Soft tissue tumors are a heterogeneous group of tumors, traditionally classified according to morphology and histogenesis. Molecular classification divides sarcomas into two main categories: (a) sarcomas with specific genetic alterations and (b) sarcomas showing multiple complex karyotypic abnormalities without any specific pattern. Most chromosomal alterations are represented by translocations which are increasingly detected. The identification of fusion transcripts, in fact, not only support the diagnosis but also provides the basis for the development of new therapeutic strategies aimed at blocking aberrant activity of the chimeric proteins. One of the genes most susceptible to breakage/translocation in soft tissue tumors is represented by Ewing sarcoma breakpoint region 1 (EWSR1). This gene has a large number of fusion partners, mainly associated with the pathogenesis of Ewing’s sarcoma but with other soft tissue tumors too. In this review, we illustrate the characteristics of this gene/protein, both in normal cellular physiology and in carcinogenesis. We describe the different fusion partners of EWSR1, the molecular pathways in which is involved and the main molecular biology techniques for the identification of fusion transcripts and for their inhibition. EWR1 is located on 22q12.2 chromosome and spans about 40 kb of DNA with 17 exons. The first 7 exons encode the N-terminal domain and exons 11, 12 and 13 encode the putative RNA-binding domain. The protein consists of 656 amino acids and 68.5 kDa of size and is composed of several domains. TAD domain is a transactivation domain that contains a consensus sequence repeated SYGQ and two auto regulation regions, IQ and SF1. The domain IQ binds to calmodulin, inhibiting phosphorylation by protein kinase C (PKC) and favoring the activation of EWS. On the contrary, the binding with SF1 (splicing factor 1) inhibits the activation domain TAD, negatively regulating target genes of EWS. There are also three RGG domains rich in arginine and glycine, a domain RBD that binds single filament of RNA and DNA and a ring finger domain similar to the RAN-BP2 [EWS is a member of the TET  family of RNA-binding proteins, with FUS (TLS) and TAF15 (TAFII68). The codified protein is involved in transcriptional regulation for specific genes and in mRNA splicing. In particular, EWSR1 plays a role in transcription initiation. In fact, EWSR1 is able to link with the basal transcription factor TFIID and RNA polymerase II complex.In normal cells, EWSR1 is phosphorylated by PRKC (protein kinase C) although its IQ domain, which inhibits RNA-binding EWSR1. EWSR1 is required for proper localization of aurora B during mitosis, also maintaining mitotic spindle integrity . EWSR1 aRegarding its localization, EWSR1 is ubiquity expressed , 11 withSeveral gene families, mainly encoding for transcriptional regulators, can translocate with EWSR1. The chimeric proteins have an aberrant activity interfering with different molecular pathways crucial for cell growth, differentiation and proliferation. These altered interactions are often responsible for the pathogenesis of soft tissue tumors.The ETS (E twenty-six) family Fig.  is one oFli1 (Friend leukemia integration 1 transcription factor) was identified in the mouse genome as a viral integration site common to 2 retroviruses involved in virus-induced leukemias and lymphomas. The mouse Fli1 region is close to the centromere of chromosome 9 , while hThe transcription factor Erg is essential for definitive hematopoiesis and for the function of adult hematopoietic stem cells . ChromosEWSR1/ETV1, EWSR1/ETV4 and EWSR1/FEV fusions occur in <1 % of Ewing sarcomas. FLI, ERG and FEV share 87 % identity and 98 % similarity, while ETV1 and ETV4 share 96 % identity and 100 % similarity in their DNA-binding domains. Exon 7 of EWSR1 gene can translocate to FEV exon 2, ETV1 exon 11 and ETV4 exon 9 –36. ManyThe homeobox gene superfamily Fig.  encodes POU5F1, known as OCT3/4, is a member of the POU family of transcription factors and is an important regulator of tissue-specific gene expression in lymphoid and pituitary differentiation and in early mammalian development. EWS exon 8 can translocate with POU5F1 breakpoint region, located in exon 1 . EWSR1/PPBX1 (pre-B cell leukemia transcription factor 1) is involved in the regulation of osteogenesis and it is required for skeletal patterning and programming. A chromosomal translocation, t involving this gene and TCF3/E2A gene is associated with pre-B cell acute lymphoblastic leukemia. EWS–PBX chimeric protein was found in myoepithelioma with two different isoforms, one in frame and one, not pathogenetically important, out of frame .DUX4  contains two homeodomains similar in sequence to PAX3 and PAX7 homeodomains. It is involved in myogenic differentiation and cell-cycle control. EWS–DUX4 chimeric protein was found in rhabdomyosarcoma, interfering with normal muscle cell proliferation  and in uZinc finger proteins Fig.  are amonWT1 gene, deleted in individuals with Wilm’s tumor, encodes a zinc finger DNA-binding protein that acts as a transcriptional activator or repressor, mainly regulating normal formation in the genitourinary system and mesothelial tissues. The chimeric transcript EWS/WT1 was only described in desmoplastic small round cell tumor [ll tumor , 49. EWSll tumor  and tranll tumor .Patz1 encodes a zinc finger protein responsible repression of basal transcription as well as repression of RNF4-mediated activation and transcriptional activation of c-Myc. Deregulation of Patz1 has been described in colorectal cancer [l cancer  and in tl cancer . PATZ1 gl cancer .Transcription factor ZNF384/CIZ/NMP4 plays a role in bone metabolism and spermatogenesis. It is recurrently involved in translocations in acute lymphoblastic leukemia, where it can produce a translocation transcript with EWSR1 or with its homolog, TAF15 .CHN is a member of the steroid/thyroid receptor gene superfamily, with central bipartite zinc finger DNA-binding domain. The protein is implicated in the control of cell proliferation, differentiation and apoptosis, with a prevalent expression in the central nervous system. The specific chromosomal translocation EWSR1/CHN has been observed in extrascheletric myxoid chondrosarcoma , 56.Leucine-zipper transcription factors family consists of a positively charged segment linked with a sequence of heptad repeats of leucine residues (leucine zipper) Fig. . LeucineATF1 is a transcription factor (c-AMP dependent) belonging to leucine-zipper proteins. The ability of this gene to produce a fusion transcript with EWS has been demonstrated in clear-cell sarcoma, defined in the past as malignant melanoma of soft parts (MMSP) , 58 MoreCREB1 is a transcription factor Fig. , member DDIT3/CHOP is a transcription factor characterized by a carboxy-terminal region formed by a DNA-binding basic domain and a leucine-zipper dimerization domain. DDIT3 negatively regulates C/EBP heterodimers formation, preventing their binding to C/EBP sequences in the DNA. DDIT3 is strongly implicated in adipogenesis, but also in erythropoiesis and in the induction of growth arrest . CHOP isUQCRH (ubiquinol-cytochrome c reductase hinge protein) is a component of the ubiquinol-cytochrome c reductase complex associated with the mitochondrial respiratory chain, in which mediates the formation of the complex between cytochromes c and c1.Deregulation of this gene has been described in several cancer cell lines, while a fusion transcript with EWSR1 has been detected in a panel of cancer cell lines of a small round cell sarcoma .NFATC2  is a transcription factor implicated in control T-cell activation and function. Specifically, the transcription factor NFATc2 affects the regulation of cell differentiation and growth and plays a critical role in the development of colonic inflammation. Its deregulation was described in colon cancers , in pancSP3 is a transcription factor belonging to the Sp/XKLF family able to recognize GCrich DNA motifs, found in many promoters and enhancers of housekeeping genes. Moreover, sp3 is involved in cell-cycle regulation and hormone induction. Two different breakpoints have been identified for production of EWSR1/SP3 transcripts, involving EWS exon 7 with SP3 exon 6 and EWS exon 8 with SP3 exon 6. The fusion transcripts have been detected in undifferentiated small round cell sarcomas .SMARCA5 encodes for a Helicase that possesses intrinsic ATP-dependent nucleosome-remodeling activity. The protein is mainly involved in chromatin remodeling and regulation of transcription. Its deregulation has been described in gastric cancer . More reFluorescence in situ hybridization (FISH) is a rapid diagnostic test using molecular cytogenetic techniques. FISH technique supplements conventional cytogenetics and in some cases provides additional information, which is not detected by karyotyping. In the case of EWSR1 gene, several commercial probes has been developed by different companies (Vysis (Abbott Molecular); ARUP LABORATORIES; KREATECH DIAGNOSTIC; Cytocell Ltd.) that allow to specifically identify the breaking point in the 22q12 region of the gene Fig. . HoweverRecently, other commercial probes have been developed for detecting EWSR1/FLI1 translocation, made of a mixture of two FISH DNA probes. One of these, the Vysis LSI EWSR1 (22q12) Dual Color, Break-Apart Rearrangement Probe consists of a mixture of a ~500-kb probe labeled in SpectrumOrange, flanks the 5′ side of the EWSR1 gene and extends inward into intron 4 and a second ~1,100-kb probe labeled in SpectrumGreen, flanks the 3′ side of the EWSR1 gene (Abbott). Another FLI1/EWSR1 probe mix consists of green probes flanking the breakpoint region at the EWSR1 gene locus and red probes flanking the breakpoint region at the FLI1 locus (Cytocell Ltd.).The technique more widely used for the detection of fusion transcripts is represented by RT-PCR. Many studies are reported in literature, which design specific primers for the fusion partners of EWS, able to detect all fusion transcripts. The most numerous are those associated with the detection of EWRS/FLI , 81–87  For ERG Finally, one of the most common problems associated with this detection technique is represented by the quality of archival biological sample, in fact, RNA fragmentation is more frequent, thus better pre-analytical procedures of sampling and storage must be developed . FurtherIn recent years with the advent of quantitative PCR (qPCR), many molecular diagnostic tests were oriented toward this innovative system that could provide additional quantitative information. In this technique, PCR is usually performed using primers labeled with fluorescent chromophores and a suppressor molecule. Recently, this technique has been used for the detection and quantization of the fusion transcripts, and in many cases, it is more sensitive and reliable than qualitative PCR , 102. ReDifferent systems based on q RT-PCR have been developed for the detection of fusion transcripts in soft tumors, in particular for synovial sarcoma with Syt translocation –105. A rThere are few information about the use of western blotting for the detection of translocations in soft tissue tumors. Wang et al.  applied Currently, sequencing represents, actually, the system for molecular investigating safer also for the detection of fusion transcripts. In fact, in most of the studies that have been carried out on the detection of EWSR1 fusion transcripts in soft tissues tumors, the sequencing of the transcript produced by PCR amplification, not only confirmed the efficiency of the investigation system used, but also the diagnosis.The identification of specific fusion transcripts, including several variants of spicing of EWSR1 gene could help in establishing new biotechnological systems to block their aberrant activity in cancer cells. The most modern technologies in recent years have shown the effectiveness of “silencing systems” that could be used also to establish specific target therapies for soft tissue tumors. First of all, there is an extensive literature concerning the use of siRNA in blocking fusion transcripts in several human malignancies. The use of this blocking system has also been described for numerous fusion transcripts, primarily associated with inhibition of production of protein transcripts Syt/SSX –110.Even for the main fusion transcript of EWSR/FLI1, several studies have been carried out showing the effectiveness of this technique both in vitro and in vivo –113. MorThere are many different strategies to investigate EWS gene status. For diagnostic purpose, the choice of a technique relies mainly on the technological support and the “know-how” available to the pathologist. In a not too distant future, blocking strategies may be implemented at all levels, from pre-transcriptional to post-translational stages, interfering with aberrant activity of chimeric transcripts and allowing to establish new and more focused therapies for soft tissue tumors.


