CYC1 and ADH1, contain cryptic promoters that generate abundant convergent antisense-transcription in Saccharomyces cerevisiae. Moreover we show that aberrant, truncating 3′ –end formation is often associated with regulated transcription in TAP-tagged strains. Importantly, the steady-state level of both 3′ –truncated and antisense transcription products is locus dependent. Using TAP and GFP-tagged strains we show that the transcriptional state of the gene-of-interest induces changes to 3′ –end formation by alternative polyadenylation and antisense transcription from a universal 3′ UTR. This means that these 3′ UTRs contains plastic features that can be molded to reflect the regulatory architecture of the locus rather than bringing their own regulatory paradigm to the gene-fusions as would be expected. Our work holds a cautionary note for studies utilizing tagged strains for quantitative biology, but also provides a new model for the study of promoter driven rewiring of 3′ –end formation and regulatory non-coding transcription.Epitope-tagging by homologous recombination is ubiquitously used to study gene expression, protein localization and function in yeast. This is generally thought to insulate the regulation of gene expression to that mediated by the promoter and coding regions because native 3′ UTR are replaced. Here we show that the 3′ UTRs,  Saccharomyces cerevisiae genetic model is beloved by all who have worked with it. Easy transformation and efficient homologous recombination has meant that researchers working with baker's yeast were among the first to embrace designer genomic knock-out  it means that collision is not the only mechanism of regulatory control imposed by non-coding antisense RNA  tag is prone to truncating cleavage and adenylation. In these genetically modified epitope-tagged strains, both aberrant 3′ -end formation and activation of 3′ UTR cryptic promoters is locus specific and dependent on the transcriptional state of the local genetic landscape. Together, our data provide a practical caution with regards to the use of expression plasmids and tagged strains for quantitative studies, but also conceptually promote the idea that 5′ driven regulation imposes reinforcing regulatory control over 3′ UTR dynamics.More than 30% of yeast transcripts show evidence for 3′ -end overlap –20. The MATa his3Δ0 leu2Δ0 met15Δ0 ura3Δ0) cells were grown in rich selective media  to exponential phase (OD600∼0.6). Transcription of Galactose (GAL) regulated genes was induced by addition of galactose (2%) and harvested after 1 h, snap frozen and stored at −80°C. For TAP-tagged strains, the cells were grown in YPEG  to an OD600 of ∼0.6 and harvested for RNA analysis. For GFP-tagged strains the cells were grown either in YPD  or YPE . Additional mutations were introduced into GFP and TAP tagged strains by homologous recombination of deletion cassettes re-amplified from confirmed mutant strains from the yeast deletion collection. The pGal plasmid was generated from a pRS423 backbone plasmid and 822 bases of the GAL1 promoter, a was a gift from Dr G. Perrone . The full-length open reading frame of NGL3 was amplified from genomic DNA  and ligated both in the forward and in the reverse orientation into the Bam HI site of this plasmid.For plasmid expression, BY4741 -Test (PAT) 3′ RACE assays on RNA isolated from cells before and after galactose induction. A version of the PAT assay, called ePAT and its sister assay TVN-PAT, are assays for detection of adenylated 3′ ends of RNA with either the full native poly(A)-tail or a minimal poly(A12)-tail, respectively  and 3′ UTR. Sanger sequencing of this amplification product revealed that cleavage and polyadenylation occurred at −176 bases upstream of the Gal1 start codon .The d Figure . The laty Figure ). Using d Figure . This suCYC1 3′ UTR might have driven transcription of the full-length NGL3 transcript in this reverse orientation. Being fully in reverse complement, such a transcript would contain no premature stop codons or other obvious triggers of decay. Whereas any other antisense transcription from this 3′ UTR would be expected to hit numerous such sites and be rapidly destabilized by nonsense mediated decay in the empty plasmid or NGL3-forward plasmid. To determine if antisense transcription did indeed proceed from within the CYC1 3′ UTR and termination sequence, we designed an additional primer + 61 bases after the native CYC1 stop codon but in reverse complement. A strong signal (second panel) from the empty plasmid, the NGL3-forward plasmid, and the NGL3-reverse plasmid confirmed that abundant antisense transcripts proceeded from the CYC1 3′ UTR. The site of 3′ cleavage and adenylation was different in each case , the adenylation sites presumably reflecting the different DNA sequences upstream of the CYC1 3′ UTR region in each plasmid. Galactose mediated induction of forward transcription did not appear to influence the level of reverse transcription products generated from this multicopy (2 micron) plasmid. This was illustrated in the pGAL-NGL3-F samples where forward transcription was strongly induced by galactose, but the level of the ∼300 bp antisense product driven from the cryptic promoter in the 3′ UTR remained at a constant level  emanating from the CYC1-UTR1 intragenic region . Interestingly, the convergent transcription and overlap of the 3′ UTRs of CYC1 and UTR1 was described in the earliest literature describing the use of this 3′ UTR . Albeit in this case we found no prior evidence for a promoter activity in the 210 bases of ADH1-MHF1 intragenic sequence. Nonetheless, we asked if this 3′ UTR inherited a propensity for overlapping 3′ UTRs when utilized in heterologous contexts, and turned to the GFP-tagged collection (CIT1-GFP and CIT3-GFP) as indicated. Previous work from our lab and by others showed that expression of both CIT1 and CIT3 was induced by starvation  in addition to the major full-length transcript , was associated with strong antisense transcription (bottom panel). Such antisense transcription is not normally monitored, and thus remains unaccounted for by standard measures of gene expression. To ask if it quantitatively influenced apparent forward transcription, we performed quantitative reverse transcription PCR (qPCR) with primers designed towards 3 positions across the GFP fusion as indicated by the schematic . Therefore, to determine if the protein was expressed, but at levels too low for fluorescence visualization, we performed western blot analysis comparing the accumulation of Cit1-GFP and Cit3-GFP. To capture the expression difference indicated by quantitation of mRNA abundance  suggested a carbon-source dependent inverse relationship between CIT3 expression and convergent transcription of SUT822, a non-coding RNA that initiates from a cryptic promoter ∼2/3rd into the CIT3 open reading frame  or ethanol (YPE) as a carbon source  and is also observed in the untagged CIT3 gene (data not shown). As predicted, the induction of CIT3-GFP expression reduced the expression of the cryptic antisense transcript (second panel) and the increase in forward transcription in the Δfkh2 mutant was associated with a decrease in reverse transcription. Finally, placement of a primer (P5) to detect the last ∼150 bases of the endogenous SUT822 non-coding RNA . The adenylation sites for SUT822 captured here were ∼75 bases upstream of the CIT3 start codon. CUTs and SUTs often display heterogeneous 5′ and 3′ ends  from previous work . The amplicon intensity representing each of the different 3′ UTR-isoforms appeared to be strain specific, suggesting that features within the local genomic landscape influence the steady-state level of these truncated products. That is, RGI1-TAP was biased towards accumulation of the shortest truncating-isoform whereas HXT7-TAP is biased towards the full-length transcript correctly processed from cleavage and adenylation within the ADH1 3′ UTR.To confirm the identity of the truncation products and to determine the exact position of the adenylation sites, two additional primers were designed towards elements universal to the TAP-tagged strains; the start of the Calmodulin Binding Protein (CBP) sequence, and straddling the duplicated protein-A sequence . Sanger sequencing of the 3 distinct amplicons that result from the P2 primer Figure , confirmhxk2/HXK1-TAP strain to search for expression of the hypothetical tagless-Hxk1 -tails. The sequencing supported this, with the average sequenced tail of the truncated mRNA being 12 A-residues (n = 8), whereas the average sequenced length for the correct distal-most adenylation site was 38 A-residues (n = 2). Short poly(A)-tails such as these are associated with non-coding RNA, transcriptional-stall products and decay intermediates . Because1 Figure . We coul1 Figure , and any1 Figure .RGI1-TAP strain. The literature provides evidence for transcriptional regulation of the RGI1 locus by the general transcriptional repressor Tup1/Cyc8 -tailed 3′ truncated transcripts. They could be products of transcription generated by induction of premature cleavage and adenylation. Alternatively they could be generated during failed translation by activation of cytoplasmic surveillance such as no-go or non-stop decay ,35. To bup1/Cyc8 ,29. To ae Figure . WhereasADH1 3′ UTR that terminates the TAP and GFP tagged strains used in this study. The adenylation-site choice and antisense induction from the ADH1 3′ UTR is locus specific  in comparison to the non-coding transcripts that run antisense to either the empty plasmid or the forward oriented NGL3 gene -tailed and presumably stably translated in the cytoplasm, the nonsense antisense products are slightly less abundant, short-tailed, and likely subject to rapid decay after synthesis. The transcription initiation rates from the cryptic promoter are not expected to be different in the different plasmids, but there must exist an early metabolic branch-point with built-in recognition of RNA that encodes protein from the RNA that does not.It is not yet clear what mechanistically differentiates the early metabolism of coding from of non-coding RNA. The outcome of cryptic transcription from the e Figure . Whereaset al., (2011) show both in human genome-wide data and gene-by-gene plasmid-based experiments that transcriptional activity influences 3′ UTR choice (Drosophila, e.g. the rate of transcription elongation controls adenylation-site choice in the transcription factor Polo, with slower elongation favouring a proximal site for adenylation (The close physical association of promoters with sites for 3′ -end processing by gene loops provides a convenient mechanism for RNA polymerase II recycling, and in general for the insulation of the regulation of individual genomic loci . There iR choice . Albeit,R choice  where trnylation . It willThe induction of truncating 3′ end formation at the start of each of the duplicated Protein A moieties of the TAP tag Figure . Indeed bone-fide 3′ -end formation is not easily distinguished from 3′ -ends generated by the adenylation that is associated with mRNA surveillance, or stalling of translation and associated stimulation of no-go decay (One caveat to the interpretation of inappropriate truncating adenylation is that go decay . We alsogo decay  data notIn sum, our data provide yet another novel application for the yeast genomic resource of TAP and GFP tagged libraries. That is, as new models to understand the regulatory paradigms for promoter driven control of 3′ dynamics, antisense convergent transcription and 3′ -end formation.


