We found that full RKIP promoter activity requires the region −56 to +261 relative to the transcription start site. Within the full promoter region, there are two motifs rich in G/C that responded to transcription factor Sp1, one cAMP-responsive element that responded to the transcription factor CREB, and one docking site for the histone acetylase p300. In human melanoma A375 cells and human cervical cancer HeLa cells, mutation or deletion of each of these cis-acting elements decreased promoter activity. In A375 cells, knockdown of the corresponding transcription factors Sp1, CREB, or p300 decreased RKIP promoter activity, whereas overexpression of CREB and p300 increased RKIP promoter activity. The results obtained with HeLa cells also supported the idea that Sp1 and CREB play positive roles in the regulation of RKIP transcription. These findings suggest that regulators of the expression or activity of Sp1, CREB, and p300 are involved in regulating RKIP transcription.The Raf kinase inhibitory protein (RKIP) is down-regulated in multiple types of human cancers. Decreased RKIP transcription activity may be one of the major mechanisms responsible for the downregulation of RKIP expression in human diseases. To test this hypothesis, we need to gain basic knowledge of the transcriptional regulation of RKIP. To achieve this objective, we made a systematic effort to identify  Activation of receptor tyrosine kinases by growth factors and cytokines promotes cell proliferation, survival, and migration through activation of the Ras-Raf-MEK-ERK cascade cis-acting elements and the trans-acting factors that regulate RKIP promoter activity.Raf kinase inhibitory protein  is a well-characterized inhibitor of Raf kinase cis-acting elements and trans-acting factors that regulate gene transcription cis-acting elements and corresponding transcription factors that regulate RKIP promoter activity. Our results demonstrated, for the first time, that Sp1, CREB, and p300 are among the critical transcription factors that positively regulate RKIP transcription.Luciferase-based reporter activity assay and electrophoretic mobility shift assay (EMSA) are well-established approaches to identifying Pol-II-containing transcription machinery can be assembled In a typical gene, the region −40 to +50 relative to a transcription start site constitutes the core promoter region in which the To further define the promoter region, we performed 5′- and 3′-deletions of the inserted RKIP sequence and determined the effect on its promoter activity by luciferase assay in A375 cells and in HeLa cells. From the 5′ end, deletion of the region −813 to −194 increased luciferase activity by ∼2-fold in both cell lines, whereas the next deletion (region −193 to −57) produced minor and inconsistent effects in these cell lines . Howevercis-acting elements within region −56 to +261 of the inserted RKIP sequence, we searched the sequence for potential transcription factor binding sites. Although no TATA box sequence was identified, two putative Sp1 binding sites (−17 to −6 and −5 to +5), one putative CREB binding site (−28 to −17) and one p300 binding site (+108 to +121) were identified within this region  or the −5 to +5 site (Sp1 II) with nuclear extracts of A375 or HeLa cells resulted in multiple electrophoretic mobility shifts  and in HeLa cells  . These rThe critical roles of these two Sp1 binding sites in RKIP promoter activity indicate that Sp1 plays a positive role in RKIP transcription. To test this prediction, we transfected A375 or HeLa cells with Sp1-specific siRNA or negative control siRNA and determined the effect of Sp1 knockdown on both the promoter activity of the reporter construct and the level of endogenous RKIP transcript. In both cell lines, as determined by immunoblotting, transfection with Sp1-specific siRNA reduced the expression of Sp1 to ∼50% of its original level . This le32P-labeled oligonucleotide with nuclear extracts resulted in one obvious gel mobility shift band. The shift band was greatly reduced by adding a large excess (20- to 80-fold) of the cold WT oligonucleotide but not by adding the same concentration of the mutant oligonucleotide , one functional CREB binding site upstream of the Sp1 binding sites (region −28 to −17), and one p300 binding site downstream of the Sp1 binding sites (region +108 to +121). All three kinds of A minimal promoter activity requires the assembly of general transcription machinery by binding of an initiating protein near the transcription initiation site The efficiency of the assembly of the general transcription machinery is greatly enhanced by recruitment of co-activators that both bind to regions close to the minimal promoter region and interact with components in the general transcription machinery The rate of the transcription conducted by the assembled transcription machinery is known to be greatly affected by chromatin structures Identification of these three kinds of transcription factors that positively regulate the RKIP promoter activity suggests that the expression or function of these transcription factors regulates RKIP expression. Thus far, the transcriptional activity and stability of Sp1 can be regulated by posttranslational modifications, including phosphorylation, acetylation, sumoylation, ubiquitylation and glycosylation 2 in Dulbecco’s modified Eagle’s medium (GIBCO) supplemented with 10% fetal bovine serum (HyClone).The A375 (CRL-1619) and HeLa (CCL-2) cell lines were purchased from American Type Culture Collection and cultured at 37°C in 5% COKpn I and Bgl II sites for directed cloning. Site-directed mutations were introduced using mutant primers (Genomic PCR was performed by using Phusion high-fidelity DNA polymerase (New England Biolabs) and human genomic DNA (Promega) as the template. The RKIP gene transcription regulatory region was cloned into the pGL3 Basic vector (Promega), and various deletion constructs were prepared by PCR with added  primers . All PCRsiRNAs used for silencing endogenous expression of Sp1 and p300 were synthesized by GenePharma according to the sequence information previously reported 5′-CGGAATTCGGTAACTAAATGACCA-3′  and 5′-CGGGATCCATCCCAAATTAATCT G-3′ . The PCR products were inserted into the pcDNA3.1 (-) vector (Invitrogen) and the construct was confirmed by DNA sequencing.Generation of CREB plasmid was engineered using a PCR technique. We amplified a 1059-bp fragment of the human genomic cDNA and reverse-transcripted it to mRNA by using M-MLV reverse transcriptase (Promega) according to the manufacturer’s protocol. The following primers were used for amplification: 5 cells/well in 24-well plates. On the following day, transfection was performed with 0.6 µg of the indicated promoter-luciferase constructs or the empty vector pGL3-Basic together with 0.2 µg of pCMV-β-galactosidase plasmid. Transfection was performed using Lipofectamine2000 transfection reagent (Invitrogen) according to the manufacturer’s instructions. For RNAi experiments, specific siRNA or control RNA was co-transfected into A375 or HeLa cells with the reporter plasmids respectively, and the cells were further cultured for 48 hr. Cells were then lysed with Promega’s cell culture lysis reagent , and luciferase activity level was measured using Promega’s luciferase assay system (Cat.#E1500) and normalized to β-galactosidase activity Cells were plated at a density of 1.6×10Protein extraction from A375 or HeLa cells, SDS-PAGE, and immunoblotting were performed as previously described 32P] ATP. Nuclear extracts were incubated with 32P-labeled oligonucleotide probes with or without unlabeled oligonucleotide competitors (20- to 80-fold molar excess of labeled probes). The samples were then separated with 4% non-denaturing polyacrylamide gel electrophoresis at 300 V in 0.5×TBE buffer for 2 hr. The gel was dried and exposed to X-ray film (Kodak) overnight at −80°C.Nuclear extracts were prepared by using a Fermentas cytoplasmic and nuclear protein extraction kit. Complementary strands of oligonucleotides were synthesized by Invitrogen . EMSA wa5′-AGACCCACCAGCATTTCGTG-3′ and 5′-GCTGATGTCATTGCCCTTCA-3′; p300, 5′-AGGTCTTCTTTGTGATCCG-3′ and 5′-CCAACCACACCAGTCCG-3′; GAPDH, 5′-GCACCGTCAAGGCTGAGAAC-3′ and 5′-TGGTGAAGACGCCAGTGGA-3′.Total RNA was extracted from A375 or HeLa cells using TRIZOL reagent (Invitrogen) according to the manufacturer’s protocols. For reverse transcription (RT), 2 µg of RNA was annealed to oligo (dT) at 65°C for 5 min and cooled to room temperature. The RNA-oligo (dT) mixture was then incubated with M-MLV reverse transcriptase (Promega) and dNTPs at 42°C for 1 hr. For the PCR step, RT products were amplified by PCR under log phase conditions. The sequences of the primers for RT-PCR were as follows: RKIP, www.cbrc.jp/research/db/TFSEARCH.html) using the default parameters.Analysis of the transcription factor binding sites was done by TFSEARCH online Click here for additional data file.Table S2Oligonucleotides for EMSA.(DOC)Click here for additional data file.


The mammalian Peg3 domain harbors more than 20 evolutionarily conserved regions (ECRs) that are spread over the 250-kb genomic interval. The majority of these ECRs are marked with two histone modifications, H3K4me1 and H3K27ac, suggesting potential roles as distant regulatory elements for the transcription of the nearby imprinted genes. In the current study, the chromatin conformation capture (3C) method was utilized to detect potential interactions of these ECRs with the imprinted genes. According to the results, one region, ECR18, located 200-kb upstream of Peg3 interacts with the two promoter regions of Peg3 and Zim2. The observed interaction is most prominent in brain, but was also detected in testis. Histone modification and DNA methylation on ECR18 show no allele bias, implying that this region is likely functional on both alleles. In vitro assays also reveal ECR18 as a potential enhancer or repressor for the promoter of Peg3. Overall, these results indicate that the promoters of several imprinted genes in the Peg3 domain interact with one evolutionarily conserved region, ECR18, and further suggest that ECR18 may play key roles in the transcription and imprinting control of the Peg3 domain as a distant regulatory element. In mammals, a small subset of autosomal genes are expressed mainly from one parental allele due to an epigenetic mechanism termed genomic imprinting . These gcis-regulatory elements similar to other imprinted domains . The converted DNA was used for PCR amplification using the two following primers: ECR18-bis-a  with the DMEM plus GlutaMAX medium containing 5% fetal bovine serum and 1% antibiotic-antimycotic (GibcoBRL). On the following day, the cells were transfected with 1 µg of each reporter construct using 2.5 µL Lipofectamine 2000 (Invitrogen). Fresh complete media was added 6 hrs post transfection, and total cell lysates were harvested in 100 µL of 1X reporter lysis buffer 48 hrs post transfection (Promega). The luciferase assay was performed for each promoter construct in triplicate according to the company’s protocol (Promega). We also performed a similar set of transfection experiments using an independent β-Geo reporter construct to monitor the transfection efficiency. Thus, the initial values from the luciferase assay were normalized with the β-Gal activity.For promoter assays, we first modified the promoterless β-Geo reporter  by replaMaterial S1Sequence information for 18 ECRs and oligonucleotides used for 3C and ChIP analyses.(DOCX)Click here for additional data file.