However, no genome-wide analysis of MYC-binding sites by chromatin immunoprecipitation (ChIP) followed by next generation sequencing (ChIP-Seq) has been conducted in BL so far.MYC is a key transcription factor involved in central cellular processes such as regulation of the cell cycle, histone acetylation and ribosomal biogenesis. It is overexpressed in the majority of human tumors including aggressive B-cell lymphoma. Especially Burkitt lymphoma (BL) is a highlight example for MYC overexpression due to a chromosomal translocation involving the ChIP-Seq was performed on 5 BL cell lines with a MYC-specific antibody giving rise to 7,054 MYC-binding sites after bioinformatics analysis of a total of approx. 19 million sequence reads. In line with previous findings, binding sites accumulate in gene sets known to be involved in the cell cycle, ribosomal biogenesis, histone acetyltransferase and methyltransferase complexes demonstrating a regulatory role of MYC in these processes. Unexpectedly, MYC-binding sites also accumulate in many B-cell relevant genes. To assess the functional consequences of MYC binding, the ChIP-Seq data were supplemented with siRNA- mediated knock-downs of MYC in BL cell lines followed by gene expression profiling. Interestingly, amongst others, genes involved in the B-cell function were up-regulated in response to MYC silencing.The 7,054 MYC-binding sites identified by our ChIP-Seq approach greatly extend the knowledge regarding MYC binding in BL and shed further light on the enormous complexity of the MYC regulatory network. Especially our observations that (i) many B-cell relevant genes are targeted by MYC and (ii) that MYC down-regulation leads to an up-regulation of B-cell genes highlight an interesting aspect of BL biology. Once bound to its target promoter, MYC can interact with histone modifiers, such as histone acetylases (HATs), GCN5/PCAF, P300/CBP, TIP60 or HAT-associated proteins (e.g. TRRAP), resulting in local hyper-acetylation of histones P15, P21, P27) by blocking the respective activation factors such as SMAD, YY-1, SP1, MIZ-1, TFII-I and NF-Y MYC can also inhibit transcription of genes (e.g. MYC. This translocation leads to a fusion of MYC to one of the three immunoglobulin (Ig) loci MYC was first discovered in Burkitt lymphoma (BL), which harbors a chromosomal translocation of MYC-binding sites were previously analyzed by ChIP-on-chip (Chromatin immunoprecipitation in combination with promoter tiling arrays) and by ChIP-PET (Paired end sequencing of the precipitated DNA fragments) analysis, in a single BL cell line and one MYC-inducible lymphoblastoid cell line (P493-6), respectively, latter serving as BL model The primary goal of the present study thus was the generation of a genome-wide map of MYC-binding sites in BL genomes. To this end, we carried out ChIP in 5 human BL cell lines, employing a MYC-specific antibody followed by next generation sequencing of the precipitated DNA fragments. We identified 7,054 MYC-binding sites and used the associated genes to detect functionally relevant gene sets. In addition we performed MYC knock-down experiments in BL cell lines followed by gene expression profiling. Similar functional groups of genes were found by ChIP-Seq and by MYC silencing. Of special interest was, however, our finding that – in addition to already established MYC target genes – genes important for the function and immunogenicity of B-cells are targeted by MYC binding and are up-regulated by MYC inhibition.2 in RPMI 1640  supplemented with 10% Sera Plus  at 37°C.Five human Burkitt lymphoma (BL) cell lines  were purchased from the German Collection of Microorganisms and Cell Cultures  and cultured with 5% COA polyclonal MYC antibody (N-262: LOT E2308) Santa Cruz, CA, USA) was employed for ChIP. The specificity and suitability of this antibody for ChIP has already been shown by previously published work NPM1, an already well defined MYC target gene Successful enrichment (threshold >20-fold) of MYC-bound DNA fragments was determined by real-time DNA-PCR with primers for PRAME gene were employed, with the exception of the Ramos cell line where the PRAME 3′- end could not be amplified, and therefore primers annealing to the ACTIN gene were used for normalization. Furthermore, selected MYC-binding sites discovered by the ChIP-Seq analysis described below were validated by real-time DNA-PCR. All primers employed were tested to display an efficiency of approximately 100% (+/−10%). Primer sequences are available from Relative quantification of real-time DNA-PCR results was calculated using the comparative ΔΔCT method Approximately 200 ng of ChIP-DNA was used as template for generating an Illumina sequence library . The DNA was not further size fractionated and directly taken for adaptor ligation, using a standard Illumina genomic library preparation kit. Briefly, DNA was end-repaired using a mix of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment and dATP to yield a protruding 3′-‘A’ base for ligation of Illumina adapters which have a single ‘T’ base overhang at the 3′- end. After adapter ligation fragments of ∼250–350 bp (insert plus adaptor sequences) were isolated from an agarose gel and were PCR amplified with Illumina primers for 15 cycles. The purified DNA was captured on an Illumina flow cell for cluster generation. These libraries were submitted to high-throughput sequencing on the Illumina Genome Analyzer II (GAII).http://bowtie-bio.sourceforge.net/manual.shtml#the-n-alignment-mode) for mismatches were considered for further analysis (Bowtie option –m 1).The resulting sequence reads were mapped to the human reference genome  using Bowtie The detection of genomic regions enriched by ChIP versus input control was conducted with HOMER (v2.6) for each experiment individually All discovered putative peaks were merged into one list of putative peak regions that were detected in at least one experiment. A matrix containing the number of reads for every experiment in each putative peak region was assembled. DESeq (v1.4.0) was employed to test the number of reads for being differential over all ChIP versus input samples http://aug2010.archive.ensembl.org/index.html) www.ncbi.nlm.nih.gov/geo/) under the accession number GSE30726.The remaining peaks were ranked by their FDR and annotated with ChIPpeakAnno using Ensembl Biomart employing the Ensembl Genes 59 database and the GCRh37 (hg19) dataset (http://genome.ucsc.edu/cgi-bin/hgLiftOver).In order to compare our ChIP-Seq data to previously published MYC-binding sites, we additionally analyzed the data provided by Zeller et al. http://fantom3.gsc.riken.jp/) using the oligo capping method, (ii) to determine the occurrence of E-boxes, (iii) to perform an orthologous region search in 6 placental mammalian (Eutheria) species  and (iv) to determine the overrepresentation  of the MYC motive as discovered by CoreSearch.The overlap of genomic intervals determined by ChIP-Seq with genomic features, such as transcriptional start sites or exons, was calculated utilizing RegionMiner  de novo calculation of a MYC-binding motive using the 100 highest ranked genomic intervals CoreSearch (Genomatix) with default settings was employed for a MYC smart pool siRNA and control siRNA , respectively. Resulting down-regulation of MYC protein expression was monitored by immunoblot analysis SiRNA-mediated knock-down of MYC was performed employing the BL cell lines Raji, BL41 and Blue1 in order to detect changes of MYC-driven gene expression. For this purpose, the cells were Amaxa-transfected using MYC siRNA-treated and control siRNA-treated cells was extracted employing the RNeasy Midi kit according to standard protocols . Affymetrix GeneChip analysis (HG-U133A) was performed according to the manufacturer's recommendations, starting with 5 µg total RNA. CEL files were generated with the help of the GCOS 1.3 software (Affymetrix). The CEL files of all experiments are available via the Gene Expression Omnibus (GEO) of the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov/geo/) under the accession number GSE30726.Total RNA of MYC siRNA treatment using an empirical Bayesian method. P-values were corrected for multiple testing by the method proposed by Benjamini and Hochberg For analysis of Affymetrix data the expression levels were normalized using VSN The differential expression of selected genes was confirmed by Western blotting as previously described http://www.bioconductor.org/packages/2.5/bioc/html/ChIPpeakAnno.html). The nearest transcription start region (TSR) was used as reference point. By default, the distance is calculated as the distance between the start of the binding site and the TSR that is the gene start for genes located on the forward strand and the gene end for genes located on the reverse strand.Ensembl annotations  closest to or overlapping with the 7,054 genomic intervals were determined employing the Bioconductor package ChiPpeakAnno (MYC siRNA