In the Metazoan nucleus, core histones assemble the genomic DNA to form nucleosome arrays, which are further compacted into dense chromatin structures by the linker histone H1. The extraordinary density of chromatin creates an obstacle for accessing the genetic information. Regulation of chromatin dynamics is therefore critical to cellular homeostasis, and histone chaperones serve as prominent players in these processes. In the current study, we examined the role of specific histone chaperones in negotiating the inherently repressive chromatin structure during transcriptional activation.Using a model promoter, we demonstrate that the human nucleosome assembly protein family members hNap1 and SET/Taf1β stimulate transcription in vitro during pre-initiation complex formation, prior to elongation. This stimulatory effect is dependent upon the presence of activators, p300, and Acetyl-CoA. We show that transcription from our chromatin template is strongly repressed by H1, and that both histone chaperones enhance RNA synthesis by overcoming H1-induced repression. Importantly, both hNap1 and SET/Taf1β directly bind H1, and function to enhance transcription by evicting the linker histone from chromatin reconstituted with H1. In vivo studies demonstrate that SET/Taf1β, but not hNap1, strongly stimulates activated transcription from the chromosomally-integrated model promoter, consistent with the observation that SET/Taf1β is nuclear, whereas hNap1 is primarily cytoplasmic. Together, these observations indicate that SET/Taf1β may serve as a critical regulator of H1 dynamics and gene activation in vivo.These studies uncover a novel function for SET that mechanistically couples transcriptional derepression with H1 dynamics. Furthermore, they underscore the significance of chaperone-dependent H1 displacement as an essential early step in the transition of a promoter from a dense chromatin state into one that is permissive to transcription factor binding and robust activation. In the Metazoan nuclei, the extraordinary compaction of the genetic material is achieved through organization of the chromosomal DNA into extensively folded chromatin structures. Nucleosomes, the most basic unit of chromatin, are formed by wrapping 147 bp of DNA around two copies each of the core histones H2A, H2B, H3, and H4  were incubated in the presence of purified, recombinant activators , the indicated histone chaperone (0.67 µM), and Ac-CoA 100 µM) , and was present through termination (stop). To assess whether hNAP1 functioned optimally at a specific, temporally-distinct step in the transcription assay  at the beginning of the PIC assembly step. Figure 0 repressed activated transcription (lanes 5–7). However, addition of increasing amounts of hNAP1 reversed rH10 repression in a dose-dependent manner (lanes 8–10). Addition of rH10 to reactions performed on naked DNA templates produced only a modest effect on transcription, indicating that H1-repression requires a chromatin context . To verify the incorporation of the linker histone into our chromatin templates, we measured salt-dependent chromatin condensation ) by salt deposition as described previously . Briefly2, 10 % glycerol, 0.5 mM EDTA, 0.5 mM DTT). RNA synthesis was initiated by addition of 224 μM ATP, 224 μM CTP, and 0.75 μM [α-32P] UTP (3000 Ci/mmol), and the elongation reaction incubated for an additional 40 min. Reactions were stopped, RNA was isolated following digestion with Proteinase K, and transcripts were separated by denaturing PAGE and analyzed using ImageQuant® software. Molecular weight markers (radiolabeled HpaII-digested pBR322) were used to estimate the sizes of the RNA products. A labeled 622-bp DNA fragment was added to each reaction mixture as a recovery standard. Unless otherwise indicated, constant amounts of the following purified, recombinant proteins were used: pCREB (0.03 μM), Tax (0.06 μM), p300 (0.02 μM), hNAP1 (0.67 μM), SET/Taf1β (0.67 μM) and Ac-CoA (100 μM). Experiments in which the chaperone was added and removed at a specific step were performed as follows: after the indicated incubation, the supernatant was removed by magnetic isolation of the immobilized transcription templates, and replaced with supernatant generated in parallel, but lacking the histone chaperone. Using this approach, the overall steady-state concentrations of each reaction component remained constant. All transcription experiments were performed at least three times, with each replicate giving essentially identical results.The immobilized chromatin-assembled promoter templates  were used in transcription assays as previously described , 18. BriBinding reactions were prepared as described for the in vitro transcription assays, except the reactions were increased 20-fold (1 μg of DNA) to enable visualization of template-bound proteins by Coomassie-stained gels. Unless otherwise indicated, reactions contained purified, recombinant pCREB (0.03 μM), Tax (0.06 μM), p300 (0.02 μM), hNAP1 or SET/Taf1β (0.67 μM), and Ac-CoA (100 μM) in 0.5× TM buffer. At the completion of the binding reaction, the bead-bound templates were isolated by magnetic separation, and washed twice with 0.5× TM buffer. Template-bound proteins were separated by 15 % SDS-PAGE and analyzed by Western blot Fig. a, b or C2 and subjected to centrifugation for 10 min at 16,100g. DNA from both the pellet and supernatant fractions was purified by phenol/chloroform extraction and ethanol precipitation, and analyzed by 1 % agarose gel electrophoresis followed by ethidium bromide staining. DNA from each fraction was quantified using ImageQuant® software and plotted as percentage of total DNA in each reaction.Magnesium-dependent chromatin condensation assays were performed as previously described , 84. Chr2, 1 mM EDTA, 20 % glycerol, 0.5 % NP40, 0.1 % Chaps, and 100 ng/μl BSA) for 1 h at 4 °C, followed by incubation with an equal concentration of rH1.0 for an additional 16 h at 4 °C. The beads were washed extensively, and GST-bound proteins analyzed by 15 % SDS-PAGE and Coomassie blue staining   and Alexa Fluor® 594 goat anti-mouse IgG (H+L)  secondary antibodies. An Olympus IX81 spinning disk confocal microscope with 60×/1.42 NA objective and a Photometrics Cascade II CCD camera with SlideBook (Intelligent Imaging Innovations) software were used for fluorescence images. Metamorph software was used to process images.Western blot and immunofluorescence labeling were performed according to the manufacturer’s instructions. Anti-hNAP1 , anti-SET/Taf1β , anti-p300 , anti-H1 , anti-HA (Santa Cruz Biotechnology #SC-805), and anti-GAPDH (Santa Cruz Biotechnology #SC-137179) were used for western blot analysis. Immunofluorescence labeling in HEK-293T cells was performed using anti-hNAP1  and anti-SET/Taf1β  primary antibodies and Alexa Fluorsame gel) was spliced to either remove irrelevant lanes or enhance presentation of the data. Minimal and uniform brightness/contrast was applied to some gel images.Solid vertical lines on some gels indicate where the image (from the


Transient induction of the Src oncoprotein in a non-transformed breast cell line can initiate an epigenetic switch to a cancer cell via a positive feedback loop that involves activation of the signal transducer and activator of transcription 3 protein (STAT3) and NF-κB transcription factors.We show that during the transformation process, nucleosome-depleted regions ) are largely unchanged and that both before and during transformation, STAT3 binds almost exclusively to previously open chromatin regions. Roughly, a third of the transformation-inducible genes require STAT3 for the induction. STAT3 and NF-κB appear to drive the regulation of different gene sets during the transformation process. Interestingly, STAT3 directly regulates the expression of NFKB1, which encodes a subunit of NF-κB, and IL6, a cytokine that stimulates STAT3 activity. Lastly, many STAT3 binding sites are also bound by FOS and the expression of several AP-1 factors is altered during transformation in a STAT3-dependent manner, suggesting that STAT3 may cooperate with AP-1 proteins.These observations uncover additional complexities to the inflammatory feedback loop that are likely to contribute to the epigenetic switch. In addition, gene expression changes during transformation, whether driven by pre-existing or induced transcription factors, occur largely through pre-existing nucleosome-depleted regions.The online version of this article (doi:10.1186/1756-8935-8-7) contains supplementary material, which is available to authorized users. Oncogenic transformation is often driven by the genetic alteration of protein kinases, which results in inappropriate activity of downstream transcription factors (TFs). These TFs cause changes in gene expression that ultimately mediate the phenotypes of a transformed cancerous cell type such as invasion, metastasis, loss of contact-mediated growth inhibition, uncontrolled proliferation, and tumor formation. The signal transducer and activator of transcription 3 protein (STAT3) is an important mediator of the transcriptional changes during this process, and it is implicated in many types of cancers –4.705) and serine 727 (Ser727) in response to many cytokines and growth factors [705 phosphorylation is critical for dimerization, nuclear localization, and gene activation, while Ser727 phosphorylation plays a minor role in modulating STAT3 activity. The oncogenic kinase SRC phosphorylates STAT3 in vitro and co-immunoprecipitates with STAT3 from cellular extracts [705-phosphorylated STAT3. Inhibition of STAT3 activity impairs proliferation and induces apoptosis [STAT3 is a DNA-binding transcription factor (TF) that is part of a larger family consisting of seven members. STAT3 contains an SH2 domain, and it is phosphorylated at tyrosine 705 -seq or DNase-seq reveals that human cells typically have approximately 100,000 such open chromatin regions, with many differences in the genomic location of such regions among different cell types , 16. Celde novo formation of FAIRE