Cockayne syndrome is an inherited premature aging disease associated with numerous developmental and neurological defects, and mutations in the gene encoding the CSB protein account for the majority of Cockayne syndrome cases. Accumulating evidence suggests that CSB functions in transcription regulation, in addition to its roles in DNA repair, and those defects in this transcriptional activity might contribute to the clinical features of Cockayne syndrome. Transcription profiling studies have so far uncovered CSB-dependent effects on gene expression; however, the direct targets of CSB's transcriptional activity remain largely unknown. In this paper, we report the first comprehensive analysis of CSB genomic occupancy during replicative cell growth. We found that CSB occupancy sites display a high correlation to regions with epigenetic features of promoters and enhancers. Furthermore, we found that CSB occupancy is enriched at sites containing the TPA-response element. Consistent with this binding site preference, we show that CSB and the transcription factor c-Jun can be found in the same protein-DNA complex, suggesting that c-Jun can target CSB to specific genomic regions. In support of this notion, we observed decreased CSB occupancy of TPA-response elements when c-Jun levels were diminished. By modulating CSB abundance, we found that CSB can influence the expression of nearby genes and impact nucleosome positioning in the vicinity of its binding site. These results indicate that CSB can be targeted to specific genomic loci by sequence-specific transcription factors to regulate transcription and local chromatin structure. Additionally, comparison of CSB occupancy sites with the MSigDB Pathways database suggests that CSB might function in peroxisome proliferation, EGF receptor transactivation, G protein signaling and NF-κB activation, shedding new light on the possible causes and mechanisms of Cockayne syndrome. Cockayne syndrome is a devastating inherited disease, in which patients appear to age prematurely, have sun sensitivity and suffer from profound neurological and developmental defects. Mutations in the CSB gene account for the majority of Cockayne syndrome cases. CSB is an ATP-dependent chromatin remodeler, and these proteins can use energy from ATP-hydrolysis to alter contacts between DNA and histones of a nucleosome, the basic units of chromatin structure. CSB functions in DNA repair, but accumulating evidence reveals that CSB also functions in transcription regulation. Here, we determined the genomic localization of CSB to identify its gene targets and found that CSB occupancy displays high correlation to regions with epigenetic features of promoters and enhancers. Furthermore, CSB is enriched at genomic regions containing the binding site for the c-Jun transcription factor, and we found that these two proteins interact, uncovering a new targeting mechanism for CSB. We also demonstrate that CSB can influence gene expression in the vicinity of its binding sites and alter local chromatin structure. Together, this study supports the hypothesis that defects in the regulation of gene expression and chromatin structure by CSB might contribute to the diverse clinical features of Cockayne syndrome. The additional proteins that form complexes with ATP-dependent chromatin remodelers are often involved in enhancing the specific activity of the remodeler or targeting the remodeling complex to specific genomic regions Cockayne syndrome is a devastating inherited disease in which patients have features of premature aging, display increased sun sensitivity, and suffer from profound neurological and developmental defects In vitro, the CSB remodeler appears to interact with DNA in a sequence-independent manner, and CSB displays both DNA and nucleosome stimulated ATP hydrolysis activity CSB is best known for its function in transcription-coupled DNA repair, a process that preferentially removes bulky DNA lesions that stall transcription, such as those created by UV irradiation In addition to its well-documented function in DNA repair, accumulating evidence indicates that CSB also participates in transcription regulation To understand better how CSB carries out its diverse functions and to gain potential insights into the underlying mechanism of Cockayne syndrome, we performed a genome-wide study of CSB occupancy to elucidate the mechanisms of CSB targeting and to understand the impacts of CSB occupancy on transcription regulation.To identify genomic regions that CSB occupies, we performed chromatin immunoprecipitation using a monoclonal antibody raised against CSB, followed by deep sequencing. CS1AN-sv cells that were reconstituted with CSB were used for these experiments, as CS1AN-sv cells do not express the alternatively spliced CSB-PiggyBac fusion protein  [30]; thThe resulting sequencing reads were mapped to the human genome (HG19 assembly) using the Bowtie aligner We subsequently classified peaks unique to CSB or CSBΔN1 . To do tWe then classified the CSB and CSBΔN1 occupancy sites into seven functional categories see , using tGiven that CSBΔN1 is more significantly enriched at promoters and 5′ UTRs than CSB  , we calcWe next classified the genomic localization of the common and unique occupancy sites into the seven functional categories described above . We founTo gain further insight into the potential functions of CSB, we classified CSB and CSBΔN1 occupancy sites according to the 15 chromatin states defined by Ernst et al. (2011), which are largely based on the presence or absence of specific histone marks  [33], . ThTo better understand the mechanisms by which CSB is targeted to specific genomic regions during replicative cell growth, we used HOMER to determine if binding motifs for sequence-specific transcription factors were enriched at sites of CSB occupancy . This anGiven that CSB does not bind to DNA in a sequence-specific manner We next used shRNA-mediated RNA interference to directly examine the impact of c-Jun on the targeting of CSB to genomic regions containing a TPA-response element. As shown in CSB-PGBD3 is a fusion protein that arises from alternative splicing between sequence encoding the N-terminal 465 amino acids of CSB with sequence encoding a piggybac transposase that lies within the fifth intron of the CSB gene Gene ontology analysis using GREAT revealed the top three “Biological Processes” associated with common CSB and CSB-PGBD3 occupancy sites were tissue development, positive regulation of developmental processes and positive regulation of catecholamine secretion . GREAT wWe next determined the extent to which CSB occupancy can impact the expression of nearby genes. Using reverse transcription coupled with quantitative PCR (RT-qPCR), we compared RNA expression levels of 10 genes, which displayed significant CSB occupancy, in CS1AN-sv cells expressing CSB to those in CS1AN-sv cells harboring an empty vector ZNFX-NC1 and MCPH1 are two examples of genes that were positively regulated by CSB . The ZNFAlso shown in To identify, on a global level, potential direct transcriptional targets of CSB, we compared our ChIP-seq data to publicly available CSB microarray data In the CS1AN/hTERT cells, Newman et al. (2006) identified 188 CSB up-regulated genes and 205 down-regulated genes. As shown in The Bailey et al. (2012) study also examined the transcription profile of USS1KO cells coexpressing CSB and CSB-PGBD3 as well as USS1KO cells expressing CSB-PGBD3 alone. When we compared the CSB occupancy data with those transcription profiling data  and S4, To determine whether the remodeling activity of CSB is required for its function in transcription regulation, we used RT-qPCR to examine the effect of the remodeling defective CSBΔN1 protein on RNA expression To further demonstrate that the chromatin remodeling activity of CSB is important for regulating ZNFX-NC1 expression, we performed micrococcal nuclease (MNase) sensitivity assays. MNase is a nuclease that preferentially digests naked DNA and leaves nucleosomal DNA intact. ENCODE data obtained from the extensively studied K562 and GM12878 cell lines indicates that there are two MNase-resistant regions adjacent to the CSB occupancy site in the ZNFX-NC1 gene  [33]: onAs shown in We next examined the MNase sensitivity of a region of the PRMT5 promoter near a CSB occupancy site. Remodeling by CSB is partially required for suppression of PRMT5 expression . As depiWe also examined MNase sensitivity near the site of CSB occupancy in an intron of the MAD1L1 gene, where several MNase-resistant regions were predicted based on ENCODE data Lastly, we examined MNase sensitivity at the promoter of the WDR74 gene, where remodeling by CSB was dispensable for CSB-dependent suppression of WDR74 expression. Cells expressing CSB or CSBΔN1 displayed very similar patterns of MNase sensitivity, agreeing with the results of our RT-qPCR analysis , which rOur results reveal that the mechanism that targets CSB to chromatin for transcription regulation is distinct from the mechanism that targets CSB during transcription-coupled DNA repair Recently, Gray et al. showed that the N-terminal region of CSB mediates the interaction between c-Jun and CSB-PGBD3 CSB and CSB-PGBD3 both interact with c-Jun, and 45% of peaks common to CSB and CSB-PGBD3 contain a TPA-responsive element  [39]. ThFrom comparisons between ChIP-seq and transcription profiling data, it can be seen that the majority of CSB occupancy sites are not associated with known CSB-responsive genes –6. This The composition of the dimeric AP-1 transcription factors that bind to TPA-response elements varies; for instance, there are three Jun and four Fos family members, and some of these members have variants that result from alternative splicing Saccharomyces cerevisiae revealed that this remodeler is primarily targeted to specific loci by sequence-specific transcription factors; however, more than half of the transcription factor-dependent occupancy sites did not contain a cognate binding motif Approximately 15% of the total CSB occupancy sites contain TPA-response elements. The mechanism that underlies the targeting of CSB to regions of the genome that do not contain TPA-response elements is not yet clear, but it is likely that CSB is delivered to or stabilized at these regions through associations with other DNA-binding proteins. A recent study examining the targeting of Isw2 in Our ChIP-seq data revealed that 36% of the CSB peaks were unique to CSB and 24% of the CSBΔN1 peaks were unique to CSBΔN1 . We do nin vitro; therefore, CSB likely repositions nucleosomes in vitroBy examining the effect of CSB on several genes that lie close to CSB occupancy sites, we provide evidence that CSB can directly influence local gene expression mediated by RNA polymerase II. CSB is a member of the SWI2/SNF2 ATP-dependent chromatin remodeling protein family and displays ATP-dependent chromatin remodeling activity 2), approximately 90% of the CSB population can become stably associated with chromatin During replicative cell growth, approximately 10% of CSB associates with chromatin, and this likely represents the CSB population that participates in normal transcription regulation. However, in the presence of UV-induced DNA damage  Mission shRNA targeting c-Jun  was used to decrease c-Jun protein levels. A non-targeting shRNA  was used as a negative control. Virus was produced by cotransfecting a 10 cm plate of ∼90% confluent 293T cells with third generation lentivirus packaging plasmids . A total of 20 µg of plasmid was transfected, with the individual plasmids at an equal molar ratio. The culture medium was changed 24 hours post-transfection, and virus-containing medium was collected 24 hours later. Medium from one plate of virus-producing cells was distributed to six 10 cm dishes of target cells: CS1AN-sv/CSB. The confluence of the target cells at the time of infection was approximately 20%. Infected cells were harvested 36–48 hours post-infection for RNA preparation and western blot analysis.Total RNA was prepared using TRIzol (Invitrogen). AMV reverse transcriptase and random primers were used for first strand cDNA