each sample for incubation at 37°C for 2 h prior to absorbance measurement at 450 nm on a MTP-310 photospectrometer (Corona).Colorectal adenocarcinoma LS180 and SW480 cells were cultured and maintained at 37°C, 5% CO3 and 3 mM biotin at 65°C for 4 h under agitation.LS180 and SW480 cultures were treated with 500 nM KR12 in 0.05% DMSO for 6 h before washing, detachment in ice-cold phosphate-buffered saline and collection by centrifugation. Pellets were resuspended in a lysis buffer of 5 mM PIPES, 0.5% NP40 and 1% Complete Protease Inhibitor, incubated on ice for 10 m to extract nuclei by ultracentrifugation. After resuspension in nuclease lysis buffer of 50 mM Tris-HCl, 10 mM EDTA, 1% SDS and 1% Complete Protease Inhibitor at pH 8.1, samples were again incubated on ice for 10 m prior to overnight storage at -80°C. Since KR12 formed alkylation adducts with DNA, crosslinking was not performed during the DNA enrichment process. Genomic DNA was fragmented by sonication on a Covaris M220 for two 10 m runs of 200 cycles  before precipitation in a solution of 300 mM NaOAc in 70% ethanol  at 30°C and isolation of genomic DNA by Micro-Vac evaporation . Samples were resuspended in nuclease-free water (Millipore) for 15 m before collecting the supernatant for enrichment with Dynabeads MyOne streptavidin C1 beads (Invitrogen) over 15 m. Beads were washed twice in a buffer of 20 mM Tris-HCl, 0.1% SDS, 1.1% Triton X-100, 2 mM EDTA and 500 mM NaCl at pH 8.0, and five additional times in detergent-free buffer  prior to elution with 2% SDS, 0.1 mM NaHCOEnriched DNA was purified by RNase A  for 10 m at 37°C, followed by proteinase K  treatment at 55°C for 40 m. Samples were mixed with glycogen (Ambion), resuspended prior to phenol extraction. The supernatant was retained, from the phenol layer, after centrifugation in 1:1 TE:NaCl and stored as aliquots in absolute ethanol at -20°C prior to precipitation by centrifugation . Samples were evaporated in open atmosphere, reconstituted in 20 μL TE and quantified by Qubit 2.0 Fluorometer (ThermoFisher). Samples were end-repaired by 30 m incubation in the supplied end-repair buffer and enzyme (2 μL) in Ion XpressTM Plus Fragment Library Preparation Kit; the reaction mixtures were suspended with AMPure XP Reagent Beads (Agencourt), washed in ethanol (70%) and eluted in Low TE buffer before adapter ligation for 30 m and again purified with AMPure beads. Nick repair and amplification with Platinum PCR SuperMix High Fidelity mix (ThermoFisher) proceeded at 72°C for nick repair (20 m), 95°C for denaturation (5 m), 18 cycles of 97°C denaturation, annealing at 60°C and 70°C extension . Library preparation proceeded with 1.68 ng of enriched DNA with Ion Plus Fragment Library Kit (ThermoFisher) and AMPure XP Reagent Beads (Agencourt) per manufacturer’s recommendations. Library sizes were validated on a Bioanalyzer 2100 (Agilent) and quantified using the Ion Library Quantitation Kit (ThermoFisher); after emulsion PCR of the prepared sample on Ion OneTouch 2 (ThermoFisher), a final quantity of 0.8 fmol DNA was used for sequencing on an Ion Proton sequencer (ThermoFisher). Data acquisition and alignment to hg19 genome were performed using Torrent Suite 5.0.4.Ambiguous reads, i.e. mapping quality = 0, were removed from the BAM files before summarizing base calls of aligned reads (mpileup) with samtools, with BAQs recalculated on the fly and genotype likelihood output in uncompressed BCF format. Sequencing data (treatment and control) were then converted to BED format before differential calling by diffReps with hg19 as the genome size, method of chi-square, fragment size of 0 and no annotations; window sizes varied from run to run within the range of 400–1200 bp. Sequences were regenerated over such diffReps-assigned regions from the source FASTA file with indels ignored , by bedtools, ignoring strand information. Matches to the KR12 motif (“TGWWGGCGW” on the (+) strand), in both forward and reverse complementary orientations, were identified by searching against the reconstructed sequence files, followed by a back-transformation to hg19 coordinates. Positions were again inspected against pileup results for indels; sequences from such sites were reconstructed by variant calling with bcftools over given regions by multi-allelic calling, with compressed output containing only variant sites. Indel information was used to reconstruct a small region for motif searches and translated to hg19 coordinates. Transcript positions and gene symbols from hg19 RefFlat (last accessed Feb 2015) were used to generate an annotation including transcript locations and promoter regions, defined as 1000 bp region upstream of transcription start site (TSS), based on RefFlat strand information. KR12 candidates were subsequently annotated to produce a list of all positions of candidate KR12 sites from sequencing results annotated by their respective gene or promoter symbols.p-values were adjusted for multiple comparisons by Benjamini-Hochberg corrections. Statistical significance was assessed based on the adjusted p-value at the 99.9th percentile against a pre-defined α-level of 0.05; sites with a p-value of 0.05 < p < 0.055 were considered marginally significant and likewise annotated. Fold enrichment was calculated based on the log2 ratio of the maximum coverage within a given window in the pulldown and input samples. Results from multiple runs of varying