Saccharomyces cerevisiae, entry into meiosis depends on multiple signals converging upon IME1, the master transcriptional activator of meiosis. Here we studied the regulation of the cis-acting regulatory element Upstream Activation Signal (UAS)ru, which resides within the IME1 promoter. Guided by our previous data acquired using a powerful high-throughput screening system, here we provide evidence that UASru is regulated by multiple stimuli that trigger distinct signal transduction pathways as follows: (i) The glucose signal inhibited UASru activity through the cyclic AMP (cAMP/protein kinase A (PKA) pathway, targeting the transcription factors (TFs), Com2 and Sko1; (ii) high osmolarity activated UASru through the Hog1/mitogen-activated protein kinase (MAPK) pathway and its corresponding TF Sko1; (iii) elevated temperature increased the activity of UASru through the cell wall integrity pathway and the TFs Swi4/Mpk1 and Swi4/Mlp1; (iv) the nitrogen source repressed UASru activity through Sum1; and (v) the absence of a nitrogen source was detected and transmitted to UASru by the Kss1 and Fus3 MAPK pathways through their respective downstream TFs, Ste12/Tec1 and Ste12/Ste12 as well as by their regulators Dig1/2. These signaling events were specific to UASru; they did not affect the mating and filamentation response elements that are regulated by MAPK pathways. The complex regulation of UASru through all the known vegetative MAPK pathways is unique to S. cerevisiae and is specific for IME1, likely because it is the master regulator of gametogenesis.The choice between alternative developmental pathways is primarily controlled at the level of transcription. Induction of meiosis in budding yeasts in response to nutrient levels provides a system to investigate the molecular basis of cellular decision-making. In  Transcriptional regulation is the key mechanism that controls cell fate in response to internal and external stimuli. This is often achieved through a transcriptional regulatory cascade controlled by a master regulator Saccharomyces cerevisiae provides an experimental system for studying developmental decisions made by eukaryotic cells in response to nutrient deprivation. In the presence of any carbon and nitrogen sources cells vegetative growth is in oval shape. This organism prefers glucose as a carbon source and has evolved diverse regulatory mechanisms to ensure its survival in nature where the levels of glucose fluctuate widely. In response to nitrogen depletion, diploid S. cerevisiae has two alternative developmental pathways. The absence of glucose triggers cells to undergo meiosis and spore formation, which is referred to as sporulation  RNA in cells expressing the haploid MATa or MATα mating types but not in diploid MATa/α cells IRT1) inhibits IME1 expression in cis at the IME1 promoter by inducing the formation of a repressive chromatin structure comprising Set2 histone methyltransferase and the Set3 histone decarboxylase g types   but is nIME1 promoter is unusually long compared with other Saccharomyces upstream regulatory elements  and comprises at least 10 distinct positive- and negative-control elements (reviewed in IME1 promoter IME1 promoter by molecularly dissecting the upstream activation sequence (UASru), a glucose-responsive regulatory element that is essential for the robust transcription in the absence of glucose The 2.1 kb 7 cells/ml), harvesting, washing once with water, and re-suspending in SPM. The β-galactosidase activity was assayed as described 7 or 3×108 cells were harvested during exponential phase for β-galactosidase or ChIP assays, respectively.SD, minimal glucose, SLAD (synthetic glucose medium with glutamic acid as a nitrogen source), PSP2 , SPM, and SPO  media were prepared as reported 8 cells using the hot acidic phenol method RNA was extracted from 10UASru-his4-lacZ; ACT1∶5′-ATCACCGCTTTGGCTCCAT-3′ and 5′-CCAATCCAGACGGAGTACTTTCTT-3′.Primers: UASru: 5′CGTTGATGTCATCCGCTATT-3′and either 5′- GACCCAAGAAGCCACCATGA-3′ for the genomic sequence, or 5′-CATACCTCGACATCACATGCT-3′ for COM2, SWI4, TEC1, DIG1, and STE12 or to SKO1, HOG1, SUM1, MLP1, MPK1, respectively, and were inserted in place of their cognate endogenous genomic allele, and their expression was regulated by their natural promoters. Following IP, 100 ng genomic DNA was analyzed using qPCR for specific (IP), nonspecific , and IP without primary antibody (IP w/o Ab) according to the manufacturer’s instructions . Three primer sets were used for qPCR as follows: 1) UASru (UASru-his4-lacZ chimera). This construct, integrated into the genome, was used to examine the binding of the various TFs. This construt was also used to examine its expression level in response to various perturbations. This allowed us to conclude about binding to the specific tested element, rather than adjacent regions within IME1 promoter; 2) control TEL1 or POL1; and 3) positive control . The extent of enrichment was calculated as the ratio of specific (IP) to nonspecific (WCE) DNA, and the data were normalized to that of IP w/o Ab. Antibodies: Mouse anti Myc epitope (9E11) (Santa Cruz), 0.33 µg per sample; mouse anti-HA epitope (12CA5) (Roche), 0.8 µg per sample. Primers: ChIP assays were performed essentially as described IME1 open reading frame IME1 promoter, we deleted UASru from the genome. This decreased the levels of IME1 mRNA by approximately 2-fold throughout the meiotic pathway (IME1 is regulated by multiple UASs UASru is located between nucleotide positions −1198 to −1370 from the initiation codon of the  pathway . This efime1-lacZ reporter in which lacZ transcription is driven by the IME1 promoter. Cells deleted for UASru and grown in SA (acetate without glucose) and SPM (acetate without glucose or a nitrogen source) , consistent with the results of our previous study To determine whether this effect was caused by inhibition of transcription, increased mRNA turnover, or both, we deleted UASru from a chimeric  source)  expresse source)  in SD, lhis4-lacZ reporter . The rational for using heterologous reporters rather than observing an IME1 expression levels, was to overcome the presence of the additional elements in its promoter. These elements could mask our ability to observe response to specific signals. The activity of the ABC construct (the entire UASru) was as reported Because the UASru element is relatively large (173 base pairs (bp), it may contain multiple UAS, or URS (Upstream Repression Sequence) elements, or both, that are mutually inhibitory. To ensure that only carbon sources regulate UASru, we divided it into three segments, designated A, B, and C see . We geneHIS4 UAS in the his4-lacZ reporter (strain Y2029 compared with Y2028) resulted in 10.7, 11.2, and 22-fold reductions in reporter expression in SD, SA, and SPM media, respectively (data not shown). These results support the conclusion that UASru-C acts as a negative regulatory element. Further work is required to reveal why nitrogen depletion did not promote the expression of this reporter.The UAS activity of the C construct was relatively low compared with those of the others. Moreover, its activity was increased 6.5-times when cells were grown in SPM , suggestThe activity of UASru-BC was similar to that of UASru-ABC and UASru-AB. Because this construct was not significantly affected by the presence of a nitrogen source, we suggest that a sequence in region B, or one that was disrupted or removed from the B and C elements, masked detection of a response of the BC element to a nitrogen source.IME1 promoter. We normalized the expression of these reporters to a constitutively expressed RFP reporter. The data was transformed to Z-scores, and P-values on the basis of a normal distribution  that affect the activity of UASru We previously used the R-SGA approach GPR1 (G protein-coupled receptor), RAS2 , and CDC25 (Ras2 guanine nucleotide exchange factor) on the expression of a UASru-lacZ reporter construct. Deletion of GPR1 or the SH3 domain of CDC25 (required for its association with adenylate cyclase RAS2 had a similar effect but only in SD (The R-SGA screen identified nine genes that encode components of the cAMP/PKA pathway  [27]–. Becdetected . In contAB)-lacZ , suggestRLM1 did not detectably affect transcription of UASru-GFP. In contrast, deletion of SWI4 significantly reduced its expression , which differs from the SBF consensus site at one position. This suggests that the Swi4/Mlp1 or Swi4/Mpk1 complexes bind to and directly activate this element, which is consistent with the results of the qChIP assay demonstrating specific binding of Swi4, Mlp1, and Mpk1 to UASru . We concSUM1 deletion mutants cultured in SD but not in SPM . These results suggest that the effect of Tec1 in diploid cells grown in SD -regulated genes, inhibit their expression wn in SD . In cellid cells . These ri) Dig1 and 2 will not bind UASru, because they function only as positive regulators; and (ii) Ste12 will also function as a positive regulator. The results of qChIP assays confirmed the first prediction, because Dig1 was not detected bound to UASru (FUS1 promoter in a control experiment (data not shown). Deletion of STE12 caused a significant decrease in UASru activity in haploid cells  The effect of Dig1 is more prominent in haploids, consequently the level of Ste12 is increased and the effect of deleting STE12 is elevated; and (ii) in MATa/MATα diploids, the transcription of STE12 is repressed Tec1 binds to its target genes (FRE) as a heterodimer with Ste12 TEC1 deletion mutants. The results of qChIP analysis revealed that, independent of ploidy, deletion of TEC1 resulted in loss of Ste12 binding in SD but not in SA (i) In the presence of glucose, it binds UASru as a heterodimer with Tec1; and (ii) in the absence of glucose and the presence of acetate, it binds DNA as a homodimer. Nonetheless, our results cannot exclude the possibility that in SA it also binds as a heterodimer with Tec1.Ste12 binds DNA either as a homodimer or heterodimer with Tec1 ot in SA . These rThe activities of Ste12/Ste12 and Tec1/Ste12 are regulated by mating and filamentation signals, respectively, which are transmitted through MAPK cascades that share many components STE7 and kinase-dead (kd) alleles of either KSS1or FUS3 reduced the expression of UASru-lacZ  activate the FRE and/or PRE elements? To answer these questions, we determined the response of reporter genes regulated by UASru, FRE, or PRE elements in response to these signals.FRE-lacZ was induced in cells grown in SLAD, which contains a poor nitrogen source and glucose as the sole carbon source  and UASru-AB were significantly increased, while the B region had no detectable UAS activity. We found that the cAMP/PKA pathway transmits this signal to UASru  and 3 ani) In haploid cells Kss1 activates invasive growth in response to glucose limitation ii) the activity of UASru in haploid cells expressing a kss1 kd allele was decreased in cells grown in SD and increased in cells grown in SA (iii) Tec1 must bind UASru in cells grown in SD, because its deletion reduced binding of Ste12 (i) Deletion of TEC1 did not affect activity in cells grown in SD and caused only a minor inhibitory effect in cells grown in SA; (ii) UASru-AB does not carry a putative binding site for Tec1/Ste12 or Ste12/Ste12; and (iii) Fus3 and Ste12 are more potent activators than Kss1 or Tec1  Deletion of Hog1 pathway components, including HOG1, significantly affected the activity of UASru (ii) Hog1 binds to UASru depending on exposure to NaCl  and is required for the efficient IME1 expression The level of NDT80 and certain mid meiosis-specific genes IME2, suggesting a specific mechanism that inhibits Sum1 activity, which depends on the Set3 histone deacetylase complex UASru-C functions both as a URS and a UAS that are regulated by nitrogen. Repression is mediated by Sum1, which binds UASru-C under all growth conditions and represses UASru-C activity only in the presence of a nitrogen source . Sum1 ali) Nitrogen limitation in the presence of glucose activates the Kss1 MAPK pathway ii) UASru activity is reduced in cells expressing a kd allele of either Kss1 or Fus3; (iii) Tec1 and Ste12, which activate the transcription of FRE-regulated genes, bind to and activate UASru (iv) Ste12 binds UASru in the absence of TEC1; and (v) an imperfect binding sites for Tec1 and Ste12 is present in UASru-C  St UASru-C .MAPK pathways present in all eukaryotes transmit specific signals to diverse targets using shared components through an unknown mechanism. Here we show that UASru (meiosis), PRE (pheromone response), and FRE (filamentation response), share pathway components. UASru is activated in the absence of glucose but is not activated by either pheromones or nitrogen limitation in the presence of glucose , 6, 7, 8IME1 (or only UASru) and FLO11 (required for filamentation) carry responsive elements for both the MAPK and cAMP/PKA pathways (FLO11 transcription IME1) represent alternative developmental pathways. Thus, repression of UASru by PKA prevents activation by Kss1 and Tec1/Ste12, allowing UASru to specifically respond to the meiotic signal. However, we used a FRE-driven reporter gene that includes only the binding site for Tec1/Ste12, and, therefore, this mechanism does not apply to the lack of response of FRE to the meiotic signal.Therefore, our results raise additional questions regarding signaling specificity. The pathways  that regpathways , 3 but aTable S1List of plasmids.(DOCX)Click here for additional data file.Table S2List of strains.(DOCX)Click here for additional data file.Table S3List of oligonucleotides.(DOCX)Click here for additional data file.