synthesis (Roche). cDNA was analyzed by real-time PCR using a MyiQ thermal cycler and SYBR green (BioRad). Expression was first normalized against β-actin and fold over vector-only control was then calculated using ΔΔCt method 2, 20 mM HEPES pH 7.8, 10% Glycerol, 0.5% Triton X-100) and soluble CSB was separated from chromatin by centrifugation at 15,000 RPM for 5 min at 4°C. The resulting pellets were resuspended in Buffer B and fixed with 1% formaldehyde for 10 min at room temperature. Cross-linked cells were sonicated at 40% amplitude  using the Branson 101-135-126 Sonifier. Chromatin IP (ChIP) was performed using a monoclonal anti-CSB antibody (1B1) that recognizes the N-terminal 507 amino acids of CSB To increase ChIP efficiency we removed soluble CSB before cross-linking DNA and proteins http://ngsc.med.upenn.edu/). The Next-Generation Sequencing Core at the University of Pennsylvania performed DNA sequencing using Illumina hiSeq2000 sequencers for single-end sequencing with a read length of 50 bps. The resulting sequencing reads were mapped to the human genome (HG19 assembly) using Bowtie version 0.12.7. Peaks were identified using HOMER version 4.1 (Hypergeometric Optimization of Motif EnRichment) with a default option  on ChIPed samples against matching input DNA samples. Raw and processed files (GSE50171) have been deposited at the Gene Expression Omnibus (GEO) repository. (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=vfmfryagmygcony&acc=GSE50171).10 ng of ChIPed DNA was used to prepare libraries for deep sequencing using the multiplexed ChIP-Seq sample preparation protocol described on the website of the Next-Generation Sequencing Core, Perelman School of Medicine, University of Pennsylvania  of the CSB and CSBΔN1 signals within a 200 bp region around a peak center. If the difference between signal intensities was 4-fold or greater and the p-value for that difference was ≤0.0001, the peak was classified as unique. The remaining signals were classified as common. CSB and CSBΔN1 peaks were classified as follows: (1) promoter (from −1 kb to the transcription start site), (2) TTS (from the transcription termination site to +1 kb), (3) 5′ UTR, (4) 3′ UTR, (5) exon, (6) intron, and (7) intergenic (the remainder). The source of gene annotation was UCSC RefGene. The CEAS package The Genomic Regions Enrichment of Annotations Tool  was used for pathway analysis of CSB occupancy sites, using the “MSigDB pathways' category” To compare the genomic occupancy of CSB to CSB-PGBD3, we downloaded the ChIP-seq data for CSB-PGBD3 and the matching input from the GEO repository (GSE37919) 2, 1 mM DTT, 0.5% triton X-100 and 20% glycerol) with protease inhibitors  and centrifuged at 15,000 rpm for 5 min at 4°C. The supernatant was used for co-immunoprecipitation assays. CSB-containing complexes were recovered using the monoclonal anti-CSB antibody 1B1 (1 hour at 4°C) and protein G agarose beads (30 min at 4°C). The resulting immunocomplexes were washed four times in buffer B60, and the immunoprecipitated proteins were eluted with Laemmli buffer. Beads-only control immunoprecipitations were conducted in parallel.CS1AN cells expressing CSB were lysed in Buffer B60 (20 mM HEPES (pH 7.9), 60 mM NaCl, 0.5 mM MgCl260 = 500) were incubated with MNase  at 37°C for 10 min and reverse cross-linked at 65°C for 16 hours. After phenol-chloroform extraction, digested DNA was resolved on a 1.2% agarose gel in 1×TAE at 100 V for 3.5 hr. Mononucleosome-size DNA fragments were purified from gel slices and subjected to qPCR analysis  and CSBΔN1 peaks were classified using the epigenomic information derived from NHLF  cells, via different chromatin features using chromHMM To compare the CSB ChIP-seq data with the microarray data, we used the lists of up- and down-regulated genes generated by Newman et al. (2006) and Bailey et al. (2012) Figure S1CSB-PGBD3 is not expressed in CS1AN-sv cells reconstituted with CSB. Whole-cell lysates from 293T cells, CS1AN-sv cells, and CS1AN-sv cells reconstituted with CSB were subjected to immunoprecipitation using the monoclonal anti-CSB N-terminal antibody 1B1. The immunoprecipitated material, along with the input lysates, was resolved in a 7% Tris-Acetate gel (NuPAGE) and the western blot was probed with the polyclonal anti-CSB N-terminal antibody. The monoclonal antibody immunoprecipitated both CSB and CSB-PGBD3 from 293T cells, but this antibody only immunoprecipitated CSB from CS1AN-sv cells reconstituted with CSB. Nothing was immunoprecipitated from CS1AN-sv cells. The band marked with an asterisk, present in lanes 1, 2 and 3, is of unknown identity; however, this protein could not be immunoprecipitated with 1B1 and is, therefore, likely cross-reacting with the polyclonal antibody.(TIFF)Click here for additional data file.Figure S2Genomic distributions of CSB and CSBΔN1. The genome was divided into seven categories defined by the UCSC RefSeq gene annotation. The genomic distributions of CSB and CSBΔN1 were determined using the CEAS package and are presented as pie charts (TIFF)Click here for additional data file.Figure S3CSB and CSBΔN1 occupancy positions relative to transcription start sites. The number of CSB and CSBΔN1 binding sites were plotted as a function of distance from transcription start sites (TSS) using a bin size of 500 bp.(TIFF)Click here for additional data file.Figure S4Genomic distributions of peaks common or unique to CSB and CSBΔN1. Distributions were determined using the CEAS package and are presented as pie charts. The table includes p-values for these distributions.(TIFF)Click here for additional data file.Figure S5Genomic distribution of CSB peaks containing TPA-response element. Distributions were determined using the CEAS package and are presented as a pie chart. The table includes p-values for these distributions.(TIFF)Click here for additional data file.Figure S6Preparation of mononucleosomal DNA for MNase sensitivity assays. (A) After limited MNase digestion of formaldehyde cross-linked nuclei, the cross-links were reversed, and the DNA was purified and resolved in an agarose gel. (B) Mononucleosomal DNA purified from (A) and used in the qPCR assays.(PDF)Click here for additional data file.Figure S7MNase-qPCR analysis of the WDR74 promoter region. Upper panel from top to bottom: screen shots from the UCSC genome browser (GRCh37/hg19 Assembly) showing the RefSeq gene and direction of transcription (arrow head), the position of CSB occupancy (CSB ChIP), the position of CSBΔN1 occupancy (CSBΔN1 ChIP), ENCODE MNase-seq data obtained from K562 cells (MNase), and the amplicons used in the MNase-qPCR assays (Amplicon). The chromosome coordinates shown are chr11:62,608,860-62,609,234. Lower panel contains bar graphs showing results from the MNase-qPCR assays. The primers used in the MNase-qPCR assays are listed in (TIFF)Click here for additional data file.Table S1Genomic annotation of peaks common to CSB and CSBΔN1.(XLSX)Click here for additional data file.Table S2Genomic annotation of peaks unique to CSB.(XLSX)Click here for additional data file.Table S3Common sites of CSB and CSB-PGBD3 occupancy.(XLSX)Click here for additional data file.Table S4Lists of genes correlated in (XLSX)Click here for additional data file.Table S5Primers used in ChIP-qPCR assays.(DOCX)Click here for additional data file.Table S6Primers used in RT-qPCR assays.(DOCX)Click here for additional data file.Table S7Primers used in MNase-qPCR assays.(DOCX)Click here for additional data file.


Vibrio cholerae carries a chromosomal superintegron, composed of an integrase, a cassette integration site (attI) and an array of mostly promoterless gene cassettes. We determined the precise location of the promoter, Pc, which drives the transcription of the first cassettes of the V. cholerae superintegron. We found that cassette mRNA starts 65 bp upstream of the attI site, so that the inversely oriented promoters Pc and Pint (integrase promoter) partly overlap, allowing for their potential co-regulation. Pint was previously shown to be induced during the SOS response and is further controlled by the catabolite repression cAMP-CRP complex. We found that cassette expression from Pc was also controlled by the cAMP-CRP complex, but is not part of the SOS regulon. Pint and Pc promoters were both found to be induced in rich medium, at high temperature, high salinity and at the end of exponential growth phase, although at very different levels and independently of sigma factor RpoS. All these results show that expression from the integrase and cassette promoters can take place at the same time, thus leading to coordinated excisions and integrations within the superintegron and potentially coupling cassette shuffling to immediate selective advantage.Chromosome 2 of  Vibrio cholerae, a marine bacterium, is the causative agent of cholera in humans, a severe diarrheal disease often fatal by dehydration. Its genome is characterized by the presence of two chromosomes: a large one which contains the vast majority of genes essential for cell functions and pathogenicity, and a small one which contains a large number of genes of hypothetical function and of exogenous origins Vibrio species, called a superintegron (SI) intI, a primary recombination site, attI, and a strong promoter, Pc, located upstream of attI, either in the non coding sequence upstream of intI or in intI itself , in a single cassette array. While most of the cassettes found in the Vibrio chromosomal integrons have unknown functions, the few that were characterized comprise phage-related proteins, toxin/antitoxin systems, acetyltransferases, sulfate-binding proteins, lipases, polysaccharide biosynthesis proteins, dNTP pyrophosphohydrolases, DNA modifying enzymes and virulence factors I itself . The genV. cholerae alternates between various environmental growth conditions: freshwater - which is regarded as its reservoir - brackish water, planktonic growth, biofilm formation on crustacean shells in the sea and transit through the “gastric acidity barrier” before colonizing the intestinal tract during human infection During the course of its life cycle, attC site and the adjacent cassette, as it has been described in the chromosomal integron of Vibrio sp. DAT722. This can explain how a portion of the cassette array is transcribed attI itself, as in class 2 integrons intI gene, as in class 1 and 3 integrons V. cholerae most uncharacterized cassettes carry an open reading frame with a potential start codon located very close to the recombination site, leaving no space for a promoter. As for the characterized cassettes, their transcription relies on the Pc promoter carried in the platform. The Vch IntIA integrase allows cassette excision through recombination between two VCR sites and cassette integration through attI x VCR recombination V. cholerae SI, no Pc cassette array promoter has yet been characterized and the question of how cassette genes are expressed remains unanswered. However, it was recently found that the catB9 cassette Several cassettes of large chromosomal integrons, such as those carrying toxin-antitoxin gene pairs, have been found to carry their own promoter V. cholerae integron integrase, has been characterized and shown to be directly controlled by LexA and induced during the SOS response, and also by the cAMP-CRP complex intIA by LexA and cAMP-CRP occurs via specific regulatory boxes located in the superintegron integrase (intIA) promoter region On the other hand, the Pint promoter, which controls the expression of the V. cholerae superintegron cassettes, we first characterized the mRNA start site and determined the location of the cassette promoter (Pc). We then explored how various growth conditions impact the Pc and Pint activities, in order to define favorable environmental factors that increase the expression of the cassette array and/or of the integrase. The role of the SOS response and of the cAMP-CRP complex in cassette array expression was also investigated.In order to shed light on the conditions which regulate the expression of the lacZ, crp or recA deletions were introduced in the V. cholerae N16961 chromosome by