window sizes for diffReps were compiled to produce the final output of 3,343 KR12 binding sites.For statistical validation, a list of nonbinding regions was generated by subtracting candidate KR12 sites from the list of hypothetical sites via bedtools (removing entire feature if any overlap was present). Randomizations were performed to generate null foreground and background positions in R . Per-base coverage was calculated over candidate regions as well as paired null foreground and background regions from sequencing data, before unity normalization of each region. A differential distribution was determined by subtracting per-base coverage between the input and pulldown groups; for the null differential distributions, shuffling was performed to pair randomly the null foreground with a background to increase randomness. For each of the candidate differential distribution, two-sample Kolmogorov-Smirnov test was performed for each of the null differential distribution under the hypothesis that there was no difference between the distributions; resultant 4 LS180 cells/well were treated with 500 nM KR12 for 6 h before RNA extraction with RNAeasy plus mini kit (Qiagen) and reverse transcription of 500 ng RNA to cDNA by the SuperScript VILO cDNA Synthesis System (Invitrogen) for experiment as previously described [RPS18: 23 cycles; KRAS, PIK3CA, GUSB: 28 cycles); 72°C, 5 m, ending with holding at 4°C. Primer sets used were as follows: KRAS, 5’-GGAGAGAGGCCTGCTGAA-3’ (sense) and 5’-TGACCTGCTGTGT CGAGAAT-3’ (antisense); RPS18, 5’-GAGGATGAGGTGGAACGTGT-3’ (sense) and 5’-TCTTCAGTCGCTCCAGGTCT-3’ (antisense); PIK3CA, 5’-AGTCGCCACCTACCACAGAG-3’ (sense) and 5’-GCTGACCCTCATGGCTGT-3’ (antisense); GUSB, 5’-GGTGGTTCATTGCTGCTGAC-3’ (sense) and 5’-TAGAACAGAGAGCGCCATTG-3’ (antisense).Cultures of 9.6 × 10escribed . Polymer4 cells per sample were plated in a 6-well microtiter plate for overnight attachment prior to treatment with 500 nM KR12 or 0.05% DMSO for 6 h. After RNA extraction with RNeasy Plus Mini Kit (Qiagen), samples at 100 ng were labeled with RNA Spike-In Kit and analyzed on SurePrint G3 Human GE 8x60K V2 microarrays per Agilent Technologies’ recommendations. Arrays with sample replicates  for each condition  were scanned on an Agilent SureScan microarray scanner, with differential expressions calculated by the LIMMA package [t-test under the null hypothesis that there was no difference between their sample means against a predefined α-level of 0.05. For the purpose of comparison, genes with promoter region (defined as 1000 bp upstream of transcription start site) binding were also annotated with corresponding mRNA expressions.LS180 and SW480 cultures at 9.6 × 10 package  , distance to the closest H3K27Ac feature was determined similarly.DHS, H3K27Ac and nucleosome structure information were obtained as described in the General Computational Tools Section. ChromHMM tracks from eight cell lines  were combined to identify common overlaps, and heterochromatin regions with ≥ 5/8 overlaps were retained to create a consensus heterochromatin track for comparison. LS180 H3K27Ac data were reprocessed in MACS 1.4.2 to generate a region file of signature peaks with default parameters and an effective genome size of 2.7 × 10α-level of 0.05, Bonferroni corrections against a reference list of Homo sapiens genes). A list of downregulated KR12-bound genes was selected by cross-referencing microarray expression results for negative log2FC values for interaction analysis by STRING 10.0 with the following parameters: highest confidence of 0.900, no additional nodes, custom limit of 0 interactors and all active prediction methods [Gene set enrichment analysis was performed with PANTHER 10.0  statistical overrepresentation test, using the list generated from sequencing results as well as genes belonging to the Ras pathway  und methods  using STKRAS for possible involvement to assess off-target effects of KR12.The KEGGREST package  in R wasseco-CBI moiety [The last eight bases of the KR12 motif (TGWWGGCGW on the (+) strand) were randomly scrambled for 100 permutations to check for genomic binding sites in hg19, with coding region annotations extracted from the Table Browser from UCSC. To simulate 3’ adenine alkylation by the conjugated I moiety  for the KRAS transcript (chr12: 25357722–25403865) was also extracted for comparison. Tantan [Annotations for lncRNA (colon lincRNA RNA-Seq Reads ) and miR. Tantan  was usedSequence read datasets for the KR12-enriched ("pulldown") and unenriched ("input") LS180 and SW480 genomes are available in the NCBI Sequence Read Archive (SRA) under BioProject PRJNA342228; expression microarray datasets are available at the NCBI Gene Expression Omnibus (NCBI GEO) database (accession number GSE86599).KR12 N/B) with biotin to create KR12  and those without ; this further suggested that KR12 did not have a direct role in influencing large-scale gene transcription. Furthermore, out of the 161 transcription factors, only 67 contained predicted KR12 binding sites, and only 12 contained sites not located in the introns  and a small fraction of coding regions Click here for additional data file.S2 Appendix(GZ)Click here for additional data file.S3 Appendix(PDF)Click here for additional data file.S4 Appendix(PDF)Click here for additional data file.S1 Fig(TIFF)Click here for additional data file.S2 Fig(SVG)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.