Seven linker histone H1 variants are present in human somatic cells with distinct prevalence across cell types. Despite being key structural components of chromatin, it is not known whether the different variants have specific roles in the regulation of nuclear processes or are differentially distributed throughout the genome. Using variant-specific antibodies to H1 and hemagglutinin (HA)-tagged recombinant H1 variants expressed in breast cancer cells, we have investigated the distribution of six H1 variants in promoters and genome-wide. H1 is depleted at promoters depending on its transcriptional status and differs between variants. Notably, H1.2 is less abundant than other variants at the transcription start sites of inactive genes, and promoters enriched in H1.2 are different from those enriched in other variants and tend to be repressed. Additionally, H1.2 is enriched at chromosomal domains characterized by low guanine–cytosine (GC) content and is associated with lamina-associated domains. Meanwhile, other variants are associated with higher GC content, CpG islands and gene-rich domains. For instance, H1.0 and H1X are enriched at gene-rich chromosomes, whereas H1.2 is depleted. In short, histone H1 is not uniformly distributed along the genome and there are differences between variants, H1.2 being the one showing the most specific pattern and strongest correlation with low gene expression. Eukaryotic DNA is packaged into chromatin through its association with histone proteins. The fundamental repeat unit of chromatin is the nucleosome, which consists of 146 bp of DNA wrapped around an octamer of core histone proteins H2A, H2B, H3 and H4. Linker histone H1 sits at the base of the nucleosome near the entry and exit sites and is involved in the folding and stabilization of the 30-nm chromatin fiber, allowing a higher degree of DNA compaction . HistoneUnlike core histones, the H1 histone family is more evolutionary diverse and many organisms have multiple H1 variants or subtypes, making the study of these proteins more complex. In humans, the histone H1 family includes 11 different H1 variants with 7 somatic subtypes , three testis-specific variants  and one oocyte-specific variant (H1oo). Among the somatic histone H1 variants, H1.1 to H1.5 are expressed in a replication-dependent manner, whereas H1.0 and H1X are replication-independent. H1.2 to H1.5 and H1X are ubiquitously expressed, H1.1 is restricted to certain tissues, and H1.0 accumulates in terminally differentiated cells .It is still far from clear why there are so many H1 variants and great efforts have been made recently to elucidate whether they play specific roles or have redundant functions. Single or double H1 variant knock-out studies in mice did not identify any specific phenotype and this was attributed to the compensatory upregulation of other subtypes, favoring the view that there is redundancy between H1 variants . Despitein vivo. Initial biochemical and microscopy-based approaches suggested a nonuniform distribution of H1 in the cell nucleus and found differences between variants  combined with quantitative polymerase chain reaction (qPCR), tiling promoter arrays and high-resolution sequencing. We combined the use of specific antibodies for some variants and hemagglutinin (HA)-tagged recombinant H1 variants expressed in cell lines to study the genome-wide distribution of H1.0, H1.2 to H1.5 and also H1X, a more recently identified and distantly related H1 variant. H1.1 was omitted from our analysis, it being the only somatic H1 variant not present in many cell types, including the cells used here. We also compared H1 distribution with the nucleosome distribution in our T47D human breast cancer cell lines, by H3 immunoprecipitation. Our data support the view that all H1 variants occur across the genome, but also uncover specific features for H1.2, both at promoters and genome-wide. Interestingly, H1.2 enrichment correlates the most closely with gene repression, structural domains of chromatin such as lamina-associated domains (LADs) and regions of low GC content. Overall, the distribution of H1.2 along chromosomes differs from that of other variants including H1.0 and H1X, the two variants most structurally distant within the somatic H1 family. This work represents a comprehensive attempt to investigate for the first time the occurrence and relevance of the different histone H1 variants in the genome of human cancer cells, and provides valuable data to clarify our understanding of the functionalities and heterogeneity of H1.2 in RPMI 1640 medium, supplemented with 10% FBS, 2 mM l-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin, as described previously  and 1% penicillin/streptomycin. MCF7 cell line was grown at 37°C with 5% CO2 in Minimum Essential Medium (MEM) medium containing 10% FBS, 1% penicillin/streptomycin, 1% nonessential amino acids, 1% sodium pyruvate and 1% glutamine.Breast cancer T47D-MTVL cells (carrying one stably integrated copy of luciferase reporter gene driven by the MMTV promoter), or derivative cells stably expressing HA-tagged H1 variants (H1-HA), were grown at 37°C with 5% COeviously . HeLa ceFor Phorbol myristate acetate (PMA) experiments, serum-containing Roswell Park Memorial Institute (RPMI) 1640 media was replaced by serum-free media. After 24 h under serum-free conditions, cells were treated with PMA (100 nM) for the indicated time at 37°C.Generation of T47D-MTVL stably expressing HA-Tagged H1 variants was achieved as described previously . BrieflyChIP assays were performed as described previously . BrieflyThe following antibodies were used in this study: anti-H1.2 (Abcam 4086), anti-H1X (Abcam 31972), anit-H3 (Abcam 1791) and anti-HA tag (Abcam 9110).Real-time PCR was performed on ChIP and input DNA using EXPRESS SYBR GreenER qPCR SuperMix Universal (Invitrogen) and specific oligonucleotides in a Roche 480 Lightcycler. ChIP values were corrected by the correspondent input chromatin sample. All oligonucleotide sequences used for the amplifications are available on request.At least 10 ng of ChIP and input DNA was amplified using GenomePlex Complete Whole Genome Amplification Kit (Sigma) and eluted with GenElute PCR Clean-Up Kit (Sigma). For ChIP-on-chip experiments we used Nimblgen HG18 Refseq Promoter 3x720K array. One microgram of ChIP and input DNA was directly labeled by Klenow random priming with Cy5 and Cy3 nonamers with Nimblegen Dual-color DNA Labeling Kit following manufacturer's user's guide Chip-chip arrays v6.2, and the labeled DNA was precipitated with 1 volume isopropanol. Hybridization mix including 15 µg of labeled DNA was prepared using Nimblegen Hybridization Kit. Arrays were hybridized in Nimblegen Hybridization System 4 Station for 16–18 h at 42°C, and then washed in 1× Wash solution I, II and III. Hybridization buffers and washes were completed using manufacturer's protocols. Arrays were scanned on a Nimblegen MS 200 Scanner per manufacturer's protocol.ChIP-on-chip raw data was normalized and differential intensity of each probe compared with input control was calculated using the Nimblegen software DEVA. Average fold change (ChIP versus input) each 50 bp bin for a range of −3.2 kb upstream and 800 bp downstream window from RefSeq transcription start sites (TSS) were calculated using in-house Perl script. LOESS smoothed line plot around the TSS were plotted using in-house script written in R statistical programming language. For ChIP-signal heat map, similarly fold change average for each individual RefSeq transcript was calculated and then data were visualized with Java Treeview . FunctioLibrary preparation for sequencing: ChIP and genomic library preparation was performed using standard Illumina protocols. Libraries were prepared with the ChIP-seq Sample Preparation Kit (Illumina) according to the manufacturer’s instructions. Briefly, 10 ng of ChIP and input DNA were repaired to overhang a 3′-dA and then adapters were ligated to the end of DNA fragments. DNA fragments with proper size  were selected after PCR amplification, obtaining qualified library for sequencing.Supplementary Table S1. Peak caller program for histone, SICER (version 1.1)  reference genome (human hg18) using the program SOAP (version 2.21) . Sequencion 1.1) , was useBinding sites to gene feature annotation: Enriched peaks were annotated to nearest gene (RefSeq genes) using Bioconductor package ChIPpeakAnno . DistribRegulatory regions, histone modification peaks, CpG and LADs abundance: Input-subtracted normalized average H1 variants read density in each enriched locations of regulatory regions, histone modification peaks, CpG and LADs were calculated, and representation in box-plot were made using in-house scripts. As a control, a random sample of genomic windows with equal width was used to perform the significance test (Kolmogorov–Smirnov test).http://genome.ucsc.edu/cgi-bin/hgLiftOver).Publicly available genome-wide location data analysis: Public ChIP-seq data, which includes H3K4me1, H3K4me2, H3K4me3, H3K27me3, H3K27ac, H3K9me3, H3K9ac, H3K36me3, P300, CTCF, FAIRE and DNase enriched genomic locations, are taken from ENCODE project. CpG island genomic location information (hg18) and the coordinates of LADs  were takOverlap analysis: Overlap of genomic position range data was done using BedTools . OverlapAverage ChIP signal profile: For sequencing data, ChIP signal around center of each given genomic location were calculated by using normalized input subtracted-average tags number in each 50 bp bins in a set window. Relative distance of each tag from above-mentioned position and average signal was determined by using ‘Sitepro’ script of CEAS package  and plotOccupancy of H1 variants at individual chromosomes: Occupancy of H1 variants at all human chromosomes is an average of the input-subtracted ChIP-seq signal in 50 bp windows. Heat map and dendrogram were done with in-house R scripts. Correlation between the occupancy of H1 variants  and gene expression and gene richness coefficient was done with in-house R scripts. Gene expression for each chromosome was computed as the average of the expression of all the available expressed genes. The gene-richness coefficient (GRC) for each chromosome was calculated as the ratio between the percentage of total genes present in each chromosome and the percentage of base pairs of each chromosome to the total human genome.in vitro transcribed into cRNA in the presence of Cy3-CTP using the Low input quick Amp kit (Agilent). Labeled samples were purified using RNeasy mini spin columns (Qiagen). Then, 600 ng of cRNA were preblocked and fragmented in Agilent fragmentation buffer and mixed with Agilent GEx Hybridization mix. Hybridization mix was laid onto each sector of subarray gasket slide and sandwiched against an 8 × 65K format oligonucleotide microarray  inside a hybridization chamber, which was hybridized