homologous recombination as previously described Bacterial strains and plasmids used in this study are listed in Total RNA was purified as previously described 5′ RACE assay was performed with 2 µg total RNA isolated from strainω8320, using the standard protocol of the SMARTER RACE cDNA amplification kit (Clontech) and primer GSP1 . PCR waslacZ, using primer pair Elrork7_1Fw/Elrork7_1Rw, and inserted into EcoRI and BamHI sites of p4730 giving rise to p7476. pA089 was used to create the CRP binding site mutant and p9327 for the Pint LexA box mutant. Overlapping sequences were realized to confirm correct fusion insertion in the different mutant strains context. crp deletion was introduced in the chromosome of V. cholerae by homologous recombination using plasmid p8348, as previously described rpoS deletion was introduced using the same protocol and plasmid pB410.Strains were constructed as previously described E. coli. Mutations were confirmed by sequencing.Plasmids p9327 (LexA box mutant) and pA089 (cAMP-CRP complex box mutant) were constructed by amplifying the complete plasmid p7476 using Herculase (Stratagene) and primer pairs 24/25 and crpmut5/crpmut3, respectively. After column purification, the amplification product was digested by DpnI to remove the native p7476 and then transformed into β-galactosidase activities were determined by the method of Miller intI gene or in the region between the attI recombination site and the intI translational start site V. cholerae. Determining the position of the transcriptional start site is crucial to study the expression of the cassette array in the V. cholerae superintegron. Using a 5′RACE-PCR assay, we located the cassette mRNA start 65 bp upstream of the attI recombination point and 132 bp upstream the intIA transcription start site , marine environment  and the human gastrointestinal tract . To investigate the role of these parameters on the expression of the cassette array and of the intIA gene, we constructed lacZ transcriptional fusions of both Pc and Pint promoters , marine broth  and LB broth (rich medium) at 29°C . The two promoters were generally less active in marine broth while the highest expression levels were observed in rich medium for Pc and in both M63 and LB broth for Pint . intIA expression was found to be 2.7-fold higher at 37°C than at 29°C and, in this condition, its expression was favoured by the absence of NaCl  and found a moderate 1.5 fold induction at 42°C compared to 37°C, identical in both mitomycin C induced SOS response or no induction conditions , grown until the end of exponential phase in Bacto-Tryptone medium supplemented with 1% glucose or succinate, as previously described To explore this hypothesis, we performed β-galactosidase assays using strain ω8320 (Pc-crp strain was constructed (strain ω9369), and the lacZ fusion expression level was compared with those obtained in the wild type context. Deletion of crp led to a 6-fold decrease of the cassette array expression at the end of exponential phase in LB with high concentration of NaCl  strains grown in LB medium, with high level of NaCl, during the entry in stationary phase at 42°C. No significant difference was observed (2216 vs 1855 U/dry weight: 1.17-fold), suggesting that RpoS has no direct or indirect role in the activation of the Pc promoter in these conditions.As the cAMP-CRP complex role only explains the Pc regulation by the entry in stationary phase, we investigated the involvement of RpoS in all the Pc regulations. V. cholerae N16961 superintegron cassette array promoter, Pc, which insures the expression of the first cassettes of the array, and found that it is located upstream of the superintegron primary recombination site attI  is highly conserved in all sequenced V. cholerae genomes and similar regulation mechanisms and expression conditions can be expected for the SI integrase and the cassettes of all V. cholerae strains. In addition, both the regulatory LexA and CRP boxes, as well as the Pc and Pint sequences are also conserved in the V. cholerae closest relative, Vibrio mimicus, and it is likely that similar regulatory mechanisms are at play for both promoters in this species , high (332 mM) or no NaCl, from 29°C to 42°C), the cassette expression reaching its maximal level at the beginning of the stationary phase Click here for additional data file.


Drosophila melanogaster, recruitment of PcG proteins to targets requires the presence of at least one polycomb response element (PRE). Although the sequence requirements for PREs are not well-defined, the presence of Pho, a PRE-binding PcG protein, is a very good PRE indicator. In this study, we identify two PRE-containing regions at the PcG target gene, giant, one at the promoter, and another approximately 6 kb upstream. PRE-containing fragments, which coincide with localized presence of Pho in chromatin immunoprecipitations, were shown to maintain restricted expression of a lacZ reporter gene in embryos and to cause pairing-sensitive silencing of the mini-white gene in eyes. Our results also reinforce previous observations that although PRE maintenance and pairing-sensitive silencing activities are closely linked, the sequence requirements for these functions are not identical.Polycomb-group (PcG) proteins are highly conserved epigenetic transcriptional regulators. They are capable of either maintaining the transcriptional silence of target genes through many cell cycles or enabling a dynamic regulation of gene expression in stem cells. In  Drosophila melanogaster  genes were initially identified as repressors of Hox genes in nogaster . In receDrosophila PcG protein complexes have been described, Polycomb repressive complex 1 (PRC1), PRC2, and Pleiohomeotic (Pho)-RC. Additional Drosophila complexes that contain PcG proteins dRAF and PR-DUB also have been identified  , and Grainyhead (Grh), , but the exact sequence requirements for PRE activity remain elusive (In s (PREs) , Dorsal  elusive . This is elusive . The othUltrabithorax (Ubx) or engrailed (en) produce ectopic expression of a lacZ reporter, in addition to expression within the normal domains of Ubx and en, respectively. Inclusion of a PRE within the transgene prevents ectopic expression and maintains lacZ expression within the normal boundaries of the endogenous gene  is a zygotic gap gene that affects the development of the head and abdominal regions in Drosophila. Its identification as a PcG target gene was based on the isolation of dominant E(z) alleles that suppress the nanos (nos) maternal effect (nos) phenotype was shown to be attributable to failure to maintain repression of the gap genes gt and knirps initiated by maternal Hunchback (Hb). ChIP-on-chip studies have confirmed the presence of PcG proteins at gt in embryos  (−1.3 to −2.5 kb) produces the posterior stripe; gt_(-10) (−8.8 to −10.5 kb) produces the major anterior stripes; gt_(-6) (−4.3 to −6.5 kb) produces the anterior tip expression; and gt_(-1) (−0.05 to −1.3 kb) produces both the posterior and major anterior stripes. The role played by the apparent redundancy of gt_(-1) with gt_(-10) and gt_(-3) in regulation of the endogenous gt gene is not clear. Beyond gastrulation, gt primarily is expressed in the head region . To initiate a more detailed analysis of PcG regulation of gt, we have mapped the locations of gt PREs.l effect . The Su(observed . This exd region . Throughch as Hb . Howeverhttp://flystocks.bio.indiana.edu) unless otherwise specified. To produce the SD10 constructs, gt genomic fragments were PCR-amplified from CH322-101F2 or CH322-5H16 BAC clones (BACPAC resources: http://www.pacmanfly.org/) using primers that included FRT sequences and NspI sites . Amplified fragments were digested with NspI and ligated to the vector SphI site located between the en enhancer and promoter (in the same orientation relative to the en promoter as in the gt promoter). SD10-gt constructs were injected into 1118w embryos by Genetics Services  or BestGene . Additional lines were obtained by transposon mobilization using P[Δ2-3]  and SD10CL (5′-GTTTTCCCACTCACGACGTTG-3′) primers. To examine β-galactosidase expression in a PcG-mutant background, SD10-gt males were crossed to 401 ph-p602 w1/FM7cph-d females. To test for pairing-sensitive silencing, males were aged 2 days after eclosure and the eye colors of flies that were homozygous for the transgene were compared to those of flies that were heterozygous for the same transgene.Strains are described at the Bloomington Drosophila Stock Center web site . Embryos were dehydrated and mounted in Permount (Fisher Scientific). Images were obtained using a Ziess Axiovert 200M microscope.Embryos were collected at 25° then were fixed and processed essentially as previously described  with theOregon-R embryos were collected for 30 min, aged for 170 min at 25°, and then dechorionated in 50% hypochlorite bleach, briefly washed with water, and fixed in 2% formaldehyde in PBS:heptane (1:3) for 20 min. Fixation was quenched by addition of glycine to a final concentration of 50 mM. Embryos were then washed, weighed, flash-frozen, and stored at −80°. Embryos were homogenized in 50 mM Tris (pH 8.1) and 10 mM EDTA supplemented with 1.25× complete EDTA-free protease inhibitor cocktail (Roche). SDS was added to a final concentration of 1%. After 10 min of incubation on ice, samples were sonicated in a cup horn sonicator (Misonix sonicator 3000) at 4° to produce DNA fragments that were predominantly within the range of 0.3 to 0.7 kb. An equivalent of 2 mg of embryos was set aside as input genomic DNA and incubated with 0.2 M NaCl overnight at 65° to reverse the crosslinks. The remainder of the supernatant was diluted 10-fold with ChIP dilution buffer, and chromatin from the equivalent of 3.2 mg of embryos was incubated with 2.5 μl rabbit anti-Pho antisera (The protocol was performed as indicated in the ChIP assay kit (Millipore) with the following modifications. Table S2. PCR reactions were performed in triplicate for each ChIP experiment. The data presented are the average of three independent ChIP experiments from independent chromatin preparations. Percent input values were calculated using Rotor Gene software by comparing Ct values of samples to standard curves established by the Ct values of total chromatin controls.Quantitative PCR was performed with PerfeCTa SYBR Green SuperMix (Quanta Biosciences) using a Rotor Gene RG3000 thermocycler (Corbett Research). Immunoprecipitated DNA from 100 μg of embryos was used for each PCR reaction. Sequences of primers are in P values were calculated using unpaired t test to determine statistical significance.Eye pigmentation was quantified by homogenizing a total of 10 male heads (4 days after eclosion) from each fly group in 0.5 ml of 0.01 M HCl in ethanol. Homogenate was left overnight at 4°, warmed for 5 min at 50°, and then centrifuged. The absorbance of the supernatant was measured at 480 nm   . ChIP ason of gt . The firon of gt . These r embryos . Even thgt genomic fragments were inserted into the SD10 P-element vector, which contains an engrailed (en) enhancer and promoter upstream of the lacZ reporter gene. The en enhancer produces lacZ expression in 14 stripes and resembles endogenous en expression. Without the presence of a PRE, ectopic expression of lacZ is detected between en-like stripes. However, the addition of an en PRE (invected (inv) or Ubx (bxdd) genes  enhancer; gt2 contains the entire gt_(-3) enhancer; gt3 does not include an identified enhancer and does not show Pho binding by ChIP; gt4 contains the gt_(-6) enhancer; and the gt4 and gt5 fragments overlap by 1.2 kb and contain the second Pho-positive region. The constructs were used to produce multiple transgenic lines. The inserts were deleted from a subset of these lines by FLP recombinase. Stage 14 embryos containing intact transgenes and their deletion derivatives were tested for β-galactosidase expression.Guided by the locations of Pho-positive ChIP regions and the locations of nhancers : gt1 incgt1, SD10-gt4, or SD10-gt5 showed restricted en-like patterns of β-galactosidase expression, indicative of the presence of a PRE within those