Basal-like breast cancers (BLBCs) are aggressive breast cancers associated with poor survival. Defining the key drivers of BLBC growth will allow identification of molecules for targeted therapy. In this study, we performed a primary screen integrating multiple assays that compare transcription factor expression and activity in BLBC and non-BLBC at the RNA, DNA, and protein levels. This integrated screen identified 33 transcription factors that were elevated in BLBC in multiple assays comparing mRNA expression, DNA cis-element sequences, or protein DNA-binding activity. In a secondary screen to identify transcription factors critical for BLBC cell growth, 8 of the 33 candidate transcription factors (TFs) were found to be necessary for growth in at least two of three BLBC cell lines. Of these 8 transcription factors, SOX11 was the only transcription factor required for BLBC growth, but not for growth of non-BLBC cells. Our studies demonstrate that SOX11 is a critical regulator of multiple BLBC phenotypes, including growth, migration, invasion, and expression of signature BLBC genes. High SOX11 expression was also found to be an independent prognostic indicator of poor survival in women with breast cancer. These results identify SOX11 as a potential target for the treatment of BLBC, the most aggressive form of breast cancer. Breast cancer is a heterogeneous disease that can be divided into clinically defined subtypes including estrogen receptor (ER)-positive, HER2-positive, and triple-negative breast cancer  and HER2), or molecularly defined subtypes including luminal A, luminal B, HER2-enriched and basal-like breast cancer (BLBC). Identifying critical regulators specific to these subtypes has led to the development of targeted therapies; particularly, endocrine therapy for ER-positive breast cancer patients , and HERMicroarray expression studies are useful in defining distinct breast cancer subtypes. The contrasting mRNA expression in BLBC versus other breast cancer subtypes suggests that transcription factors (TFs) may be critical for the development and progression of these breast cancer subtypes. The important role of transcription factors in promoting breast tumor growth is seen in the luminal subtype which is predominantly defined by the activity of the ER-alpha transcription factor.In this study we hypothesized that specific transcription factors are critical for BLBC growth and maintenance of BLBC phenotypes. We utilized an integrated genomic screen to identify transcription factors overexpressed or active in BLBC compared to non-BLBC. For this study, we used three independent approaches in our primary screen to identify critical transcription factors by determining the: (1) transcription factors differentially expressed at the mRNA level in TNBC compared with non-TNBC tumors; (2) transcription factor DNA motifs overrepresented among the promoters of selected BLBC genes; and (3) transcription factor motifs which are more highly bound by nuclear proteins present in BLBC versus non-BLBC cells. We then integrated the results of these three independent approaches and identified 33 transcription factors that are highly active in BLBCs. We then performed a secondary screen to discover whether the individual transcription factors identified in our primary screen are critical for growth of BLBC cells, and found that inhibition of 8 of the 33 candidates resulted in reduced growth of at least two of three BLBC cell lines tested. Of the 8 transcription factors critical for BLBC growth, inhibition of SOX11 had the most BLBC-specific effect, suppressing growth in all three BLBC cell lines, while minimally affecting growth of three non-BLBC cell lines. Further evaluation of SOX11 demonstrated SOX11 is critical for multiple BLBC phenotypes including invasion, migration, and expression of key BLBC genes. We also found that high SOX11 expression is an independent prognostic marker of poor survival in women with breast cancer. Taken together, these results identify SOX11 as a critical regulator of gene expression in BLBC and a promising target for the treatment of these aggressive breast cancers.in silico and in vitro analyses of mRNA expression, DNA sequence, and protein activity  to perform a median-rank-based meta-analysis of differential expression in TNBC vs. non-TNBC. The p-values of each differentially expressed gene in each of the 15 datasets are shown using a colorimetric scale in Figure p-value from the dataset that had the median gene rank across all datasets. The p-value of the median ranked gene is shown colorimetrically in the right column labeled “Median Rank p-value”. Of the 702 transcription factors queried, we identified 132 transcription factors that are significantly overexpressed  in TNBC compared to non-TNBC tumors. The top 25 genes are shown in Figure For our RNA-based screen Figure , we focup-value <0.01) in three previously published, independent breast tumor microarrays [p-value <0.05 using Fisher's Exact Test  cutoff criteria to select transcription factor motifs that had significantly increased binding by protein from BLBC cells  compared to non-targeting siLuc control siRNA in at least two of three BLBC cell lines. Of these 8 critical BLBC growth regulators, three  were also critical for growth in two of three non-BLBC cell lines tested, demonstrating that these factors are most likely general growth regulators, not uniquely specific to BLBC. An additional four transcription factors  were moderately specific for growth of BLBC, with siRNA causing growth reduction in at least two BLBC cell lines and only one non-BLBC cell lines. MYC  and FOXM3% comparSOX11 was one of the transcription factors found to have higher expression in TNBC compared to non-TNBC , but a dSeveral potential mechanisms may be responsible for the elevated SOX11 expression in breast cancer compared to normal breast tissue. One possibility is that SOX11 is methylated in normal adult mammary cells, and becomes hypomethylated in cancer, particularly Her2-positive and basal-like breast cancers. Hypomethylation of oncogenic genes has been associated with many cancer types ; and metin-vitro in a panel of ER-negative/HER2-negative , ER-negative/HER2-positive , ER-positive/HER2-positive (BT474), and ER-positive/HER2-negative (MCF7 and ZR751) breast cancer cell lines as well as non-transformed breast cell lines (HME-hTERT and MCF10A). SOX11 knockdown significantly reduced growth of each of the ER-negative breast cancer cell lines tested, including both HER2-negative and HER2-positive, ER-negative cell lines, as well as triple-negative cell lines representative of the claudin-low subtype. , a marker of mitotic cells, was measured in MCF7 and MDA-MB-468 cells which had been treated with siRNA targeting SOX11 or non-targeting control siRNA. SOX11 knockdown did not significantly decrease the proportion of pH3-positive cells in MCF7 cells, but did have a significant effect, reducing the percent of cells which stained positive for phosphorylated Histone H3 in MDA-MB-468 cells Figure , demonstWhile investigating the role of SOX11 depletion in ER-negative breast cancer cells, we noted an altered morphology of MDA-MB-231 cells following treatment with siRNA targeting SOX11. MDA-MB-231 cells typically have a mesenchymal-like appearance with long protrusions; however, following SOX11 depletion, the cells appear more cuboidal . The ability of the invasive, ER-negative breast cancer cells lines to migrate towards serum supplemented media either uninhibited  test, which analyzes expression of 50 genes to separate breast tumors into specific subtypes [Breast tumors can be subtyped into BLBC and the other molecular subtypes using the subtypes . We nextIn the non-BLBC cell line MCF7, most signature BLBC genes tested were either undetectable by qRT-PCR, or did not change following SOX11 depletion. The exception to this was anillin, actin binding protein (ANLN), which was reduced in both the MCF7 cell line and the BLBC cell line MDA-MB-468 following SOX11 depletion. In MDA-MB-468 cells, the majority of signature BLBC genes were reduced following inhibition of SOX11. This set of genes included forkhead box C1 (FOXC1), keratin 14 (KRT14), cyclin E1 (CCNE1), melanoma inhibitory activity (MIA) and secreted frizzled-related protein 1 (SFRP1), while expression of CDCA1 and MELK, (neither of which were detectable in MCF7), did not change following SOX11 depletion. These results show that SOX11 regulates the expression of many genes that define “basal-ness” in BLBC.p=0.0775), and was most likely due to the small sample size of patients with ER-negative breast cancer available for the disease specific survival analysis. While SOX11 levels are comparatively low in ER-positive tumors , and unWe identified 8 transcription factors for which siRNA inhibition reduced growth in multiple BLBC cell lines in our secondary screen. Of these, only SOX11 was required for growth of all 3 BLBC cell lines, but not for the growth of the non-BLBC cell lines. SOX11 is a member of the SOX (SRY-related HMG-box) family of transcription factors. Other members of this family, including SOX9 , SOX10 , and SOXSeveral studies have shown that SOX11 can regulate differentiation. In MCL and in osteoblasts, SOX11 promotes proliferation of progenitor cells and blocks terminal differentiation , 40. OurOur results demonstrating that SOX11 inhibition affected multiple BLBC phenotypes suggest that SOX11 acts as a key signaling molecule in these breast cancers. This theory is supported by our finding that several PAM50 markers of BLBC, including the FOXC1 transcription factor, are reduced following SOX11 inhibition. High FOXC1 mRNA expression is frequently seen in BLBCs . FOXC1 hOur results showed that high SOX11 expression is correlated with worse clinical outcome in breast cancer patients. However, other studies in ovarian cancer  and in gFor this study we used a novel, comprehensive, and integrated approach utilizing RNA-, DNA- and protein-based assays to identify a set transcription factors that are differentially activated in BLBC and may be critical for BLBC development and progression. The discovery that SOX11 is critical for the growth, migration, and invasion of BLBC cells identifies a novel molecule that can be targeted for the treatment of BLBC. Further study of SOX11 and the other transcription factors identified here will be essential to understanding their role controlling BLBC growth and tumorigenicity. Such studies will provide additional therapeutic targets to effectively treat this aggressive form of breast cancer.Breast cancer cell lines were purchased from American Type Culture Collection  and their identities were confirmed by short tandem repeat (STR) DNA fingerprinting using the AmpFℓSTR Identifiler kit . as described previously . Cells woncomine.com, Compendia Bioscience, Ann Arbor, MI) to perform a meta-analysis of differential expression between TNBC and non-TNBC across 15 breast tumor datasets which contain TNBC status annotation  for higher expression in TNBC vs. non-TNBC tumors. The p-values for each dataset are shown using a colorimetric scale in Figure p-value of the dataset showing the median gene rank across all datasets is shown on the right in the column labeled “Median Rank p-value.” The median gene rank and associated p-value across the set of 15 datasets for each investigated transcription factor was then used to select candidate transcription factors which had increased expression in TNBC with a cutoff of p<0.05.Expression of 702 genes previously identified as DNA-binding transcriptional regulators  was compp-value <0.01 in BLBC tumors compared to non-BLBC tumors. The intersection of the three resulting sets gave a 117-gene BLBC gene set. A set of 1500 control genes were randomly selected from the set of genes not significantly more highly expressed in BLBC tumors. The online tool, CORE_TF [p<0.05 was used for significance.mRNA expression and tumor subtype classifications from three previously published breast tumor microarray studies –8 were u CORE_TF , was the CORE_TF  in the rhttp://rsb.info.nih.gov/ij/macros/toolsets/DotBlotAnalyzer.txt), with the intensity of each spot calculated relative to TFIID. The experiment was performed in triplicate for each cell line. The BLBC average  and the luminal average  were calculated and compared using the Student's t-test. Spots which had a fold change greater than 1.4 and with a p-value less than 0.05 were included for follow up.Nuclear proteins were collected from breast cancer cell lines using the NE-PER kit . The protein DNA-binding assay was performed using the Combo Protein/DNA Array  according to manufacturer instructions. Briefly, 10ug of nuclear protein was combined with biotin-labeled DNA probe mix representing 345 consensus binding motifs. After incubation, the protein/probe mixture was put through spin columns to remove unbound probes. The protein/probe mixture was heat denatured, and the previously bound probes were hybridized to membranes containing corresponding consensus transcription factor binding motifs. Detection was performed with streptavidin-HRP using BioRad ChemiDoc. TIF images were quantified using ImageJ Dot Blot Analyzer (available in IJ macros toolsets repertory at For the promoter analysis and the protein DNA-binding analyses which result in transcription factor motifs, we used TRANSFAC  annotatisiRNA, qPCR and growth assays were performed as previously described . qPCR seThe phospho-histone H3 immunofluorescence assay was performed as previously described  with priet al. [et al. [et al. [et al. [mRNA expression and survival data from Curtis et al. , Desmedt [et al. , Esserma [et al. , and Hat [et al.  were use