overnight at 65°C. Subsequently array chambers were disassembled submerged in Agilent Gene Expression Buffer 1 and washed 1 min in another dish with the same solution with a magnetic stirrer at 200 rpm at room temperature, followed by 1 min in Agilent Gene Expression Buffer 2 with a magnetic stirrer at 200 rpm at 37°C and immediate withdrawal from the solution and air drying. Fluorescent signal was captured into TIF images with an Agilent scanner using recommended settings with Scan Control software (Agilent). Signal intensities were extracted into a tabulated text file using Feature Extraction software (Agilent) using the appropriate array configuration and annotation files. The normalized log2 intensities were obtained using quantile method with normalized expression background correction the Bioconductor Limma package in R.Total RNA was extracted using High Pure RNA isolation Kit (Roche) according to the manufacturer’s instructions. cDNA was obtained from 100 ng of total RNA using SuperScript VILO cDNA synthesis Kit (Invitrogen). High RNA integrity was assessed by Bioanalyzer nano 6000 assay. For each sample, 100 ng of total were reverse transcribed into cDNA with a T7 promoter and the cDNA was The correspondence of the human H1 variants nomenclature with its gene names is as follows: H1.0, HIF0; H1.1, HIST1H1A; H1.2, HIST1H1C; H1.3, HIST1H1D; H1.4, HIST1H1E; H1.5, HIST1H1B; H1X, HIFX.Supplementary Figure S1). In ChIP-qPCR experiments, an anti-HA antibody was used to specifically pull down H1-associated chromatin fragments in cells expressing H1-HAs . H1-associated chromatin included gene promoters, coding regions and repetitive DNA, irrespective of which H1-HA variant was immunoprecipitated . A few differences were observed between variants, e.g. there were relatively less H1.3 but more H1.4 and H1.5 at alphoid repeats.To determine whether the genomic distribution of human histone H1 differs between variants, we used ChIP combined with semiquantitative PCR (ChIP-qPCR), promoter array hybridization (ChIP-on-chip) and massive sequencing (ChIP-seq). Because there is a limited number of H1-variant–specific ChIP-grade antibodies (only H1.2 and H1X in our hands), we developed T47D-derived cell lines stably expressing HA-tagged versions of each of the five somatic H1 variants expressed in most cell types   . These cSupplementary Figure S4). Interestingly, the TSS-associated H1 valley was not observed at genes inactive in these cells, i.e. OCT4 and NANOG . Furthermore, under stimulating conditions H1 depletion at the TSS was increased at inducible promoters, such as steroid hormone responsive promoters (MMTV) or genes induced by mitogenic agents (JUN and FOS) . Noteworthily, in these early response genes, there was already an H1 valley in noninducing conditions and this became deeper on stimulation .The specificity of H1 variant distribution was investigated in more detail at gene promoters previously shown to contain H1 in distal regions located 10 kb upstream of their TSS and depletion of H1 at the TSS  . All thend NANOG C, while To explore the genome-wide distribution of the different H1 variants across gene promoters, we hybridized ChIP material obtained with variant-specific antibodies or corresponding to HA-tagged H1 variant-associated chromatin with a promoter tiling array containing probes for 30 893 transcripts (−3200 to +800 bp to the TSS) arising from 22 542 human promoters. The average log2 ratio of probe intensity for all transcripts was plotted against the relative distance to the TSS for each variant and an H1 valley close to the TSS was apparent in all cases. Interestingly, in the two H1.2 samples (endogenous H1.2 and H1.2-HA), the valley was more pronounced and slightly shifted toward the TSS, compared with that for the other H1 variants (endogenous H1X and H1.0/3/4/5-HA) A.FigureSupplementary Figure S8), and heat maps representing binding intensity were constructed for each variant, ranking promoters from highest to lowest gene expression .Subsequently, this ChIP-chip data was combined with gene expression data for ca. 20 000 of the transcripts, obtained with the parental cell line in a human expression array (Agilent) .In addition to protein-coding genes, the promoter array contained 1145 noncoding transcripts, including structural RNAs and transcribed pseudogenes, that overall presented a low expression rate compared with the complete transcriptome. An H1 valley at the TSS was only apparent on the ChIP-chip heat maps for endogenous and HA-tagged H1.2, in agreement with our observation that an H1.2 valley occurs even at weakly expressed promoters (Supplementary Table S2).Noteworthily, H1.2 abundance at distal promoter regions (−3200 to −2000 bp from TSS) was inversely proportional to gene expression, being more abundant at repressed promoters C. This wSupplementary Figure S11). This indicates that there is a preferential binding of H1.2 in some promoters (mostly repressed genes) compared with the other variants, and vice versa, many promoters are devoid of H1.2 but contain other H1 variants.Moreover, H1.2 abundance at distal promoter regions was inversely correlated with H3, H1X and H1-HA abundance, while H1.2-HA showed an intermediate pattern . The largest overlaps were between low2/highX promoters (553 genes), mainly corresponding to expressed genes . Taken together, our data indicated that promoters having few H1.2 variants are loaded with large amounts of other variants, not only with exogenously expressed H1.0-HA but also endogenous H1X. Expression analysis of such groups of genes found that genes with few H1 variants at distal promoters are highly expressed, and vice versa, but also that H1.2 content is the strongest predictor of gene expression .Venn diagrams were drawn for the top 10% genes with high or low H1.2 and high or low H1X at the distal promoter to identify genes presenting high2/lowX and vice versa . HeLa cells showed results similar to T47D, i.e. H1.2/H1X ratios were higher in COL4A3 and CUGBP2 genes than TMEM204 and TUBGCP5, although ratios in all genes were higher than in T47D reflecting a higher relative abundance of H1.2 in HeLa cells . On the other hand, H1.2/H1X ratios in MCF7 were similar in all four genes, due to higher H1X signals in COL4A3 and CUGBP2 genes. This result indicated that relative abundances between variants at promoters were not fully conserved between cell types, although the patterns in T47D and HeLa were similar. In relation to this, ChIP-chip of H1.2 and H1X in HeLa confirmed that these two variants do not coexist at exactly the same distal promoters .The universality of the relative H1.2/H1X abundance at representative genes was tested in two additional cell lines by ChIP-qPCR . The observed clustered distribution was well conserved between cell lines, but differed between H1 variants. H1X and H1.0-HA abundances were not clustered with the same pattern as gene expression. Rather, these variants were abundant at promoters located on gene-rich chromosomes 17 and 19, and depleted on the gene-poor chromosome 13 . In summary, H1.2 content at promoters is the best H1 reporter of gene expression.Next, we plotted heat maps of H1.2 abundance at the promoters of genes ranked according to their position along several human chromosomes A. IntereSupplementary Table S1). To confirm the results obtained by ChIP-chip, we focused first on the input-subtracted normalized average ChIP signal obtained around coding regions of genes grouped according to basal expression as before  of up to 50 million reads per sample . When we calculated the input-subtracted coverage of H1 variants across the peaks of selected core histone modifications, depletion of H1.0 and H1.2, and to some extent of H1.4 but not H1X, was associated with positive histone marks linked to strong enhancers such as H3K4me1, H3K4me2 and H3K27ac . H1 abundance at H3K4me3 and H3K9ac sites, enriched at TSS of active promoters, differed between variants, reflecting H1.2 depletion at the TSS of most genes but local enrichment of the other variants immediately after the TSS. No strong enrichment of H1 was found at negative histone marks such as H3K9me3 or H3K27me3. It is also worth noting that H1.2 abundance was lower at active marks than at those related with repression and chromatin compaction, in agreement with the observed correlation between H1.2 content and gene repression.In addition to the local displacement of H1 from active promoters, H1 variants were markedly depleted from other regulatory regions along the genome, namely, CCCTC-binding factor (CTCF) binding sites corresponding to insulators, and p300 binding sites associated with enhancers, but little affected at DNase hypersensitivity sites and FAIRE-identified regions representing open chromatin . When the input-subtracted coverage of H1 variants across LADs was calculated, H1.2 was the only variant showing enrichment . Interestingly, the H1.2 pattern was the most different from the other variants, endogenous H1X and HA-tagged variants showing patterns that were similar to each other and to that of H3, while the pattern for HA-tagged H1.2 was more similar to endogenous H1.2 than to other HA-tagged H1s. It is worth noting that long genome patches of low GC content were found to be devoid of all H1 variants except H1.2, which was enriched. We next performed genome-wide correlation analysis between the input-subtracted H1 variant signal and GC content. Low CG content was associated with high occupancy of H1.2 but low occupancy of the other variants, including H1X, and vice versa .We then examined individual chromosomes for the presence of the input-subtracted signal of the different H1 variants. Abundance of H1 was heterogeneous along chromosomes, showing extensive patches of enrichment or depletion of H1 compared with the input . Notably, H1 variants were clustered differently depending on whether they were replication-independent (H1.0 and H1X) or synthesized over the course of DNA replication only . Further, H1.0 and H1X had a more heterogeneous distribution between chromosomes (data not shown).Further comparison of the overall abundance of H1 variants at each individual chromosome revealed unique patterns creating corresponding clusters of chromosomes and H1 variants . InteresSupplementary Table S3). Most H1-enriched regions were inside genes (arbitrarily defined as from −5 kb upstream to +3 kb downstream of the TTS), whereas H1.2 peaks were more abundant at intergenic regions . On the other hand, all H1-depleted regions were more abundant inside genes, especially for H1.2. Within genes, H1.2-enriched regions were disfavored at promoters (−5 kb to +1 kb flanking TSS) compared with other H1 peaks, whereas H1.2-depleted regions were strongly favored, in agreement with ChIP-chip data presented in Supplementary Figure S20). Next, we analyzed the overlap between H1-enriched and depleted regions