regions, but rather due to an increase in signal at sites that already had low-amplitude FAIRE enrichment  in non-transformed ER-Src cells, presumably representing a basal level of ER-Src signaling and hence STAT3 function. Upon transformation, STAT3-bound sites  more than double at 4 h , 12 h , and 36 h  post induction. In accord with STAT3 binding in macrophages [STAT3 is critical for oncogenic transformation, and the STAT3 motif is overrepresented within FAIRE regions associated with differentially regulated genes . The enrichment of AP-1 motifs within FAIRE regions  is a direct transcriptional target of STAT3. These additional complexities to the inflammatory feedback loop are likely to contribute to the epigenetic switch.http://genome.ucsc.edu/ENCODE/. Cultures were grown to 70% confluency; then treated with either EtOH or TAM for 4, 12, 24, or 36 h, as detailed in [MCF10A-ER-Src cells were grown and ChIP DNA was isolated as per standard ENCODE protocols. A detailed protocol is available at ailed in ; and harhttp://genome.ucsc.edu/ENCODE/ and [Cells were grown as above and a full detailed FAIRE-seq protocol is available at ODE/ and . Briefly7 cells and transferred to a 2-ml screw-capped tube with rubber seal containing 1 ml of 500 μM glass beads. Cells were disrupted using a mini bead-beater  set to homogenize for five 1-min sessions with 2-min incubations on ice between sessions. Lysate was transferred to 1.5-ml tubes in 300-μl aliquots and sonicated for 15 min using a Bioruptor UCD-200 (Diagenode) set to pulse on high for 30 s followed by 30 s of rest in a 4°C water bath. Cellular debris was cleared from the lysate by spinning at 15,000 × g for 5 min at 4°C.Cells were resuspended in 1 ml of lysis buffer  per 10g for 5 min, and the aqueous fraction was transferred to a fresh 1.5-ml tube. An additional phenol/chloroform extraction was performed by adding an equal volume of phenol/chloroform to the isolated aqueous fraction. Finally, an equal volume of chloroform ) was added to the aqueous fraction and spun at 12,000 × g for 5 min.An equal volume of phenol/chloroform  saturated with 10 mM Tris, pH 8.0, 1 mM EDTA) was added to the lysate, vortexed, and spun at 12,000 × g for 30 min at 4°C, and the DNA pellet was washed with 500 μl ice-cold 70% ethanol. Upon removing the supernatant, the pellet was dried in a speed-vac. The FAIRE DNA was resuspended in 50 μl of 10 mM Tris-HCl, pH 7.5, and incubated with 1 μl of 10 mg/ml RNase A for 1 h at 37°C. The FAIRE DNA was cleaned up by performing an additional phenol/chloroform reaction and ethanol precipitation (as described above).DNA recovered in aqueous phase was precipitated by adding 3 M sodium acetate (pH 5.2) to a final concentration of 0.3 M and adding 1 μl of 20 mg/ml glycogen, which were mixed by inverting. Two volumes of 95% ethanol were added, mixed by inverting, and incubated at −20°C overnight. DNA was precipitated by spinning at 15,000 × w/v sucrose) and run at 120 V for 1 h. The brightest portion of the smear was excised (150–200 bp), and DNA was recovered using the Qiagen Gel Extraction Kit (#28704). FAIRE DNA libraries were sequenced using the Illumina GAII system to generate 36-bp single-end reads.Libraries were prepared using 100 ng of FAIRE DNA. The ends of the DNA fragments were made blunt using the End-It™ DNA End-Repair Kit (Epicentre #ER0720), an A-overhang was added using Klenow exo- (Epicentre #KL06041K), and double-stranded adapters containing a T-overhang (Illumina #1000521) were ligated to the DNA fragments using T4 DNA ligase (NEB #M0202S). Libraries were then amplified using PfuUltra II (Stratagene #600670) and primers complimentary to the adapters (Illumina #1000537 and 1000538). Amplified products were loaded into a 2% agarose gel using sample loading buffer  were aligned to the UCSC hg19 build of the human genome using Bowtie. Each altie[ZINBA, using 3tie[ZINBA and 3. T[BEDTools. Data haThe union set of FAIRE sites enriched throughout the time course were combined and analyzed to identify sites with either an increase or a decrease of FAIRE signal. The occurrence of each active regulatory element was evaluated to determine the extent to which it differed with respect to the other time points, either present at one time point and absent at the others or vice versa. The count of reads aligning to each region identified in the union set was recorded for each time point and normalized by the total number of sequencing reads for that sample. Here, the mean normalized values for the replicates for each time point were compared to the mean values of the replicates for the remaining time points and divided by the sum of standard deviations. To estimate the differences expected by chance, the individual replicates were randomly permuted and the scores were calculated as above. Those FAIRE sites whose scores exceeded the scores calculated at random (0.05) were called as differential. Thus, a small fraction of called differential sites are likely to be false positives.HOMER[Motif enrichment for the set of sequences within all FAIRE sites discovered throughout the time course and those identified as differential was performed using HOMER. For allP < 0.005). These randomly selected gene groups contained the same number of genes.Motif enrichment for differentially expressed genes was calculated by identifying the set of FAIRE sites within ±500 kb of the genes in each group. The sequence within these sites was compared to the set of FAIRE sites identified from nearby randomly selected gene groups using HOMER (http://genome.ucsc.edu/ENCODE/) and was immunoprecipitated with anti-phospho-STAT3 antibody  and anti-FOS antibody , or input DNA  were end repaired with calf intestinal alkaline phosphatase  and sent for sequencing to the Stanford Center for Genomics and Personalized Medicine. ChIPs for anti-NF-κB  anti-E2F4 (SC-866x), and anti-MYC (SC-764) were prepared as above but using one biological replicate. Library preparation and Illumina (USA) sequencing were carried out as per Illumina protocols, and a detailed protocol is available at http://genome.ucsc.edu/ENCODE/. Sequence reads (32 nucleotides) were mapped to the Homo sapiens genome (hg19) using Bowtie[MACS v1.4 [P value threshold of 10−9, “auto” redundant read setting, using input to control for local genomic biases. PeakSplitter[MACS called peaks into subpeaks of local maxima using default settings. STAT3 subpeaks were called as differential if they did not overlap a STAT3-bound subpeak in the non-transformed control population  and had a fold change greater than the mean + 1× standard deviation of all peaks within the population. Fold change in STAT3 ChIP-seq signal was calculated as read counts per region per million mapped reads divided by the corresponding control ChIP signal of that region. A smoothing value of 10 was added to the read count of each region in the transformed and control samples. Data has been submitted to the NCBI SRA database under project PRJNA270300 [see Additional files ChIP DNA  was prepared as above , intronic, exonic, 5′ UTR or 3′ UTR) was calculated and compared to the percentage of the genome within each category. Significance was calculated using the single-sided binomial test as implemented in the binom function in R.STAT3 peak summits, defined as the local maxima of read counts within a peak, were mapped to the nearest RefSeq TSS, incorporating strandedness, using an in-house script. Histograms were plotted using the P value) STAT3 or FOS ChIP-seq subpeak summit locations were determined and the sequence ±50 bp was extracted and repeat masked. A 5-order Markov model was used as the background set and was extracted from the repeat-masked, non-redundant set of FAIRE-seq cis-regulatory elements. Parallel MEME was run with the following settings: zoops, revcomp, minw = (4–26), and maxw = (6–30). For STAT3 and FOS, the top motif corresponded to the respective known canonical motif. The motif position-specific frequency matrices derived from MEME were used as input to FIMO[P value threshold of 10–4.The top 10,000 , gene probe IDs, with the corresponding log2 fold change, were uploaded into and analyzed by IPA  using default settings. Molecular signaling pathways were visualized using IPA where a gray-shaded node represented a subpeak binding site located within the putative regulatory region, as defined by GREAT, of that gene/molecule. The biological relationship between two molecules is represented as a line and is based on professionally curated literature findings. The relationships can be direct or indirect.Genomic locations of subpeak summits were submitted to the annotation tool GREAT using thMCF10A-ER-Src cells were reverse transfected with Dharmacon siRNAs  according to the manufacturer’s protocol. Briefly, cells were seeded in six-well plates containing 25 nM ON-TARGETplus SMARTpool STAT3 or ON-TARGETplus Non-targeting Pool and after 48 h of growth were treated with EtOH or tamoxifen (1 μM) for 4 or 24 h as per . For WesRNA  was prepared for arrays using the 3′ IVT Express Kit  as per manufacturer protocol—100 ng RNA and 15 amplification cycles. Amplified RNA was given to the Boston Children’s Hospital microarray core facility for hybridization to GeneChip Human Genome U133 plus 2.0 gene expression arrays  for hybridization and imaging as per manufacturer protocols. Data has been submitted to the NCBI GEO database under accession GSE64536.Membranes were probed using mouse-derived anti-phospho-STAT3 antibody from Cell Signaling Technologies, USA, and anti-β-actin from Sigma, USA. Bands were detected with IRDye 800-labeled goat anti-mouse IgG  and imaged using an Odyssey infrared imaging system .GREAT[DAVID[P value <10−4 and with an absolute log2 fold change >0.5. Those genes determined to be STAT3 and transformation regulated were determined by comparing EtOH to TAM samples under both siSCM and siSTAT3 conditions. Genes were selected as STAT3 independent if their differential expression was statistically insignificant upon siSTAT3 and had an absolute log2 fold