PRDE-1 is required specifically for the production of piRNA precursors from genomic loci containing an upstream motif, the Ruby motif. Intriguingly, the expression of a second class of motif-independent piRNAs is unaffected in prde-1 mutants. This study defines a new paradigm for two distinct classes of piRNAs and provides insights into the biogenesis and function of piRNAs in gene regulation.Piwi-interacting RNAs (piRNAs) are small regulatory RNAs with essential roles in genomic maintenance. Weick et al. identify  Caenorhabditis elegans piRNAs are transcribed from two genomic clusters that likely contain thousands of individual transcription units; however, their biogenesis is not understood. Here we identify and characterize prde-1 (piRNA silencing-defective) as the first essential C. elegans piRNA biogenesis gene. Analysis of prde-1 provides the first direct evidence that piRNA precursors are 28- to 29-nucleotide (nt) RNAs initiating 2 nt upstream of mature piRNAs. PRDE-1 is a nuclear germline-expressed protein that localizes to chromosome IV. PRDE-1 is required specifically for the production of piRNA precursors from genomic loci containing an 8-nt upstream motif, the Ruby motif. The expression of a second class of motif-independent piRNAs is unaffected in prde-1 mutants. We exploited this finding to determine the targets of the motif-independent class of piRNAs. Together, our data provide new insights into both the biogenesis and function of piRNAs in gene regulation.Piwi-interacting RNAs (piRNA) are small regulatory RNAs with essential roles in maintaining genome integrity in animals and protists. Most  Drosphila and mammals, piRNAs are produced from long precursor transcripts, which subsequently undergo processing to mature piRNA sequences with a length distribution of 24–32 nucleotides (nt) and a strong preference for a 5′ uracil. In conjunction with their Piwi protein partners, these small RNAs can target transposons by base-pairing and instigate secondary amplification cycles to ensure robust silencing of repetitive elements.Piwi-interacting RNAs (piRNAs) have an evolutionarily conserved role in maintaining the genetic and epigenetic integrity of the germline in many organisms . piRNAs Caenorhabditis elegans (C. elegans encodes two Piwi clade Argonaute (AGO) superfamily proteins, PRG-1 and PRG-2, although PRG-2 has likely little or no function  response   . piRNA-m2G-RNAs) .C. elegans germline that becomes independent of prg-1 (Endogenous protein-coding gene and transposon transcripts exhibit Piwi-dependent endo-siRNAs (22G-RNAs) at sites complementary to piRNAs and are derepressed in Piwi mutants. Intriguingly, piRNA-mediated silencing can establish a multigenerational silencing memory in the of prg-1 . The sigDrosophila or mammals  as the first such factor. We show that prde-1 is a nuclear factor associated with piRNA clusters on chromosome IV defining the site of piRNA precursor generation. Intriguingly, prde-1 is specifically required for the accumulation of both mature piRNAs and their precursors that come from genomic loci containing a Ruby motif. piRNAs that do not come from loci associated with a Ruby motif are not affected in prde-1 mutants. Thus, we used prde-1 mutants as a tool to assess the different targets of motif-dependent and motif-independent piRNAs. Together, our results clarify the distinct stages of piRNA biogenesis in C. elegans and extend classification of piRNAs and their targets.Despite these recent advances into the understanding of the mechanism of C. elegans using a “piRNA sensor” transgene expressing a histone-GFP fusion protein in the germline that is responsive to the endogenous piRNA 21UR-1. In a wild-type background, the sensor transgene is efficiently silenced, whereas in mutants of piRNA pathway factors, such as prg-1 and, for example, mutator class genes, the GFP transgene is derepressed  and prde-1(mj258) mutants, expression of the mCherry-PRDE-1 fusion protein largely restored piRNA sensor silencing, as assessed by microscopy and flow cytometry  in prde-1 mutants. We found that both of these piRNA targets were up-regulated in prde-1 mutants relative to wild type to an extent similar to in prg-1 mutants . Finally, maintaining prde-1 mutant families for successive generations in the laboratory, we noticed instances of complete sterility where adult animals were devoid of embryos . These data support a prominent role for PRDE-1 in germline integrity in C. elegans.ood size . Scoringransgene . These de  . In contrast, at the restrictive temperature, prde-1 mRNA was not detected from glp-4(bn2ts) and glp-1(e2144lf) mutant animals, which are devoid of germ cells (prde-1 mRNA was still detected in RNA from fem-1(hc17ts)   . prde-1 rm cells . However(hc17ts)  and fem-22sd,ts)  mutants C. elegans small RNA pathways demonstrated that genes required for transposon silencing are often also involved in RNAi-related phenomena (mut-7 and mut-2), which act downstream from prg-1 in the piRNA pathway . These data in conjunction with the localization data shown below in prde-1 mutants are also competent in heritable RNAi, whereas the downstream Piwi/piRNA pathway gene hrde-1 is not  promoter  . Thus, reduction in PRG-1 levels in prde-1 mutants is likely to be a post-transcriptional effect. Interestingly, any residual GFP-PRG-1 still detectable in prde-1 mutants after image processing still appears to be localized correctly to P granules. Together, these data suggest that PRDE-1 is directly or indirectly required for PRG-1 protein stability. In contrast, the mCherry-PRDE-1 fusion protein expressed from a heterologous promoter  remains unchanged in expression level and localization in prg-1 mutant animals as compared with animals carrying a rescuing GFP-PRG-1 transgene . We therefore concluded that PRDE-1 acts upstream of PRG-1.To place PRDE-1 within the Piwi/piRNA pathway, we next considered whether PRDE-1 and PRG-1 are required for the localization or stability of each other. PRG-1 protein levels are strongly reduced in extracts , a findiextracts . Importa animals . Moreovet panel)  shows a prde-1 mutant animals in the distal germline . Similarly, piRNAs were also absent in two different prde-1 mutant lines  . Thus, we concluded that PRDE-1 acts as an upstream piRNA biogenesis factor.Given the distinct localization of PRG-1 and PRDE-1, a direct effect of PRDE-1 on PRG-1 protein stability seemed unlikely. Therefore, we considered the possibility that PRDE-1 was acting instead on the small RNA component of the PRG-1 complex , the piRld type) . We confprde-1 mutants could reflect a role in either piRNA stability or the generation of piRNAs. Recently, evidence has suggested that individual piRNA loci in C. elegans are transcribed separately by RNA Pol II to produce individual capped long piRNA precursors, which are subsequently processed into mature piRNAs . As mature piRNAs are absent in prde-1, this loss of capped, 5′-extended sequences provides for the first time strong genetic evidence that these species are indeed the first intermediate product of generation of mature 21U-RNAs. Moreover, based on the length profile of precursors detected , the presence of any longer precursor species is unlikely. In conclusion, our data show that PRDE-1 is required for the biogenesis of piRNA precursors.Interestingly, the piRNA precursor sequences were still present in ibraries . EquallyC. elegans is dependent on an upstream Ruby motif . Taken together with the absence of 21U-RNAs defined as having a motif score of >7 . On the other hand, piRNAs that are motif-independent can be produced without PRDE-1 activity. Indeed, analysis of the motif scores for the extended set of 21U-RNAs showed a bimodal distribution of motif scores in the wild type, indicative of two distinct populations of piRNAs. The motif scores for 21U-RNAs present in prde-1 mutants showed only a single peak with a modal motif score of −10 (C. elegans piRNAs into Ruby motif-dependent (and prde-1-dependent) and motif-independent (and prde-1-independent) piRNAs.As biogenesis of classical piRNAs in by motif , we wondby motif . Recentlby motif . We therby motif  and recaby motif . We groutability . In prdey absent , recapity absent . Strikinild type . In addire of >7 , this sure of >7 . In addie of −10 . Based oprg-1 and prde-1 mutants. We identified genes that were up-regulated by more than fourfold  in prg-1 mutants or either of two independent prde-1 alleles relative to wild type using microarray expression analysis . Known piRNA target genes such as bath-45 . C. elegans innate immune genes  overrepresented in prde-1-independent prg-1-regulated genes . Moreover, gene set enrichment analysis using the entire array data confirmed statistically highly significant up-regulation of innate immunity genes in prg-1 relative to both wild type and prde-1 (permutation estimated P < 0.01) . We therefore hypothesized that the different sets of genes up-regulated in prg-1 versus prde-1 mutants might reflect Ruby motif-dependent and motif-independent piRNA targets. Sixty-four percent of genes up-regulated in both prg-1 and prde-1 and 59% of genes up-regulated only in prg-1 were targeted by at least one piRNA, allowing up to three mismatches, compared with 47% of all genes . However, using direct sequence matching to known piRNA sequences is likely an insensitive method to identify possible targets . Both prg-1-specific and prg-1/prde-1 shared genes had robust levels of antisense 22G-RNAs mapping to them in wild type, although, interestingly, prg-1-specific genes had fewer 22G-RNA reads  . Both categories showed strong reductions in 22G levels in prg-1. However, in prde-1 mutants, prg-1/prde-1 shared genes showed clear reductions in the 22G-RNA level  , arguing that the motif itself is more important in differentiating between classes of piRNAs. This supports recent work showing that a transgene containing the piRNA motif inserted on chromosome II could nevertheless produce robust levels of piRNAs  . Our claf piRNAs .prg-1 mutants but not in prde-1 mutants and, moreover, are able to produce 22G-RNAs at their target sites  . Thus, there might be selective pressure on genes to avoid Ruby motif-independent piRNAs, and a “snapshot” of these targets reveals only the genes that move most rapidly through the sequence space. Alternatively, the ability of the piRNA pathway to target fast-evolving genes may be beneficial; for example, by contributing to the fine-tuning of expression levels of newly evolved genes.Our analysis of the targets of Ruby motif-dependent and motif-independent piRNAs shows that piRNAs from the two types of genomic loci tend to target different types of genes. Since the mechanism of silencing appears to be the same for both types of piRNAs, different sequence properties of the piRNAs themselves may be important. One possible distinction between motif-dependent and motif-independent piRNAs is that motif-independent piRNAs derived from transcribed protein-coding genes may be under more sequence constraint than the motif-dependent piRNAs. However, the target genes of motif-independent piRNAs appear to be enriched for innate immunity genes , which evolve extremely rapidly in  elegans . Indeed,While the enforced repression of pathogen response genes in the germline may be puzzling at first, it is conceivable that such genes need to be repressed in normal environmental conditions but may be derepressed upon immunogenic insult. Indeed, down-regulation of somatic small RNA pathways upon infection can lead to relief of repression of pathogen response genes . A similprde-1 mutants has confirmed the importance of both Ruby motif-dependent and motif-independent piRNAs in gene expression control in C. elegans. This study provides a starting point for a more detailed examination of the