with CpG islands. CpG islands were enriched at H1.2-depleted regions and at regions enriched for the other variants, confirming the inverse correlation between CpG islands and H1.2 described above . As expected, regions overlapping with CpG sites were preferentially located at promoters. For example, 42% of H1.0- or H1X-enriched regions located at promoters overlapped with a CpG island, while this was the case for only 4–8% of regions enriched in these variants located at intergenic regions.Next, we searched specific regions of the genome either enriched or depleted for each H1 variant signal over input DNA with a fold change ≥2 using SICER software (Supplementary Table S3). Overlap analysis disclosed the number of genes containing peaks of a single variant or several variants , and expression analysis revealed that genes with only H1.2 peaks were less expressed than target genes containing peaks of any other H1 variant (Supplementary Table S3). On the other hand, genes presenting H1.2-depleted regions were highly expressed, while genes with depleted regions of H1.0, H1.4 or H1X were expressed at lower levels than the total transcriptome average . Except for those for H1.2, H1 peaks were as abundant in introns as in distal intergenic regions. On the other hand, depleted regions were similarly distributed across compartments in the different H1 variants, except H1.2-depleted regions, which were more abundant at promoters and less so at intergenic regions.We further investigated whether the identified H1-enriched regions fell within genes, proximal regulatory regions or distal intergenic regions using CEAS software . Again, In summary, our data shows that histone H1 is not uniformly distributed along the genome and there are differences between variants, H1.2 being the one showing the most specific pattern and strongest correlation with low gene expression in breast cancer cells.Herein, we have investigated the distribution of all somatic histone H1 variants present in breast cancer cells, i.e. H1.0, H1X and H1.2 to H1.5 by combining ChIP with genomic technologies such as tiling promoter array hybridization and high-resolution sequencing. After testing several H1 variant-specific antibodies that we and others have produced, only H1.2 and H1X commercial antibodies were found to be useful in the ChIP-qPCR experiments, and variant specific, as shown by performing ChIP experiments in H1.2 and H1X knockdown (KD) cells. Consequently, we generated stable cell lines expressing HA-tagged versions of the H1 variants at protein levels close to or below endogenous levels, despite mRNA levels of exogenous H1 forms being higher (data not shown). This suggests that H1 is tightly posttranscriptionally regulated to control the overall levels of H1 and the proportion between variants, which vary considerably across cell types and cell lines. HA-tagging allowed us to perform ChIP of all variants with the same antibody, ruling out the variability being due to diverse antibody specificity or affinity. We found that all the H1 variants studied are widely distributed along the genome and within promoters with few differences between HA-tagged H1.0, H1.3, H1.4 and H1.5. In contrast, endogenous H1.2 presents striking differences. We rule out the possibility that the differential distribution is due to antibody usage or protein overexpression, as endogenous H1X presented an occurrence similar to HA-tagged variants and exogenous H1.2-HA resembled its endogenous counterpart more closely than the other H1-HAs.On this basis, we report that, in the cell line investigated, H1.2 presents a variant-specific distribution and may have differential functions. In fact, we reported elsewhere that H1.2 KD produces unique effects, namely, cell cycle arrest at G1 and decreased nucleosome spacing, not seen in other H1 KDs, and these were observed not only in T47D cells but also in MCF7 cells . NonetheWe have noticed that H1.2-HA was not distributed in exactly the same way as endogenous H1.2 and showed intermediate features somewhat similar to the other H1-HAs. We believe that this recombinant protein has the H1.2 structural features that direct it to the natural H1.2-occupied sites, but owing to its overexpression it may also locate at distinct sites normally occupied by other H1 variants. We have observed, by ChIP, that on knock down of endogenous H1.2, H1.2-HA occupancy increased (data not shown), suggesting a relocation to H1.2 sites. Overall, we believe that caution should be taken when interpreting data generated with exogenous histone variants fused either to the Dam domain or to peptide tags.Our analysis has also shown that all H1s are removed from active promoters, with maximum depletion close to TSS but extending several nucleosomes upstream, beyond the reported NFR, and within the coding regions. These regions containing nucleosomes but not H1 may coincide with H2A.Z and H3.3-containing nucleosomes, as both H2A.Z and H3.3 have been reported to locate at active promoters surrounding the NFR, where they positively regulate transcription . AdditioInterestingly, we have found that immediate-early responsive promoters, under nonstimulating conditions, are prepared to respond to stimuli by keeping the TSS free of H1, indicating that mechanisms other than transcription initiation might dictate H1 clearance. In this case, there is also histone H3 depletion at the TSS compared with at the distal promoter in the absence of stimuli, indicating that the NFR might be maintained to allow rapid response after stimulation. Supporting our hypothesis, it has been recently proposed that transcription factors interact with DNA in a dynamic way, and some transcription factor–DNA interactions are established before the stimuli, especially at immediate-early genes .Comparison of H1 occupancy with H3 has shown that all H1s except H1.2 follow the distribution of the core histone, whether this represents nucleosome enrichment, stability or defined positioning through the cell population. Nonetheless, H1 depletion at promoters and regulatory sites (CTCF or p300 binding sites) is more extensive than H3, denoting that nucleosomes might be ejected from delimited sites such as the NFR at the TSS, but H1 might be depleted from larger regions encompassing several nucleosomes. This is in agreement with previous reports showing that dips of low H1 occupancy at TSS and regulatory sites are not due to a lack of nucleosomes as they show enrichment of the core histone variant H3.3 . MoreoveInitial ChIP-qPCR experiments indicated that all H1 variants were present at all tested promoters. Nonetheless, hybridization of ChIP material with a promoter array revealed that promoters might present differential H1 variant abundance . The mosMyc and H1dFLAG in ESCs, the closest orthologs of human H1.2 and H1.3, by Li et al. for human H1.5 in differentiated IMR90 fibroblasts, and by Izzo et al. for human Dam-H1.2 to H1.5 in IMR90 cells also  was more closely related to GC-rich sequences and LINES, and H1c (H1.2) to AT-rich sequences, Giemsa-positive regions and satellite DNA. It is conceivable that there are at least two groups of H1 variants with different distributions in each cell type, such that taken together histone H1 variants cover the whole genome, being present in most of the nucleosomes.The negative correlation observed between gene activity and H1.2 content found at promoters extended upstream toward the whole genomic region. Patches of H1.2 enrichment seem to be associated with gene repression, gene-poor regions , low GC content or LADs, features related to chromatin compaction . Moreovells also . HoweverWhether a single variant may present distinct features in different cell types rather than having intrinsic properties is an intriguing question. Factors involved may be the relative and absolute abundance of each variant and whether a genome needs more plasticity or is progressively silenced, i.e. pluripotency versus terminal differentiation. In this sense, Li et al. described the existence of zones of H1.5 enrichment in differentiated fibroblasts but not in ESCs , and it Cancer is another cellular state in which global chromatin rearrangement is observed. In fact, alterations in nuclear morphology are one of the characteristics of cancer cells. Tumor-originated cells accumulate genetic and/or epigenetic differences compared with nontumor cells, and chromatin is reorganized leading to altered gene expression programs and higher plasticity. The hallmark of cancer is dedifferentiation and gene dysregulation. DNA methylation and histone modifications are two epigenetic mechanisms that are altered in cancer cells. Moreover, large organized chromatin K (lysine) modifications are reduced in cancer , and genTumor cells are characterized by a different methylome from that of normal cells [reviewed in ]. There Similarly, within a long region of genomic sequence, genes are often characterized by having a higher GC content than the background GC content of the entire genome. We found that H1 variants except H1.2 are associated with higher GC content regions, consistent with the preferential location of H1-enriched regions within genes. H1.2 presents an inverse correlation with GC content at a genome-wide level and H1.2-enriched regions associate with lower GC content than other variants. In our analysis, H3 also associates preferentially with higher GC-content regions, in agreement with reports describing greater nucleosome-space occupancy coinciding with active transcription and higher GC contents .et al.  has been found to locate around the TSS of active genes . InsteadSeveral studies have compared the chromatin binding affinity and residence time on chromatin of the different H1 subtypes in different organisms or cell lines, as well as its nuclear localization, obtaining diverse, if not controversial, results on the functional heterogeneity of H1 variants. In general, H1.2 is among the variants presenting intermediate or low affinity for chromatin and, consequently, elevated mobility. Instead, H1.4 has been mostly associated with high affinity, low mobility and colocalization with heterochromatin ,101–103.Drosophila cells, five principal chromatin types have been described, H1 being present in all of them in different proportions  database under the accession number GSE49345.Supplementary Data are available at NAR Online.Ministerio de Ciencia e Innovación of Spain (MICINN) and FEDER [BFU2011-23057 to A.J.]; Ministerio de Economía y Competitividad [SAF2012-36199 to N.L.-B.]; Generalitat de Catalunya [2009-SGR-1222 to A.J.]; JAE-Doc contract from CSIC-MICINN (to J.-M.T.); TA contract from CSIC-MICINN (to R.M.); FPU predoctoral fellowship from MICINN (to L.M.-A.). Funding for open access charge: Ministerio de Ciencia e Innovación of Spain (MICINN) and FEDER [BFU2011-23057 to A.J.].Conflict of interest statement. None declared.