change of <0.5 upon siSTAT3. The number of STAT3-dependent and STAT3-independent genes does not equal the total number of genes considered differential by transformation as many genes could not be unambiguously defined as “dependent” or “independent”.Gene expression arrays were background corrected and normalized and probe set expression values determined by the mas5 algorithm [see Additional files GREAT or DAVIDEAT[DAVID. Genes dDAVID, and probe sets annotated to the term “transcription factor activity” (GO:0003700) were selected. Normalized expression values for each gene are expressed as log2 fold change over siSCM 4-h EtOH-treated samples. Hierarchical clustering of the resultant expression matrix was carried out using the Pearson correlation and average linkage using the software package TMEV[All probe set IDs that were differentially regulated during transformation (see above) were submitted to kage TMEV.P value. “Promoter” regions are defined as −2,500 to +500 bp from RefSeq TSS. “Upstream/downstream” regions are defined as ±50 kbp from the RefSeq TSS, excluding the promoter region. The number of transformation-specific differential STAT3 ChIP-seq peaks were counted within these regions, normalized to peaks per 1 kbp, and plotted using a 1,000 gene rolling mean.Differentially expressed genes were defined as above. Non-redundant probe sets were used, discarding the probe set with the highest Supplementary Figures S1 to S12. Supplementary supporting evidence. For a description of each figure, see its associated legend. (PDF 8 MB)Additional file 1: All FAIRE sites. Genomic locations of all FAIRE-seq sites discovered in transformed and non-transformed MCF10A-ER-Src cells. (XLS 2 MB)Additional file 2: Differential FAIRE sites. Genomic locations of all FAIRE sites discovered in MCF10A-ER-Src cells that were determined to be differential during the time course of transformation. (XLS 481 KB)Additional file 3: MCF10A-ER-Src STAT3 ChIP-Seq 4-h TAM. Genomic locations of all STAT3 genomic binding sites, at a significance P value ≤1e − 09, discovered by ChIP-Seq in MCF10A-ER-Src cells treated with tamoxifen for 4 h. (XLS 3 MB)Additional file 4: MCF10A-ER-Src STAT3 ChIP-Seq 12-h TAM. Genomic locations of all STAT3 genomic binding sites, at a significance P value ≤1e − 09, discovered by ChIP-Seq in MCF10A-ER-Src cells treated with tamoxifen for 12 h. (XLS 2 MB)Additional file 5: MCF10A-ER-Src STAT3 ChIP-Seq 36-h TAM. Genomic locations of all STAT3 genomic binding sites, at a significance P value ≤1e − 09, discovered by ChIP-Seq in MCF10A-ER-Src cells treated with tamoxifen for 36 h. (XLS 3 MB)Additional file 6: ER-Src STAT3 peaks differential. Genomic locations of all STAT3 genomic binding sites determined to be differential during the time course of tamoxifen treatment. (XLS 147 KB)Additional file 7: MCF10A-ER-Src STAT3 ChIP-Seq EtOH. Genomic locations of all STAT3 genomic binding sites, at a significance P value ≤1e − 09, discovered by ChIP-Seq in MCF10A-ER-Src cells treated with EtOH. (XLS 990 KB)Additional file 8: MCF10A-ER-Src FOS ChIP-Seq 4-h TAM. Genomic locations of all FOS genomic binding sites, at a significance P value ≤1e − 09, discovered by ChIP-Seq in MCF10A-ER-Src cells treated with tamoxifen for 4 h. (XLS 6 MB)Additional file 9: MCF10A-ER-Src FOS ChIP-Seq 12-h TAM. Genomic locations of all FOS genomic binding sites, at a significance P value ≤1e − 09, discovered by ChIP-Seq in MCF10A-ER-Src cells treated with tamoxifen for 12 h. (XLS 6 MB)Additional file 10: MCF10A-ER-Src FOS ChIP-Seq 36-h TAM. Genomic locations of all FOS genomic binding sites, at a significance P value ≤1e − 09, discovered by ChIP-Seq in MCF10A-ER-Src cells treated with tamoxifen for 36 h. (XLS 5 MB)Additional file 11: ER-Src FOS peaks differential. Genomic locations of all FOS genomic binding sites determined to be differential during the time course of tamoxifen treatment. (XLS 129 KB)Additional file 12: MCF10A-ER-Src FOS ChIP-Seq EtOH. Genomic locations of all FOS genomic binding sites, at a significance P value ≤1e − 09, discovered by ChIP-Seq in MCF10A-ER-Src cells treated with EtOH. (XLS 5 MB)Additional file 13: Differential gene expression at 4-h TAM. Genes determined to be differentially regulated between MCF10A-ER-Src cells treated with ethanol or tamoxifen for 4 h and with a control scrambled siRNA. (XLS 75 KB)Additional file 14: Differential gene expression at 24-h TAM. Genes determined to be differentially regulated between MCF10A-ER-Src cells treated with ethanol or tamoxifen for 24 h and with a control scrambled siRNA. (XLS 263 KB)Additional file 15: STAT3-dependent differential gene expression at 4 h. Genes determined to be differentially regulated between MCF10A-ER-Src cells treated with ethanol or tamoxifen for 4 h and with a STAT3-specific siRNA. (XLS 27 KB)Additional file 16: STAT3-dependent differential gene expression at 24 h. Genes determined to be differentially regulated between MCF10A-ER-Src cells treated with ethanol or tamoxifen for 24 h and with a STAT3-specific siRNA. (XLS 120 KB)Additional file 17: RNA expression values. Raw RNA MAS5 expression values for MCF10A-ER-Src cells treated with tamoxifen or ethanol, and control siNEG (siSCM) or siSTAT3 at various time points. (XLS 19 MB)Additional file 18:


Despite a high sequence homology among four human RNAi-effectors Argonaute proteins and their coding sequences, the efficiency of ectopic overexpression of AGO3 and AGO4 coding sequences in human cells is greatly reduced as compared to AGO1 and AGO2. While investigating this phenomenon, we documented the existence of previously uncharacterized mechanism of gene expression regulation, which is manifested in greatly varying basal transcription levels from the RNApolII promoters depending on the promoter-proximal downstream sequences. Specifically, we show that distinct overexpression of Argonaute coding sequences cannot be explained by mRNA degradation in the cytoplasm or nucleus, and exhibits on transcriptional level. Furthermore, the first 1000–2000 nt located immediately downstream the promoter had the most critical influence on ectopic gene overexpression. The transcription inhibiting effect, associated with those downstream sequences, subsided with increasing distance to the promoter and positively correlated with promoter strength. We hypothesize that the same mechanism, which we named promoter proximal inhibition (PPI), could generally contribute to basal transcription levels of genes, and could be mainly responsible for the essence of difficult-to-express recombinant proteins. Finally, our data reveal that expression of recombinant proteins in human cells can be greatly enhanced by using more permissive promoter adjacent downstream sequences. Small non-coding regulatory RNAs including siRNAs and miRNAs mediate their function in association with proteins of Argonaute (AGO) family13467995131516in vivo. Thus, some authors suggested that differential distribution of rare codons over AGO3 and AGO4 coding sequences may enhance hydrolysis of their mRNAs in the cytoplasm during translation18In mammalian tissues Argonaute genes are expressed in different proportions depending on cell type and differentiation stage215161891820In this work, we confirm that AGO3 and AGO4 coding sequences are overexpressed with considerably lower efficacy as compared to AGO1 and AGO2 on both mRNA and protein levels in human cells. Furthermore, we demonstrate that impaired overexpression efficiency of AGO3 and AGO4 protein coding transcripts is independent on their translation on ribosomes and is manifested due to distinct velocity of mRNA synthesis in the cell nucleus. We also confirm that sequence-specific targeting via endogenous RNAi pathways cannot be responsible for this phenomenon. Interestingly, different regions of human AGO1, AGO2, AGO3 and AGO4 coding sequences had strong and varied impact on the amount of transcripts generated from CMV promoter when subcloned immediate downstream the promoter. Strikingly, the transcription efficacy among even highly homologous sequences varied up to 10–50 folds. At the same time, the observed impacts of conventional regulatory DNA sequences, including upstream enhancer of CMV promoter, Sp1 and Ap1 binding regions, on the transcription rate were much more modest. Finally, the inhibitory effects of downstream sequences were strongly dependent on their distance to the promoter and positively correlated with promoter strength. Based on this data, we suggest a hypothesis that retardation of RNApolII elongation on certain promoter-proximal DNA sequences could cause its collision with the following polymerases, thus prematurely terminating transcription. While in more distant regions from the promoter the processing rate of RNA polymerase could become more even or robust, this effect is only evident at the promoter proximal downstream sequences. It is feasible that besides the strength of a promoter, promoter-proximal downstream sequences could significantly contribute to basal transcription rate of many genes.We initiated this study after making an independent observation of the markedly different overexpression efficiency of human Argonautes coding sequences from an ectopic CMV promoter, both on mRNA and protein level in several common human cell lines 918. Desp9Previously, Grimm and colleagues hypothesized that a suboptimal rare codons distribution throughout the AGO3 and AGO4 mRNAs could explain the reduced translation efficiency of these proteins22We next tested whether nuclear mechanisms could contribute to uneven expression of ectopic AGO mRNAs. For this purpose, nuclear and cytoplasmic RNA fractions were isolated from U2OS cells 24 hours post-transfection with pAGO(1–4)LS-nk plasmids . SubsequAlternative polyadenylation is one of the well-known mechanisms of differential expression of pre-mRNAs in the nucleus. However, it could not explain the observed differences in exogenous AGO expression, because relative abundance of 5′-end promoter-proximal transcripts were similar to the abundance