evolutionary history of this ancient yet extraordinarily dynamic component of the animal kingdom's genome defense armory.Overall, our examination of C. elegans were grown under standard conditions . All strains used are listed in Supplemental Table S1. Additional standard C. elegans experimental procedures, including RNAi and progeny counts, are detailed in the Supplemental Material.nditions  at 20°C mcherry∷prde-1 fusion construct, we cloned F21A3.5 from N2 cDNA, including the STOP codon  into pDONR221. The par-5 3′ untranslated region (UTR) was generated from N2 genomic DNA (655 nt) and inserted into pDONR-P2R-P3. The sequence of all pDONR constructs was confirmed by sequencing. To generate transgenic animals, germline transformation was performed as described  for imaging of live specimen and immunostaining of isolated gonads using confocal microscopy as well as information on FISH in combination with wide-field fluorescence imaging are provided in the Supplemental Material.Synchronized populations of worms were grown on 90-mm NGM agar plates to the gravid adult stage, and generation of protein lysates and Western blotting were performed according to standard procedures. The primary antibodies used were custom rabbit anti-PRG-1 (1:1000) , rabbit Worms synchronized by L1 starvation arrest were grown on 90-mm plates to the required stage, washed several times in M9 buffer, snap-frozen in TRISure (Bioline) at a ratio of 1 vol of worms to 10 vol of TRISure, and cracked open by five freezing–thawing cycles in liquid nitrogen. RNA extraction was performed using standard procedures.qRT–PCR experiments and oligonucleotides for qRT–PCR are described in the Supplemental Material .prde-1(mj207) (SX2470), piRNA sensor; prde-1(mj258) (SX2471) and prg-1(n4357); and piRNA sensor (SX1888). RNA samples (4 × 3) were used to generate cDNA libraries followed by microarray hybridization. Sample processing from total purified RNA and Affymetrix C. elegans 1.0 gene arrays were performed by the EMBL Genomic Core Facilities . Data were processed using the Affymetrix Expression console. Processed microarray data are available in comma-separated table format (.csv) as Supplemental Data File 1. A description of further data analysis is in the Supplemental Material.RNA was extracted as described above from each of the following strains in three biological replicates: wild-type piRNA sensor (SX1316), piRNA sensor; Five micrograms of total RNA was pretreated with either 20 U of TAP (Epicentre) or 20 U of RNA 5′ polyphosphatase (Epicentre) in a volume of 20 µL for 45 min at 37°C or used directly. After standard extraction, RNA was used for TruSeq small RNA library preparation according to the manufacturer's instructions (Illumina), including 15 cycles of PCR. Libraries were sequenced on a MiSeq instrument (Illumina). A detailed description of processing and analysis of the data thus generated are in the Supplemental Material.Purification of nuclear short RNAs was performed as described . Brieflyhttp://www.ncbi.nlm.nih.gov/geo) under the accession number GSE49220.The sequencing data reported in this study have been deposited in the Gene Expression Omnibus (GEO) database (


Vibrio cholerae normally enters the developmental program of natural competence for transformation after colonizing chitinous surfaces. Natural competence is regulated by at least three pathways in this organism: chitin sensing/degradation, quorum sensing and carbon catabolite repression (CCR). The cyclic adenosine monophosphate (cAMP) receptor protein CRP, which is the global regulator of CCR, binds to regulatory DNA elements called CRP sites when in complex with cAMP. Previous studies in Haemophilus influenzae suggested that the CRP protein binds competence-specific CRP-S sites under competence-inducing conditions, most likely in concert with the master regulator of transformation Sxy/TfoX.The human pathogen qstR and comEA as an example of the complex process that controls competence gene activation in V. cholerae. We identified previously unrecognized putative CRP-S sites upstream of both genes. Deletion of these motifs significantly impaired natural transformability. Moreover, site-directed mutagenesis of these sites resulted in altered gene expression. This altered gene expression also correlated directly with protein levels, bacterial capacity for DNA uptake, and natural transformability.In this study, we investigated the regulation of the competence genes qstR and comEA and therefore for natural transformability of V. cholerae even though the motifs might not reflect bona fide CRP-S sites.Based on the data provided in this study we suggest that the identified sites are important for the expression of the competence genes  Vibrio cholerae is a Gram-negative bacterium that often lives in aquatic environments in association with the chitinous exoskeleton of zooplankton comEA [comEA was constructed by cloning the PCR-amplified insert [−134]comEA into the NcoI site of pBR-Tet_MCSII [The majority of the plasmids carrying the wn]comEA  as templet_MCSII .Table 2V. cholerae strains on chitin flakes with a medium change on day two  site_qstR) abolished qstR and comEA expression, ComEA production, DNA uptake and, consequently, natural transformability. Remarkably, the change to a CRP-0 site again slightly exaggerated those same phenotypes  Figure . To inveing site  was delecomEA and qstR as important genes of the competence regulon of V. cholerae, this study contributes to the understanding of the regulatory network driving natural competence. For comEA we showed that the region up to 134 bp upstream of the start codon is sufficient to drive comEA expression in this organism. But what initiates transcription of comEA? Trying to answer this question, we followed a common assumption, namely that the expression of the competence genes of V. cholerae is TfoX- and CRP-cAMP-dependent and that competence genes are preceeded by so-called CRP-S sites as previously suggested for H. influenzae [in silico predicted CRP-S sites [qstR, which likewise was required for qstR expression and natural transformability. Interestingly, our site-directed mutagenesis approach resulted in unexpected but interesting phenotypes. CRP-mediated activation at the CRP-S site is expected to be different from that at the canonical CRP binding site . We therefore assumed that exchanging the putative CRP-S site for a CRP-N motif would enhance competence gene expression. Notably, and in contrast to this assumption, the expression of comEA and qstR was significantly reduced when preceeded by a CRP-N site , the comEA/qstR genes were expressed at higher levels, which correlated well with increased levels of ComEA protein, enhanced DNA uptake, and higher transformation frequencies  binding of the RNA polymerase (RNAP) holoenzyme to the promoter region and 2) formation of an open complex of the DNA. Promoters that are non-constitutively active  require one or several activating protein(s), such as the CRP protein, that directly interact with the RNAP and assist the holoenzyme in the steps preceding transcriptional initiation . Using cfluenzae ,49. And -S sites  had a ne Figures  and 6. M Figures  and 6. WcomEA. A sequence (TGCGA-N6-AAGCA) centered at −115.5 from the transcription start point and located between −147 bp and −132 bp upstream the comEA start codon, was recently discussed  by Antonova et al. The authors suggested that this sequence serves as a potential CRE element  also leads to questions regarding its functionality with respect to the regulation of comEA.Upon visual inspection we did not identify other motifs resembling the CRP-S site within the putative promoter region of  element ; the for element . AlthougcomEA we raised the possibility that TfoX and CRP-cAMP only indirectly regulate comEA via the intermediate transcriptional regulator QstR. Indeed, we have previously shown that QstR is necessary for the expression of comEA and comEC but not of the DNA-uptake pilus-encoding genes [qstR in trans only low levels of comEA transcript were measured in accordance with the production of low levels of the ComEA protein (Figure comEA by TfoX/CRP-cAMP still occurs but involves a CRP-S binding site that significantly differs form the consensus that was in silico predicted for the Vibrionaceae [in vivo binding sites of CRP-cAMP, QstR, and potentially also of TfoX in naturally competent V. cholerae cells.Given the absence of any obvious alternative CRP-S site within the 134 bp upstream of ng genes ,22. Howeionaceae . Further


We have recently shown that T-antigen binding to Site I results in the replication-dependent introduction of H3K9me1 into SV40 chromatin late in infection. Since H3K9me2 and H3K9me3 are also present late in infection, we determined whether their presence was also related to the status of ongoing transcription and replication. Transcription was either inhibited with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidizole (DRB) or stimulated with sodium butyrate and the effects on histone modifications early and late in infection determined. The role of DNA replication was determined by concomitant inhibition of replication with aphidicolin.We observed that H3K9me2/me3 was specifically introduced when transcription was inhibited during active replication. The introduction of H3K9me2/me3 that occurred when transcription was inhibited was partially blocked when replication was also inhibited. The introduction of H3K9me2/me3 did not require the presence of H3K9me1 since similar results were obtained with the mutant cs1085 whose chromatin contains very little H3K9me1.Our data suggest that methylation of H3K9 can occur either as a consequence of a specific repressive event such as T-antigen binding to Site I or as a result of a general repression of transcription in the presence of active replication. The results suggest that the nonproductive generation of transcription complexes as occurs following DRB treatment may be recognized by a ‘proof reading’ mechanism, which leads to the specific introduction of H3K9me2 and H3K9me3. Five distinct but potentially related elements are thought to contribute to the epigenetic regulation of eukaryotic gene expression: DNA methylation, nucleosome location, histone variation, covalent histone modifications, and interactions by regulatory RNA. Of these elements, the post-translational modification of histones has been of particular interest because of the diversity of the available forms of modification and the number of target amino acids and their physical location within histones. Importantly, there is an abundance of published reports demonstrating the association between certain forms of histone modification and activation or repression of transcription in one or more well characterized biological systems.In general, it is thought that acetylation of certain lysines in histones is associated with active biological processes like transcription while methylation of lysines or arginines may be associated with either activation or repression of gene transcription. For example, in the nucleosome core, histone H3 acetylation on lysine 9 and 14 (H3K9 and H3K14) have been shown to be associated with transcription along with methylation of lysine 4 (H3K4), while methylation of lysine 9 (H3K9) has been shown to be associated with repression.While the association between the methylation of H3K9 and gene repression has been well established, much less is known about the circumstances that lead to the introduction of methylated H3K9 into chromatin. What has added to this uncertainty is the complexity of methylation, including mono-, di-, and tri-methylation, and the involvement of multiple methylating enzymes[In order to better understand the factors that contribute to epigenetic regulation, we have been investigating the role of histone modifications in the regulation of gene expression during the Simian Virus 40 (SV40) life cycle. In a reDuring the course of these studies, we investigated the introduction of all three methylated forms of H3K9 during replication. We noted that unlike the introduction of H3K9me1 which absolutely required replication, the introduction of H3K9me2 and H3K9me3 appeared to occur even when replication was substantially blocked by inhibitor. These rIn order to address these questions in SV40 chromatin, we have investigated the effects of a general inhibition or stimulation of transcription on the introduction of H3K9 methylation in the presence or absence of active replication. Transcription was inhibited with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidizole (DRB), a well-Since we previously observed that the introduction of H3K9me2 and H3K9me3 was not directly related to repression by T-antigen binding to Site