PTEN. Repression of this gene is critical to mediate the anti-apoptotic function of MAD1. Under certain conditions it also antagonizes the functions of the oncoprotein MYC. Previous studies have demonstrated that MAD1 expression is controlled by different cytokines and growth factors. Moreover we have recently demonstrated that the MAD1 promoter is controlled by the cytokine granulocyte colony-stimulating factor (G-CSF) through the activation of STAT3, MAP kinases and C/EBP transcription factors.The MAD1 protein, a member of the MYC/MAX/MAD network of transcriptional regulators, controls cell proliferation, differentiation and apoptosis. MAD1 functions as a transcriptional repressor, one direct target gene being the tumor suppressor MAD1 mRNA and protein in promyelocytic tumor cells. Moreover we found that C/EBP and SP transcription factors cooperated in regulating the expression of MAD1. This cooperativity was dependent on the respective binding sites in the proximal promoter, with the CCAAT boxes being bound by C/EBPα/β heterodimers. Both C/EBP and SP transcription factors bound constitutively to DNA without obvious changes in response to TGFβ1. In addition SMAD3 stimulated the MAD1 reporter, cooperated with C/EBPα and was bound to the core promoter region. Thus SMAD3 appears to be a potential link between TGFβ1 signaling and C/EBP regulated promoter activity. Moreover TGFβ1 stimulated the phosphorylation of polymerase II at serine 2 and its progression into the gene body, consistent with enhanced processivity.We observed that in addition to G-CSF, the cytokine transforming growth factor β (TGFβ1) rapidly induced the expression of MAD1 gene that participate in mediating signal transduction events emanating from different cytokine receptors. SMAD3, a target of TGFβ1 signaling, appears to be functionally relevant. We suggest that a key event induced by TGFβ1 at the MAD1 promoter is the recruitment or activation of cofactors, possibly in complex with C/EBP, SP, and SMAD3 transcriptional regulators, that control polymerase activity.Our findings suggest that C/EBP and SP factors provide a platform of transcription factors near the core promoter of the  The MYC/MAX/MAD network of transcriptional regulators is essential to control many aspects of cell physiology . MYC wasPTEN [PTEN promoter directly [Mad1 revealed increased sensitivity to pro-apoptotic conditions [MAD proteins are alternative binding partners of MAX . Six difPTEN . MAD1, wPTEN -12, reprdirectly . This conditions , furtherMad1 results in a differentiation defect of granulocytes [MAD1 gene is highly regulated, generally reciprocal to the regulation of MYC genes [MAD1 expression [MAD1 promoter is organized and how signals of these differentiation factors control gene expression. The MAD1 promoter contains a CpG island as part of a roughly 400 bp proximal promoter region highly conserved between humans and rodents [MAD1 promoter by G-CSF is in agreement with the described role of this cytokine and of Mad1 in the control of granulocyte differentiation and survival [In addition to the anti-apoptotic function, MAD1 has been suggested to control proliferation and differentiation antagonistically to MYC . Indeed ulocytes ,7,13-15.pression -20. Thes rodents . This resurvival .MAD1 in U937 and in HaCaT keratinocytes, respectively [MAD1 induction were observed in a clone of U937 promyelocytes that stably express a viral version of MYC (U937-myc6). In these cells a weak induction was observed in response to TGFβ by 8 hrs, possibly as a result of constitutive MYC expression [MAD1 gene expression, we addressed how this cytokine affects MAD1 promoter activity. It appears that TGFβ1 stimulates MAD1 through elements proximal to the core promoter.Cytokines of the TGFβ family have broad activities in controlling cell physiology, including proliferation, differentiation and survival -23. TGFβectively ,19. In bpression . To undeMAD1 gene is regulated by multiple signaling pathways. One of the regulatory cytokines is TGFβ1, which is known to induce MAD1 in keratinocytes and in U937-myc6 promyelocytes [MAD1, we performed time course experiments. TGFβ1 rapidly activated MAD1 mRNA expression in U937 cells  since the TGFβRI inhibitor SB505124 blocked MAD1 activation  in comparison to a control , indicating that these transcription factors as well as C/EBP proteins are constitutively interacting with the MAD1 promoter.In addition to CCAAT boxes, the proximal promoter region of the s Figure . To tests Figure . Bindings Figure . MoreoveMAD1 promoter  . The SMAD3 responsive region was mapped to the promoter fragment that contains the two C/EBP half sites and one SP binding site, i.e. GC box1  resulted in almost complete loss of SMAD3 responsiveness   Figure . All the) Figure  and 6C. s Figure . We furt1 Figure . These rs Figure . Consistr Figure . Finally) Figure . HoweverMAD1 promoter is activated, we analyzed acetylation of histone H3 (H3ac) and trimethylation at Lys 4 of histone H3 (H3K4me3) before and after TGFβ1 stimulation. Both are marks for active promoters. We observed H3ac throughout the locus and H3K4me3 at the promoter, however, none of these marks was significantly modified by TGFβ1 stimulation . We suggest that C/EBP and SP transcription factors form a platform for incoming signals as exemplified by G-CSF [MAD1 expression. Consistent with this interpretation, SMAD3 cooperated with C/EBP proteins to activate MAD1 promoter reporter genes. The finding that SMAD3 was bound to the MAD1 promoter suggests that SMAD3 is directly recruited to the MAD1 promoter by binding to C/EBPs or C/EBP associated factors. Because the GC box was also relevant, we propose that a large transcription factor/cofactor complex interacts with the identified promoter proximal region, including SMAD3. However, we point out that we cannot exclude direct binding of SMAD3 to the MAD1 promoter. Although no obvious binding sites could be detected, SMAD binding sites are rather short and leave the possibility open that SMAD3 forms a dimeric or multimeric complex with other factors, in which SMAD3 might bind directly to DNA.We observed that C/EBP and SP transcription factors bind constitutively to the proximal lf sites . Consistby G-CSF  and possMAD1 promoter translate into the activation of Pol II as measured by its progression into the gene body and the concomitant change in the phosphorylation of the C-terminal domain of Pol II. This is consistent with recent observations on many genes, which have provided evidence that Pol II phosphorylated at Ser-5 is located at the promoter in a preactivated or paused mode. The switch to Ser-2 phosphorylation, possibly by the recruitment and activation of the P-TEFb kinase CDK9, results in the activation and promoter clearance of Pol II [MAD1 promoter prior to stimulation with cytokines. Thus at least in U937 tumor cells, the MAD1 promoter is preoccupied by Pol II and thus allows for rapid activation by multiple signals. It will now be of interest to specifically dissect how different cytokines use the C/EBP-SP transcription factor platform to activate the paused Pol II.The signals that are integrated at the proximal f Pol II . Thus thMAD1 promoter reporter gene constructs has been reported previously [+-C/EBPα, and pCB6+-C/EBPβ are found in [The cloning of eviously . Descripfound in ,35; pCDNfound in ; pCL-neofound in .2. U937 cells were treated with TGFβ1 (Peprotec) at a concentration of 2.5 ng/ml and with 5 μM SB505124 (Sigma-Aldrich) as indicated. Proliferation and viability of U937 cells were analyzed using Trypan Blue staining and the CASY cell counting system (Innovatis).HEK293 (ATCC CRL-1573) and HeLa (ATCC CCL-2) cells were cultured in DMEM (Gibco) with 10% fetal calf serum (Gibco) and penicillin/streptomycin (Seromed). U937 (ATCC CRL-1593.2) promyelocytes were grown in RPMI 1640 (Gibco) with 10% fetal calf serum and penicillin/streptomycin. All cells were cultured at 37°C and 5% COTransient transfection of HEK293 and HeLa cells were performed using the calcium phosphate co-precipitation method as described previously . HeLa ceC/EBPβ mRNA and the resulting sequences were analyzed via the BLAST algorithm. The hybridized oligonucleotides were cloned into the pSuper vector (obtained from R. Bernards) linearised with BglII and HindIII [The online program siDirect (genomics.jp/sidirect) was used to design shRNA oligonucleotides targeting the  HindIII .Sense: 5'GATCCCCAGCACAGCGACGAGTACAATCTTCAAGAGAGATTGTACTCGTCGCTGTGCTTTTTTGGAAA;Antisense: 5'AGCTTTTCCAAAAAAGCACAGCGACGAGTACAATCTCTCTTGAAGATTGTACTCGTCGCTGTGCTGGG.MAD1 QantiTect primer assay (Qiagen) and β-GLUCURONIDASE-f 5'-CTCATTTGGAATTTTGCCGATT-3', β-GLUCURONIDASE-r 5'-CCGAGTGAAGATCCCCTTTTTA-3'. The relative quantification of MAD1 mRNA was calculated by the comparative CT method and normalized to β-GLUCURONIDASE using the Software RelQuant.The RNAeasy Mini Kit (Qiagen) was used for total RNA extraction, according to the manufacturer's instruction and residual genomic DNA was removed by DNase (Qiagen) digestion. 