of 3′-end transcripts . PrematuNext, we aimed to determine which parts of the Argonautes coding sequences of approximately 2.5 kb total length could be responsible for their differential ectopic transcription. For this purpose we generated FLAG-AGO overexpression plasmids containing one of the three sequential deletions within their corresponding AGO encoding sequences . ExpressThe fact that the restoration of AGO4 mRNA was weaker after deleting the promoter-proximal 1 kb region of AGO4 cDNA indicates that the adjacent middle portion of the AGO4 coding sequence could also be inhibitory for transcription when located immediately downstream the promoter. To test this idea we cloned the portions of AGO coding sequences corresponding to the deleted AGO regions directly under the CMV promoter upstream the full-length luciferase gene (pAGO(1–4)(i-iii), We further found that the overexpression efficiency of recombinant full-length AGO transcripts varied 2-fold depending on whether a short 200 bp EGFP gene fragment was cloned in either forward or reverse orientation immediately downstream the CMV promoter . This unTo further corroborate that promoter-adjacent downstream sequences can critically affect transcription rate, we investigated whether the inhibitory effects of AGO3 and AGO4 sequences can be exempted by increasing their distance to the promoter. In accordance with this hypothesis, cloning of either truncated EGFP CDS  or protein-null full-length EGFP CDS (720 bp) upstream the FLAG-AGO encoding sequences restored the ectopic expression of both AGO3 and AGO4 approximately proportionally to the length of the upstream insert . SpecifiThese observations were also confirmed on the protein level. The overexpression capacities of recombinant FLAG-AGO3 and FLAG-AGO4 proteins were also restored to the levels of FLAG-AGO1 and FLAG-AGO2 after the full-length 714 bp EGFP gene fragment devoid of all internal ATG codons was inserted into pAGO(1–4)-wt plasmids directly downstream the promoters . ImportaThe fact that the expression of recombinant FLAG-AGO3 and FLAG-AGO4 sequences increased with their distancing from the ectopic CMV promoter further confirms the ability of promoter-adjacent downstream DNA to significantly affect the expression of the whole transcript. Notably, the currently well-characterized mechanisms of gene expression regulation in the nucleus including alternative polyadenylation, alternative splicing and RNA interference would not explain the observed phenomena, since none of those mechanisms relies significantly on the proximity to the promoter.To confirm that the inhibitory effect of AGO3 and AGO4 sequences was not restricted to only cytomegalovirus promoter, we have overexpressed human FLAG-AGO sequences from several other constitutive RNApolII promoters, including human elongation factor-1 alpha (EF1α), human ubiquitin C (UBC), mouse phosphoglycerate kinase 1 (mPGK) and herpex simplex virus thymidine kinase (TK) promoters, which are the most commonly used promoters for gene expression in mammals. We first compared the basal strengths of those promoters by measuring overexpression efficiency of AGO1 coding sequence in U2OS cells . The EF1After transient overexpression of all protein-null FLAG-AGO sequences under EF1α + e1, EF1α + e1 + i1, UBC + e1, UBC + e1 + i1, PGM or TK promoters in U2OS cells, the relative levels of each Argonaute transcript was analyzed with luciferase TaqMan assay . SurprisOur data also suggests that common downstream transcription factors binding sites including Sp1 and Ap1 had rather marginal effect on transcription from EF1α promoter. The presence of endogenous EF1α intron1 carrying five Sp1 and one Ap1 binding site increased the recombinant FLAG-AGO1 expression from EF1α promoter only 1.5-fold . The traDespite a significant (70–76% on DNA level) homology between four human full-length Argonaute CDSs  the rema27This work is primarily aimed to point out a surprising phenomenon which we encountered while investigating the putative mechanisms of differential overexpression capacity of Argonautes coding sequences in transfected human cells. Specifically, we demonstrate the existence of a hitherto uncharacterized mechanism of gene expression regulation which manifests in strong interference of promoter-proximal downstream DNA sequences in basal transcription rate from several constitutive RNApolII promoters. We found that different regions of human AGO1, AGO2, AGO3 and AGO4 coding sequences had strong and heterogenous impact on the amount of transcripts generated from CMV and other promoters when located downstream in close proximity to the promoter. Astoundingly, the difference in transcription efficacy among even highly homologous sequences was up to 10–50 folds. None of the currently known mechanisms could adequately explain this phenomenon, and we suggest a general hypothesis of how promoter proximal downstream DNA regions can affect transcription rate. Because an inhibitory effect of a given downstream sequence declined proportionally with the increasing distance to the promoter, this mechanism must harness the particularities of promoter adjacent sequences. Furthermore, inhibitory effects of downstream DNAs strongly correlated with the strength of the tested promoters, and only marginally manifested under very weak promoters. We hypothesize that certain downstream DNA sequences could facilitate RNA polymerase slowing or pausing, what in turn could lead to the increased incidence of collisions between leading and following RNA polymerases . This th283032As a second aspect, our data suggest an efficient and easy approach to boost the production of difficult-to-express recombinant RNA and proteins. We show that certain protein coding DNA sequences can be suppressive for transcription especially from strong promoters and, as in the case of AGO4 (and to a lesser extent AGO3), the expression could be drastically increased by cloning a more permissive downstream DNA sequence between a promoter and a coding sequence. As an example we demonstrate that the full-length EGFP coding sequence devoid from ATG codons can serve as a highly permissive promoter-adjuscent sequence that considerably restores/improves both mRNA and protein expression rates.Finally, our work introduces an update to the current knowledge on the regulation of endogenous human Argonautes genes. In almost all tissues and cell lines, the expression of AGO3 and AGO4 genes is lower as compared to AGO1 and AGO2. Initially, it was hypothesized that the same mechanism could be responsible for the impaired expression of both endogenous AGO3 and AGO4 mRNAs and ectopic AGO3 and AGO4 coding sequences182. U2OS and HeLa cells were grown in DMEM supplemented with 10% FBS, 1% non-essential amino acid and penicillin/streptomycin at 37 °C in 5% CO2. For the overexpression of AGO constructs cells were transiently transfected with 300 ng of corresponding plasmid DNA using Turbofect transfection reagent (Fermentas) according to the manufacturer’s protocol. In some experiments 100 ng pEGFP-C1 vector was co-transfected with plasmids overexpressing wild-type Argonautes. Next day after transfection cells were lysed in either Laemmli loading buffer or Qiazol reagent and used for either Western blot analysis or total RNA isolation. Actinomycin D was purchased at Sigma-Aldrich (cat. A1410), dissolved in DMSO and added to cells at 10 μg/mL final concentration. Isolation of nuclear and cytoplasmic fraction was performed as described inAll cell lines were obtained from the American Type Culture Collection (ATCC). In addition, all cell lines have been genetically authenticated by DKFZ (Heidelberg) core facility. MCF7 cell line was grown in αMEM supplemented with 10% FBS, 1% non-essential amino acid and penicillin/streptomycin at 37 °C in 5% COTo allow accurate quantification of Argonautes overexpression FLAG-AGO coding sequences were amplified from pIRESneo-FLAG/HA-AGO1, pIRESneo-FLAG/HA-AGO2, pIRESneo-FLAG/HA-AGO3 and pIRESneo-FLAG/HA-AGO4 plasmids originally described inTotal RNA was isolated with miRNAeasy kit (Qiagen) and treated with TurboDNAse (Ambion) for 40 min at 37 °C in a final volume of 20 μL. DNAse was inactivated by incubating samples with DNAse inactivating reagent (Ambion) for 20 min on a shaker at 37 °C and diluted 5 times in water. Next, 7.7 μL of the diluted DNAse-treated RNA was used for the cDNA synthesis and subsequent qPCR. Reverse transcriptase reactions contained 7.7 μL of purified DNAse-treated total RNA, 2.5 μM random hexamer primers, 1 × RT buffer, 0.25 mM each of dNTPs, 3.33 U/μl MultiScribe reverse transcriptase and 0.25 U/μl RNase inhibitor. RT reaction  were incubated for 10 min at 25 °C, 30 min at 48 °C, 5 min at 85 °C and then held at 4 °C. RT products were was further diluted ten times with RNAse-free water. All TaqMan probes and primers were designed using online Primer3 software and are listed in Cultured U2OS cells transfected with corresponding plasmid DNA (or U2OS cytoplasmic or nuclear lysates) were lysed in Laemmli loading buffer , separated SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked in Odyssey blocking buffer (LI-COR) or 5% non-fat milk containing 1xPBST for 1 h at RT. Incubation with anti-FLAG , anti-LaminB1 + B2 , anti-AGO1 , anti-EGFP , anti-α-tubulin , anti-β-actin , anti-GAPDH  was performed at 4 °C overnight in a 1:1000 dilution. After incubation with primary antibodies, membranes were washed twice in 1 × PBST for 10 min and incubated for 1 h at RT with infrared fluorescent anti-mouse IgG  or anti-rat IgG  diluted 1:10000 in Odyssey blocking buffer (LI-COR); or HRP-conjugated anti-mouse IgG  diluted 1:1000 in 5% non-fat milk containing 1xPBST. Membranes were washed four times for 10 min in PBST and signals were then acquired with Odyssey infrared imaging system (LI-COR) or luminescent detection.How to cite this article: Turchinovich, A. et al. Interference in transcription of overexpressed genes by promoter-proximal downstream sequences. Sci. Rep. 6, 30735; doi: 10.1038/srep30735 (2016).