experiments (http://david.abcc.ncifcrf.gov/) Ensembl annotations next to significant MYC-binding sites  and the eriments  were uplIG-MYC translocation positive BL cell lines  were analyzed after MYC chromatin immunoprecipitation (MYC-ChIP) by next generation sequencing, leading to a total number of more than 16 million and 12 million sequence reads in the MYC immunoprecipitated (ChIP) and input samples, respectively. Alignment to the human reference genome  using Bowtie default settings mapped 10,882,577 ChIP reads and 8,495,924 input reads uniquely, which were used for further analysis. More details for read numbers and identified peaks at different stages of data processing are given in DNA-fragments obtained from five The software package HOMER (v2.6) NME1 (rank position 5) and NPM1 (rank position 47) revealed a higher enrichment as compared to lower ranked genes such as BOB1 or MS4A1 (CD20) , thereby confirming the reliability of our ChIP-Seq data  of the identified MYC-binding sites were located in close proximity to transcriptional start sites within +/−1000 bp . AdditioMoreover, we found 2,320 exact matches for the canonical E-box sequence CACGTG in our 7,054 genomic intervals . This corresponds to a 4.6 fold higher enrichment as compared to the 194 occurrences in 590 intervals described by Zeller et al.  de-novo from our ChIP-Seq data  and is shown in The typical MYC-binding E-box motive could also be calculated Pan troglodytes, Macaca mulatta, Bos tarus, Canis familaris, Mus musculus and Rattus norvegicus; 5,558 of the 7,054 (78.8%) binding sites are evolutionary conserved with orthologous regions in at least 4 out of 6 placental mammalian genomes  displayed MYC binding in BL. 10 of these (71.4%) had orthologous regions in at least 4 of 6 placental mammals , CD21 (CR2), CD22, CD79a/b, SYK, LYN, BLK, BLNK, BOB1 (POU2AF1) and PAX5) were identified as active MYC-binding sites  with MYC-specific siRNAs resulted in a reduction of the MYC protein of approx. 75% , as well as for NF-kB associated proteins , which are down-stream components of the B-cell receptor pathway .The understanding of MYC biology is of paramount importance to elucidate its role in the pathogenesis of Burkitt lymphoma (BL), a disease characterized by a consistent high MYC protein expression due to a genomic translocation. Therefore we undertook a comprehensive approach to identify MYC-binding sites by chromatin immunoprecipitation with subsequent deep sequencing (ChIP-Seq). This led to a ranked list of 7,054 MYC-binding genomic intervals in BL .NPM1, NME1) and identification of MYC-binding sites associated with miRNAs known to be regulated by MYC   Confirmation of the exact MYC-binding position in the promoters of previously published MYC target genes (e.g. r-29b-2)  [24], [4r-29b-2) . (iii) Tr-29b-2) .Our genome-wide approach largely extends previous MYC ChIP-analyses from one BL cell line using promoter tiling arrays (ChIP-on-chip) covering only 4,839 proximal promoters myc gene along with the E-box containing RiBi core-promoter signature MYC is a prerequisite for maintenance of this precise cell number, most likely by a lack of MYC-driven cell proliferation.Gene set enrichment analysis using the Ensembl annotations related to MYC binding  revealedHox genes BMI1 as MYC-collaborating oncogene EZH1/2, EED and BMI1. Given the important role of PcG in B-cell development, lymphomagenesis, and tumor stem cell development Another important finding of our study is the binding of MYC to many Polycomb group (PcG) genes in BL. PcG proteins were identified in Drosophila more than 30 years ago as regulators of anterior-posterior body patterning through the repression of CD19, CD20 (MS4A1), CD21 (CR2), CD22, PAX5, SYK, LYN, BLK, BLNK, BOB1 (POU2AF1), CD79a/b) as active MYC target genes  are illustrated for the 5′- ends of the NME1 gene by using the UCSC genome browser (http://genome.ucsc.edu/). Reads in red map to the forward strand and blue reads to the reverse strand. The location of real-time DNA-PCR ( DNA-PCR  is schem DNA-PCR .(PDF)Click here for additional data file.Figure S2MYC-binding sites in the NPM1 gene. ChIP-Seq reads obtained after MYC ChIP-Seq and from input controls analyzing 5 BL cell lines  are illustrated for the 5′- ends of the NPM1 gene by using the UCSC genome browser (http://genome.ucsc.edu/). Reads in red map to the forward strand and blue reads to the reverse strand. The location of real-time DNA-PCR ( DNA-PCR  is schem DNA-PCR .(PDF)Click here for additional data file.Figure S3MYC-binding sites in the BYSL gene. ChIP-Seq reads obtained after MYC ChIP-Seq and from input controls analyzing 5 BL cell lines  are illustrated for the 5′- ends of the BYSL gene by using the UCSC genome browser (http://genome.ucsc.edu/). Reads in red map to the forward strand and blue reads to the reverse strand. The location of real-time DNA-PCR ( DNA-PCR  is schem DNA-PCR .(PDF)Click here for additional data file.Figure S4MYC-binding sites in the PAX5 gene. ChIP-Seq reads obtained after MYC ChIP-Seq and from input controls analyzing 5 BL cell lines  are illustrated for the 5′- ends of the PAX5 gene by using the UCSC genome browser (http://genome.ucsc.edu/). Reads in red map to the forward strand and blue reads to the reverse strand. The location of real-time DNA-PCR ( DNA-PCR  is schem DNA-PCR .(PDF)Click here for additional data file.Figure S5MYC-binding sites in the MS4A1 gene. ChIP-Seq reads obtained after MYC ChIP-Seq and from input controls analyzing 5 BL cell lines  are illustrated for the 5′- ends of the MS4A1 gene by using the UCSC genome browser (http://genome.ucsc.edu/). Reads in red map to the forward strand and blue reads to the reverse strand. The location of real-time DNA-PCR ( DNA-PCR  is schem DNA-PCR .(PDF)Click here for additional data file.Figure S6NOP56 intron 8 (negative control). ChIP-Seq reads obtained after MYC ChIP-Seq and from input controls analyzing 5 BL cell lines  are illustrated by using the UCSC genome browser (http://genome.ucsc.edu/). Reads in red map to the forward strand and blue reads to the reverse strand. The location of real-time DNA-PCR ( DNA-PCR  is schem(PDF)Click here for additional data file.Figure S7CCR7 (negative control). ChIP-Seq reads obtained after MYC ChIP-Seq and from input controls analyzing 5 BL cell lines  are illustrated by using the UCSC genome browser (http://genome.ucsc.edu/). Reads in red map to the forward strand and blue reads to the reverse strand. The location of real-time DNA-PCR ( DNA-PCR  is schem(PDF)Click here for additional data file.Figure S8SEMA4C (negative control). ChIP-Seq reads obtained after MYC ChIP-Seq and from input controls analyzing 5 BL cell lines  are illustrated by using the UCSC genome browser (http://genome.ucsc.edu/). Reads in red map to the forward strand and blue reads to the reverse strand. The location of real-time DNA-PCR ( DNA-PCR  is schem(PDF)Click here for additional data file.Figure S9Significantly enriched KEGG pathways detected by ChIP-Seq analysis  in relat(TIF)Click here for additional data file.Table S1Primer sequences for real-time DNA-PCRs.(XLS)Click here for additional data file.Table S2Complete list of Ensembl annotations  annotated with the number of detected MYC-binding sites for each Ensembl annotation.(XLS)Click here for additional data file.Table S3Differential gene expression after siRNA mediated knock-down of MYC. Three BL cell lines  were analyzed employing U133A Affymetrix GeneChip hybridization.(XLS)Click here for additional data file.Table S4Number of reads in the Fastq files for all MYC ChIP-Seq and input samples mapped to the human hg19 reference genome. The number of reads within the putative peaks discovered by HOMER and within the final list of 7,054 merged peaks (DESeq) is shown in the far right column.(DOC)Click here for additional data file.Table S57,054 peak regions (i.e. MYC-binding sites) with a FDR below 1e-4 are ranked by their FDR. Their closest or overlapping gene annotation  is given.(XLS)Click here for additional data file.Table S6MYC.01 Transcription factor binding matrix. The MYC.01 binding matrix was generated de novo by CoreSearch using the top 100 sequences in (XLS)Click here for additional data file.Table S7Biological annotations associated with genes next to the 7,054 genomic intervals (i.e. MYC-binding sites). Interesting terms are highlighted in red.(XLS)Click here for additional data file.Table S8Biological annotations associated with 5,558 of the 7,054 genomic intervals with orthologous regions in at least 4 of 6 mammalian species. Genomatix RegionMiner with default parameters was employed for the search of orthologous genomic regions. Interesting terms are highlighted in red.(XLS)Click here for additional data file.Table S9Biological annotations associated with genes differentially expressed after siRNA-mediated MYC knock-down in 3 BL cell lines . Interesting terms are highlighted in red.(XLS)Click here for additional data file.