1 μg total RNA was reverse transcribed into cDNA using the Transcriptor First Strand cDNA Synthesis Kit (Roche) and analyzed by quantitative real time PCR using a LightCycler (Roche). The real time PCR reactions were performed with the SYBRgreen Ready Mix (Qiagen) and the following primer pairs: 6 cells/ml. Following TGFβ1 treatment 5-2.5 × 107 cells/ml per IP were harvested. For immunoprecipitation 2 μg of the following antibodies were used: H3ac ; H3K4me3 ; Pol II N20 ; Pol II CTD phosphoserine 2 H5 ; Pol II CTD phosphoserine 5 H14 , C/EBPα 14AA ; C/EBPβ C19 , SP1 PEP2 , SP1 , Cytochrome C , SMAD3 (ab28379 Abcam). In addition SP1-specific antibodies were obtained from G. Suske [MAD1 gene:ChIP assays were performed as described previously . U937 ceG. Suske . The folU: 5'CCTCTTAATATACTGTCCTATGC-3';U': 5'GTCACAGCTCTCCAGAAATAGAAG-3';P: 5'AGTTGCGAATCCTGTCACCA-3';P': 5'TTCTCTTGACAGGCCAGCTT-3';G: 5'-ATATTGTAGGTGACACAAACTGC-3';G': 5'-ATCTCACTTGAAGCTTCCACAG-3'MYOD1: 5'-CCGCCGCTTTCCTTAACCACAAAT-3'MYOD1': 5'-GTAGATAGCAAAGTGCTGGCAGTC-3'For Re-ChIP assays the first immunoprecipitation was performed as above. Then the samples were washed once in ChIP RIPA buffer  and the protein-DNA complexes solubilized in release buffer . The beads were incubated at 37°C for 30 min. To the supernatant 4 volumes of RIPA-SDS  were added to perform the second immunoprecipitation.32P-ATP radiolableled and used in EMSAs:The following oligonucleotides were γCCAAT-b1 f: 5'AGCCCTCTCCCAATCGCACAAG3';CCAAT-b1 r: 5'CTTGTGCGATTGGGAGAGGGCT-3';NE-CCAAT f: 5'-TCGAGATGGGGCAATAT-3';NE-CCAAT r: 5'-ATATTGCCCCATCTCGA-3';GC box1 f: 5'-AAGTGTAGGGGCGGGGCATTCT-3';GC box1 r: 5'-AGAATGCCCCGCCCCTACACTT-3'.HEK293 whole cell extracts were prepared on ice in Frackelton-lysis buffer  Triton X-100, 10% (v/v) glycerol, 100 μM Na3VO4, 150 μM benzamidin, 0.025 U/ml α-macroglobulin, 2.5 μg/ml leupeptin, 14 μg/ml aprotinin). Whole cell extracts were incubated with the radiolabeled oligonucleotides at 30°C for 30 min and then subjected to electrophoresis as described previously . In brie7 cells/preparation), U937 cells were lysed in 20 - 30 μl FT Lysis buffer  by pipeting up and down as described previously [To generate highly concentrated U937 whole cell extracts (10eviously . The freC/EBP: CCAAT-enhancer-binding protein; ChIP: Chromatin immunoprecipitation; CTD: Carboxy-terminal domain; DMSO: Dimethyl sulfoxide; EMSA: Electrophoretic mobility shift assay; G-CSF: Granulocyte colony stimulationg factor; H3ac: acetylated histone 3; H3K4me3: trimethylated lysine 4 of histone 3; MAX: MYC-associated factor x; MYC: Myelocytomatosis viral oncogene; PTEN: Phosphatase and tensin homologue deleted on chromosome ten; Pol: Polymerase; Ser: Serine; SMAD3: Mothers against decapentaplegic homolog 3; SP: Specific protein; SPdn: Specific protein dominate negative; TGF: Transforming growth factor; TGFR: Transforming growth factor receptor; TPA:12-O-tetradecanoylphorbol-13-acetat.The authors declare that they have no competing interests.NH and BL designed the experiments. NH, KJ, and CC performed the experiments. NH, CC, and BL evaluated the data and wrote the manuscript. All authors read and approved the final manuscript.


Mycobacterium tuberculosis, the causative agent of tuberculosis, encodes a large number of putative transcriptional regulators. However, the identity and target genes of only a few of them have been clearly identified to date. In a recent study, the ArsR family regulator Rv2034 was characterized as a novel positive regulator of phoP. In the current study, we characterized the auto-repressive capabilities of Rv2034 and identified several residues in the protein critical for its DNA binding activities. We also provide evidence that Rv2034 forms dimers in vitro. Furthermore, by using DNaseI footprinting assays, a palindromic sequence was identified as its binding site. Notably, we found that the dosR promoter region contains the binding motif for Rv2034, and that Rv2034 positively regulates the expression of the dosR gene. The potential roles of Rv2034 in the regulation of lipid metabolism and hypoxic adaptation are discussed.The genome of  Mycobacterium tuberculosis, the causative agent of tuberculosis, is a leading cause of death worldwide M. tuberculosis bacilli is linked to its ability to adapt to hypoxia and persist within humans for long periods without causing any overt disease symptoms in vitro and in vivo have indicated that DosR is the key regulator that mediates hypoxic responses in M. tuberculosisMultiple studies Corynebacterium gutamicum and Streptomyces coelicolor have 12 and 23 copies of ArsR homology, respectively M. tuberculosis has 12 ArsR homologies Vibrio cholerae, the regulator HlyU regulates expression of the virulence determinant HlyA Vibrio vulnificus, the HlyU protein upregulates the expression of the essential virulence factor rtxA1Streptococcus mutansBacillus anthracisThe ArsR family regulator is widely expressed in many bacterial or archaeal species and is highly abundant in some of them M. tuberculosis have been characterized as metal sensors: KmtR (encoded by Rv0827c) and NmtR (encode by Rv3744) sense nickel-cobalt phoP and groEL2To our knowledge, four of the twelve ArsR family regulators in M. tuberculosis. The binding site of Rv2034 to its promoter DNA was mapped out and some residues critical in Rv2034 for DNA binding activity were also identified. Furthermore, a novel gene target of Rv2034, the dosR gene, was identified. Based on our analysis of its target genes, we conclude that Rv2034 is a transcriptional regulator involved in the regulation of lipid metabolism and hypoxic response in M. tuberculosis.In the present study, we have characterized the ability of Rv2034 for auto-regulation in HIS3 and aadA, to detect interaction of Rv2034 with its promoter. The coding sequence of Rv2034 was cloned into the pTRG vector. The putative promoter region of Rv2034 was cloned upstream of HIS3-aadA reporter genes of the bacterial one-hybrid reporter vector pBXcmT r) or without  the screening media  that were separated by four nucleotides (5′-CTAA-3′ or 5′-TTAG-3′) was found.The Rv2034 binding motif in its promoter DNA was characterized by DNaseI footprinting assays. As shown in ubstrate . The proubstrate . In bothWe then performed EMSA assays to confirm the significance of the motif for specific recognition by Rv2034. As shown in M. tuberculosis genome for putative Rv2034 binding motifs. Other than the promoter of Rv2034 (or Rv2033c), five promoters  were found to contain the potential Rv2034 binding site, as shown in dosR promoter DNA was co-incubated with increasing amounts of Rv2034 , clear shifted bands were observed.Characterization of the binding motif of Rv2034 made it possible for us to search putative target genes regulated by the Rv2034 protein. Putative promoter regions (upstream 250 bp) of entire open reading frames were searched in the E. coli shuttle vector pMV361 containing Rv2034 coding sequence was integrated in the bacterial genome at an attB site. The pMZ6 plasmid, in which the dosR promoter sequence was placed upstream of lacZ, and the pMZ7 plasmid, in which the dosR promoter-dosR coding sequence was cloned upstream of lacZ, were transformed into the wild type strain (M.sm) and the integrated strain (M.sm/attB::Rv2034), respectively, to create recombinant strains. In the latter, Rv2034 was expressed and functioned as a regulator for the dosR promoter in trans. As shown in dosR gene and promotes gene expression solely or in association with DosR. Taken together, our results indicate that Rv2034 is a functional regulator of the dosR gene in M. tuberculosis.We then performed beta-galactosidase reporter assays. We first created an integrated strain in which a mycobacteria-M. tuberculosisM. tuberculosis, as the regulator of DosR. We demonstrated that Rv2034 is capable of binding with the promoter DNA of DosR and positively regulating DosR expression. To the best of our knowledge, this is the first report on the characterization of a regulator of the dosR gene.DosR is the primary mediator of hypoxic response ArsR-type transcriptional regulators are known to act as metal sensors that mediate response to metal ion stress dosR, phoP, and groEL2phoP and groEL2. It is likely that the complex formed by Rv2034 binding with its promoter DNA inhibits the binding or sliding of RNA polymerase, causing auto-repression. On the contrary, binding at a further distance may be conducive to initiation or promotion of transcription. This kind of dual effects of transcriptional regulators was also observed in other report Our experiments revealed that Rv2034 acts as an auto repressor and, interestingly, positively regulates the expression of phoP). In the EMSA assays, the amount of Rv2034 protein needed to sufficiently bind with the promoter of Rv2034 is about 0.2 µM, while 2 µM of the protein was needed to bind with the promoter of phoP or groEL2M. tuberculosis. So the binding of Rv2034 has a certain extent of flexibilities. Among all the promoters, binding to its own promoter at 5′-CCGTAAGT∼N{4}∼ACTTACGG-3′ should be the strongest.It is noteworthy that the binding activity of Rv2034 to its own promoter is much stronger than to the promoter of other genes  are involved in lipid metabolism  was replaced by the hygromycin gene, is more resistant to hydrogen peroxide (H2O2), as compared with the wild type and complementary strains  and all antibiotics were purchased from TaKaRa Biotech as well. PCR primers were synthesized by Invitrogen (See 2+-nitrilotriacetate) agarose was obtained from Qiagen.The ogen See . All theA32G, E35G and Rv2034C61AC61A mutated variants were generated by site-directed mutagenesis using the overlap extension polymerase chain reaction technique (overlapping-PCR). Primers used in the overlapping-PCR are listed in supplementary Protein expression and purification were performed as previously described in vitro. The primers used for PCR are listed in Supplemental 32P] ATP or with fluorescein isothiocyanate (FITC). Mixtures containing the labeled DNA (∼10 nM) and increasing concentrations of the protein (as indicated in the legend of the corresponding figure) were incubated in EMSA buffer  for 30 min at room temperature, then subjected to 5% native polyacrylamide gel electrophoresis (PAGE). Electrophoresis was performed at 150 V at room temperature for about 1.5 hours in 0.5× Tris-borate-EDTA buffer (TBE). Radioactive gels were exposed to a storage-phosphor