An increasing number of noncoding RNAs (ncRNAs) have been implicated in various human diseases including cancer; however, the ncRNA transcriptome of hepatocellular carcinoma (HCC) is largely unexplored. We used CAGE to map transcription start sites across various types of human and mouse HCCs with emphasis on ncRNAs distant from protein-coding genes. Here, we report that retroviral LTR promoters, expressed in healthy tissues such as testis and placenta but not liver, are widely activated in liver tumors. Despite HCC heterogeneity, a subset of LTR-derived ncRNAs were more than 10-fold up-regulated in the vast majority of samples. HCCs with a high LTR activity mostly had a viral etiology, were less differentiated, and showed higher risk of recurrence. ChIP-seq data show that MYC and MAX are associated with ncRNA deregulation. Globally, CAGE enabled us to build a mammalian promoter map for HCC, which uncovers a new layer of complexity in HCC genomics. Hepatocellular carcinoma (HCC) accounts for 70%–85% of total liver cancers. The major risk factors of HCC are chronic HBV and HCV infections and alcohol . The devComprehensive transcriptome studies have revealed that a large proportion of mammalian genomes, including transposable elements (TEs), are transcribed . LTR retMdr2 knockout (KO) mouse HCC using CAGE, with special emphasis on ncRNAs distant from protein-coding genes. We show that a large proportion of the distal ncRNAs are LTR-derived in human and mouse HCC genomes. The CAGE data revealed three well-defined subclasses of human HCCs corresponding to high, intermediate, and low expression levels of a selected set of LTR ncRNAs, respectively. The LTR-high subclass was correlated with definite clinical features  and MYC pathway activation. ChIP-seq data indicate an active role for transcription factors such as MYC-based complexes in the deregulation of LTR-ncRNAs in HCC.The Cap Analysis of Gene Expression (CAGE) method has been widely used to identify transcription start sites (TSSs) of ncRNAs and messenger RNAs by capturing the capped 5′ ends of the RNAs. The reproducibility of CAGE for expression measurements has been demonstrated through many studies, including FANTOM  and ENCOWe sequenced CAGE libraries for 50 HCC tumor tissues and 50 matched nontumor (NT) tissues from patients with various etiologies, mostly HBV, HCV, and alcohol abuse. We also prepared samples from morphologically normal liver tissues collected at a distance from a liver metastasis of colon cancer in five patients. These last five samples are referred to as “normal” as opposed to the “nontumor” and control for gene signatures affected in morphologically normal liver tissue by nearby inflammation and virus activities. The total number of uniquely mapped reads is 1.7 billion—an average “mapped read count” of 16.0 million for tumors, 15.9 million for nontumors, and 19.5 million for normal tissues . CAGE peaks (corresponding to TSSs) were determined based on the 5′ position of sequenced reads using Paraclu .MALAT1. Indeed, median expression of proximal and distal transcripts are ∼20–30 times lower than coding transcripts in all the HCCs tumors, irrespective of the etiology , which is a known biomarker and a possible target for the treatment of HCC (http://www.repeatmasker.org) and RepBase  than those found for (1) non-up-regulated peaks (6.3%) , (2) up-regulated peaks in the antisense direction (7.1%) , (3) non-up-regulated peaks in the anti-sense direction (7.4%) , (4) LINE and SINE elements in sense and antisense (1%–7%) (We compared expression levels between a case group of 50 tumors and a control group of 50 matched nontumors using edgeR  to explot of HCC . About 4 RepBase  based on RepBase . Interes RepBase B. The frBase (−8 C. These n = 5) ; adult and fetus liver tissues from FANTOM5 (n = 2) ; and primary hepatocytes from FANTOM5 (n = 3) . As expected, the expression levels of LTRs in normal livers are much lower than in nontumor and tumor HCC tissues. More than half of the LTRs were not expressed at all in any of the 10 normal samples  (n = 3) and high-grade HCC (n = 4) tissues, we identified 740 significantly up-regulated distal peaks characteristic of high-grade HCC . As in the case of human HCC, ∼19% (140/740) of the up-regulated distal peaks were located within LTR elements with the same strand (Mdr2 KO mice starting from the preneoplastic inflammation stage (We previously reported that LTR activation is frequently observed in human and mouse stem cells . Given tiven HCC . We examC (>50%) . We sequon stage D. Figureon stage F. Taken −10; (2) fold change above 8.0; and (3) expression in at least 30 tumor samples. About one-third of the distal peaks (43/133) that passed the criteria coincided with LTRs  FDR threshold in human and mouse HCCs, we sought to select human ncRNA candidates appropriate for determining a molecular signature of HCC using the following stringent criteria: (1) FDR threshold below 1.0 × 10ith LTRs A. These ith LTRs C. Some oith LTRs D. In conWe submitted 15 of the 43 signature-LTRs to 3′ RACE validation , among which LTR-001 and 002 were further investigated by nested PCRs using 10 more primer pairs designed on different exons . Deep sequencing of 3′ RACE and PCR products using MiSeq successfully determined the transcript structures of 14 of them, among which 13 had at least one splice site . The 33 transcripts detected, including splice variants, were all predicted to be ncRNAs with low coding potential by the Coding-Potential Assessment Tool (CPAT)  HCC samples and in a validation set of 21 new T/NT HCC samples. The primer sequences are listed in Supplemental Table S10. Consistent with the CAGE data, the expression of these LTRs was more than 10-fold higher in ∼60%–80% of the tumor samples compared to the matched NT in the working set C. The reP = 0.011). A correlation with patient outcome was irrelevant to our purpose due to the fact that the majority of the patients with LTR-low HCC received a hepatic graft, improving their outcome compared to patients with LTR-high HCC who benefited from a partial hepatectomy.A multivariate-analysis based on the set of clinical parameters listed in MYC and AKT1 activation. These results suggest that the LTR expression level be taken as a new characteristic in the definition of HCC molecular subclasses, corresponding to an as-yet unsuspected layer of complexity in the genomics of HCC.Finally, to gain insight on the molecular pathways activated in the different HCC subclasses, we performed a gene set enrichment analysis (GSEA) from the CAGE transcriptome. We found a great enrichment of genes related to cell cycle and replication in the LTR-high subclass compared to the LTR-low one E,F. InteTo cast light on the mechanism of up-regulation of ncRNAs, we combined our data for human HCCs with the large set of data for HepG2 cells provided by ENCODE. We first examined whether the up-regulated distal peaks that we found in human HCCs see A were alP-value, and ARID3A (AT-rich interactive domain-containing protein 3), which had the highest binding counts . We then investigated which pairs of TFs shared common targets using the Jaccard index. More specifically, we generated a cobinding network, in which each node represents a transcription factor and each edge represents a cobinding pair, whose Jaccard Index is above 0.5  around the active peaks was highly enriched in transcription factor binding sites, with five or more footprints at more than half of the peaks C. We thebove 0.5 D. Six trbove 0.5 E. In addSMYD3, which encodes a lysine methyltransferase involved in the methylation of MAP3K2, increasing MAP kinase signaling and promoting the formation of Ras-driven carcinomas (SMYD3 was unchanged at 12 h but significantly lowered at 48 h (IGFBP1 (insulin-like growth factor binding protein 1) has important inhibitory role in the development and/or growth of HCC, and RBBP6 (retinoblastoma binding protein) acts as a cell-cycle regulator.We finally performed knockdown experiments to investigate whether LTR-derived ncRNAs have any functional role in the regulatory network of HepG2 cells. Because recurrently activated LTR-derived ncRNAs are enriched in the nuclear fraction of HepG2 cells F, we desrcinomas . We foun at 48 h H. HoweveMdr2 KO mouse model of HCC using CAGE. This unique resource provides a mammalian promoter map for liver cancer at single-nucleotide resolution, which enables us to connect promoter activities with surrounding DNA regulatory elements, including LTR retroviral promoters and transcription factor binding sites. The comparison of the expression levels between tumors and matched nontumor tissues revealed that more than 14,000 promoters are significantly up-regulated in HCC tumors, one-third of which are at least 5 kb away from the promoters of known protein coding genes. Remarkably, 20% (935/4756) of the up-regulated distal peaks are LTR retroviral promoters, which are mostly silenced in normal hepatocytes and liver tissues. Nevertheless, a subset of the LTRs is strongly expressed in reproduction tissues, such as testis and placenta, indicating a highly tissue-specific regulation of the LTRs. The mechanisms of regulation of the LTRs was previously investigated in iPS and ES cells, which express high levels of specific LTR families, namely, LTR7 and HERVH. An appropriate activation of these LTRs was shown to be essential for iPS reprogramming . This shows that young repetitive elements are not largely activated in liver cancer.In this study, we used tags uniquely mapped to the genomes. This strategy might miss transcripts derived from young ERV elements that accumulate few mutations. To assess the potential impact of multimap tags that mapped to multiple genomic loci , we have performed analyses of repetitive elements at the family level. We measured expression values for each repeat family defined by RepeatMasker in the human and mouse genomes based on all tags, including multimap tags, and compared tumors with nontumors using edgeR. We confirmed that various LTR families are significantly up-regulated in tumors, whereas most young ERV elements, in particular, human ERVK (LTR5_Hs), SVA, GPC3 activation  in the vast majority of the HCCs studied. For example, LTR-007, which was totally suppressed in normal liver and most other healthy tissues, was more than 10-fold up-regulated in ∼80% of the HCC tumors. This high frequency of LTR-007 activation is comparable to the frequencies of the tivation  and TP53s in HCC . In addis in HCC , suggestLiver tissues were collected from 50 patients resected for HCC and five patients resected for metastatic liver colon cancer. The latter were used as control samples. The ethics evaluation committee of the Inserm  and the ethical review committee of RIKEN (H24-4) approved the use of human liver samples in this study. Informed consent was obtained from each subject. Total RNAs were extracted from the tissues using a mirVana kit (Ambion). The quality of the RNA samples was assessed using an Agilent RNA 6000 Nano kit . We prepared CAGE libraries following the protocol described in detail in http://compbio.gsc.riken.jp/rnadust.html). We then aligned the extracted CAGE tags to the human genome (hg19/GRCh37 assembly) using BWA v0.5.9  and FVB/NJ  mice were purchased from the Jackson Laboratory. Mouse colonies were maintained in a specific pathogen-free animal facility. Tumor growth in Mdr2 KO livers is multinodular, and HCC tends to develop within an adenoma (as a focus of tumor progression). The histological composition of grossly detectable hepatic nodules was semiquantitatively determined based on reported classification criteria  and the guidelines of the European Commission Recommendation 2007/526/EC-June 18, 2007. The project has been reported to the Italian Ministry of Health (project n. 106/11). Founders of the FVB.129P2-Abcb4tm1Bor/J  using BWA v0.5.9  with defhttp://fantom.gsc.riken.jp/5/datafiles/latest/basic/). This process was done for the CAGE libraries of 135 tissues, 432 primary cells, and 241 cell lines . To seeThe graphical representation of CAGE LTR expression profile and unsupervised classification of HCC samples were performed with the