Divergence of transcription factor binding sites is considered to be an important source of regulatory evolution. The associations between transcription factor binding sites and phenotypic diversity have been investigated in many model organisms. However, the understanding of other factors that contribute to it is still limited. Recent studies have elucidated the effect of chromatin structure on molecular evolution of genomic DNA. Though the profound impact of nucleosome positions on gene regulation has been reported, their influence on transcriptional evolution is still less explored. With the availability of genome-wide nucleosome map in yeast species, it is thus desirable to investigate their impact on transcription factor binding site evolution. Here, we present a comprehensive analysis of the role of nucleosome positioning in the evolution of transcription factor binding sites.sensu stricto yeasts at nucleosome occupied regions and nucleosome depleted regions. The binding sites at nucleosome occupied regions exhibited a consistently higher evolution rate than those at nucleosome depleted regions, corroborating the difference in the selection constraints at the two regions. Finally, through site-directed mutagenesis experiment, we found that binding site gain or loss events at nucleosome depleted regions may cause more expression differences than those in nucleosome occupied regions.We compared the transcription factor binding site frequency in nucleosome occupied regions and nucleosome depleted regions in promoters of old (orthologs among Saccharomycetaceae) and young (Saccharomyces specific) genes; and in duplicate gene pairs. We demonstrated that nucleosome occupied regions accommodate greater binding site variations than nucleosome depleted regions in young genes and in duplicate genes. This finding was confirmed by measuring the difference in evolutionary rates of binding sites in Our study indicates the existence of different selection constraint on binding sites at nucleosome occupied regions than at the nucleosome depleted regions. We found that the binding sites have a different rate of evolution at nucleosome occupied and depleted regions. Finally, using transcription factor binding site-directed mutagenesis experiment, we confirmed the difference in the impact of binding site changes on expression at these regions. Thus, our work demonstrates the importance of composite analysis of chromatin and transcriptional evolution. The chromatin of eukaryotic genomes is compacted into several levels. Nucleosomes, which form the lowest level of compaction, are made up of ~147 bp of DNA wrapped around a histone protein complex and interspersed by ~50 bp of exposed linker DNA. In recent years, the occupancy of nucleosome positions in yeasts has been investigated by using different approaches , which employs micrococcal nuclease (MNase) digestion . The rescis-regulatory sequences; and at the chromatin level, where the chromatin allows the chromosomal segments to switch between activated state and suppressed states of transcription [et al. [It is known that the transcription mechanism in eukaryotes functions at different levels, e.g. at the DNA sequence level, transcription factors interact with cription ,18. The cription ,20. For cription ,21,22. Mcription . As an e [et al.  characteet al. [cis-effects). Moreover, the sequences that quantitatively affect nucleosome occupancy were found to evolve under compensatory dynamics while maintaining heterogeneous levels of AT content [cis-regulatory elements [Recent studies have identified the evolutionary features related to nucleosome organization in yeasts ,25. For et al.  indicate content . Consideelements -32, undeelements . With thelements , it is tSaccharoymyces cerevisiae, Candida glabrata, and Kluyveromyces lactis (Saccharomycetaceae); and 2) those that specifically regulate S. cerevisiae (Saccharomyces specific) genes, which represent young genes. We found that TFBS locations in orthologous genes are dominant in NDRs, but those in Saccharomyces specific genes appear more frequently in NRs. To further validate this evolutionary tendency, we investigated the distribution of TFBSs in NRs and NDRs in duplicate gene pairs of yeast that might have undergone relaxation of selection pressure. Since TFBS variations are due to difference in consensus sequences and nucleotide substitutions can promote diversification of regulatory elements [To determine the evolutionary features of TFBSs constrained by nucleosome occupancy, we first investigated the distribution of TFBSs in NRs and NDRs that regulate 1) orthologous genes of elements ,36, theselements . The evoSaccharomyces sensu stricto clade, namely, S. cerevisiae, S. paradoxus, S. mikatae, S. kudriavzevii and S. bayanus, were retrieved from the MYBS database http://cg1.iis.sinica.edu.tw/~mybs/[The genome sequence and the gene and chromosome annotations of the yeast species examined in this study were obtained from a recent compilation in the Saccharomyces Genome Database (SGD) . The tartw/~mybs/ , and then mapped to genomic coordinates obtained from http://www.yeastgenome.org[et al. established rules governing genomic nucleosome organization in S. cerevisiae. They also developed a statistical model to predict nucleosome positions in terms of nucleosome occupancy, and identified well positioned and fuzzy nucleosomes. In this work, we consider both well positioned and fuzzy nucleosomes.Genome-wide nucleosome occupancy data Figure  of S. ceyces.html. Mavrichl[et al.  used MNaenome.org. FurtherS. cerevisiae, with respect to nucleosome occupancy. For this task, we collected the genome sequences of three diverged yeast species, namely, S. cerevisiae, C. glabrata, and K. lactis, from SGD [S. cerevisiae, we downloaded the "orthologous" genes in C. glabrata and K. lactis from the OrthoMCL-DB [S. cerevisiae, but not in C. glabrata or K. lactis, are called "Saccharomyces specific" genes. Additional file We first examined the differential relationship between the frequency distribution of TFBSs in orthologous genes (in Saccharomycetaceae) and in genes only present in the descendent species from SGD . Then, fhoMCL-DB   and nucleosome depleted regions (NDRs) of each gene Table  based onrevisiae   of reciprocal best hits during the BLASTP search should be < 10-20. Second, the length of the alignable region (L) between the two sequences should be greater than half of the length of the longer protein. Third, their similarity should be ≥ I, where I = 30% if L ≥ 150 amino acids (a.a.); and I = 0.06 + 4.8L-0.32(1 + exp(-L/1000)) if L < 150 a.a.. Furthermore, all overlapping pairs and transposons containing genes were excluded to ensure that each gene pair only occurred once in our dataset. Moreover, only gene pairs with at least 150 informative codons were retained for further analysis.We compiled a list of 1,048 independent duplicate pairs in the u et al. . First, n of SGD . To idenp-value < 0.01 , and contaminated DNAs were removed by treatment of DNaseI in the same kit.A yeast strain (BY4741 (BY), a descendant of S288C) was grown in yeast extract-peptone-adenine-dextrose (YPAD) medium [S. cerevisiae promoters for site-directed mutagenesis. Further, we identified the nucleotides that cause TFBS gain or loss in each gene for site-directed mutagenesis. The constructions were performed by PCR-based mutagenesis, which involved two sequential steps [URA3 cassette with about 45 bp flanking homologous regions to the gene of interest at both ends. To perform the first transformation, we used the LiOAc/SS Carrier DNA/PEG method [URA3 in the TFBS region was confirmed by diagnostic PCR and sequencing. The inserted URA3 was then replaced by a second transformation with the appropriate fragment of BY's PCR-based TFBS-modified sequence (where the specific transcription factor could not bind) in the URA3-inserted strain. The second transformation was performed by electroporation based on the user manual of MicroPulser™ electroporator (BIORAD). The transformants were selected by 5-Fluoroorotic Acid (5-FOA) counter selection. Only the strains  that carried the desired sequence (where the specific transcription factor could not bind) survived and formed colonies on the media with 5-FOA(4 g/ml). The constructions in the TFBS region were confirmed by diagnostic PCR and sequencing.To determine the effects of TFBSs that have recently evolved in related yeast species on expression difference, we randomly chose TFBSs that have undergone gain or loss events  from NRsal steps . First, G method , and theACT1 gene  is defined as the number of cycles required for the fluorescent signal to cross the defined threshold). In addition, the amplification efficiency of each primer pair was tested by using two-fold serial dilutions of the templates as suggested by ABI. Finally, the mRNA levels of the candidate genes were compared using a paired t-test.To compare the mRNA levels of the candidate genes (the genes in the mutagenesis and control groups), we used SYBR green core reaction to perform quantitative PCR . Before performing real-time PCR, total RNAs were first reverse transcribed by a high-capacity cDNA reverse transcription kit (Applied Biosystems) using oligo dT primers as reverse transcription primers. Real-time PCR was performed on the final volume of 25 μL containing 50 ng of the cDNA sample, 50 nM of each gene-specific primer, and 12.5 μL of the SYBER green Taq premixture . The PCRS. cerevisiae that had orthologs in both C. glabrata and K. lactis from OrthoMCL-DB [p-value of the two-sided 2χ-test is ≤ 0.05, which indicates a significant association between TFBSs and nucleosome occupancy, rather than random expectation . These results suggest that young genes found only in the descendent S. cerevisiae species exhibit more TFBS variation and frequently occur in NRs, indicating a possible source of the vicissitude in their regulatory sequences.To understand the differences in the selection constraints due to nucleosome occupancy of the regulatory sequences in yeast promoters, we first compared the distribution of TFBSs in orthologous genes of Saccharomycetaceae and Saccharomyces specific genes in NRs and NDRs. For this task, we downloaded 2,152 genes of hoMCL-DB  with 23,p-value < 10-40) indicated that the duplicate pairs that have undergone relaxation of the selection constraint [To verify the evolutionary tendency of TFBSs with respect to nucleosome occupancy, we examined the distribution of TFBSs in the promoters of duplicate gene pairs at NRs and NDRs. Our results . Nevertheless, experimental errors in determining nucleosome positions and TFBS prediction might be the possible source of the broad scatter in the data and could bias our result.Previous studies analyzed the dependence of nucleotide substitution rates in the yeast genome by comparing their positions on a map of nucleosome locations ,25. A res Figure  and 1h. S. cerevisiae promoters for site-directed mutagenesis. The TFBSs corresponding to gain or loss events were removed from the laboratory strain , significant expression changes were found between the mutant and wild type strains in three TFBSs in NDRs (50%), while only one out of six TFBSs in NRs demonstrated expression differences  were frequently located at NDRs, while TFBSs in younger Saccharomyces specific genes were dominant at NRs. Furthermore, genes that have undergone duplication are known to be under lower purifying (stabilizing) selection ,58. In aof TFBSs ,59. Thesof TFBSs -63. Thisof TFBSs  and coulof TFBSs . This stof TFBSs .via stochastic mutational processes [The availability of whole genome nucleosome maps has facilitated research on the regulatory process. As a result, some studies have hinted that the existence of competition and co-operation between nucleosomes and transcription factors may contribute to the regulatory effects on expression divergence ,65-67. Srocesses ,71. Furtrocesses .via TFBS modification and expression analysis  used in the site-directed mutagenesis experiment along with their promoter and target gene information.Click here for file


Gene transcription is constrained by the nucleosomal nature of chromosomal DNA. This nucleosomal barrier is modulated by FACT, a conserved histone-binding heterodimer. FACT mediates transcription-linked nucleosome disassembly and also nucleosome reassembly in the wake of the RNA polymerase II transcription complex, and in this way maintains the repression of ‘cryptic’ promoters found within some genes. Here we focus on a novel mutant version of the yeast FACT subunit Spt16 that supplies essential Spt16 activities but impairs transcription-linked nucleosome reassembly in dominant fashion. This Spt16 mutant protein also has genetic effects that are recessive, which we used to show that certain Spt16 activities collaborate with histone acetylation and the activities of a Bur-kinase/Spt4–Spt5/Paf1C pathway that facilitate transcription elongation. These collaborating activities were opposed by the actions of Rpd3S, a histone deacetylase that restores a repressive chromatin environment in a transcription-linked manner. Spt16 activity paralleling that of HirC, a co-repressor of histone gene expression, was also found to be opposed by Rpd3S. Our findings suggest that Spt16, the Bur/Spt4–Spt5/Paf1C pathway, and normal histone abundance and/or stoichiometry, in mutually cooperative fashion, facilitate nucleosome disassembly during transcription elongation. The recessive nature of these effects of the mutant Spt16 protein on transcription-linked nucleosome disassembly, contrasted to its dominant negative effect on transcription-linked nucleosome reassembly, indicate that mutant FACT harbouring the mutant Spt16 protein competes poorly with normal FACT at the stage of transcription-linked nucleosome disassembly, but effectively with normal FACT for transcription-linked nucleosome reassembly. This functional difference is consistent with the idea that FACT association with the transcription elongation complex depends on nucleosome disassembly, and that the same FACT molecule that associates with an elongation complex through nucleosome disassembly is retained for reassembly of the same nucleosome. While FACT has recently been implicated in the transcription of rRNA genes by RNA polymerase I and small-RNA genes by RNA polymerase III The packaging of DNA into chromatin, mainly in the context of nucleosomes, restricts access to the DNA template. Several proteins or protein complexes regulate access to DNA for transcription in vitro transcription by dissociating one histone H2A–H2B heterodimer from the rest of the nucleosome 2 tetramer 2 interactions FACT has several activities during transcription. Most notably, FACT is involved in diminishing the nucleosomal barrier to transcription that is encountered by RNAPII, and in this way facilitates transcription elongation. This outcome is achieved in part by the destabilization of normal nucleosomal structure, which can involve the reorganization of normal histone protein interactions and/or the displacement of histones FACT also participates in the nucleosome reassembly that takes place in the wake of the transcription complex, as evidenced by the transcription-dependent loss of histones from transcribed regions in yeast cells mutant for FACT subunit Spt16 in vivo, while in vitro assays show that FACT facilitates the cooperative binding of TBP and TFIIA to promoter sequences that are in a nucleosomal configuration FACT has effects on transcription initiation that are more direct. The yeast FACT subunit Spt16 physically interacts with the general transcription factor TFIIE, and shows genetic interactions with the general transcription initiation factor TFIIA and with TATA-binding protein TBP, which binds TFIIA and promoter DNA for transcription initiation spt16 mutations that were identified because they cause temperature sensitivity for yeast cell proliferation spt16 mutations destabilize the mutant Spt16 protein, making it difficult to distinguish inadequate but normal Spt16 function from mutationally impaired function SPT16 gene that affect function in a dominant manner; our reasoning was that a mutant Spt16 protein with a dominant effect would be expected to be relatively stable spt16-E857K and spt16-E763G substitution mutations were identified through the impaired control over inappropriate transcription initiation that they cause as a consequence of defective nucleosome reassembly in the wake of transcribing RNAPII: each of these mutations, in dominant (or co-dominant) fashion, activates functional transcription from cryptic promoters within certain reporter genes Significant advances in understanding Spt16 functions, and thus most likely FACT functions, have been made through the study of spt16-E857K mutant allele was used here in a genetic study to explore functional interactions with several components that influence transcription elongation. These experiments indicated that Spt16, and therefore FACT, has activities that parallel those of other conserved factors that facilitate transcription elongation, including the elongation factors Bur1–Bur2, Spt4–Spt5 and Paf1C, and the HirC corepressor of histone gene expression. Furthermore, several deleterious genetic interactions involving spt16-E857K were alleviated by preventing the Rpd3S-mediated deacetylation of histones that normally occurs during transcription elongation. These findings suggest not only that Spt16 carries out chromatin-related activities during transcription elongation, but also that these activities overlap functionally those of a Bur kinase/Spt4–Spt5/Paf1C pathway.The SPT16 gene that, in dominant fashion, allow effective transcription from cryptic promoters in certain reporter genes spt16 mutant alleles  within the bodies of certain genes. In an earlier study we described the identification and partial characterization of novel mutant alleles of the prGAL1-FLO8-HIS3 gene, which was constructed to have functional transcription of its HIS3 ORF possible only through transcription initiation at a cryptic internal promoter, within FLO8 ORF sequences, that is normally repressed by chromatin (nucleosome) structure GAL1 promoter driving this FLO8-HIS3 reporter gene allows evaluation of the requirement for transcription across the cryptic internal promoter from the upstream GAL1 promoter, as expected for a situation of promoter activation through faulty nucleosome reassembly in the wake of RNAPII passage. All plasmid-borne spt16 mutant alleles listed in prGAL1-FLO8-HIS3 reporter gene in dominant fashion, in a manner dependent on GAL1 promoter activity. Similarly, in cells lacking the chromosomal SPT16 gene the novel spt16 mutant alleles that supply essential function activated this reporter gene in a manner dependent on GAL1 promoter activity  contains an extra G residue (coding strand) following ORF position 253, causing an R85K substitution and a frameshift that brings into register a stop codon, resulting in a truncated protein. This Bur2 fragment is unlikely to be functional: SPT16 bur2-fs1 mutant cells have a slow-growth phenotype analogous to that of bur2Δ cells  