screen  overnight at room temperature. Images of gels were acquired using a Typhoon Scanner .Large DNA segments with more than 70 base pairs were acquired using polymerase chain reaction (PCR), and short DNA segments were synthesized and directly annealed HIS3 and aadA) in the pBXcmT plasmid Escherichia coli XR competent cells. Co-transformants were grown on selective medium containing 20 mM 3-amino-1, 2, 4-triazole (3-AT), 16 µg/mL streptomycin, 15 µg/mL tetracycline, 34 µg/mL chloramphenicol, and 50 µg/mL kanamycin. Plates were incubated at 30 degree centigrade for 3–4 days. Co-transformants containing the pBX-MthspXp and pTRG-Rv3133c plasmids Bacterial one-hybrid assays were performed as previously described The substrate DNAs used in this assay were about 220 base pairs long, including 140 base pairs upstream from the start codon and the first 82 base pairs of the Rv2034 coding region. Both the coding and non-coding strands were amplified by PCR using their specific primers labeled with Fluorescein Isothiocyanate (FITC) see . PCR prolacZ were cloned into the pMV261 backbone step by step  were then collected and washed with cold PBS buffer  once or twice. For cell permeabilization, 600 µl of the Z buffer  was dispensed into the microcentrifuge tube to resuspend the cells. Three hundred microliters of resuspended cells was saved to determine the A600 value. After adding 50 µl of 0.1% SDS (sodium dodecyl sulfonate) and 100 µl of chloroform, the mixture was vortexed and incubated at 28°C for 5 min. A total of 200 µl of ONPG  was then added to a final concentration of 0.66 mg/ml. Each tube was incubated at room temperature for 5–10 min. Five hundred microliters of 1 M Na2CO3 was added to end this reaction. Each tube was centrifuged at 1200 rpm for 5 min and the supernatant was used to determine the A420 and A550 values. β-galactosidase activities were calculated as described previously β-galactosidase activity assays were performed as previously described step See . Taking Figure S1Search for putative target genes regulated by the Rv2034 protein using the binding motif of Rv2034. Putative promoter regions (upstream 250 bp) of entire open reading frames in the M. tuberculosis genome were searched based on the Rv2034 binding motif  that we identified. Other than the promoter of Rv2034 (or Rv2033c), we found that five promoters  contained the potential Rv2034 binding site. The promoter regions were aligned and mismatches are shown on the right.(TIF)Click here for additional data file.Figure S2Pearson correlation coefficient values of Rv2034/Rv2035, Rv2034/Rv2036, and Rv2035/Rv2036 pairs. Correlation coefficient values were obtained from TBDB. Co-expressed genes are likely to have higher coefficients than ones that are not co-expressed (from 0 to 1).(TIF)Click here for additional data file.Figure S32O2).Construction of an ArsR-knockout strain and the growth determination in the presence and absence of hydrogen peroxide (H (A) Construction of the Ms6762 knockout strain of M. smegmatis and Southern blot assays. Ms6762 is the homologous gene of Rv2034 in M. smegmatis. Left panel: Using a recombination-based strategy, the coding sequence of Ms6762 was replaced by a hygromycin resistance gene (indicated as hyg in the schematic representation). The restriction sites for NarI are indicated with arrows. The DNA probes used in Southern blotting are indicated with black bars. Right panel: Southern blot assays. The DNA fragment corresponding to the region upstream of Ms6762 in M. smegmatis was obtained by PCR and labeled with digoxigenin dUTP . The probe was used to detect the change in size of the NarI-digested genomic fragment of M. smegmatis wild type  and knock-out  strains. The knock-out, the wild type and the complement strains were cultured in the presence of 0 mM (B), 15 mM (C) and 20 mM (D) of hydrogen peroxide, as indicated on the top. The complement strain was generated by transforming an inducible expression vector of pMind-Ms6762 into the verified Ms6762 knock-out strain. To induce the expression of Ms6762 in the complement strains, ∼25 ng/ml of the inducer (tetracycline) was added in the corresponding culture. The experiments were performed thrice with similar results.(TIF)Click here for additional data file.Table S1Primers used in this study.(DOC)Click here for additional data file.Table S2Plasmids used in this study.(DOC)Click here for additional data file.Table S3DNA substrates used in this study.(DOC)Click here for additional data file.


This may not only affect the efficiency of protein synthesis in vitro, but also limit our understanding of the functions and interactions of biomolecules involved in this fundamental biological process.Cell-free protein expression (CFPE) comprised of Renilla luciferase in wheat germ extract as a model system, we investigated the CFPE under macromolecular crowding environments emulated with three different crowding agents: PEG-8000, Ficoll-70 and Ficoll-400, which vary in chemical properties and molecular size. We found that transcription was substantially enhanced in the macromolecular crowding solutions; up to 4-fold increase in the mRNA production was detected in the presence of 20% (w/v) of Ficoll-70. In contrast, translation was generally inhibited by the addition of each of the three crowding agents. This might be due to PEG-induced protein precipitation and non-specific binding of translation factors to Ficoll molecules. We further explored a two-stage CFPE in which transcription and translation was carried out under high then low macromolecular crowding conditions, respectively. It produced 2.2-fold higher protein yield than the coupled CFPE control. The macromolecular crowding effects on CFPE were subsequently confirmed by cell-free synthesis of an approximately two-fold larger protein, Firefly luciferase, under macromolecular crowding environments.Using cell-free synthesis of Three macromolecular crowding agents used in this research had opposite effects on transcription and translation. The results of this study should aid researchers in their choice of macromolecular crowding agents and shows that two-stage CFPE is more efficient than coupled CFPE. This is followed by investigation into synthesis of an approximately two-fold larger protein, Firefly luciferase , to test the general applicability of macromolecular crowding effects to CFPE. The crowding environments in vitro are emulated by three inert macromolecular crowding agents, polyethylene glycol (PEG)-8000, Ficoll-70 and Ficoll-400, which vary in chemical properties, molecular size and morphology. While the PEG-8000 occurs as a flexible long-chain polyethylene glycol with sparse and short branches, the two Ficoll molecules are highly branched copolymers of sucrose that have more spherical and compact structures The present study provides an extensive investigation of the CFPE under macromolecular crowding conditions by using as a model system the synthesis of a reporter protein in vitro transcription under macromolecular crowding conditions using a pIVEX1.3-RL plasmid harboring Rluc gene  of transcription. In the presence of 20% (w/v) of Ficoll-70 transcription proceeded much faster than in the dilute solution with a ∼3-fold increase in the initial transcription velocity . Furtherst step, transcription was terminated at 3 hr and allowed to proceed for a further 5 hr (step 2). Ficoll-70 was either present in both steps (I) or only in step 1 (II) and its effects compared to a dilute control (III). It was interesting to find that transcription was retriggered in the macromolecular crowding environment and could proceed for additional 3–5 hr. The final yield of the Rluc mRNA generated from these two steps was close to that from the reaction under macromolecular crowding conditions in both steps. In contrast, in the absence of the crowding agent in the 2nd step no significant increase in the mRNA yields was observed  . PEG shoin vitro translation (The western blots verified that Rluc was produced from the nslation  and enabin vitro translation system (Considering the opposite effects of the crowding agents on transcription (enhancement) and translation (inhibition), we continued to investigate the overall effects of the macromolecular crowding conditions on the protein synthesis in the coupled transcription/translation system. Unexpectedly, the yields of the Rluc protein  consistently decreased in the presence of more than 1.0% (w/v) of PEG or 2.0% (w/v) of two Ficoll molecules, similar to those observed in the n system . Again, in vitro transcription and the coupled transcription/translation systems should be attributed to the difference in the temperature and salt concentrations, particularly the Mg2+ concentrations, of these two systems. The effects of macromolecular crowding on transcription varied significantly with the temperature and salt concentrations of the reaction , could interfere with the studies of the macromolecular crowding effects. Therefore we turned to a new CFPE system, PURExpress™, that is entirely reconstituted from the defined and purified components necessary for E. coli translation in vitro translation system and the coupled transcription/translation system, but then inhibited the translations when more crowding agents were added  concentrations. In the coupled CFPE system, the reaction conditions used are usually optimized for the translation such as relatively low macromolecular crowding and high Mg2+ concentration, which are suboptimal for transcription process. Currently, establishing a coupled CFPE system in which transcription and translation proceeds under different molecular crowding conditions remains technically challenging. However, this can be easily achieved with an uncoupled