constructs, whereas all SD10-gt2 and SD10-gt3 lines showed distinct ectopic expression of the lacZ reporter. Representative embryos for each construct are shown for each in en-like expression pattern was lost upon excision of gt1, gt4, and gt5 inserts  mutant (401 ph-p602ph-d double mutants) and β-galactosidase expression was compared to the same transgenic lines in a wild-type PcG background . This suggests that the region shared by gt4 and gt5 is necessary, but not sufficient, for pairing-sensitive silencing, and that additional sequences contained within gt5, but not gt4, are needed. Quantitative assays performed with the same fly lines shown in Because some, but not all, of the previously tested PREs exhibit pairing-sensitive silencing , all SD10 vector . The eyece assay , three ot3 lines . Curiousilencing . Of the gt locus. One is near the promoter and a second is ∼6 kb upstream. This organization is most similar to the PRE distribution at the invected (inv) gene, which has two PREs located at similar positions relative to the inv transcription start site  gene. en has two closely linked PREs that are located in an interval spanning ∼0.4 to 2.4 kb upstream of the transcription start site (even skipped (eve) gene also has two PREs; one is near the promoter and the second PRE is ∼9 kb downstream (inv, en, and eve may contribute to physical interactions between remote cis-regulatory regions and promoters (en PREs than by either PRE alone suggests that they are only partially redundant (gt PREs may be functionally redundant remains to be determined. It is also possible that gt PREs may independently regulate distinct enhancers or may somehow cooperate in the establishment and/or maintenance of PcG-mediated repression.We have identified and mapped the locations of two PRE-containing regions at the Mcp PRE, which regulates the Abdominal-B gene of the Bithorax complex. The 800-bp Mcp core fragment behaves as a functional PRE in reporter maintenance assays but is unable to demonstrate pairing-sensitive silencing unless supplemented with additional flanking sequences or possibly other regulatory sequences from other genes (gt4 and gt5 may be similar to the Mcp core fragment in that both SD10-gt4 and SD10-gt5 maintain repression of en enhancer-driven reporter expression, but only SD10-gt5 exhibits pairing-sensitive silencing. It may be that sequences contained in gt5, but not gt4, include binding sites for proteins needed for pairing-sensitive silencing, but not for maintenance assays. Definition of the sequence requirements for pairing-sensitive silencing will require further dissection of this portion of the gt regulatory region and mutational analysis of potential transcription factor binding sites.Even though most PREs that test positive in maintenance assays also show pairing-sensitive silencing, it has been shown that pairing-sensitive silencing does not always correlate with PRE function. In some cases, a pairing-silencing element, conferring pairing-sensitive silencing, is distinct from a PRE. One example is the Although attempts have been made to identify PREs based on their sequences (gt1, gt4, and gt5 sequences reveals that, although Pho consensus sites coincide with the ChIP-defined localization of Pho in embryos (gt2 and gt3 fragments have eight and seven Pho consensus sites, respectively, but are negative for Pho binding in ChIP assays and fail to behave as PREs in the maintenance and pairing-sensitive silencing assays. The extended Pho-consensus sequence (gt regulatory region. It seems probable that a PRE is more likely to be determined by the presence of multiple binding sites for an array of DNA-binding proteins, and that Pho binding may involve cooperative interactions with some combination of other DNA binding factors (gt cis-regulatory region at later developmental stages.Examination of the  embryos , Pho doe embryos . For exa factors . It is agt PRE-containing regions within the gt cis-regulatory region. Consistent with other characterized PREs, both gt PRE-containing regions correspond to localized binding by Pho in vivo. The presence of Pho at a single location within each of these regions indicates that it is likely that each contains just one PRE. However, at this time we cannot rule out the possibility that one or both regions may contain multiple closely linked PREs.In conclusion, we have demonstrated the presence of two


Here we examine the evolution of mRNA degradation rates between two closely related yeast species. Surprisingly, we find that around half of the evolutionary changes in mRNA degradation were coupled to transcriptional changes that exert opposite effects on mRNA levels. Analysis of mRNA degradation rates in an interspecific hybrid further suggests that opposite evolutionary changes in transcription and in mRNA degradation are mechanistically coupled and were generated by the same individual mutations. Coupled changes are associated with divergence of two complexes that were previously implicated both in transcription and in mRNA degradation (Rpb4/7 and Ccr4-Not), as well as with sequence divergence of transcription factor binding motifs. These results suggest that an opposite coupling between the regulation of transcription and that of mRNA degradation has shaped the evolution of gene regulation in yeast. cis or trans). This analysis indicated that coordinated changes in transcription and mRNA degradation are likely to be driven by the same individual mutations, and thus directly coupled. Finally, we suggest several mechanisms that may mediate this coupling, including complexes which are involved in both processes (Rpb4/7 and Ccr4-Not) and promoter regulatory regions. These results suggest that a direct coupling between the regulation of transcription and mRNA degradation is a common phenomenon employed by approximately 10% of the genes in yeast.The regulation of mRNA levels in the cell is important to ensure, for instance, timely cellular responses to changes in the environment. mRNA transcription and mRNA degradation directly affect mRNA levels and it would make sense to have a system in place that would coordinate these opposing processes. Previous studies suggested that regulation of transcription in the nucleus may be linked to regulation of mRNA degradation in the cytoplasm, yet the details of this connection are poorly understood. In this study, we took an evolutionary approach to address this question by comparing both transcription and mRNA degradation between two yeast species. We found that evolution of these distinct processes is coordinated, as genes that diverged in mRNA degradation tend to also diverge in transcription. Interestingly, the coordination is counterproductive, as increased transcription is linked to increased mRNA degradation. We analyzed a hybrid between the two yeast species to classify evolutionary differences according to the type of underlying mutation ( Work on the regulation of mRNA levels has traditionally focused on transcription, although mRNA levels reflect the balance between transcription and mRNA degradation. Recent studies have shown that regulation of mRNA degradation also has a central role in control of gene expression, and in certain systems might be as important as transcription regulation S. cerevisiae to environmental perturbations and found coordinated changes in mRNA degradation and transcription Although both transcription and mRNA degradation individually contribute to the regulation of mRNA levels, they are ultimately integrated to form a coherent regulatory system, and several studies provided evidence for crosstalk between the regulation of transcription and mRNA degradation. First, two conserved and general regulatory complexes, the Rpb4/7 dimmer, which is composed of two subunits of RNA polymerase II Taken together, these observations suggest that transcription and mRNA degradation are often coordinated. However, this coordination remains poorly understood, raising several important questions. What is the scope of this coordination? What mechanisms underlie this coordination and are they directly or indirectly influencing both processes? What is the mode of coordination—is transcriptional induction mostly coordinated with decreased degradation, increased degradation, or both? What is the functional significance of such coordination?S. cerevisiae and S. paradoxus. These species diverged from a common ancestor ∼5–10 million years ago and maintained similar physiology and genomic sequences (∼90% identity), yet as we have shown previously cis and trans, by allele-specific analysis of the interspecific hybrid, suggests a direct mechanistic coupling whereby individual mutations influence both transcription and mRNA degradation. These mutations seem to involve Rpb4/7, Ccr4-Not, as well as additional unknown factors.To address these questions, we set out to examine the coordination between transcription and mRNA degradation from an evolutionary perspective, by comparing two closely related yeast species, 2 mRNA levels with time  above 0.94, while genes with lower R2 were excluded from further analysis. The calculated mRNA degradation rates of S. cerevisiae genes were highly reproducible among two biological repeats and between probes that were designed for different positions of the same genes, and were consistent with previous measurements of mRNA degradation that utilized a PolII mutant strain to block transcription  and above 1.4-fold , or in a previous work A levels . This inrevisiae .Technical biases do not seem to have a significant effect on the observed coupling. First, the coupling is observed for large differences in mRNA degradation (red section), but not for genes with very small changes in degradation, which are more dependent on technical variations , while trans-mutations occur in other loci and indirectly influence the affected gene through the activity of another protein . Importantly, the genome-wide contributions of cis- and trans-mutations can be uncovered by analysis of inter-species hybrids: cis-mutations discriminate between two hybrid alleles that reflect orthologs from the two species, while trans-mutations do not discriminate between the two hybrid alleles, as the alleles are in the same nucleus and thus exposed to the same set of trans-regulators. This approach has previously been used to assess the contribution of cis- and trans-mutations to total mRNA levels Transcription and mRNA degradation are controlled by different mechanisms and are thus expected to diverge through a separate set of mutations. However, the strong coupling that we observe suggests the intriguing possibility that individual mutations may influence both transcription and mRNA degradation, generating opposing effects on mRNA levels. Although we cannot identify the effect of individual mutations, this possibility can be examined by differentiating between the contributions of S. cerevisiae and S. paradoxus, with two biological repeats and using the same method as described above for the two species. For each gene whose mRNA degradation rate differs between the species, we examined whether this difference is maintained (cis) or abolished (trans) between the corresponding two hybrid alleles. This analysis indicated that ∼60% of the differences in mRNA degradation reflect primarily cis-mutations, while ∼40% reflect trans-mutations  and to trans-effects in transcription (cis-trans combination). However, if transcription and degradation changes are mechanistically coupled and the observed opposite effects are generated by the same individual mutations, then these coupled changes would be generated by a single effect, either in cis (cis-cis combination) or in trans (trans-trans combination), but not by cis-trans or trans-cis combinations. Consistent with this, a strong coupling is observed only for cis-cis and trans-trans combinations but not for cis-trans or trans-cis combinations  suggests that divergence of upstream regulator(s) has influenced both processes. We thus searched for enrichment of 85 high-confidence trans-coupled genes with targets of 116 transcription factors (TFs) p<0.05) among the trans-coupled