was also relieved by increased Bur1 expression from a low-copy BUR1 plasmid, although not by several partially functional Bur1 mutants spt16-E857K bur2 double-mutant cells is due to inadequate Bur kinase activity.Bur2 is involved in transcription elongation as the regulatory subunit of the Bur1–Bur2 cyclin-dependent kinase; Bur1 is the catalytic subunit. Deletion of bur1 mutations spt16-E857K, using a plasmid-shuffling approach. In combination with spt16-E857K none of the bur1 mutations was lethal, but growth was impaired by two mutations with decreased Bur kinase activity: the temperature-sensitive mutant allele bur1-23 and bur1-T240A, mutated to eliminate an activating phosphorylation site . This observation is consistent with other work showing that CTDK-1 and Bur kinase have distinct functions: they are recruited to the transcription complex through distinct sequential mechanisms, and phosphorylate distinct spectra of substrates in addition to the RNAPII CTD spt16-E857K and Bur kinase may therefore implicate a phosphorylation substrate other than the RNAPII CTD.One Bur kinase substrate is RNAPII. Bur kinase phosphorylates the largest subunit of RNAPII on serine-2 of its C-terminal-domain (CTD) repeat to stimulate transcription elongation spt16-E857K mutation does not impair Rad6 or COMPASS function (Another target of Bur kinase is Rad6. Phosphorylation of Rad6 promotes histone H2B ubiquitination and the consequent recruitment of the histone methyltransferase COMPASS and trimethylation of histone H3 on lysine 4 (K4), a hallmark of transcribed regions function .spt16-E857K mutation might impair an Spt16 activity that overlaps that of H2B ubiquitination or H3K4 methylation by analyzing double-mutant derivatives created by standard genetic procedures. The rad6Δ mutation itself causes both cold sensitivity and temperature sensitivity rad6Δ single-mutant segregants exhibited sensitivity to high and low growth temperatures, to the same degree as the double-mutant derivatives , which is involved in transcription elongation and counteracts the tendency for nucleosomes to cause transcription arrest function . We also of Spt5 . Thus SpA downstream effector of Spt4–Spt5 is the multi-subunit complex Paf1C, which is involved in the elongation stages of transcription, associates with FACT, Spt4–Spt5, and RNAPII, and localizes throughout the coding regions of genes. Bur kinase, Spt5 phosphorylation, and the Spt4 protein are important for this Paf1C localization and for Paf1C association with RNAPII, suggesting that Spt4–Spt5 is a platform for Paf1C recruitment spt16-E857K cells lacking Paf1C components  eliminating members of a histone-modification pathway distinct from the COMPASS pathway described above The growth of spt16-E857K bur2Δ synthetic lethality reported here could therefore reflect different but complementary roles played by FACT and Bur kinase in overcoming the repressive effects of deacetylated histones resulting from Rpd3S activity. This hypothesis was tested genetically.The observations that Rpd3S deacetylation activity presents a significant problem for cells with impaired Bur kinase suggest that Bur kinase fosters transcription in part by counteracting the effects of a repressive chromatin environment. The spt16-E857K, we assessed double-mutant cells harboring spt16-E857K and either set2Δ, sin3Δ, eaf3Δ, or rco1Δ. In each case, the double mutant grew as well as each single mutant (data not shown). Standard genetic procedures were then used to determine whether Set2-mediated H3K36 methylation is inhibitory in cells mutant for both Bur kinase and Spt16. We found that, in contrast to the lethality of spt16-E857K bur2Δ double-mutant cells, spt16-E857K bur2Δ set2Δ triple-mutant segregants formed colonies, showing that Set2 is indeed inhibitory in spt16-E857K cells with impaired Bur-kinase activity . These genetic interactions suggest that Rpd3S histone deacetylation makes Spt16 activity important, and imply that the Spt16-E857K mutant protein operates inefficiently with deacetylated histones.The  mutants . In contutations . This rcbur2, spt4, and cdc73 (Paf1C) deletion mutations shown here to have deleterious genetic interactions with spt16-E857K are also deleterious in combination with mutations inactivating HirC, a histone chaperone spt16 mutant alleles spt16-E857K were due to the substitution mutation hir2-L747R; this L747R substitution affects the Hir2 HIRA domain, conserved among HIRA orthologs and thought to be important for protein function. In another derivative the genetic interaction was due to the mutation hir1-L760P; this L760P substitution affects the part of Hir1 involved in Hir2 binding HIR2 plasmid alleviated the synthetic lethality caused by hir1-L760P (data not shown). Our screen also identified a mutation affecting the HirC subunit Hir3 (data not shown). HirC mutations cause increased histone gene expression spt16-E857K cells with normal HirC activity  that acetylate the histones deacetylated by Rpd3S during the previous round of transcription. The HATs SAGA and NuA4 are known to mediate transcription-linked histone acetylation sas3Δ) elp3Δ) gcn5Δ) ESA1 gene is essential, so we used the substitution mutation esa1-L254P, which enfeebles Esa1 spt16-E857K in combination with sas3Δ, elp3Δ, gcn5Δ, or esa1-L254P (data not shown). This is another indication that spt16-E857K exerts more selective effects on Spt16 activity than does a temperature-sensitive mutation such as spt16-11, which has deleterious genetic interactions with the same HAT mutations Sas3, Elp3 and Gcn5 are the catalytic subunits of HAT complexes, and deletion of their genes eliminates the HAT activity of NuA3  spt16-E857K esa1-L254P sas3Δ triple mutants grew as well as the single or double mutants, indicating a lack of genetic interaction among these mutations (data not shown). In marked contrast, spt16-E857K esa1-L254P gcn5Δ triple-mutant cells grew very poorly, indicating a common functional role for the affected proteins . It is therefore likely that the growth defects of esa1-L254P gcn5Δ double-mutant and spt16-E857K esa1-L254P gcn5Δ triple-mutant cells result from impaired transcription.To test the possibility of overlapping HAT functions, we evaluated triple-mutant cells containing proteins . A mildeSpt16 exists in complexes with RNAPII and many other proteins involved in transcription elongation Spt16 can bind H2A–H2B dimers Co-purification of histone H2B with Spt16 could be the result of interactions with H2A–H2B dimers and/or with intact nucleosomes. However, no histone H3 was detected in any of the Spt16 pull-downs . Thus ouspt16-E857K, also compromises an important histone-related activity for transcription elongation, as indicated by biochemical and genetic interactions. Co-purification studies showed that the Spt16-E857K mutant protein, while unaffected for interaction with RNAPII, is impaired for interaction with histone H2B, which probably reflects decreased interaction with the H2A–H2B heterodimer. The spt16-E857K mutation has deleterious genetic interactions with mutations affecting the transcription elongation factors Bur kinase, Spt4–Spt5, and Paf1C. Most of these mutations, like spt16-E857K itself, also impair transcription-linked nucleosome reassembly as indicated by cryptic-promoter activity spt16-E57K cells harboring other nucleosome-reassembly mutations could in principle reflect an additive effect resulting in growth inhibition due to progressively more impaired nucleosome reassembly. However, our finding that an Rpd3S histone-deacetylase mutation, which also activates cryptic promoters spt16-E857K cells that are also mutant for Bur kinase or Paf1C is more consistent with models that emphasizes the importance of histone acetylation for disassembly of a nucleosome encountered by the transcription elongation complex. Bur kinase, Spt4–Spt5, and Paf1C form a functional pathway: Bur phosphorylates Spt4–Spt5, which in turn recruits Paf1C to the transcription complex. Our findings indicate that this Bur/Spt4–Spt5/Paf1C pathway and FACT have overlapping nucleosome-related activities that are important for transcription elongation.The histone chaperone FACT is involved in the elongation stages of RNAPII-mediated gene transcription, facilitating the dismantling and reassembly of nucleosomes encountered by the transcription complex in vitro transcription elongation on a chromatin template spt16-E857K plus a mutation affecting Paf1C or an upstream pathway component, resulting in impaired transcription elongation and consequent growth effects. Conversely, Rpd3S deacetylase inactivation, which increases the acetylated state of nucleosomes spt16-E857K, in Rpd3S-dependent fashion, and the Bur/Spt4–Spt5/Paf1C pathway have deleterious genetic interactions with the histone chaperone HirC  More striking SPT16 ORF was subjected to mutagenesis in the identification of the spt16 mutant alleles studied here and listed in spt16-E857K and spt16-E763G, alter only a limited segment approximating the middle domain of the large Spt16 polypeptide Although almost the entire set1Δ deletion strain spt4Δ strain JS34 was obtained from a cross of strain Y2454 spt4Δ::kanMX4 deletion-collection strain, which eliminated an extra, uncharacterized kanMX4 insertion. All deletion strains used for direct analysis were confirmed using PCR. Strain AFO400 was derived by three backcrosses of strain KanB316-A4HA and derivatives to strain GHY1088, followed by transformation of an appropriate haploid segregant with pRS314-spt16-E857K. To create strain bur1Δ68Δ3C, one copy of BUR1 was replaced with