CFPE system. In order to evaluate the potential of CFPE with the macromolecular crowding effects under consideration, we explored a two-stage CFPE , followed by the addition of translation mixture to a total volume of 50 µl for protein synthesis. Using this technique mRNA synthesis is presumably enhanced by the addition of high concentration of crowding agents. In the subsequent translation step, the high concentrations of crowding agents and salts in transcription stage are subjected to a 1∶10 dilution and thus would not exert inhibitory effects on the translation process. For comparison, a conventional coupled CFPE and a control two-stage CFPE (without addition of crowding agent) were also tested.Obviously, the age CFPE , in whicWith the same amount of DNA template input and the same incubation time (2 hr), the control two-stage CFPE produced slightly (18%) more protein than the coupled CFPE. When a crowding agent, Ficoll-70 was added in transcription stage, then the two-stage CFPE produced 2.2-fold and 1.8-fold more protein than the coupled CFPE and the control two-stage CFPE, respectively . Obviousin vitro transcription and translation using the pT7CRL-FL template (In vitro translation and coupled transcription/translation showed that the expressions of Fluc protein were improved by the addition of 2% to 5% (w/v) of Ficoll-70, but inhibited in the presence of >10% (w/v)  , which i0% (w/v) , showingin vitro transcription and translation using coupled and two step CFPE in order to find out which of these gave the best protein yields.CFPE allows studies on the complex process of genetic message transfer from DNA to protein in which a number of biomolecules and their conformational rearrangements are involved in vitro transcription, the enhanced association of T7 RNA polymerase (T7 RNAP) with DNA template under macromolecular crowding conditions could be seen on an agarose gel following electrophoresis of transcription samples  new Rluc mRNA than the retriggered transcription containing 20% (w/v) of Ficoll-70   but alsoicoll-70 . ObviousEscherichia coli, rabbit reticulocytes or wheat germ (WG) have still been used in most CFPE applications. We focused on the eukaryotic WG-based system since it contains less inhibitors and allows ready application to high-throughput expression E. Coli-based translation systems are often supplemented with 2–5% (w/v) of PEG Compared with transcription and many other enzymatic reactions that have been investigated under macromolecular crowding conditions, translation is a much more sophisticated process in which more than 100 molecules including ribosome, mRNA, tRNAs, tRNA synthetases and amino acids, etc. work in concert to generate polypeptides from mRNA templates E-Coli S30-based translation solutions was earlier reported to induce protein precipitation Presence of more than 5% (w/v) of PEG in the The purpose of setting up the two-stage CFPE  was to eThe macromolecular crowding effects on CFPE were subsequently confirmed by cell-free synthesis of a larger Fluc protein using an unrelated template pT7CRL-FL. In fact it was found that the crowding agent Ficoll-70 improved transcription and two-stage CFPE more significantly with the pT7CRL-FL template than with the pIVEX1.3-RL template . This inin vitro transcription was significantly enhanced by the high concentrations of crowding agents, particularly Ficoll-70, the translation was generally inhibited. A two-stage CFPE integrating the macromolecular crowding effects could improve the efficiency of CFPE. Our results have profound implications in system and synthetic biology and will allow us to better reproduce the gene transcription and translation process in an in vitro setting.Finally, it is worth pointing out that only the macromolecular crowding effects on CFPE were discussed in this research. In fact, the efficiency of CFPE is also limited by other factors such as energy supply and inhibitory by-products generated from translation process PEG-8000 (molecular weight: ∼8 kDa) was purchased from Fisher . Ficoll-70 and Ficoll-400  were purchased from Spectrum Chemicals & Laboratory Products . A 50% (w/v) stock solution of each crowding agent was prepared in nuclease-free water before being used in experiments.Apa I and EcoICR I, respectively, and then purified with phenol: chloroform: isoamyl alcohol extraction and ethanol precipitation before being used for in vitro transcription.The cell-free protein expression vector pIVEX1.3-RL harboring the Rluc gene  was cons2, 0.1 M spermidine, 0.5 M NaCl), 0.2 µl 1 M DTT, 0.5 µl RNasin® Plus RNase Inhibitor , 0.5 µl (for transcription of Rluc gene) or 1.2 µl (for transcription of Fluc gene) ribonucleotide triphosphates , 0.4 µg pIVEX1.3-RL plasmid or 0.2 µg pT7CRL-FL plasmid, 0.5 µl T7 RNA polymerase (RNAP)  and nuclease-free water or crowding agents to a final volume of 20 µl, and incubated at 37°C for various period of time. After the reaction, the DNA template was removed by digestion with 0.5 µl DNase I  at 37°C for 15 min. Total mRNA was then quantified with the Quant-iT™ RiboGreen® RNA Reagent and Kit  according to the manufacturer's instructions.Transcription was carried out by mixing 0.4 µl 50× transcription buffer , 0.5 µl amino acid mixture (1 M each), 0.4 µl RNasin®, 0.45 µg Rluc mRNA or Fluc mRNA , and nuclease-free water or crowding agents to a final volume of 20 µl, and incubated at 30°C for 2 hr. Protein synthesis with the TNT® Coupled Transcription/Translation system (Promega) was carried out by mixing 10 µl WG lysate, 0.8 µl TNT® reaction buffer, 0.5 µl amino acid mixture (1 M each), 0.4 µl RNasin, 0.5 µl T7 RNAP, 2 µg pIVEX1.3-RL template or pT7CRL-FL template, and nuclease-free water or crowding agents to a final volume of 20 µl, and incubated at 30°C for 2 hr.in vitro transcription were treated with DNase I; the samples from the coupled transcription/translation were treated with DNase I first, and then extracted with phenol/chloroform solution and ethanol precipitation. Then 5 µl of each sample was mixed with 3 volumes of formaldehyde loading dye (New England BioLabs) and incubated at 65°C for 15 min. The samples were then electrophoresed on a 1% agarose/formaldehydel gel. Post-staining was done with SYBR® Green II RNA Gel Stain . Images were captured with a Foto/Analyst PC Image .The samples from the NcoI-XmaI fragment of the plasmid pIVEX1.3-RL) as a template. After agarose gel electrophoresis, RNA was transferred to a positively charged nylon membrane . The methods for RNA transfer, pre-hybridization, hybridization with a DIG-labeled probe, low stringency washing and high stringency washing were carried out following the NorthernMax® protocol . The DIG label was then detected by chemiluminescence (exposed to X-ray films) according to the manufacturer's manual (Roche).For Northern blotting, a digoxigenin (DIG)-labeled DNA probe was synthesized by random primed labeling with a DIG-High Prime Labeling and Detection kit (Roche) using the Rluc gene (in vitro translation solutions was separated on a 12% SDS polyacrylamide gel (SDS-PAGE). The proteins were then electrically transferred to a PVDF membrane for Western blotting analysis. The primary antibody was a rabbit anti-Renilla luciferase or a rabbit anti-Firefly luciferase polyclonal antibody  and the secondary antibody was a goat anti-Rabbit IgG (whole molecule) conjugated with Peroxidase . The protein bands on PVDF membranes were visualized on X-ray films following exposure of the membranes to SuperSignal® West Pico Chemiluminescent Substrate .Five µl each of the Renilla luciferase Assay System kit  according to the manufacturer's manual. The relative luminescence unit (RLU) for each sample was measured with a Modulus™ system . The measured RLU was converted to weight concentration according to a calibration curve obtained with the Rluc standard.Total Rluc protein was quantified based on Western blotting analysis as before Figure S1Northern blotting analysis of the Rluc mRNA produced in coupled transcription/translation. Each reaction solution (after 2 hr incubation) was treated with DNase I to remove DNA template. The Rluc mRNA was then extracted with phenol/chloroform and ethanol precipitation before being subjected to electrophoresis.(DOCX)Click here for additional data file.Figure S2Effects of PEG-8000 (PEG), Ficoll-70 (F-70) and Ficoll-400 (F-400) on in vitro transcription under different conditions. (a) In transcription buffer at 30°C; (b) In coupled transcription/translation buffer at 30°C; (c) In transcription buffer at 37°C; (d) In coupled transcription/translation buffer at 37°C. In all panels, each data point is the mean of triplicate values; error bars indicate the standard deviation from the mean.(DOCX)Click here for additional data file.Figure S3Cell-free protein expression under macromolecular crowding conditions in the PURExpress™ system. (a) Active Rluc protein yields from in vitro translation; (b) Active Rluc protein yields from coupled in vitro transcription/translation; (c) Northern blotting analysis of the Rluc mRNA in coupled in vitro transcription/translation (after 2 hr incubation).(DOCX)Click here for additional data file.Figure S4Agarose gel separation of the in vitro transcription solutions with (+) or without (−) Ficoll-70  added. Two control samples, one without T7 RNAP (control #1) and the other without rNTPs (control #2) were loaded for comparison. The band corresponding to the DNA/RNA-RNAP complex was absent in control #1, and appeared in control #2 but was not as dense as that observed in the samples (at 10 min).(DOCX)Click here for additional data file.Table S1Estimation of the macromolecular concentrations of different in vitro translation solutions.(DOCX)Click here for additional data file.