I, we hypoSV40 minichromosomes were isolated and purified from cells infected with wild-type 776 virus following treatment with DRB or no treatment. When minichromosomes were isolated at 2 hours, cells were pretreated with DRB for 2 hours prior to infection, while for minichromosomes isolated at 48 hours post-infection infected cells were treated from 24 to 48 hours. In these experiments, we observed a 112 ± 35 fold increase in the size of the pool of SV40 chromosomes present in glycerol gradient fractions following purification from cells infected for 48 hours compared to 24 hours post-infection. While the increase was somewhat variable we always observed at least a tenfold increase. This increase was reduced 10 ± 6 fold following treatment of the SV40 infected cells with DRB from 24 to 48 hours post-infection. The latter analysis was determined by comparing the increase in the presence and absence of inhibitor and indicates that on average there was about a tenfold reduction in the pool of SV40 minichromosomes following inhibition. As expected, treatment with DRB substantially inhibited the generation of mRNA (data not shown).The effect of DRB on the introduction of methylated H3K4 and H3K9 was determined by subjecting treated and untreated samples of intact SV40 minichromosomes obtained at 2 hours and 48 hours post-infection to ChIP analyses with antibodies that recognize mono-, di-, or tri-methylated H3K4 or H3K9. Intact SV40 minichromosomes were used because they are easily obtained in relatively large amounts and yield the maximum PCR signal compared to fragmented chromatin. Because the SV40 genome was intact, this analysis only yielded information relative to changes in the numbers of minichromosomes carrying a particular modification following treatment. No information was obtained concerning the location of any specific histone modifications. The results of these analyses are graphically shown in Figure Since we have previously shown that the introduction of H3K9me2 and H3K9me3 into SV40 chromatin late in infection does not require DNA replication, we nextSince H3K9me1 can be present in as much as 22% of the SV40 minichromosomes present at late times[In order to determine whether H3K9me2 and H3K9me3 were being added to minichromosomes that contain RNAPII following treatment with DRB, we analyzed SV40 minichromosomes that contain RNAPII for the presence of the two methylated forms of H3K9 following treatment with DRB using our ISFIP procedure. In thisSince the introduction of H3K9me2 and H3K9me3 appeared to be associated with repression of transcription, we then determined whether stimulation of transcription would have the opposite effect. Sodium butyrate is a well-known inhibitor of many histone deacetylases and for this reason has been used extensively to investigate the relationship between histone acetylation and various biological processes including transcription, 12. We SV40 wild-type infected cells were harvested at 24 hours post-infection, 48 hours post-infection, or 48 hours post-infection following treatment with 50 μM sodium butyrate from 24 to 48 hours post-infection to determine the effects of sodium butyrate during the period of active replication. In a parallel analysis, infected cells were harvested at 12 hours post-infection and 12 hours following a 12-hour treatment with sodium butyrate to determine the effects of sodium butyrate in the absence of replication. The minichromosomes were purified and subjected to ChIP analyses with antibodies to methylated H3K4 and H3K9 with minichromosomes isolated late in infection but only methylated H3K9 when isolated at very early times. The reason for this was our previous observation that relatively little of the SV40 chromatin contained methylated H3K4 at the very early times[Since DRB had a major effect on transcription and the introduction of H3K9me2 and H3K9me3, we also determined whether it affected the proportion of minichromosomes carrying RNAPII and was, therefore, potentially capable of transcription. We have previously shown that with short-term treatment with DRB there was little effect on the percentage of minichromosomes carrying RNAPII. HoweverAs indicated above, between 24 and 48 hours post-infection we observed a 112 ± 35 fold increase in the size of the minichromosome pool. Following treatment with aphidicolin the pool size at 48 hours post-infection was essentially the same as at 24 hours post-infection indicating substantial inhibition by the drug. Following treatment with DRB we again observed a substantial increase in the pool of minichromosomes although the increase was 10 ± 6 fold less than observed in the absence of the inhibitor. Treatment with sodium butyrate was seen to increase the size of the pool of minichromosomes by 10 ± 4 fold above the level of increase without the added sodium butyrate. The latter two results indicate that the inhibitors had a modest effect on pool sizes when replication was occurring.SV40 minichromosomes from treated or untreated infections were subjected to ChIP analysis with antibody to RNAPII with the results shown in Figure The introduction of H3K9me2 and H3K9me3 into SV40 chromatin following repression of transcription through the inhibition of RNAPII elongation by the inhibitor DRB is consistent with previous reports showing that the presence of H3K9me2 and H3K9me3 in chromatin is associated with transcriptional repression . H3H3K9me2 and H3K9me3 are typically introduced into chromatin by a member of the SET domain family of histone methyltransferases. The relThere are two well-characterized mechanisms for the introduction of H3K9me2 and H3K9me3. H3K9me2 is associated with repression of nuclear hormone receptor regulated transcription (reviewed in). In thiThe introduction of H3K9me3 into heterochromatin has also been extensively characterized (reviewed in). The inIt seems unlikely that either of these mechanisms is responsible for the introduction of H3K9me2 and H3K9me3 reported here. With respect to the introduction of H3K9me2, SV40 transcription does not appear to be regulated by a nuclear hormone type mechanism as previously described for H3K9me2. Similarly the observed increase in H3K9me2 occurred as a consequence of the inhibition of transcription by DRB not the binding of normal transcription factors. The introduction of H3K9me3 also is unlikely to occur by the same mechanism as described for heterochromatin. Notably, SV40 DNA in chromatin is not known to be methylated because of an absence of DNA methylase substrates in the DNA. Also our observed increase in H3K9me2 and H3K9me3 appeared to be only secondarily a result of DNA replication since the increase was observed on minichromosomes containing RNAPII.To explain our results we propose the following model. As DNA replication takes place, some of the newly replicated minichromosomes are committed to transcription through the binding of specific and general transcription factors. Concurrently, for reasons as yet unknown, some of the preexisting transcribing minichromosomes stop transcription. These nonfunctioning minichromosomes are recognized as being repressed, and H3K9me2 and H3K9me3 are introduced into their chromatin as a mark of repression. We propose that following replication and the activation for new transcription there is a ‘proofreading mechanism,’ which ensures that the newly initiated minichromosomes are capable of productive transcription. In the event that this is not the case, the minichromosomes are again recognized as repressed, as with the nonfunctioning minichromosomes, and marked by H3K9me2 and H3K9me3. Since treatment with the inhibitor DRB results in a large increase in the number of minichromosomes in which transcription is stopped, presumably at initiation, there is a corresponding increase in the incorporation of H3K9me2 and H3K9me3.This proofreading mechanism could be specific to SV40 and other similar viruses in which case it could function to ensure that the size of the pool of transcribing minichromosomes is tied to the level of replication and encapsidation. Conversely, this mechanism could be true for higher eukaryotes in general and serve as a way to ensure that when genes are activated for transcription, only those genes correctly activated are allowed to persist in the activated state.Assuming that the introduction of H3K9me2 and H3K9me3 following the inhibition of elongation by RNAPII reflects a normal biological process for SV40, we believe it is likely to play a role in two aspects of normal SV40 molecular biology. First, the introduction of H3K9me2 and H3K9me3 could play a critical role in regulating the pool size of transcriptionally competent minichromosomes during an infection. We have presented evidence that the pool size is dynamic with new transcribing minichromosomes entering the pool as a result of replication and old minichromosomes leaving the pool. We believe that old minichromosomes leaving the pool of transcribing minichromosomes would be labeled with H3K9me2 and H3K9me3 in order to prevent their reactivation.Second, H3K9me2 and H3K9me3 could also play a critical role during the initiation of a subsequent infection. We have shown that minichromosomes in virions contain significant amounts of H3K9me2/3[in vivo.Because DRB treatment blocks RNAPII elongation, we believe that it is a way to model the effects of repression of transcription in order to characterize the subsequent changes in epigenetic modifications. However, we recognize that the DRB treatment, as well as the treatment with the other inhibitors themselves, may be responsible for the observed effects on the levels of H3K9me2 and H3K9me3 and may not accurately reflect the epigenetic changes occurring Methylation of H3K9 can occur during active expression either as a consequence of a specific repressive event such as T-antigen binding to Site I or as a result of a general repression of transcription. Moreover, these results suggest that the nonproductive transcription complexes which are generated by DRB treatment may be recognized by a ‘proof reading’ mechanism, which leads to the specific introduction of H3K9me2 and H3K9me3.Wild-type and mutant SV40 minichromosomes were prepared in the monkey kidney BSC-1 cell line (ATCC) using either wild-type 776 virus (from Dr. Daniel Nathans) or cs1085 virus (from Dr. Daniel Nathans).BSC-1 cells were maintained and infected as previously described–7. An RNSV40 minichromosomes were isolated at the indicated times from 2 hours to 48 hours post-infection as described for each of the analyses using our standard purification protocols–7 with oChIP kits were obtained from Millipore  and the protocol was followed as previously described. The antChIPs were performed as previously described–7 with tPreparation of DNA: Samples were prepared for PCR using an MP Bioscience  Geneclean Spin Kit (#111101-200) with the following modifications. The glassmilk reagent (100 μl) was mixed with 50 μl of sample in a 1.5-ml centrifuge tube. The tube was mixed by repeated inversion at 2 minutes and again at 4 minutes of incubation. Following 5 minutes of room temperature incubation, the samples were centrifuged at 6,000 rpm for 30 seconds in a Micro One (Tomy) to pellet the glass. The supernatant was discarded and 200 μl of the wash buffer was added to the tube. As the wash buffer was being added, the pipette tip was used to break up the pellet by physically rubbing the pellet and vigorously pipetting up and down. The samples were inverted twice and centrifuged at 6,000 rpm for 30 seconds to again pellet the glass. The supernatant was discarded and the pellets were dried in a vacuum for 5 minutes. The glass pellet with bound DNA was resuspended in 25 μl Tris EDTA (TE) buffer.For most of the amplifications, DNA was amplified from the promoter region of the SV40 genome using the primers 5′ TTG CAA AAG CCT CCA AA 3′ and 5′ TGA CCT ACG AAC CTT AAC CGA GGG 3′ in a CFX Connect Real Time System thermal cycler  using ‘SSO Advanced DNA polymerase . In the immune selection fragmentation experiment, DNA was amplified from the early region using the primers 5′TGCTCCCATTCATCAGTTCC3′ and 5′CTGACTTTGGAGGCTTCTGG3′ because the promoter region is extremely sensitive to sonication. Immediately before use, the primers and DNAse free water were added, and 28 μl of the mix was used per sample. 2 μl of the resuspended glass milk in TE buffer was added per sample. Samples were amplified by PCR in triplicate with a melt curve applied afterwards to ensure specific amplification. All sample preparation for PCR was done in either a Nuaire biological safety cabinet Model NU_425-400 or an AirClean 600 PCR Workstation (ISC BioExpress).This research did not involve the use of any human subjects, materials, or data.