a natMX4 cassette in strain KanBd and this derivative was transformed with pRS316-A4 and pGP161 and sporulated. Strains JS312 through JS329 are plasmid-shuffle derivatives in which pGP161 was replaced by versions of BUR1 on pRS315-based plasmids, after which pRS316-A4 was replaced by versions of SPT16 on pRS314-based plasmids. Before use, esa1-L254P segregants derived from strain LPY3500 were backcrossed three times into the Y2454 genetic background. Strains JS337 and JS338 have rco1Δ::kanMX4 replaced by rco1Δ::Ura3MX4 using the marker-swap plasmid pAG60 rad6Δ effects, the rad6Δ::kanMX4 gene-replacement allele from the BY4741-derived deletion strain was amplified by PCR and transformed into Y2454-E857K×BY4741 diploid cells. The resulting spt16-E857K rad6Δ heterozygous diploid cells, confirmed using PCR, were put through meiosis and sporulation, and fresh rad6Δ single-mutant and spt16-E857K rad6Δ double-mutant segregants were identified at 30°C and analyzed. Standard procedures were used for cell transformations, PCR, tetrad analysis, plasmid loss, and plasmid preparation.Strains used in this work are listed in SPT16, spt16-E857K, and spt16-E763G were created by subcloning from the pRS316-based versions 6 tags from pET32, obtained using primers with XbaI adaptors, into an engineered XbaI site at the 3′ end of the SPT16 ORF. The low-copy LEU2 genomic library p366 was a gift from B. Andrews (University of Toronto). The low-copy BUR1 TRP1 plasmid pGP161 was a gift from G. Prelich (Albert Einstein College of Medicine). pRS315-based BUR1 and bur1 mutant plasmids The plasmids used in this study are listed in SPT16 gene that, in dominant fashion, bring about transcription from otherwise repressed ‘cryptic’ promoters were created and selected as described HindIII restriction site, allowing additional gap-repaired plasmids with the desired dominant effect to be screened by restriction analysis. Plasmids lacking a codon-763 mutation harbored a range of dominant mutant alleles, and fragment-swap plasmid reconstructions were used to eliminate substitutions with neutral effects. Phenotypes of mutant alleles, on low-copy pRS316-based plasmids, were assessed in FY2393 (SPT16) and KanB (spt16Δ) cells. Despite the dominant effect on reporter-gene expression used for selection, lower-case nomenclature is used here for these novel spt16 mutant alleles because several phenotypes described below and previously Plasmid-borne mutant alleles of the SPT16 but with viability maintained by separate SPT16 and spt16-E857K plasmids, were subjected to UV mutagenesis, grown at 30°C, and screened for red/white colony sectoring as described SPT16 ADE3 URA3 GAL1pr-CEN4 plasmid pSLCDC68, and thus failed to produce red/white sectored colonies; galactose growth medium was used to destabilize pSLCDC68 inheritance spt16-E857K gene were responsible for the reliance on pSLCDC68. To identify the chromosomal gene responsible for the synthetic lethality with spt16-E857K, derivatives were transformed with the LEU2-based centromeric yeast genomic library p366 and screened for complementation of the lethal phenotype as indicated by restoration of colony sectoring. Plasmid-borne genomic inserts were identified by insert-vector junction sequencing. Mutated genomic loci were retrieved by gap-repair Cells of strain AFO400, chromosomally deleted for 6 cells/ml) were harvested by centrifugation  and protein extracts were obtained by glass-bead lysis in disruption buffer  containing PMSF (10 µl/ml) and protease inhibitors . For pull-down experiments, cells were washed once with ddH2O and resuspended in 500 µl of Co-IP Buffer  containing protease inhibitors and PMSF, and protein extracts were obtained by glass-bead lysis. Protein concentrations were quantified using the Bradford assay (BioRad) with a bovine serum albumin (BSA) standard.Mid-log phase cells . Membranes with associated proteins were blocked overnight at room temperature in 10% milk (Carnation non-fat milk powder) or 5% BSA (when using antibodies against the RNAPII CTD) in 0.1% TBS-T buffer , and washed 3×10 min in 0.1% TBS-T. Incubation with primary antibodies (in 0.1% TBS-T) was then carried out as follows: anti-HA  1∶2000 in 1% milk, 2 h; anti-H3  1∶5000 in 1% milk, 1.5 h; anti-H3K4triMe  1∶2000 in 1% milk, 2 h; anti-RNAPII  1∶15,000 in 5% BSA, 2 h; and S-protein-HRP (Novagen) 1∶2000 in 1% milk, 2 h. Membranes were then washed 3×10 min in 0.1% TBS-T, followed by incubation with secondary antibodies : goat anti-rabbit-HRP  at 1∶5000 (anti-H3 and anti-H3K4me3 blots), or goat anti-mouse-HRP (Santa Cruz) at 1∶2000 (anti-HA and anti-RNAPII blots). Membranes were then washed 3×10 min in 0.1% TBS-T, and bound antibody was detected using either the LumiGlo Chemiluminescent (Kirkegaard and Perry) or Western Lightning Chemiluminescent Reagent Plus  substrate system.Table S1Yeast strains.(DOC)Click here for additional data file.Table S2Plasmids.(DOC)Click here for additional data file.


It is not only the most accepted marker for that cell population but is also considered lineage determinative. Chromatin immunoprecipitation (ChIP) of TFs in combination with genomic tiling microarray analysis (ChIP-on-chip) has been shown to be an appropriate tool for identifying FOXP3 transcription factor binding sites (TFBSs) on a genome-wide scale. In combination with microarray expression analysis, the ChIP-on-chip technique allows identification of direct FOXP3 target genes.The transcription factor (TF) forkhead box P3 (FOXP3) is constitutively expressed at high levels in naturally occurring CD4ChIP-on-chip analysis of the human FOXP3 expressed in resting and PMA/ionomycin–stimulated Jurkat T cells revealed several thousand putative FOXP3 binding sites and demonstrated the importance of intronic regions for FOXP3 binding. The analysis of expression data showed that the stimulation-dependent down-regulation of IL-22 was correlated with direct FOXP3 binding in the IL-22 promoter region. This association was confirmed by real-time PCR analysis of ChIP-DNA. The corresponding ChIP-region also contained a matching FOXP3 consensus sequence.Knowledge of the general distribution patterns of FOXP3 TFBSs in the human genome under resting and activated conditions will contribute to a better understanding of this TF and its influence on direct target genes, as well as its importance for the phenotype and function of Tregs. Moreover, FOXP3-dependent repression of Th17-related IL-22 may be relevant to an understanding of the phenomenon of Treg/Th17 cell plasticity. Lysed cells were centrifuged at 2500 rpm for 5 min at 4°C, and the supernatant was discarded. The chromatin material was obtained by lysing the cell nuclei with IP nuclei-lysis buffer  and incubating them on ice for 10 min. Chromatin was diluted in IP dilution buffer  and stored at -80°C.Jurkat T cells were cultivated in IMDM medium supplemented with 10% FCS (PAA Laboratories) and 100 U/ml penicillin/streptomycin. If stated, cells were stimulated with PMA  and ionomycin  for 4 h. Protein crosslinking was ensured by the addition of formaldehyde (1% v/v) and by incubation on a shaker for 10 min at room temperature. Formaldehyde was quenched for 5 min by the addition of glycine . After centrifugation, the culture supernatant was removed, and cells were washed once with ice-cold PBS. Cells were lysed in IP cell-lysis Buffer  equipped with a microtip. The following sonication conditions were applied: 60% duty cycle, maximum output power; 6 to 7 sonication cycles consisting of 30 sec on and 60 sec off. Average DNA fragment size was between 200 and 1000 bp. The IP process was performed essentially as described by the Affymetrix company: Protein G Sepharose 4B  and either 10 μg anti-human FOXP3 antibody  or 10 μg of isotype control antibody  were used per IP, respectively.ChIP chromatin corresponding to 5 × 10′-GTTTCCCAGTCACGGTC(N)9-3′), ChIP DNA was amplified with a large-scale PCR reaction (amplification primer: 5′-GTTTCCCAGTCACGGTC-3′) involving Ampli Taq Gold DNA polymerase and partial incorporation of deoxyuridine triphosphate (dUTP). Amplified ChIP DNA was labelled and prepared for hybridization with the GeneChip WT Double-Stranded DNA Terminal Labeling Kit (Affymetrix) and was then analysed on a Human Promoter 1.0R tiling microarray (Affymetrix).After adapter ligation (adapter primer: 5http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE37256). Significance of the overlap between different lists of HGNC gene symbols was calculated using a hypergeometric test.Basic tiling array data analysis was performed with TAS software . FOXP3-bound ChIP-enriched genomic regions were identified by comparing FOXP3 IP to a matched isotype control IP from the same cell material. Only continuous genomic regions exhibiting a p-value higher than the 95th percentile of all p-values were included in subsequent analyses. Both resting and PMA/ionomycin–stimulated conditions were analyzed in independent duplicate experiments. Each replicate experiment was analyzed separately. Calculated ChIP intervals were visualized with IGB software . The web23 primer (5′-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG(T)23-3′; Metabion, Planegg, Germany) and SuperScript II reverse transcriptase . A second-strand cDNA synthesis was then performed with DNA polymerase I (Invitrogen) and E. coli DNA ligase (Invitrogen). After purification of double-stranded cDNA, the first amplification round was performed in an in vitro transcription reaction by using the Promega P1300 RiboMax Kit for T7 amplification ; this procedure produced unlabeled cRNA. The