genes compared to uncoupled genes (trans-coupled genes (p = 2×10−7). Thus, our results are consistent with previous studies showing that these two complexes influence both transcription and mRNA degradation.Coupling between ed genes . Notablyed genes  and threed genes , which wtrans-coupled genes , and indeed we find that targets of Rpb4/7 are highly enriched among coupled trans-effects with higher S. paradoxus transcription and degradation but not among those with higher S. cerevisiae transcription and degradation (The enrichment of onserved . Increasradation .cis-changes in mRNA degradation and cis-changes in transcription (cis-coupling) suggests that mutations in a gene's promoter, coding-region, terminator or untranslated regions influenced both processes. This may reflect mutations that affect the recruitment of specific proteins to the loci of that gene, which then influence both transcription in the nucleus and degradation of the resulting mRNA following its export to the cytoplasm. To examine this possibility, we first searched for enrichment of 92 high-confidence cis-coupled genes with targets of the various regulators, as described above for the trans-coupled genes. Only one of the 170 datasets was enriched among the cis-coupled genes with a p value below 0.01 (cis-coupling (p = 7×10−5), suggesting that cis-mutations may have influenced the recruitment of Rpb4/7 to many genes. At a p value of 0.05, only one additional target gene-set was enriched (Hap3), while ∼9 sets would be expected by pure chance (0.05×173).Coupling between low 0.01 . This dacis-coupled genes, suggesting the existence of other mechanisms for cis-coupling. We next examined the sequence divergence between S. cerevisiae and S. paradoxus at various predicted and known cis-regulatory elements. Analysis of diverged 3′-UTR sequences that were predicted to influence mRNA stability cis-coupled genes . This enrichment was found both for known S. cerevisiae TF binding sites S. paradoxus and for predicted S. paradoxus TF binding sites that are not conserved in S. cerevisiae  suggesting a mechanistic coupling. (iv) Trans-coupling is associated with regulators that are known (Rpb4/7 and Ccr4-Not) to control both transcription and mRNA degradation, while cis-coupling may be associated with diverged TF motifs.Our systematic comparison of mRNA degradation among two yeast species demonstrated the following: (i) Degradation rates differ among ∼11% of the orthologs, compared to ∼50% that differ in transcription or in mRNA levels. (ii) Differences in mRNA degradation are often coupled to opposite differences in transcription, and this coupling constitutes around half of the changes in mRNA degradation but only ∼10% of the changes in transcription. (iii) Coupled changes in transcription and degradation are generated by the same type of mutations , suggesting that these may also serve as coordinators of transcription and mRNA degradation, and possibly of additional steps.The association of ling see , wherebytrans-regulators can cause similar evolutionary changes across many co-regulated target genes. Indeed, trans-coupling includes a set of respiration-related genes, all with higher transcription and mRNA degradation rates in S. paradoxus than in S. cerevisiae, likely reflecting a module that coherently diverged through one or few trans-mutations. While this module is known to be transcriptionally co-regulated, these results suggest that it is also post-transcriptionally co-regulated, thus representing an “RNA regulon” S. cerevisiae and an associated optimization of anaerobic fermentation trans-coupled genes are highly enriched with the respiration module (p = 10−10), this enrichment accounts only for a quarter (21/85) of these genes, suggesting that additional RNA regulons might have evolved by parallel (and opposite) changes in their transcription and mRNA degradation.Notably, divergence of individual trans-regulators may affect transcription and mRNA degradation in parallel, cis-acting sequences are likely to be more specific to one of these processes, for example, by mediating the binding of TFs to promoters or that of RBPs to mRNAs. We thus propose that cis-coupling may work by sequential coupling (cis-acting elements affect one process (transcription or degradation) and this in turn signals to the other process, thereby causing an additional effect. The enrichment of cis-coupling with diverged TF motifs, but not RBP  motifs, suggests a mode of sequential coupling that is directed from transcription to mRNA degradation. This possibility is consistent with a shuttling mechanism, as previously proposed for Rpb4/7 cis-coupled genes, but this accounts only for a small proportion of cis-coupling, suggesting the existence of additional factors for sequential coupling by a similar shuttling mechanism or by other mechanisms.While coupling , wherebycis-regulatory elements would affect both transcription and mRNA degradation of the associated genes.Alternatively, the enrichment of TF motifs, but not stability motifs, may reflect the bias in current knowledge, as fewer motifs are known for RNA-binding proteins and these may rely more heavily on structural properties. Sequential coupling may thus initiate by binding of RBPs to yet unknown motifs and regulate mRNA degradation, followed by signaling back to the nucleus that influences transcription of that gene or perhaps of a set of genes. This possibility is consistent with the notion that RBPs are highly inter-connected and coordinate multiple regulatory events trans-mutations may have driven the evolution of many target genes  and by that bias our results. Importantly, however, cis-coupled genes are each affected by distinct sets of mutations; the only exception is of neighboring genes which may diverge through the same mutations in cis, but these encompass only up to 5% of the observed cis-coupled genes. Therefore, our results imply ∼140 independent cases in which cis-acting mutations affected both transcription and mRNA degradation, generating opposite effects on mRNA levels  transcriptional regulation is much more prevalent than regulation of mRNA degradation, although the exact proportion is difficult to quantify as differential mRNA degradation is more difficult to identify than differential transcription; (ii) coupling constitutes approximately 10% of the regulation of transcription but almost half of the regulation of mRNA degradation. (iii) Coupling occurs almost exclusively between opposite effects on mRNA levels (increased transcription is associated with increased mRNA degradation and vice versa).It is tempting to further speculate that This last prediction is especially surprising given that previous studies have highlighted a coherent mode of coupling whereby changes in mRNA levels may be driven by both transcription and mRNA degradation acting in the same direction This proposed mode of opposite coupling appears counterintuitive and inefficient, as transcription and degradation effects would compensate one another. What then may be the benefits of such coupling? One possibility is that an opposite coupling may enable transient responses to environmental changes: upon stress conditions, cells cease to grow and mount an transcriptional response, but at the same time increase the degradation rates of upregulated genes, thereby facilitating their return to basal expression levels and normal growth Another plausible advantage of such coupling is that it may decrease the effect of genetic or environmental perturbations on mRNA abundance, as changes in one level of regulation would be compensated by another level. Such intrinsic “negative feedback” could increase the robustness of gene regulation and thus reduce cell-to-cell variability. Surprisingly, however, we observe the exact opposite: genes that display coupled evolution in our data or that are targets of coupling mechanisms  have a considerably higher cell-to-cell variability in protein abundance (expression noise S. cerevisiae (BY4741) and S. paradoxus (CBS432) strains, by transient HO activation and selection for diploid strains. The hybrid strain was generated by mating the same parental haploids. These three diploid strains  were grown to log-phase at rich media (YPD medium at 30°C).To facilitate comparison to the diploid hybrid, we generated diploid homozygote yeast strains of the two species, thus avoiding both potential differences between haploids and diploids, and potential heterozygosity within normal diploid strains, which could confound inter-species comparisons. Diploid homozygote strains were generated from the haploid Two to five different 60-mer probes were designed for most genes in each of the two species, and each probe was placed at two different positions (duplicates) on an Agilent custom (two-species) microarray. Probes were selected both by general criteria for probe selection  and by preference for low sequence similarity between the two species in order to avoid cross-hybridization .S. cerevisiae, S. paradoxus, and their hybrid were subjected to 150 µg/ml of 1,10-phenanthroline at log-phase and sampled after 0, 20, 40, and 60 min. Total RNA was extracted using MasterPure Yeast RNA purification Kit (EPICENTRE), amplified with Agilent's Low RNA Input Amplification Kit and hybridized with Agilent's standard protocols and kits to the two-species microarrays. S. cerevisiae and S. paradoxus samples were pooled and hybridized together and the hybrid was hybridized separately, both with biological repeats. Arrays were scanned using Agilent microarray scanner and feature extraction software. Raw and processed microarray data are available at the GEO database (GSE28849).2 of the total (or average) abundance of all mRNAs should decrease linearly by 1 unit every 25 min, and thus decrease by 0.8 every 20 min . We thus scaled the data by centering the four consecutive time points  on 0, −0.8, −1.6, and −2.4, respectively.During the time-course, transcription is arrested and total mRNA levels are decreasing, but this decrease is masked by the experimental protocol, as equivalent amounts of total RNA are extracted from each sample. Previous studies that used a PolII mutant strain could circumvent this problem since mRNAs constitute only a minor fraction of the total RNAs in a yeast cell, and the transcription of other RNAs (by PolI and PolIII) was not inhibited 2-intensities. All probes with an R2 value smaller than 0.94 were excluded from further analysis. For each gene, the absolute value of the median slope of all remaining probes was defined as its degradation rate.For each probe, we averaged the hybridization intensities from the duplicate microarray spots, and fitted a linear slope to the logt test, comparing the three estimates of each probe  between the two species. Genes for which the median p value from the t tests of the different probes was below 0.05 were further examined. p values reflect both the degree of differential degradation and the consistency among the three estimates (even a negligible difference can be identified as significant if the three measures are highly similar within each species). We thus further examined the extent of differential degradation and retained only those genes in which the ratio between the faster and lower degradation rates (from the two species) is higher than 1.4.Since the four time-points are evenly spaced  the difference between mRNA levels at consecutive time-points should be approximately constant and reflect the mRNA degradation rates. To identify differential degradation rates among orthologous probes, we thus performed a two-sample The first time-point reflects mRNA levels during exponential growth and before transcriptional arrest. It therefore reflects an approximate steady-state mRNA level. A potential caveat is that if the first time-point is used to measure both mRNA levels and mRNA degradation, then measurement errors could generate artificial coupling between mRNA levels and degradation. For example, if the first time point is increased due to technical noise, then estimates of both mRNA level and mRNA degradation would increase and result in apparent