MeV (MultiExperiment Viewer) software, version 4.9.0 using the Euclidean distance metric with average linkage algorithm during classification . The obtP-value of the test for heterogeneity < 0.05 and if the random effects on the forest plot are well centered.The detection of LTR level expression allowed us to distinguish two groups of tumor samples by CAGE analysis (high level and low level of LTR discriminated by unsupervised clustering). The odds ratio of each clinical and biological criterion was calculated for each class of patients (high-LTR and low-LTR). Meta-analysis performed with the totality of clinical and biological criteria taken into account during the study allowed testing of the heterogeneity of the calculated odds ratios. A forest plot was drawn with odds ratios and interval of confidence at 95% for each criterion. Significance of the meta-analysis was retained if the http://genome.ucsc.edu/ENCODE/) to determine which tumor TSSs are active in HepG2. First, all CAGE tags produced for different compartments  were combined. Secondly, narrow peaks, defined by the ENCODE pipeline, of DNase-seq rep1, rep2, and FAIRE-seq  were combined. Finally, tumor TSSs were determined to be active in HepG2 if at least one CAGE tag was found in the TSS and at least one narrow peak of the open chromatin signals was found within 1 kb of the TSS.We used CAGE, DNase-seq, and FAIRE-seq data of the HepG2 cell line, downloaded from the ENCODE website  to test associations between TF bindings and up-regulations. For each TF, a 2 × 2 contingency table was generated in which one factor is whether or not distal TSSs are significantly up-regulated and the other factor is whether the TSSs have a binding site of the TF (midpoint of the peak) in a range from −600 to +400 bp. A 2. For the qPCR experiment, 80,000 cells were seeded in 12-well plates, and for the CAGE experiment, 160,000 cells were used in 6-well plates. Transfection was done according to the Lipofectamine RNAiMAX protocol. We used LNA longRNA GapmeRs by Exiqon, which are antisense oligonucleotides (ASOs) containing a central stretch of DNA monomers flanked by LNA blocks. Target RNAs are cleaved by RNase H, activated by ASOs. The ASOs were transfected into HepG2 cells at a concentration of 20 nM. Lipofectamine alone and scramble ASOs were used as controls. Knockdown efficiency was measured by qPCR with specific primer sets  for biological triplicates at 12, 24, 48, and 72 h after the transfection. GAPDH was used for normalization.HepG2 cells were maintained in Dulbecco's modified Eagle's medium, 10% FBS, and 100 units/mL penicillin streptomycin (Gibco) at 37°C, 5% COhttp://www.ncbi.nlm.nih.gov/gap/) under accession number phs000885.v1.p1 for human HCC and the Gene Expression Omnibus  under accession number GSE60982 for mouse HCC.CAGE data from this study have been submitted to the NCBI database of Genotypes and Phenotypes (dbGaP;


Chromatin regulation in eukaryotes occurs through complex and interconnected mechanisms that ensure heterochromatin maintenance and compartmentalization of chromosome domains, genome stability, chromatin conformational changes before and after mitosis, gene silencing and transcriptional activation and chromatin remodeling at specific promoters. We refer to these events as a whole using the term “chromatin dynamics.” Chromatin dynamics involves a number of protein families including epigenetic enzymes, DNA repair factors, heterochromatin proteins, transcription factors and transcriptional regulators. Although lamins have been involved in almost all the processes that regulate chromatin dynamics, three m


The first pair includes the HSPA1 gene promoter of camel (Camelus dromedarius) and humans. It was demonstrated that the camel HSPA1A and HSPA1L promoters function normally in vitro in human cell cultures and exceed the strength of orthologous human promoters under basal conditions. We used the same in vitro assay for Drosophila melanogaster Schneider-2 (S2) cells to compare the activity of the hsp70 and hsp83 promoters of the second species pair represented by Diptera, i.e., Stratiomys singularior and D. melanogaster, which dramatically differ in thermoresistance and the pattern of Hsp70 accumulation. Promoter strength was also monitored in vivo in D. melanogaster strains transformed with constructs containing the S. singularior hsp70 ORF driven either by its own promoter or an orthologous promoter from the D. melanogaster hsp70Aa gene. Analysis revealed low S. singularior hsp70 promoter activity in vitro and in vivo under basal conditions and after HS in comparison with the endogenous promoter in D. melanogaster cells, which correlates with the absence of canonical GAGA elements in the promoters of the former species. Indeed, the insertion of GAGA elements into the S. singularior hsp70 regulatory region resulted in a dramatic increase in promoter activity in vitro but only modestly enhanced the promoter strength in the larvae of the transformed strains. In contrast with hsp70 promoters, hsp83 promoters from both of the studied Diptera species demonstrated high conservation and universality.Heat shock gene promoters represent a highly conserved and universal system for the rapid induction of transcription after various stressful stimuli. We chose pairs of mammalian and insect species that significantly differ in their thermoresistance and constitutive levels of Hsp70 to compare  It is well known that heat shock proteins (Hsps), particularly members of the Hsp70 family, are involved in thermoprotection at the cellular level, preventing protein aggregation and the refolding or degradation of stress-damaged proteins .hsp genes leads to drastic changes in the local chromatin structure visualized in the form of large puffs in larval salivary gland chromosomes of various Diptera species including Drosophila melanogaster  basic vector (Promega). DNA fragments containing different portions of the  5’-ends , and thegularior ,21 were A1A gene . The pro cluster . The resHSP70 promoter activity, reporter constructs were expressed in the human cell lines HEK293 and HDF  strain. Flies were reared at 25°C on standard sugar-yeast-agar medium containing propionic acid as a mold inhibitor. For heat shock, larvae or adult flies were transferred into preheated 50 ml plastic conical tubes  for temperature stress in a water bath (37°C during 30 min) without anesthesia. After heat treatment, flies or larvae were frozen in liquid nitrogen immediately or after a recovery period and were used for RNA or protein extraction for 5’-RACE, Q-RT-PCR and EMSA experiments. For in situ hybridization, larvae were grown at 18°C on standard medium for 2 days before dissection, and for heat treatment, approximately 20 larvae were placed into 50 ml Falcon tubes with a moistened piece of filter paper and incubated in a water bath (37°C for 20 min).For  deleted . ProteinEcoRV-XbaI fragment from the S. singularior hsp70S3 gene (4663 bp), containing an ORF (1917 bp), a 5’-UTR and 3’-UTR (230 and 247 bp correspondingly), a 5’-flanking region (2117 bp) and a 3’-flanking (152 bp) sequence. This fragment was obtained from λ phage containing a portion of the S. singularior hsp70 cluster [The I(S) construct includes an g region 17 bp andS. singularior hsp70 promoter and 5’-UTR with a 1557 bp PCR fragment containing the promoter and 5’-UTR region of the D. melanogaster hsp70Aa gene (primer sequences are given in PstI-XbaI fragment containing the hsp70S3 ORF with a 3’-UTR and 3’-flanking sequence from the I(S) construct, ligated this fragment with the BamHI-PstI excision of the D. melanogaster PCR fragment into pBluescriptSK+, and finally subcloned the resulting sequence into pCaSpeR5. All constructs were confirmed by DNA sequencing.In construct II(M), we substituted the Escherichia coli BL21 strain was used for protein expression. His-GAGA protein purification is described in [The histidine-tagged GAF fusion protein (His-GAF) contained amino acids 5–519 of D. melanogaster GAF fused in frame with six histidine residues from the pET-28a vector . The construct was kindly provided by Dr. E.M. Baricheva of the Institute of Cytology and Genetics, Russia, Novosibirsk. The ribed in .D. melanogaster flies, S2 cells and HEK293 cells. Up to 108 S2 or HEK293 cells were collected after heat shock or from control conditions (see above) and frozen in liquid nitrogen and further processed as described in [D. melanogaster flies were taken from control conditions or stressed at 37°C and processed as in [Protein extracts were obtained from control and heat-shocked adult ribed in . D. melaed as in .hsp70 promoters from Diptera and mammalian species , respectively. EMSA with protein extracts from D. melanogaster flies and from S2 cells  was performed as in [32P-labeled oligonucleotides probes of hsp70S3, hsp70S4 and hsp70Aa sequences  were used in supershift experiments. HEK293 protein extracts (10 μg) were shifted with 32P-labelled PCR fragments from human or camel HSPA1L promoters -labeled DNA probe, and 10 ng of poly (dI-dC) in the band shift buffer [Protein extracts from S2 and HEK293 cells were used in EMSA experiments with fragments from ed as in  with 32P primers  in the pt buffer . The reaP element transposase from D. melanogaster [D. melanogaster strain 8841, which lacks all hsp70 genes [in situ hybridization with a white biotinylated probe as described in [P element and hsp70 flanking sequences, and transcription start sites of all three constructs in transformed strains were determined by 5’-RACE as described in [Plasmid DNA of the constructs I(S), I(S-GAGA) and II(M) was purified using the QIAprep Spin Miniprep Kit (Qiagen) and used for embryo injection as previously described . Transponogaster ; the rec70 genes . Adults ribed in . Transgeribed in . For prirpl32  and ef1a (elongation factor 1α) genes, and the ddCt method was used to calculate relative expression levels. Primer sequences are provided in Total RNA was isolated from larvae or adult flies using TRIzol (Invitrogen). cDNA was prepared from 1 μg of total RNA treated with DNase 1  using random decamer primers and MMLV reverse transcriptase (Evrogen). Experiments were performed using an ABI PRISM7500 Sequence Detection System . Each reaction was performed with a minimum of 3 biological replicates and performed using 2.5 X SYBR Green 1 PCR Master Mix in the presence of ROX reference dye (Syntol) in accordance with the manufacturer’s protocol. Reaction efficiency was evaluated and was at least 95%. Quantification was normalized to the ubiquitously expressed S1 Figrenilla luciferase luminescence). Signal intensity driven by constructs (pFF/pRL) is the ratio of the intensity of luminescence of firefly (pFF) and renilla (pRL) luciferase.The relative positions of major regulatory elements and boundaries of promoter regions with respect to the transcription start sites are indicated. The strength of promoters in relation to the controls under non-heat shock conditions (37°C) and after HS (43.5°C) are given in arbitrary units ((JPG)Click here for additional data file.S2 Fig(JPG)Click here for additional data file.S3 FigThe efficiency of promoters under normal physiological temperature (25°C) and after heat shock (37°C) is represented in arbitrary units resembling luminescence. Major components of promoters  are represented by boxes of different color. Thick green arrows beneath the lines indicate the sites of canonical GAGA elements insertions. Transcription starts are marked by bent arrows.(JPG)Click here for additional data file.S4 FigThe transcription direction of both genes is indicated by arrows. Transcription start points are marked by arrows. TATA-boxes and HSEs are given in red while 112 bps primate-specific sequence is colored blue.(JPG)Click here for additional data file.S5 FigD. melanogaster hsp70Aa promoter are colored red.Transcription start is marked by asterisk and a bold letter, TATA boxes, HSEs and the first ATG codon are underlined. GAGA sites within (JPG)Click here for additional data file.S1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.S3 Table(DOC)Click here for additional data file.S4 Table(DOC)Click here for additional data file.