Cyanobacteria can function as solar-driven biofactories thanks to their ability to perform photosynthesis and the ease with which they are genetically modified. In this review, we discuss transcriptional parts and promoters available for engineering cyanobacteria. First, we go through special cyanobacterial characteristics that may impact engineering, including the unusual cyanobacterial RNA polymerase, sigma factors and promoter types, mRNA stability, circadian rhythm, and gene dosage effects. Then, we continue with discussing component characteristics that are desirable for synthetic biology approaches, including decoupling, modularity, and orthogonality. We then summarize and discuss the latest promoters for use in cyanobacteria regarding characteristics such as regulation, strength, and dynamic range and suggest potential uses. Finally, we provide an outlook and suggest future developments that would advance the field and accelerate the use of cyanobacteria for renewable biotechnology. PCC 7601 RNAP transcribe the Plac and the PlacUV5 promoters in vitro have been observed  RNAP systems in vitro, it was concluded that the cyanobacterial RNAP transcribes its DNA slower but with higher fidelity , as is generally the case in bacteria. Nonetheless, its absence negatively affected the association of RNAP to the primary sigma factor, leading to the downregulation of highly expressed genes  at +1 (by definition), a −10 element (consensus sequence 5′-TATAAT-3′), and a −35 element (consensus sequence 5′-TTGACA-3′). Under normal growth conditions, type I promoters are chiefly transcribed by SigA, although group 2 sigma factors may also recognize type I promoters. Type II promoters are usually connected to stress or adaptation responses and thus are normally transcribed by group 2 sigma factors, although depending on the specific promoter they may also be recognized by SigA. Type II promoters have a −10 element but typically lack the −35 element, instead, these promoters rely on the binding of upstream transcriptional activators , the RNA helicase RhlB (for unwinding secondary structures), and the glycolytic enzyme enolase  tails. Normally though, RNA polyadenylation is handled by poly(A)-polymerase (PAP), which produces homomeric poly(A) tails  blocks translation and leaves the mRNA vulnerable to RNase attacks. Active degradation takes place when a sRNA-Hfq-RNase E complex binds an mRNA, or when the sRNA-Hfq complex binds an mRNA and thereby creates a target site for the double-stranded endonuclease RNase III, which causes cleavage of both the mRNA and the sRNA , leading to average mRNA half-lives around a few minutes in most prokaryotes  sp. PCC 7120 (Anabaena 7120) and the Synechocystis 6803 RNase E enzymes form a complex with PNPase through a nonapeptide located at the C-terminus of RNase E. Alignments of the RNase E genes from 60 different cyanobacterial strains revealed that this nonapeptide subregion is highly conserved, implying that this RNase E-PNPase complex is a general feature of cyanobacteria . Also, the transcription of the psbA2 asRNA was correlated with the expression of the psbA2 mRNA, both being upregulated by light  in cyanobacteria  for its high activity. Further, it was used to express two heterologous genes to up to 15% of the total soluble protein content. However, it is not clear to what extent the transcriptional activity of the promoter, as compared with the translational efficiency of the native 5′-UTR of cpcB, contributes to the high expression levels. Also, it would be interesting to investigate whether the strong enhancement of gene expression from the promoter fragment containing the predicted 14 TFBS is isolated to transcriptional efficiency. Pcpc560 may prove very useful, both for the design of new, “super-strong” cyanobacterial promoters, and for direct applications, as the lack of strong expression has previously been identified as a bottleneck in cyanobacterial biotechnology  that was previously shown to be very weak in Synechocystis 6803  and a range of different bacteria, thanks to its similarity to the σ70 consensus promoter sequence  were selected for characterization in Synechocystis 6803, where they were found to span a wide range of expression levels (Camsund et al., 70 promoters, they can be expected to function in a wide range of cyanobacteria.Finally, constitutive promoters may be used as an alternative when regulated promoters are not necessary, or used in expression libraries to fine-tune metabolic circuits. A strong promoter from the plant E. coli (Stanton et al., 70 promoters to ensure activity in most cyanobacteria. Examples of potentially useful repressors could be the yeast activator Gal4, which has been shown to function as a repressor in bacteria (Paulmier et al., E. coli (Zhang et al., There is clearly a need for more robust and well-regulated orthogonal promoters to help accelerate cyanobacterial biotechnology. The availability of more orthogonal promoters and TFs will decrease the dependence on strain-specific promoters and enable the sharing of parts, which is of great importance as many different cyanobacterial strains are commonly used, and because different strains grow in different environments. As done recently for Finally, the most orthogonal gene expression system is one that does not rely on the host’s own RNAP at all or otherwise minimally. By using an orthogonal RNAP that does not recognize the host’s own promoters, and for which the host’s RNAP does not recognize the orthogonal promoters, the risk for cross-talk is strongly reduced, and combined with likewise orthogonal TFs, the system is almost completely decoupled from the host’s own transcriptional systems. One such orthogonal RNAP is the phage T7 RNAP and its promoters. T7 RNAP does not recognize the host’s promoters, and vice versa, the host’s RNAP does not recognize the T7 promoters (Temme et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In our previous studies, we reported that SIRT1 prevents cellular senescence in human fibroblast, and that SIRT1-induced inhibition of cellular senescence is due to enhanced hTERT gene expression. In this study, we investigate the molecular mechanisms behind SIRT1-induced potentiation of hTERT transcription and show that FOXO3a functions downstream of SIRT1 and prevents the induction of cellular senescence by enhancing hTERT gene expression. Furthermore, we found that FOXO3a-induced potentiation of hTERT gene expression is regulated in a c-MYC/E-box dependent manner. In addition, we found that FOXO3a binds to the novel binding element in the c-MYC promoter, and this interaction activates the transcription of the c-MYC gene. The resulting increase in c-MYC leads to higher levels of c-MYC recruited to the hTERT promoter and, in turn, activates hTERT gene expression. Taken together, this pathway might constitute the molecular basis for the anti-senescence effects of SIRT1 and FOXO3a. Previous reports have shown that the activation of SIRT1 is beneficial in several age-related diseases, particularly those associated with metabolic dysregulation, through the activation of tissue/cell specific transcription factors SIRT1 is a multifunctional NADhTERT is known to play a crucial role in cellular immortalization, tumorigenesis, and the progression of cancer. Transcriptional regulation of the hTERT gene is a major mechanism underlying the cancer-specific activation of telomerase, and a large number of transcription factors have been identified to directly or indirectly regulate the hTERT promoter In our previous study, we demonstrate that the cellular senescence-inhibiting factor SIRT1 potentiates the transcription of the hTERT gene. Here, we refine the molecular mechanisms for SIRT1-induced enhancement of hTERT transcription.Normal human umbilical cord fibroblasts (HUC-F2) were obtained from Riken Bioresource Center . Cells were maintained in Dulbecco’s Modified Eagle’s Medium  supplemented with 10% fetal bovine serum .Viral supernatants were produced after transfection of 293T cells with pGag-pol, pVSV-G, and individual expression vectors  using the HilyMax reagent  as previously described 5′-GATCCCCGTGGAGCTGGACCCGGAGTTTCGAAGAGACTCCGGGTCCAGCTCCACTTTTTA-3′, shFOXO3a-1 bottom: 5′-AGCTTAAAAAGTGGAGCTGGACCCGGAGTCTCTTCGAAACTCCGGGTCCAGCTCCACGGG-3′; shFOXO3a-2 top: 5′-GATCCCCGAGCTCTTGGTGGATCATCTTCGAAGAGGATGATCCACCAAGAGCTCTTTTTA-3′, shFOXO3a-2 bottom: 5′-AGCTTAAAAAGAGCTCTTGGTGGATCATCCTCTTCGAAGATGATCCACCAAGAGCTCGGG-3′), and cloned into the pSUPER.retro vector . Viral supernatant was prepared as described above, and the cells were infected with this viral supernatant for 24 h at 37°C. Transduced cells were selected with 3 µg/mL puromycin for 3 days. Efficiency data of knockdown experiment was shown in The oligonucleotides that contain the siRNA-expressing sequence targeting FOXO3a were annealed  to the pGL3-Promoter  and named pGL3-DBE 5 cells) cells were seeded onto a 24-well dish and, the next day, were transfected with the reporter and effector constructs using the HilyMax reagent according to the manufacturer’s protocol. After 48 h, a luciferase assay was performed using the Dual-Luciferase Reporter Assay System (Promega). To evaluate the transcriptional activation ability of c-MYC, we generated GAL4-c-MYC, which contains a full-length human c-MYC cDNA (amino acids 1–440) fused to the DNA-binding domain of the yeast transcription factor GAL4  Reporter containing the DAF-16 family protein-binding element (DBE) was generated by ligation of annealed oligonucleotide ((TTGTTTAC)50). HUC-F2 cells underwent adenoviral transduction after a 1-h infection at a multiplicity of infection (MOI) of 50.We produced recombinant adenovirus using the Adeno-X Expression System  according to the manufacturer’s protocol. Recombinant adenoviruses were prepared by disrupting HEK293 cells  transfected with recombinant adenoviral DNA including SIRT1 or SIRT1-HY cDNA . All viruses were titrated using a method that measures the 50% tissue culture infectious dose . cDNA was generated from the isolated RNA using the ReverTra Ace kit . qRT-PCR was performed using the KAPA SYBR Fast qPCR Kit  and the Thermal Cycler Dice Real Time System TP-800 instrument, as described previously 5′-GGTTTTTGAGGGTGAGGGTGAGGGTGAGGGTGAGGGT-3′; tel2: 5′-TCCCGACTATCCCTATCCCTATCCCTATCCCTATCCCTA-3′; 36B4u: 5′-CAGCAAGTGGGAAGGTGTAATCC-3′; 36B4d: 5′-CCCATTCTATCATCAACGGGTACAA-3′. The telomere signal was normalized to the signal from the single-copy gene to generate a relative telomere to a single copy gene ratio indicative of relative telomere length.Genomic DNA was extracted using QIAamp DNA blood Mini kit . Telomere length was determined using qPCR as described previsouly 3), and the crosslinkage was reversed by heating at 65°C for 6 h, followed by treatment with proteinase K at 45°C for 60 min. The DNA was recovered by ethanol precipitation and used as a template for PCR to amplify the target site in the hTERT promoter and c-MYC promoter as previously described 5′-GGCCGGGCTCCCAGTGGATTCG-3′ (−293 to −272) and 5′-CAGCGGGGAGCGCGCGGCATCG-3′ (+20 to −2); c-MYC 5′-AGGGAGAGGGTTTGAGAGGG-3′ (−401 to −382) and 5′-GAAGGTGGGGAGGAGACTCA-3′ (−272 to −291). The relative amount of PCR product amplified from the ChIP assay was normalized to the input DNA and calculated as follows: Relative amount = ([IP] – [IgG])/[Input], where [IP], [IgG], and [Input] are the relative amount of PCR products from 0.25% of input DNA ([Input]), immunoprecipitated DNA with respective antibody ([IP]), and negative control IgG ([IgG]).A ChIP assay was performed using the ChIP assay kit  as described