coupling. To avoid this problem, we used only one time-course to derive estimates of mRNA degradation rates and the first time-point of the second time-course to derive an estimate of mRNA level. As additional control, we used mRNA levels as measured in a previous work and obtained similar results (unpublished data) S. cerevisiae and S. paradoxus can thus be estimated from the respective differences of degradation rates and mRNA levels: log(TRcer/TRpar) = log(Dcer/Dpar)+log(Lcer/Lpar).For each gene, we assume that the production rate of mRNAs (transcription rate) is approximately equal to the overall degradation of mRNAs, and therefore given by the steady-state level of mRNAs multiplied by their constant degradation rate. Hence, TR = D×L, where TR, D, and L are the transcription rate, degradation rate, and mRNA level, respectively. The difference in transcription rates between We note that this estimation may not be accurate as a result of possible violation of the steady-state assumption, spurious correlations with mRNA degradation due to the method of calculation, and the integration of nuclear and cytoplasmic mRNAs in our measurements. Our main conclusions do not require these estimates of transcription rates and can be inferred from direct comparison of inter-species differences in mRNA degradation to those in mRNA levels. However, since mRNA levels are inherently affected by mRNA degradation in a manner that is opposite to the observed coupling, such analysis would underestimate the scope of the coupling , the probes have large differences in hybridization intensities. This effect does not influence our ability to identify inter-species differential expression, since we always compare orthologous probes that target the same positions, but it does provide us with a control for the effect of hybridization intensities: for most genes we have multiple probes with large differences in hybridization intensities. Importantly, despite the variability in hybridization intensities, we find that changes over the time-course  are highly correlated among the different probes : as mRNA degrades and the level of mRNA decreases, the relative contribution of residual transcription on the total mRNA levels gradually increases, until a new steady-state is reached in which the degradation rate is balanced by the residual transcription. Thus, for genes with constant residual transcription, the observed rate of mRNA degradation should decrease with time, and at some point, but possibly after longer than 60 min, should reach zero . Similarly, a transient transcriptional activity would also cause genes to have variable degradation rates during the time-course. For example, if genes are transiently upregulated in response to addition of the drug but this response ceases before the end of the time-course, then during the transcriptional response the apparent degradation rate would be lower than after it ceases. A similar effect would also be expected if mRNA degradation rates are changed during the time-course, for example if degradation factors are affected by the drug or by the stress that is associated with transcriptional arrest. Taken together, residual transcription and additional potential artifacts would lead to a variable degradation rate during the time course and thus a low R2 value of the linear fit to log2 mRNA levels.A constant residual transcription activity would cause genes to have a variable degradation rate during the time-course (non-linear decrease in their log2 mRNA levels has an extremely good linear fit (R2>0.94). To further verify that this effect is not generating the observed association between transcription and mRNA degradation, we used even more stringent criteria for inclusion of genes in the analysis  and obtained similar results  is retained (cis) or abolished (trans) within the hybrid (Δhybrid), while intermediate cases are excluded from the analysis. Cis changes were defined as significant inter-species differences for which Δhybrid has the same sign as Δspecies and is larger than 1.2-fold for each of the two repeats, and the residuals (Δhybrid–Δspecies) are smaller than 1.3-fold. Trans changes were defined as significant inter-species differences for which Δhybrid has either a different sign than Δspecies or is smaller than 1.2-fold for each of the two repeats, and the residuals (Δhybrid–Δspecies) are larger than 1.3-fold. This definition is clearly threshold dependent, and other thresholds or criteria that we used led to similar proportions of cis and trans changes, typically with the percentage of cis differences between 50% and 75% (unpublished data).Classification into cis/trans-coupled genes were defined as those with a significant cis/trans mRNA degradation difference above 1.5-fold and a cis/trans mRNA level difference above 1.5-fold (in the opposite direction to that expected by the degradation difference).High-confidence sets of p<0.005 and no conservation criteria). Targets of RNA-binding proteins were defined based on RNA Immuno-precipitation, taken from Hogan et al. Targets of 116 TFs were defined based on Chromatin Immuno-precipitation and sequence analysis, taken from MacIsaac et al. p<0.005 and no conservation criteria).TF binding motifs were taken from MacIsaac et al. Diverged binding sites were defined as follows:S. cerevisiae, we searched for those in which (i) the bound S. cerevisiae promoter contains a match to the respective PSSM which exceeds a LOD score of 10 and is at least 75% of the maximal LOD score for that PSSM in the entire genome. (ii) This motif is mutated in the orthologous S. paradoxus promoter such that the LOD score drops by at least one unit, and no other motif for that TF with higher LOD score is found in that S. paradoxus promoter.Among the known TF binding events in S. cerevisiae and required the following: (i) The S. paradoxus promoter contains a match to the respective PSSM which exceeds a LOD score of 12 and is at least 90% of the maximal LOD score for that PSSM in the entire genome. (ii) This motif is mutated in the orthologous S. cerevisiae promoter such that the LOD score drops by at least 1.5, and no other motif for that TF with higher LOD score is found in that S. cerevisiae promoter.For each TF, we examined all the genes which are not bound by that TF in S. cerevisiae, our definition of diverged motifs is not symmetrical. We have identified more diverged motifs in S. cerevisiae, but these are of lower confidence as the binding in S. paradoxus is supported only based on the presence of the motif, while the binding in S. cerevisiae (for motifs that diverged in S. paradoxus) is supported by experimental binding data. We have therefore used more stringent LOD-score thresholds for identifying diverged motifs in S. cerevisiae, but this did not eliminate the bias and the difference in the number of predicted genes.Note that since we only have binding data in Total RNA was extracted with the MasterPure Yeast RNA purification Kit (EPICENTRE). One microgram of each RNA sample was reverse transcribed with Moloney murine leukemia virus reverse transcriptase  and random hexamer primers (Applied Biosystems). Real-time PCR was performed with StepOne real-time PCR machine  with Syber Green PCR supermix (Invitrogen). The primers used are described in Figure S1Frequency of interspecific differences in mRNA degradation and mRNA levels as defined by varying thresholds. (a) Percentage of orthologous gene-pairs with differential mRNA levels (blue) and differential mRNA degradation (green) defined by different thresholds of fold-difference. (b) The ratio of percentage of differences in mRNA levels to percentage of differences in mRNA degradation at different thresholds. Dashed lines indicate the same analysis when the frequency of differences in mRNA levels is estimated only among genes without differences (<1.4-fold) in mRNA degradation; this analysis thus estimates the frequency of transcriptional changes divided by the frequency of mRNA degradation changes. At small thresholds (1.1–1.2-fold difference), we find differences at most genes but many of these probably reflect technical variability. At intermediate thresholds , we find differences at 10%–15% (for mRNA degradation) and ∼50% (for mRNA levels). At higher thresholds , we find very few differences in mRNA degradation (2%) but many differences in mRNA levels (23%). This analysis suggests a much higher frequency of transcriptional changes, compared with changes in mRNA degradation, and this effect increases with the fold-difference threshold. This effect may be somewhat influenced by the more complex method required for estimation of mRNA degradation, as degradation rates are calculated by the slope of a linear fit to the time course data , while mRNA levels are estimated directly from a single time-point.(TIF)Click here for additional data file.Figure S2S. cerevisiae and S. paradoxus genes, respectively, along with linear least-square fits. (b) Comparison of differential degradation as measured by microarray and quantitative real-time PCR. In all six cases, differential degradation is consistent between the two methods although some quantitative variation is apparent.Validation of differential mRNA degradation rates of six genes using quantitative real-time PCR for the two species and for the corresponding hybrid alleles. (a) Measured mRNA levels were normalized by the zero time-point and are shown in blue and red for (EPS)Click here for additional data file.Figure S3R2 thresholds of 0.8, 0.95, and 0.995, and (ii) additional threshold for sum-of-squared residuals of 0.06, 0.03, and 0.015, for set1, set2, and set3, respectively. The resulting three sets contained 2701, 1832, and 615 genes, with set3 having the highest stringency. Notably, the percentage of opposite transcription and mRNA degradation effects increases from set1 to set3, indicating that residual transcription  does not account for the observed coupling but might in fact cause us to underestimate the effect of the coupling. Note that a similar analysis for genes with considerable deviation from exponential decay  does not reproduce the observed coupling (unpublished data).Residual transcription cannot account for the observed coupling. The analysis in (EPS)Click here for additional data file.Figure S4S. paradoxus (compared with S. cerevisiae) is associated with trans-coupled divergence of Rpb4 targets with increased transcription and mRNA degradation in S. paradoxus. Expression log2-ratios (S.cer/S.par) are shown for seven subunits of RNA PolII which are included in our analysis, demonstrating a specifically high expression of Rpb4 in S. paradoxus. Note that Rpb4 targets are enriched with trans-coupled genes for which both transcription and mRNA degradation are higher in S. paradoxus (p<10−10), but are not enriched with trans-coupled genes for which transcription and mRNA degradation are higher in S. cerevisiae (p>0.05), consistent with increased activity of Rpb4 in S. paradoxus compared to S. cerevisiae.Higher expression of Rpb4 in (TIF)Click here for additional data file.Figure S5cis-coupled and trans-coupled genes, TATA-containing and Occupied Proximal Nucleosome (OPN) genes, cis-coupled and trans-coupled genes after excluding TATA and OPN genes (T/O), and targets of three Ccr4-Not subunits, and Rpb4. These data suggest that (i) coupled genes are associated with high expression noise and that this effect is found both for genes with evolutionary coupling (cis-coupled and trans-coupled) and for other genes that are regulated by coupling mechanisms (Ccr4-Not and Rpb4/7), (ii) the high noise of coupled genes is comparable to other effects that were previously implicated in increasing expression noise (TATA and OPN), and (iii) the high noise of coupled genes cannot be accounted by enrichment with TATA and/or OPN genes.Coupling is associated with high noise in protein abundance. Cell-to-cell variability in protein abundance (noise), normalized to remove the correlation with average protein abundance , was taken from Newman et al. (EPS)Click here for additional data file.Table S1List of primers .(DOC)Click here for additional data file.