Nuclear myosin 1c (NM1) is emerging as a regulator of transcription and chromatin organization.Using chromatin immunoprecipitation and deep sequencing (ChIP-Seq) in combination with molecular analyses, we investigated the global association of NM1 with the mammalian genome. Analysis of the ChIP-Seq data demonstrates that NM1 binds across the entire mammalian genome with occupancy peaks correlating with distributions of RNA Polymerase II (Pol II) and active epigenetic marks at class II gene promoters. In mouse embryonic fibroblasts subjected to RNAi mediated NM1 gene silencing, we show that NM1 synergizes with polymerase-associated actin to maintain active Pol II at the promoter. NM1 also co-localizes with the nucleosome remodeler SNF2h at class II promoters where they assemble together with WSTF as part of the B-WICH complex. A high resolution micrococcal nuclease (MNase) assay and quantitative real time PCR shows that this mechanism is required for local chromatin remodeling. Following B-WICH assembly, NM1 mediates physical recruitment of the histone acetyl transferase PCAF and the histone methyl transferase Set1/Ash2 to maintain and preserve H3K9acetylation and H3K4trimethylation for active transcription.We propose a novel genome-wide mechanism where myosin synergizes with Pol II-associated actin to link the polymerase machinery with permissive chromatin for transcription activation.The online version of this article (doi:10.1186/s12915-015-0147-z) contains supplementary material, which is available to authorized users. Gene expression programs are activated and repressed via ATP-dependent chromatin remodeling and epigenetic modifications. During spatial and temporal activation of genes these mechanisms target nucleosomes, DNA and histone tails , 2, impaMotor proteins such as myosin are emerging as key regulators of chromatin. They coordinate global chromatin dynamics with gene-specific activities and directly affect the functional architecture of the cell nucleus . Among tWe set out to investigate whether NM1 has a fundamental function at class II promoters, contributing to the maintenance of a chromatin state required for Pol II transcription activation. Using chromatin immunoprecipitation and deep sequencing (ChIP-Seq), we show, for the first time, association of a myosin species with a mammalian genome. The results from pairwise comparisons of the genomic distributions of NM1 and SNF2h  show co-To study the distribution of NM1 across the mammalian genome, we performed chromatin immunoprecipitation and deep sequencing (ChIP-Seq) analysis using a previously characterized affinity-purified antibody specific for NM1 . BrieflyThe significance of NM1 association with intergenic sequences remains to be understood. Functional clustering of peak-related genes via Gene Ontology revealed, however, that NM1 is associated with Pol II genes involved in a wide range of biological, cellular and molecular functions  and Rpb8 (RPABC3) common to all nuclear RNA polymerases , 33 whicSince phosphorylation of the Pol II CTD occurs during transcription activation and marks Pol II engagement in the transcriptional process, the above results altogether suggest that NM1 may specifically activate transcription by maintaining Pol II in complex with actin at the gene promoter.We next asked whether the positive regulatory effect of NM1 on Pol II association with the gene promoter occurs through a chromatin-based mechanism. Since NM1 is part of the B-WICH complex , we begaThese results suggest that at those gene promoters where NM1 regulates association of both SNF2h and WSTF, NM1 may have an impact on the local structure of chromatin. We therefore began to look for NM1-dependent changes in chromatin accessibility by applying a high resolution micrococcal nuclease (MNase) assay , 34 on cWe next tested whether NM1 mediates recruitment of histone modifiers. The involvement of NM1 in the regulation of H3 acetylation and methylation became plausible when we found a correlation of the NM1 peaks of occupancy with promoter-specific acetylation and trimethylation of histone H3 Fig. . ImportaWe conclude that a functional NM1 is required to regulate recruitment of the chromatin remodeler SNF2h and histone modifiers in order to ensure an active chromatin state which is compatible with Pol II transcription.We describe, for the first time, the functional association of a myosin motor, NM1, with a mammalian genome. ChIP-Seq analysis demonstrates that NM1 binds to both non-coding and coding regions of the mouse genome. When partitioning the coding sequences into promoter, exons, introns and UTRs, we found that NM1 is particularly enriched at class II promoters, compatible with a role for NM1 in the initial phases of Pol II transcription.At the gene level, NM1 has been coupled to the coordination of pre-initiation complex (PIC) assembly and in transcription activation when Pol II becomes fully phosphorylated and the transcription machinery is cleared from the promoter . We showOur present findings also underscore the importance of the interaction between NM1 and SNF2h to start transcription at class II promoters. Genome-wide analysis by ChIP-Seq shows that NM1 and SNF2h co-localize at multiple genomic locations. The overlap between NM1 and SNF2h is not complete, consistent with a more general role of the chromatin remodeler SNF2h in the context of other nuclear functions . InteresClass 1 myosins serve as divalent crosslinkers, physically connecting and generating force between actin filaments and cargos such as membrane lipids . Our resTranscriptional activation is a fundamental cellular process essential for living organisms and it is spread across the entire genome. The association of NM1 with a large number of class II genes therefore argues that NM1 is a general factor involved in transcription activation. We speculate that this positive role in conjunction with actin and with the WSTF/SNF2h complex regulates genes that must be rapidly reprogrammed through chromatin de-repression in order to be activated. Nuclear actin and components of the SNF2 members of chromatin remodelers are important in transcriptional reprogramming –42. A reMouse embryonic fibroblasts (MEFs), wild-type HEK293T cells and HEK293T cells constitutively expressing the V5-tagged NM1 constructs V5-wtNM1, V5-RK605AA NM1 or V5-ΔC NM1 were grown in DMEM medium , supplemented with 10 % fetal bovine serum (Gibco) and a 1 % penicillin/streptomycin cocktail (Gibco) as previously performed , 26. TheMEFs transfected with NM1 RNAi duplexes or control scrRNAi duplexes were grown in 10 cm dishes for 24 h at 37 °C. Total RNA was extracted with the TRI reagent as specified by the manufacturer’s instruction manual (Sigma). For analysis of transcripts, polyA mRNA was isolated from NM1-silenced or control cells using the Oligotex mRNA Mini Kit according to the manufacturer’s protocol  and treated with DNase1. cDNA was then synthesized using Superscript II reverse transcriptase (Invitrogen) using oligo dT primers according to the manufacturer’s instructions. The concentration of cDNA was determined by nanodrop. Semi-quantitative RT-qPCR was performed using the cDNA templates prepared from MEFs treated with control scrRNAi duplexes or from MEFs treated with NM1 RNAi duplexes, a Power SYBR Green PCR kit , specific primers amplifying the mouse class II genes Rplp0, Rpl13a, Rpl19, Junb, Rad9a, Wtap, Ddx46 as well as Bad and the human class II genes RAD9A and RPL19  supplemented with protease inhibitors . For denaturation, protein extracts were incubated in Laemmli buffer at 95 °C for 10 min, separated by SDS–PAGE under reducing conditions and transferred to polyvinylidene difluoride (PVDF) membranes  by semidry blotting . Primary antibodies and dilutions used were NM1 (1:1000), β-actin , WSTF , SNF2h , PCAF , Set1/Ash2 . Immunoreactive bands were visualized by chemiluminescence .6 cells was used in each reaction, and the level of DNA was first adjusted to be in the same range in the samples from all different treatments. Several MNase concentrations were used such that the reaction occurred in the linear range of digestion. Two samples from each treatment were used for the calculations: 10 U MNase and one concentration between 10 U and 20 U MNase. The reactions were performed at 37 °C for 30 min and then stopped by adding 12.5 mM EDTA/0.5 % SDS. After three hours of proteinase K treatment, the cross-linking was reversed at 65 °C for five hours. DNA was extracted [t-test as previously described [These experiments were essentially performed as described , 34. Brixtracted  and the escribed .t-test as previously described [ChIP on growing MEFs was performed as previously described . Brieflyescribed .ChIP assays were also performed on formaldehyde crosslinked chromatin isolated from wild-type HEK293T and HEK293T cells expressing V5-wtNM1, V5-RK605AA NM1 and V5-ΔC NM1 mutants using antibodies against the V5 epitope, NM1 and histone H3 as well as non-specific rabbit IgGs. Precipitated chromatin was analyzed by PCR with primers to the EP300 gene promoter and exonic sequences and the PCR products visualized by agarose gel electrophoresis. For the primers sequences see .For ChIP-Seq analysis, crosslinked chromatin from MEFs was subjected to immunoprecipitation with antibodies to NM1. A total of 5 ng of precipitated DNA was used to prepare sequencing libraries at the Bejing Genome Institute (Hong Kong) using the Illumina HiSeq 2000 platform. The analysis procedure involved the use of the SOAP2 program to map the reads to the mouse reference genome. Sequences with more than two mismatches were discarded from further analysis. The resulting individual sequences were remapped back to the annotated UCSC MM9 reference sequence which allows for the identification of peaks corresponding to the levels of association of the ChIP target with those loci. The ChIP-Seq data sets are available for download in the Gene Expression Omnibus (GEO) database (accession number GSE66542). The ChIPseek program was used to analyze the genomic distribution of NM1 across coding and non-coding elements and around the transcription start site (TSS) . For thi