previously t-test. Statistical significance was defined as P<0.05 .All experiments were performed at least 3 times, and the corresponding data are shown. The results are expressed as mean ± standard error of mean. The statistical significance was determined using a two-sided Student’s We have previously reported that SIRT1 inhibited the onset of replicative senescence in normal human fibroblast HUC-F2 cells through transcriptional activation of hTERT Next, we aimed to clarify whether FOXO3a also inhibits the onset of replicative senescence in HUC-F2 cells. HUC-F2 cells were transduced with either FOXO3aWT or FOXO3aTM recombinant retrovirus or a control retrovirus and cultured for 40 days . ProlifeResults from the promoter assay and qRT-PCR show that FOXO3aTM increased hTERT transcription . Next, iNext, we investigated the molecular mechanisms of the FOXO3a-induced increase in hTERT transcription. We first examined the involvement of c-MYC, a dominant transcriptional activator for the hTERT promoter, in the enhancement of hTERT transcription. To clarify this, we employed a wild type hTERT promoter reporter vector (phTERTp-289) and a mutant hTERT promoter reporter vector with disrupted E-boxes (phTERTp-289EM) and performed a promoter assay. Our results indicate that the FOXO3a-induced increase in hTERT transcription is completely abolished with the disruption of the E-boxes . This su–1797TTGTTTT–1790C and –330CTGTTTA–323C) that resemble the consensus element in the human c-MYC promoter  promoter . To assepromoter . Our respromoter . Notablypromoter 1, and S3promoter .In our previous study, we found that SIRT1 prevented cellular senescence by enhancing the transcription of the hTERT gene In addition, we found that FOXO3a-induced enhancement of hTERT transcription is regulated in a c-MYC/E-Box dependent manner. FOXO3a augmented the amount of c-MYC recruited to the hTERT promoter and activated the hTERT promoter through c-MYC-dependent changes in histone acetylation, which likely involves c-MYC-binding to histone acetyltransferases Enhanced c-MYC, in turn, augments the transcription of hTERT, which exerts molecular anti-senescence effects. Previous work has shown that the constitutive expression of TERT in transgenic mice improves epithelial barriers and produces a systemic delay in aging Figure S1FOXO3a expression level in recombinant HUC-F2 cells.(PDF)Click here for additional data file.Figure S2FOXO3a expression level in HUC-F2 cells transduced with shRNA against FOXO3a.(PDF)Click here for additional data file.Figure S3SIRT1 expression level in recombinant HUC-F2 cells.(PDF)Click here for additional data file.


Histone variants establish structural and functional diversity of chromatin by affecting nucleosome stability and histone-protein interactions. H3.3 is an H3 histone variant that is incorporated into chromatin outside of S-phase in various eukaryotes. In animals, H3.3 is associated with active transcription and possibly maintenance of transcriptional memory. Plant H3 variants, which evolved independently of their animal counterparts, are much less well understood.Arabidopsis at mono-nucleosomal resolution using native chromatin immunoprecipitation. This results in the precise mapping of H3.3-containing nucleosomes, which are not only enriched in gene bodies as previously reported, but also at a subset of promoter regions and downstream of the 3′ ends of active genes. While H3.3 presence within transcribed regions is strongly associated with transcriptional activity, H3.3 at promoters is often independent of transcription. In particular, promoters with GA motifs carry H3.3 regardless of the gene expression levels. H3.3 on promoters of inactive genes is associated with H3K27me3 at gene bodies. In addition, H3.3-enriched plant promoters often contain RNA Pol II considerably upstream of the transcriptional start site. H3.3 and RNA Pol II are found on active as well as on inactive promoters and are enriched at strongly regulated genes.We profile the H3.3 distribution in In animals and plants, H3.3 organizes chromatin in transcribed regions and in promoters. The results suggest a function of H3.3 in transcriptional regulation and support a model that a single ancestral H3 evolved into H3 variants with similar sub-functionalization patterns in plants and animals. Drosophila and mammalian cells revealed specific incorporation into the gene body of active genes, into promoter regions of both active and inactive genes, as well as into regulatory elements[Histones are abundant in most eukaryotic cells where they package DNA into chromatin. Dimers of histones H2A, H2B, H3 and H4 assemble into the histone octamer core to organize 147 bp of DNA into nucleosomes, the basic building blocks of chromatin. In recent years, much has been learned about how posttranslational modifications of histones affect chromatin, such as modifying inter-nucleosomal contacts or nucleosome stability. It is now also well established that incorporation of histone variants can result in formation of chromatin with particular properties. The seqelements, supportelements. Unexpecelements,12, and elements. H3.3 iselements. RecentlArabidopsis H3.3 differs from H3.1 at positions 31, 87 and 90 but also at some additional positions[Arabidopsis [Arabidopsis H3.3 has the same epigenomic properties as the animal H3.3 variant. Recently, the genome-wide distribution of the Arabidopsis H3.3 protein At4g40040 was reported and, similar to animal H3.3, was preferentially found in the bodies of transcribed genes[Replication-coupled and replication-independent (replacement) H3 histone variants evolved independently in animals, plants, basidiomycetes, and alveolates. Similarositions. There a5g10980),17, whic5g10980),18. Howeed genes,20. In cArabidopsis genome, we generated Arabidopsis lines expressing H3.3 tagged by yellow fluorescence protein . Among several transformed lines that were phenotypically indistinguishable from wild-type control plants . We next grouped all the genes into four bins according to their relative transcript abundance, and for each group averaged H3.3 profiles were plotted along genes . Therefore, H3.3 incorporation occurs with the highest frequency at the TTSs of genes. In total, we identified 1,891 genes with only promoter-associated H3.3 nucleosomes, 10,447 genes with only TTS-associated H3.3 nucleosomes, and 3,012 genes with both promoter- and TTS-associated H3.3 nucleosomes  during the transcription of genes[Arabidopsis the role of H3.3 in nucleosomes would also be coupled to Pol II activity. Indeed, we found that in Arabidopsis H3.3 co-localizes with Pol II in the nucleus , consistent with the general trend shown in Figure P-value <2.2 × 10-16, one-tail Wilcoxon test). Therefore, H3.3 presence around TTSs is a better predictor for high transcript abundance than H3.3 presence at promoters.We next asked whether H3.3 enrichment at promoters and around TTSs was similarly related to transcription. Genes with H3.3 enriched only around TTSs and genes with H3.3 enriched at both promoters and around TTSs had significantly higher transcript abundance than the genome-wide median . In contrast, genes with H3.3 enriched only around the TTS had significantly larger expression entropies than the genome-wide median . Genes with H3.3 enriched at both promoters and around TTSs had reduced expression entropies, which were, however, much larger than those of genes with H3.3 enriched only at their promoters. These results support our hypothesis that genes with H3.3 enriched at their promoters are subject to strong transcriptional regulation.In budding yeast, Pol II was found upstream of inactive genes that could be rapidly activated upon exit from the stationary phase. We askeing data,28 . Therefore, H3.3 and mCG appear to exclude each other at promoters.Furthermore, genes with H3.3 enriched at promoters lack almost any mCG, especially in the -800 to -200 bp window where H3.3 is highest Figure C. In conIn summary, H3.3 enrichment in nucleosomes does not strongly correlate with mCG in gene bodies and is negatively correlated with mCG at promoters.Arabidopsis promoters can be categorized into three major groups depending on the core promoter element, that is, TATA, GA and coreless[P-value = 0.087, Wilcoxon’s rank test, two-tail). These results show that GA promoters are special because they are targeted by H3.3 incorporation regardless of transcriptional activity.coreless. Followiin vivo DNA bound by H3.3 nucleosomes might be more accessible than DNA bound by H3.1 nucleosomes. To test this hypothesis, we compared the accessibility of DNA[Mammalian H3.3 nucleosomes greatly impair higher-ordered chromatin folding, suggesty of DNA around Hin vitro[per se does not affect stability of mononucleosomes[H3.3 is a histone variant that differs only in four or five amino acids from the canonical H3.1 but it can have profound effects on chromatin functionality. Earlier studies had suggested that animal H3.3-containing nucleosomes isolated from native chromatin are less stable in vitro but moreleosomes. InsteadArabidopsis replacement histone variant H3.3 and found that in plants H3.3 is located in gene bodies as well and shows a positive correlation with transcriptional activity, which is consistent with earlier reports[Drosophila[in vitro after eviction of a single H2A/H2B dimer while the H3/H4 tetramer remains associated with the DNA[per se might not be sufficient to cause H3 replacement. This is consistent with the non-uniform and specific H3.3 patterns along gene bodies. Our results in Arabidopsis revealed a strong 3′ bias along gene bodies when examining plant H3.3 patterns by normalizing either to input or histone density, similar to earlier observations[per se but appears to be restricted to specific phases of the transcription process.H3.3 incorporation is generally thought to be associated with the transcription initiation and/or elongation activities of Pol II in animals and is highest in gene bodies,11,43-46 reports,20. In crosophila,8 to 3′-rosophila,10. Althrosophila. It is n the DNA,48. Comprvations,20. Our Arabidopsis, H3K4me3 is found proximal to TSSs[Arabidopsis H3.3 is highly enriched for H3K36me2[Arabidopsis[Arabidopsis that H3.3 localization extends considerably beyond the TTS indicates an even stronger connection between H3.3 and Pol II transcription termination.In animals, H3.3 levels correlate with transcriptionally active, Ser-5 phosphorylated Pol II, transcription initiation site-related mono- and tri-methylation of histone 3 lysine 4 , and transcription elongation-related tri-methylation of histone 3 lysine 36 (H3K36me3). In Arab to TSSs where H3H3K36me2, the hisabidopsis. This suArabidopsis, we found H3.3 at promoters of both active and inactive genes. It is possible that H3.3 is incorporated at promoters independently of transcription or that it is a footprint of past transcription activity of the gene. Since we could also find Pol II associated with H3.3-enriched promoters, it is possible that RNA Pol II promoter occupation or transcription caused local H3.3 incorporation. Indeed, transcription of promoter-associated short RNAs is more ubiquitous than initially thought during transcription activation[In addition to gene bodies, we found H3.3 also in plant promoters. H3 replacement at active promoters has been reported for mammals, and it tivation,55. H3.3tivation and can tivation. Alternativation-52 to fativation. The inctivation, suggesttivation,56 or prtivation. Our datArabidopsis and animal H3.3 incorporation patterns is consistent with a general association of H3.3 