Nucleosomes, which are the basic packaging units of chromatin, are stably positioned in promoters upstream of most stress-inducible genes. These promoter nucleosomes are generally thought to repress gene expression due to exclusion; they prevent transcription factors from accessing their target sites on the DNA. However, the role of promoter nucleosomes that do not directly occlude transcription factor binding sites is not obvious. Here, we varied the stability of a non-occluding nucleosome positioned between a transcription factor binding site and the TATA box region in an inducible yeast promoter and measured downstream gene expression level. We found that gene expression level depends on the occupancy of the non-occluding nucleosome in a non-monotonic manner. We postulated that a non-occluding nucleosome can serve both as a vehicle of and a barrier to chromatin remodeling activity and built a quantitative, nonequilibrium model to explain the observed nontrivial effect of the intervening nucleosome. Our work sheds light on the dual role of nucleosome as a repressor and an activator and expands the standard model of gene expression to include irreversible promoter chromatin transitions. The effect of promoter nucleosomes is generally thought to be repressive for gene expression because nucleosomes can sterically hinder access of transcription factors to the promoter during transcriptional activation Saccharomyces cerevisiae has been used extensively to study nucleosome remodeling during transcriptional activation The PHO5 promoter (PHO5pr) of When the low affinity site in the exposed region is ablated, PHO5pr activity is significantly lowered, suggesting that removal of nucleosome -2 is initiated by binding of Pho4 to the exposed binding site, and the high affinity binding site in nucleosome -2 remains inaccessible However, it is not obvious how nucleosomes would affect gene expression if they are not positioned over known regulatory sequences. This question bears physiological relevance because some promoter nucleosomes are found in non-occluding configurations For this study, we designed a synthetic promoter with a non-occluding nucleosome based on the PHO5pr in budding yeast. The wild-type PHO5pr has a low affinity Pho4 binding site in the nucleosome depleted region (NDR), a high affinity Pho4 binding site under the intervening nucleosome (nucleosome -2), and the TATA box under nucleosome -1. Thus, the intervening nucleosome in the wild-type promoter architecture occludes a transcription factor binding site. We deleted the high affinity site from nucleosome -2 to change this nucleosome to a non-occluding one and replaced the low affinity site in the NDR with a high affinity site to make the promoter highly inducible . It has To investigate the effect of this non-occluding nucleosome on gene expression level, we constructed two more Nuc -2 (+) variants with different nucleosome -2 stability. The intrinsic DNA sequence is one of the most important factors influencing nucleosome occupancy in vivo We compared the occupancy of nucleosome -2 of these Nuc -2 (+) variants under repressive condition using chromatin immunoprecipitation (ChIP). In addition to the occupancy of nucleosome -2, we also measured occupancy of nucleosomes -1 and -3 to ensure that changing the DNA sequence in the nucleosome -2 region did not perturb the position of the neighboring nucleosomes. As expected, nucleosome -2 occupancy increased with the GC% as shown by the ChIP signal at the putative center of nucleosome -2 in We then measured the GRFs of Nuc -2 (+) variants, using a doxycycline inducible strain as described in our previous study Interestingly, we found a non-monotonic relationship between GRF amplitude and the occupancy of the non-occluding nucleosome . Unlike −)) by deleting 161-bp long DNA that spans nucleosome -2 and the linker region between nucleosome -2 and -1 Because changing GC% of nucleosome -2 sequence which was similar to the wild-type sequence led to a drop in gene expression level, we explored the possibility that crucial cis regulatory elements could have been unintentionally deleted in the low or high GC% Nuc -2 (+) variant. We constructed another promoter variant which lacks the entire nucleosome -2 region , the nucleosome-bound fraction becomes 1/(DNK +1).Based on this reasoning, we assume that the promoter switches between two states with and without the intervening nucleosome -2. The chromatin remodeling complex can move the promoter state forward into a transcriptionally active state when nucleosome -2 is present, but is stalled when it is absent. In this view, nucleosomes are not bound 100% of the time, but can continuously assemble and disassemble in equilibrium. This view is seemingly inconsistent with most in vitro experiments where nucleosomes appear to be stable with the half-life of ∼0.6–4 hours rk) is a logistic function of DNK.On the other hand, nucleosome stability can generally impact gene expression in a negative manner because the remodeling rate will be limited by how frequently the contacts between DNA and the histone octamer core are broken. One can incorporate nucleosome stability into the remodeling rate by assuming that SWI/SNF uses ATP to bias the spontaneous DNA unwrapping/wrapping equilibrium of a nucleosome like a Brownian ratchet TC]/([T]+[TC]+DTK) where DTK is the equilibrium dissociation constant of transcription factor binding to DNA, and ‘’ denotes concentration. This hypothesis is similar to the squelching mechanism demonstrated by Gill and Ptashne For all three GRFs with different nucleosome occupancy, we observed that the gene expression level increased sigmoidally with increasing transcription factor level and gradually decreased after reaching the maximum level. Although the exact mechanism remains to be investigated, we speculate that titration of chromatin remodeling complexes by the transcription factor might cause this concave-down GRF at high transcription factor input level. The key idea is that binding of Pho4 alone to PHO5pr does not lead to efficient nucleosome removal. Hence, when Pho4 level is low, most of them will be taken up by SWI/SNF, and Pho4-SWI/SNF complex can bind to PHO5pr to remove nucleosomes. However, when Pho4 level is higher than that of SWI/SNF, free Pho4 will compete with Pho4-SWI/SNF for binding to PHO5pr. Effectively, free Pho4 (T) acts as a repressor, and the promoter fraction that is bound by Pho4-SWI/SNF (TC) is given by [rk) is very slow compared to other reaction steps, one can use the pre-equilibrium assumption for all reversible reactions  than Nuc -2 (+) variants can be due to multiple reasons. First, removing two nucleosomes in a row might be significantly slower than removing one nucleosome. Second, the chromatin remodeling complex is not completely processive, thus having a higher failure rate to remove the second nucleosome than the first. Third, Pho4 might be able to directly stabilize the general transcription machinery and increase the transcription rate only in Nuc -2 (−) due to proximity −) was as negligible as that of any Nuc -2 (+) in the absence of Pho4. Measuring absolute occupancy of nucleosome -1 and TATA binding proteins will shed light on this remaining question.The higher GRF amplitude of Nuc -2  alteration of nucleosome stability by changing the GC% of the underlying DNA sequence and (2) deletion of the underlying DNA. We found that destabilization of the non-occluding nucleosome by lowering the GC% from the wild-type level led to lower gene expression level. This observation is consistent with a nucleosome removal model where the chromatin remodeling complex needs to bind a nucleosome to remove other nucleosomes. Stabilization of the nucleosome also led to lower gene expression level, which raises the possibility that wild-type nucleosome DNA sequences might be evolutionarily optimized for maximum gene expression level. The obvious next step will be to correlate absolute occupancy levels of promoter nucleosomes with gene expression level genome-wide to gain further insights into the role of nucleosomes in gene regulation.All strains for GRF measurement were prepared first by generating a test strain and mating it with the base strain that contained the transcriptional circuits required for doxycycline induction of Pho4 expression and the nuclear marker as previously published Nucleosome positions in PHO5pr were estimated based on a nucleosome position database 600) of about 0.5. Each culture was further diluted by transferring to 3 ml media containing doxycycline at concentrations 0, 0.1, 0.2, 0.4 and 0.8 µg/ml and grown for another 16 hours. The measured optical densities were about 0.5 for all cultures at this point. The samples were prepared for microscopy as described previously For single-cell fluorescence measurements, cells were grown to saturation overnight at 30°C in 3 ml synthetic complete medium. Overnight cultures were sub-inoculated in a new 3 ml medium and grown for about 9–10 hours to reach an optical density at 600 nm . Harvested cells were then disrupted with glass beads in the lysis buffer with 2 mM PMSF at 4°C by vortexing on high speed for 2 hours. Disrupted cells were then separated from glass beads, and cell lysate was prepared by centrifuging them at high speed at 4°C. Resulting chromatin was sheared by sonication using a micro tip  with 20% output power and continuous pulse for 30 seconds four times. From the total sheared chromatin lysate, 10% was set aside as a normalization control (input DNA). The rest of the sample was incubated with 2.5 µg of antibody against the C-terminus of human histone H3 (abcam) overnight at 4°C. The antibody-histone complex was precipitated using Pierce Agarose ChIP kit (Thermo Scientific) following manufacturer’s instructions. The resulting immunoprepitated DNA (IP DNA) was quantified by real-time PCR.Chromatin immunoprecipitation (ChIP) was performed as described In-vivo nucleosome mapping was performed by quantitative real-time PCR (qPCR) of IP DNA using SsoFast EvaGreen Supermix (Bio-Rad Laboratories). We used five primer pairs targeting the PHO5pr region from nucleosome -3 to nucleosome -1 and one primer pair targeting the control locus REC104 for this analysis . To obtaFigure S1In-vivo nucleosome occupancy maps of the promoter variant without nucleosome -2 (Nuc -2 (−)). Relative nucleosome occupancies (in arbitrary units) were measured by quantitative ChIP at three different promoter locations (in base pairs). The nucleosome occupancy at REC104 locus was used as the reference value. The error bars represent the standard deviation of three independent measurements. Genomic coordinates of nucleosome positions (top schematic) were obtained from a compiled database for nucleosome positioning in S. cerevisiae(TIF)Click here for additional data file.Figure S2Nonequilibrium model of gene expression. The red arrows are reactions that depend on nucleosome stability. The irreversible chromatin remodeling step represented by rk brings  back to , and completes the steady-state cycle.(TIF)Click here for additional data file.Figure S3Predicted GRF. The model explains the non-monotonic change in the flux as a function of transcription factor input and as a function of nucleosome occupancy (f ). Case 1 and Case 2 show the asymmetry of the predicted GRF with respect to 1p and 2p. Case 1 is for 1p<2p, and Case 2 is for 1p>2p.(TIF)Click here for additional data file.Figure S4Fitting of GRFs. Here, the GRF is generated using a slightly different binning method than in . To generate the average GRF, CFP (output) intensity values were plotted against the common logarithm (log10) of corresponding YFP (input) intensity values. The x-data, equal to log10 (YFP intensity), were binned with a variable bin-width so that each bin contains the same number of data points . CFP and YFP intensity values within each bin were averaged, and the mean values are plotted as circles. Each GRF is fit independently with four coefficients according to Equation S12. 4p was constrained to be smaller than 1.  are  for 24%,  for 39%,  for 54%, respectively.(TIF)Click here for additional data file.Figure S5The nonequilibrium flux. The system can be driven out of equilibrium by dialing up the maximum remodeling rate (k). The flux vs. transcription factor input when k = 1 is plotted on the left with three different nucleosome occupancy values , and they closely resemble the near-equilibrium curves shown in k). Beyond the crossover point between f = 0.5 and f = 0.75, the dependence of flux on nucleosome occupancy becomes monotonic.(TIF)Click here for additional data file.Table S1Equilibrium probability of each promoter state. The promoter state is defined by the occupancy of the respective sites on DNA (D) by transcription factor (T), chromatin remodeling complex (C), and the nucleosome (N). ‘’ denotes concentration. AB indicates the complex formed between A and B. The interaction can be described by the interaction energy εAB or the equilibrium dissociation constant AB.c0K is the reference concentration at standard state that relates the two. The expressions marked in red are the approximations used for this study.(DOCX)Click here for additional data file.Table S2The fitting parameters from our quantitative model.(DOCX)Click here for additional data file.Table S3147 bp DNA sequences used to change stability of nucleosome -2.(DOCX)Click here for additional data file.Table S4Primers used for quantitative real-time PCR.(DOCX)Click here for additional data file.Text S1Detailed description of the quantitative model.(DOCX)Click here for additional data file.