with several eukaryotic chromatin remodeling processes. The presence of H3.3 on active as well as on many inactive plant promoters of strongly regulated genes suggests a function of H3.3 in transcriptional regulation.Animal and plant H3 variants evolved independently,57, but Arabidopsis  accession Columbia-0 plants. To produce 35S:H3.3-YFP lines the cDNA of HTR4 (At4g40030) was fused to the cauliflower mosaic virus (CaMV) 35S promoter at the amino terminus and the YFP cDNA sequence at the carboxyl terminus, and the fusion construct was inserted into the binary vector pCambia1380. Cloning and amplification of the plasmid was done in Escherichia coli DH5α. The plasmid was transformed into Agrobacterium tumefaciens (strain C58C1) and then transformed into Arabidopsis using the floral dip method. Transformants were selected on Murashige and Skoog medium agar plates containing hygromycin. Experimental plants were grown on soil at 21°C in dark (16 h) and 20°C in light (8 h). Plant age was recorded as days after imbibed seeds were sown on soil and transferred to the growth chamber. Leaves (leaf number 6 from about five plants per sample) were harvested after 35 days at zeitgeber time 7 , and frozen in liquid nitrogen. Note that cell division and expansion had ceased at this developmental stage in the harvested leaves. The experiment was performed with three independent biological replicates.All experiments used PP2A.Expression analysis of the H3.3 transgene was performed as described using ge35S:H3.3-YFP seedlings were ground and the powder was extracted with Buffer M (10 mM Tris-(hydroxymethyl)-aminomethan pH 7.5; 0.5% IGEPAL CA 630; 1% Triton X-100; EDTA free protease inhibitor cocktail (Roche)) plus 150 mM NaCl for 10 minutes at 4°C. The suspension was centrifuged at 16,100 × g at 4°C for 10 minutes. The pellet was subsequently extracted using Buffer M containing 500 mM NaCl, centrifuged again, and extracted once more with Buffer M containing 2 M NaCl. Extracted proteins were separated using SDS-PAGE. Total protein was transferred to PVDF-membrane . The H3.3-YFP fusion protein was detected using anti-GFP antibody  and horseradish peroxidase (HRP)-coupled anti-mouse antibody , and was visualized using Immun-Star HRP Substrate .For protein immunoblots, 50 mg of frozen 35S:H3.3-YFP line were germinated and grown for 3 days in Petri dishes on wet filter paper. For visualizing nuclear DNA in live cells, 1 μM of DRAQ5  was applied to Arabidopsis roots for 5 to 10 minutes with vacuum to facilitate penetration. DRAQ5 stain and YFP signals in roots were consecutively analyzed using a Zeiss 710 confocal laser scanning microscope.Seeds of the 4, 5 mM EGTA, pH 6.9). Root tips were digested for 10 minutes at 37°C with a PCP enzyme mixture  dissolved in MTSB) and squashed in a drop of MTSB buffer. Immunostaining was performed as described[35S:H3.3-YFP seedlings were grown on Murashige and Skoog medium for 5 days before YFP signals in roots were analyzed using a Zeiss 710 confocal laser scanning microscope .For immuno-staining, seedlings were fixed for 20 minutes with ice-cold 4% (w/v) paraformaldehyde in MTSB buffer ) for 15 minutes at 4°C. The homogenate was filtered through Miracloth , and a nuclei pellet was collected by centrifugation for 10 minutes at 1,500 × g at 4°C. Isolated nuclei were washed once in MNase buffer ), treated with 1.3 μl of RNase A, 30 μg/μl  and used for Micrococcal Nuclease  digestion for 4 minutes  in MNase buffer. The reaction was stopped with 10 mM EDTA. After a centrifugation the supernatant was collected as phase 1 chromatin preparation. The pellet was resuspended in buffer S2 ) for 30 minutes. After centrifugation the supernatant was collected as phase 2 chromatin preparation. The two phases of chromatin preparations were combined and the NaCl concentration was adjusted to 50 mM. The majority of the chromatin was of mononucleosome size (data not shown). Histone H1 was depleted by incubating the chromatin preparation with Sephadex C25-CM resin  for 1 h at 4°C[2O, Sigma-Aldrich) and collected with Dynabeads Protein A (Invitrogen). After washing, beads were re-suspended in TE buffer , and DNA was extracted using phenol-chloroform extraction and ethanol/salt precipitation. Cross-linked ChIP was performed as described[Native ChIP was performed as described with minescribed. ChIP waqPCR was performed using the ChIP-recovered DNA as template using specific primers and probes  followed by purification using MinElute PCR Purification kit .qPCR was performed for six genomic fragments before and after amplification to control for amplification bias (data not shown). Amplified ChIP DNA was fragmented, labeled and hybridized to Affymetrix AGRONOMICS1 escribed.P-values were calculated using Wilcoxon’s signed rank test.Background correction and normalization were performed as described previously. ChIP-chSupplementary raw data are available in ArrayExpress, accessibp: base pair; ChIP: chrommatin immunoprecipitation; MNase: micrococcal nuclease; PCR: polymerase chain reaction; Pol II: RNA Polymerase II; qPCR: quantitative PCR; TSS: transcription start site; TTS: transcription termination site; YFP: yellow fluorescence protein.The authors declare that they have no competing interests.HS, WG, LH designed the research; HS, MN, LB, IM, TW performed research; HS, AS, LH analyzed data; HS, WG, LH wrote the paper. All authors read and approved the final manuscript.Figure S2. ChIP-qPCR confirmation for H3.3 incorporation. Figure S3. H3.3 is present at transcribed genes and has a strong 3′ bias. Figure S4. Schematic representations of positional relation between test genes and amplicons for qPCR. Figure S5. H3.3 incorporation profiles for genes with differential H3.3 nucleosome association. Figure S6. Genes with H3.3 nucleosomes in the promoter are strongly regulated upon environmental stress. Figure S7. H3K27me3 profiles of Polycomb group target genes and genes with H3.3 in promoters. Figure S8.Arabidopsis H3.3 does not colocalize with H2A.Z. Figure S9. Identification of nucleosomes.Generation of H3.3-YFP expressing plants. Click here for fileTable S2. Primers and Universal Probes (Roche) for qPCR used in this study.List of genes with H3.3 nucleosomes in the promoter, around the TTS or both in promoters and around TTSs and list of all H3.3 nucleosomes. Click here for file


Recent studies have identified bi-directionally transcribed RNAs emanating from these enhancers known as eRNAs. However, it remains unclear how tightly coupled eRNA production is with enhancer activity. Through our systematic search for long-range elements that interact with the interferon-β gene, a model system for studying inducible transcription, we have identified a novel enhancer, which we have named L2 that regulates the expression of interferon-β. We have demonstrated its virus-inducible enhancer activity by analyzing epigenomic profiles, transcription factor association, nascent RNA production and activity in reporter assays. This enhancer exhibits intimately linked virus-inducible enhancer and bidirectional promoter activity that is largely dependent on a conserved Interferon Stimulated Response Element and robustly generates virus inducible eRNAs. Notably, its enhancer and promoter activities are fully retained in reporter assays even upon a complete elimination of its associated eRNA sequences. Finally, we show that L2 regulates IFNB1 expression by siRNA knockdown of eRNAs, and the deletion of L2 in a BAC transfection assay. Thus, L2 is a novel enhancer that regulates IFNB1 and whose eRNAs exert significant activity  Transcriptional enhancers stimulate transcription rate or transcription probability of their target genes ,2 and cain vivo  has long been a model system for studying inducible transcription –28, and in trans , chromatin state assessments and sequence analyses to identify novel putative enhancers associated with the IFNB1. Using luciferase assays, we confirmed that a transcriptional element we named ‘L2′ is a virus inducible enhancer that can regulate transcription driven by IFNB1′s proximal regulatory elements. These experiments also showed that L2 includes bidirectional promoter activity that is intimately linked with its enhancer activity. We used global run-on sequencing (GRO-seq) analysis to map eRNA production, and used luciferase assays to show that L2 retained both its promoter and enhancer functions in the absence of its associated eRNA sequences. We conclude that L2 is a novel IFNB1-associated enhancer that exhibits promoter activity that is tightly linked to its enhancer activity. Notably, the specific sequences of its associated eRNAs are dispensable in reconstituting both L2′s promoter and enhancer activities in a heterologous reporter system. This suggests that this enhancer is a complex element that may exert multiple activities that stimulate transcription.Cell lines were purchased from American Type Culture Collection and cell culture media and supplements were purchased from Life Technologies, Inc., Carlsbad, CA. IMR90 primary human lung fibroblast cells were grown in Minimum Essential Media (MEM) supplemented with Earle's salt, 10% Fetal Bovine Serum (FBS) and 1-mM Sodium Pyruvate. HEK293 cells and NIH3T3 cells were each grown in Dulbecco's modified Eagle's medium supplemented with 10% FBS. Both were grown in 37°C incubator with 5% CO2.Transfections used Lipofectamine 2000 (Life Technologies), preparing it with the DNA in Optimem (Life Technologies) per manufacturer's recommendations. The DNA–lipofectamine complexes were added to cells resuspended in growth media before plating. Transfected cells were placed back in the incubator for 3–5 h after transfection before any further treatment.Sendai Virus (SeV) (Charles River Laboratories), was centrifuged to clarify the virus and stored at –80°C. For virus infections, we used 50 μl of virus per 1 ml of culture media. Interferon-β  was used at 0.1 ng of IFN-β per 1 ml of culture media. These treatments were for 6 h, except for the Luciferase assays, which were for 24 h before prepping the cells for the assay.6 IMR90 cells and 1.2 × 106 nuclei per ligation. Small-scale 3C was performed using 1 × 106 cells at large-scale volumes through formaldehyde crosslinking and quenching, reduced to cell-proportional scale from the cell lysis and to the ligation. Additionally, the small-scale 3C ligation used 1.5 × 105 nuclei in a 1.5 ml reaction volume. Each large-scale 3C was performed once, while the small-scale 3C was performed with three biological replicates.3C experiments were performed as previously , but adjRelative-enrichment (RE) was assessed with quantitative Real-time polymerase chain reaction (PCR) using FastStart SYBR Green Master reagent  on a Mastercycler Realplex 2 , comparing the enrichment of a primer pair from 400 ng of 3C library relative to 40 ng of a randomly ligated library of Bacterial Artificial Chromosome fragments covering the region of interest. See Supplementary Table S2 for the bacterial artificial chromosome (BAC) clones used to create the BAC control library. Each Real-time PCR was done with four independent measures. Primers were designed with Primer 3 , tiled aBecause of the higher repeat density of this locus , we coulAll human genomic analysis was performed on the hg18 build, while mouse analysis was done with the mm9 build. All visualizations were done with the University of California at Santa Cruz (UCSC) Human Genome Browser . Data foThe Conserved Elements and Multiz Alignment data was previously published –49, whilAll digestion and ligation reactions were performed with enzymes from New England Biolabs. Promoter-testing constructs used enzymes KpnI and XhoI to sub-clone L2 into pGL3-basic, with XhoI and HindIII used to subclone the IFNB1 −110 region into it. Enhancer-testing constructs used SalI and BamHI to sub-clone in the fragment of interest into either pGL3-promoter or pGL3–110. In exception to this, subcloning of 4L2F utilized XhoI only, and was ligated into the compatible SalI site in pGL3-promoter. Please see Supplementary Table S1 for primers associated with each construct, with primers designed by Primer 3 .Mutagenesis was performed according to the guidelines provided in the QuikChange Site-Directed Mutagenesis Kit , with the following variations. PCR utilized a final dNTP concentration of 2 mM (Roche Applied Science). PCR was performed for 16 cycles. The final DpnI digested product was transformed into electrocompetent TOP10 cells generated by an existing protocol . Please 3 V Plate reader, Turner Biosystems Modulus Luminometer 9200–001 or the Turner Biosystems Veritas Microplate Luminometer 9100–002. For any samples with independent replicates of at least nine, the highest and lowest data points were excluded. The sample size listed in the caption accounts for the two removed data points.Luciferase assay conditions followed standard protocols associated the Dual-Glo Luciferase Assay System . Luciferase counts were read either on the Perkin-Elmer Victor6 cells per immunoprecipitation, with an SMC1A antibody , a Med1 antibody , an RNAPII antibody  and a phospho-IRF3 antibody . See Supplementary Table S1 for primer sequences, designed with Primer 3  as a negative control. We used a pool of two custom-synthesized ds-siRNAs for each eRNA, with each siRNA at equal concentration. RNA extraction and oligodT-primed cDNA synthesis was performed as previously described . Please Uninfected and SeV-infected IMR90 cells were subjected to GRO-seq, with mapping and alignment to the hg18 human genome performed with bowtie, according to previously published protocols .Recombineering was performed according to previously published protocols  using thcis-regulatory elements form chromosomal loops to their target genes  reactions per site from each of the uninfected and infected 3C libraries, as well as from the BAC control library, for a total of 2064 qPCR reactions. If the 3C-based proximal ligation between the IFNB1 promoter and an interrogated site produced any detectable product in the majority of Real-time qPCR reactions in either the uninfected or infected state, we then selected the site for subsequent validation steps. Our locus-wide 3C analysis identified seven EcoRI restriction fragments as preliminary candidate interacting sites Figure . Two of We pursued these five sites for validation and included an additional site in between sites L2 and L3 as a negative control, as no meaningful interaction was detected between IFNB1 and this restriction fragment Figure . We set We then analyzed if the status of any of the chromatin loops were specifically induced by viral infection Figure . Only thRecent work has shown that the genome is organized into large-scale topological domains, and that most chromosomal interactions occur within a single topological domain . BecauseExcluding the site L5 as a promoter for CKDN2B, we focused on sites L1–L3 as our candidate enhancers. We assessed the levels of H3K4me1 and H3K27ac enrichment, along with DNase I hypersensitivity of these sites to further evaluate these candidate enhancers. Previous work has demonstrated that, upon virus infection, p300 is recruited to sites that are pre-marked as enhancers , indicatblastn algorithm to search for similar murine genomic sequences more comprehensively  (To further assess the  (pIRF3) , RNAPII  (pIRF3) , Cohesin (pIRF3)  and Medi (pIRF3) , to L1–1 (pIRF3)  by ChIP. (pIRF3)  and C, a (pIRF3) . Med1 al (pIRF3) . In addi (pIRF3) . InteresGiven the discovery of eRNAs as an indicator of enhancer activity ,20, we weRNAs exhibit a characteristic transcriptional signature of bi-directional transcription emanating outward from the enhancer element . In the We looked for evidence of other transcriptional units at L2 that would be consistent with mRNAs. However, we found no annotated genes near L2 from either the Refseq ,64 or EnWe tested a 563-bp fragment of L1–1 that contains conserved DHS, which we call L1F, for promoter activity by reporter assays Figure  and B. IP = 2.4 × 10−6 and 6.4 × 10−4), representing 3.2-fold and 4.5-fold virus inducibility (P = 0.0018 and 9.9 × 10−4) in orientations 1 and 2, respectively. The difference in activity between orientations was not statistically significant. These results demonstrate that L2 contains orientation-independent virus-inducible enhancer activity, as expected from an enhancer associated with the IFNB1 gene.We placed a 543-bp fragment of L2 centered on the DHS, which we called L2F, downstream of the SV40 promoter-driven luciferase gene in the reporter construct to assess its enhancer activity Figure  and C, aP = 0.0021 and 1.2 × 10−5 in orientations 1 and 2, respectively). Upon virus infection, L2F drove dramatically higher promoter activity in both orientations 1 and 2 (P = 0.0035 and 2.6 × 10−4 respectively). These represented virus-inducibility of 16-fold (P = 0.0052) for orientation 1 and 14-fold (P = 0.0032) for orientation 2. We found no meaningful difference in activity between orientations. These results demonstrate that L2 contains orientation-independent, bidirectional basal promoter activity that is robustly virus-inducible.Based on our detection of eRNAs by GRO-seq, we were curious about whether or not L2 exhibited any promoter activity. We tested this by placing L2F upstream of a promoter-less luciferase gene. Figure . In bothOur sequence analysis had revealed that L2 contains a conserved ISRE Figure . In the P = 0.016 and P = 0.0067). There was no meaningful difference between the activities of the different orientations of mutant L2F. Together, these results suggest that the integrity of ISRE significantly contributes to L2′s promoter activity.Each mutated L2 construct was compared against the wild-type and empty-vector versions of the construct, first assessing promoter activity. In orientations 1 and 2 Figure  and C, wP = 0.0051 and P = 0.023, respectively). Taken together, these results demonstrate that the ISRE is a major contributor to the transcriptional activities of L2. The involvement of the ISRE sequence is consistent with our finding that IRF3 is inducibly recruited to L2 . Likewise in the infected state, the retained enhancer . This indicated that, while the 2 bp mutation had a significant impact on overall virus-inducible promoter and enhancer activities of L2, it was insufficient to completely ablate either activity from this element. Thus, mutant L2F retains low but significant residual activities that are independent of this mutation.We also assessed whether or not the residual activities of mutant L2F relative to baseline in the infected state were statistically relevant. Indeed, the infected state promoter activities Figure  and C inr Figure  and E acKnowing that L2's activity was dependent on the ISRE, it remained ambiguous whether or not L2 was regulated by IRF3 or ISGF3. Given that virus infection activates IFNβ expression that in turn activates ISGF3 , we hypoin vivo, then L2 and the previously characterized IFNB1 proximal regulatory elements . The inclusion of L2F further increased luciferase activity by more than 2-fold in both orientations . Thus, L2 is capable of enhancing the activity of the −110 element, as expected of an enhancer that would regulate IFNB1 transcription.The virus-infected state revealed that L2F could act as an enhancer for the −110 element Figure . The −11We had initially defined L2F based on the location of the DHS in the segment. However, when we mapped L2F GRO-seq transcription unit Figure , we notiP = 2.6 × 10−5 and 5.7 × 10−6 respectively). While these activities exhibited statistically significant differences to the basal promoter activities of L2F in both orientations 1 and 2 , the magnitude of these differences were weak . In the virus-infected state, 0.3L2F exhibited robustly induced promoter activity for both orientations 1 and 2 . In orientation 1, the virus-induced activity of 0.3L2F was lower than that of L2F in a statistically significant manner, but the magnitude of difference was small . Conversely, the virus-induced enhancer activity of 0.3L2F was higher than that of L2F in a statistically significant manner (P = 0.0084). However, it was also a weak difference (1.68-fold). These data show that L2F retains its promoter activity in the absence of its associated eRNA sequences.To test the necessity of the eRNA sequences to promoter activity, we compared 0.3L2F to L2F. We found that promoter activity of 0.3L2F was robust in the uninfected state for both orientations 1 and 2 , in orientation 1 . These activities were higher than those of 4L2F (P = 2.6 × 10−4 and 1.1 × 10−4), but only marginally . Taken together, we concluded that sL2F retains functional virus-inducible promoter and enhancer activities independent of the inclusion of the eRNA sequences as part of the element in our reporter assays.We then tested whether the presence or absence of associated eRNA sequences impacted the overall enhancer activity of this fragment, using 0.3L2F and 4L2F. No enhancer activity was detected for 0.3L2F in either orientation in the uninfected state Figure  and E. I1 Figure . It alsoP = 0.0048), but did not reach the nominal significance for si−eRNA (P = 0.088). In addition, si-eRNA increased the expression the +eRNA by 100% (P = 0.016), while si+eRNA appeared to increase the expression of the –eRNA by a 50% factor that did not reach statistical significance (P = 0.052). Even though the impacts on the −eRNA did not quite reach significance, we noted that siRNAs against eRNA expression has historically been relatively weak (P = 0.024), while knockdown of +eRNA reduced IFNB1 induction by 79% (P = 0.0025). From this, we concluded that the eRNAs produced from L2 are necessary for the appropriate virus-inducible expression of IFNB1 in its endogenous genomic and chromosomal contexts.Our luciferase assay data suggested the possibility that L2′s enhancer activity might be independent of the eRNAs produced by that element. To test this hypothesis directly, we designed a pool of siRNAs to the eRNAs produced off of each strand, si+eRNA and si−eRNA. We found that each siRNA reduced the expression of its target eRNA by 50% Figure , which wely weak –24. Becaely weak . We founHaving defined 0.3L2F as a minimal unit sufficient to contain virus-induced enhancer activity Figure , we wantWe have presented multiple lines of evidence demonstrating that L2 is associated with, and regulates, IFNB1. First, we observed spatial proximity with the IFNB1 promoter Figure , in a viin vivo  treatment of macrophages, which stimulates Type I interferon production, leads to p300 recruitment to sites that are already premarked for enhancer activity at the chromatin level . ENCODE'These results imply that the cell makes significant steps to establish a transcriptionally quiescent but permissive chromatin state and conformation prior to the immune response, regardless of whether or not it is an immune cell. It seems clear that this chromatin state in immune cells is established by the transcription factor PU.1 , and it The GRO-seq data have been submitted to the ArrayExpress archive. The accession number for the data is E-MTAB-2233.Supplementary Data are available at NAR Online.