amplified cRNA was subjected to a second amplification round, starting again with reverse transcription by using random hexamer primers  for first-strand synthesis of cDNA, followed by cRNA degradation with RNase H treatment. T7dT23 primers were again used for the second-strand synthesis, as described above. In the second in vitro transcription, which used the GeneChip expression 3′-Amplification Reagent Kit for labeling , biotinylated UTP was partially incorporated into the final cRNA. The quantity and quality of biotinylated cRNA were checked photometrically. Biotinylated cRNA was fragmented, hybridized to a Human Genome U133 Plus 2.0 Array (Affymetrix), washed, and stained as recommended by the manufacturer. Microarray data were analyzed with GeneSpring GX 10.0 software (Agilent Technologies) and the Robust Multi-array Analysis (RMA) normalization algorithm. Cluster analysis was performed with Genesis Software 1.7.3 [z-score transformation [http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE37256).Equal amounts of RNA were amplified with a double linear amplification protocol, starting with the synthesis of double-stranded cDNA by using the T7dTre 1.7.3  by usingormation . A list ormation ,45 and aChIP: Chromatin immunoprecipitation; FKH: Forkhead; FOXP3: Forkhead box P3; GPAT: Genomic Position Annotation Tool; Iono: Ionomycin; IP: Immune precipitation; MACS: Magnetic activated cell sorting; PMA: Phorbol 12-myristate 13-acetate; TF: Transcription factor; TFBS: Transcription factor binding site; Treg: Regulatory T cell; TSS: Transcription start site.The authors declare that they have no competing interestsAJ performed the experiments and data analyses. WH designed, cloned, and provided the retroviruses. FE performed cell isolation and quantitative real-time PCR. JB initiated the study and provided conceptual ideas. RG was principal investigator and was involved in all microarray experiments and data analysis. DB was principal investigator and wrote the manuscript together with AJ. All authors read and approved the final manuscript.Identified FOXP3 ChIP-regions. List of all identified FOXP3 ChIP regions in resting and PMA/ionomycin-stimulated FOXP3-over-expressing Jurkat T cells including chromosomal coordinates, a visual representation map of their chromosomal distribution and their genomic annotation according to the Genomic Position Annotation Tool.Click here for fileGeneGO pathway analysis of FOXP3-bound genes. Analysis of annotated FOXP3 ChIP hits for statistically over-represented pathways.Click here for fileExpression microarray data. List of 410 transcripts responding to FOXP3-over-expression in Jurkat T cells under resting and PMA/ionomycin-stimulated conditions.Click for fileComparison of human FOXP3-ChIP studies. Table containing comparison of identified FOXP3 ChIP hits with previously published human FOXP3-ChIP data.Click here for file


Eukaryotic cells package their genomes into a nucleoprotein form called chromatin. The basic unit of chromatin is the nucleosome, formed by the wrapping of ∼147 bp of DNA around an octameric complex of core histones. Advances in genomic technologies have enabled the locations of nucleosomes to be mapped across genomes . This ha In vitro a range of biochemical approaches indicate that RNA polymerase II (Pol II) can pass through a nucleosome without the need for complete dissociation of histone proteins . Howeve differs ••, raisihromatin • and it hromatin ••. As elhromatin •• and uphromatin , dissocihromatin . Howeverhromatin •, makinghromatin , but reahromatin . This mehromatin . What isKey players in the orchestration of the interplay between chromatin and transcription are the C-terminal heptapeptide repeats (CTD) of the Rpb1 subunit of Pol II and the polymerase associated PAF complex (reviewed by ). The reOne of the consequences of the pathway described above is the establishment of the characteristic distributions of histone H3 K4Me3 and H3 K36Me3 across coding regions . These mAn assortment of factors have been found to recognise histone H3 acetylated at lysine 4. These include the Sgf29 subunit of the SAGA complex ••,28•. TGAL1 where the reduced acetylation observed in the absence of SAGA is associated with increased nucleosome occupancy in the coding region and decreased mRNA production [in vitro, the combined effect of histone acetylation and remodelling by RSC can facilitate transcription through nucleosomes [The SAGA complex in addition to fulfilling a distinct function at promoters accompanies Pol II during elongation perhaps as a result of interactions between Sgf29 and H3K4Me3 and serine 5 phosphorylation of the Pol II CTD ••,34,35.oduction •. There oduction •,38. Whioduction •,39. Furleosomes •. Howeveleosomes ••,16.In addition to histone acetyltransferase activity, SAGA has a deubiquitinase (DUB) activity. As a H2B Ub is required for H3 K4Me3 which in turn recruits SAGA, this enzyme has the capability to destroy the H3K4Me3 messenger that summoned its recruitment. Furthermore, the removal of H2B Ub is required for recruitment of Ctk1 and phosphorylation of Pol II at serine 2 •. As a rThe coupling of histone acetylation mediated nucleosome dissociation with transcription potentially initiates a destabilising positive feedback loop, which could drive further nucleosome depletion and faster elongation. While this may be an advantage at genes transcribed to high levels, at genes expressed at lower levels this provides an opportunity for transcription from cryptic promoters normally occluded by chromatin. To counter this effect histone acetylation is short lived as a consequence of coupling histone deacetylase activity with transcription as described above. Where nucleosomes have been removed, chromatin assembly pathways are required to reassemble nucleosomes.in vitro [in vitro [in vitro [in vivo [Histone chaperones are prime candidates for a role in this chromatin assembly reaction . Recent in vitro . Functioin vitro •,49. Thein vitro . Althougin vitro • and FAC[in vivo •.Spt6 acts similarly to FACT in the reassembly of chromatin following transcription •,54. HowThe reassembly of chromatin following transcription is not restricted to coding mRNAs. Transcription of non-coding RNAs is also associated with chromatin assembly and in some cases this has been found to play regulatory roles .in vitro, in the absence of other factors, the positions adopted by nucleosomes do not fully replicate those observed in vivo [CHD1 mutations with other elongation factors suggest that Chd1 acts to reduce the efficiency of elongation in a similar fashion to the Rpd3S histone deacetylase complex [CHD1 or ISW1 it is possible that these proteins function with partial redundancy in chromatin assembly [When nucleosomes are assembled  in vivo . An ATP  in vivo ••. Prime in vivo  and Chd1 in vivo . Both Is in vivo . The gen complex ••. As crassembly ••. Furthassembly  and thatassembly . The estassembly •• and thassembly . In addiassembly .Some doubt remains as to how tightly coupled the nucleosome spacing reaction is to transcription. Favouring close links to transcription are the observation that nucleosome spacing decays with distance from the +1 nucleosome whose positioning is likely to be established by other factors , and thae.g. FACT, SAGA, SWI/SNF, RSC, NuA4, Chd1 and Isw1). Where a strong phenotype is conferred by one mode of action, for example promoter remodelling, it may confuse interpretation relating to transcriptional elongation. Another reoccurring issue is the presence of parallel pathways that confer partial redundancy which greatly complicates the interpretation of genetic interactions. For example, due to overlapping functions, multiple HDACs and chromatin remodelling enzymes must be removed to observe defects [The very fact that Pol II moves across genes during elongation complicates studies of its localisation in comparison to activities which are recruited to fixed loci such as promoters. Furthermore, many genes are actively transcribed for relatively short periods of time, making it even harder to study factors involved in elongation using chromatin immunoprecipitation assays. Genetic studies of elongation factors are complicated as a result of them often having distinct roles in other processes such as chromatin reconfiguration at promoters ( defects •,68.Over the last decade important insights into many of the factors involved in transcribing through chromatin provide the opportunity to take a step back and consider the overall organisation of the pathway . The patHighly transcribed genes are observed to be enriched for distinct patterns of histone modifications . The difThe above description is no doubt a simplification. There is considerable variation in the ways that different genes respond to the loss of different component's of the transcriptional machinery . There iIn summary, the process of transcription through chromatin is becoming clearer as a result of a huge effort to characterise each of the steps involved. While there are undoubtedly many aspects that still remain to be discovered, overall the process provides a means of ensuring that the dynamic events occurring at the site of transcription are restored. In this way a substantial proportion of the chromatin landscape can be considered as being directed towards discretely covering the tracks left by the passage of RNA polymerase.• of special interest•• of outstanding interestPapers of particular interest, published within the period of review, have been highlighted as:


Orphan nuclear receptor ERRγ is a member of nuclear receptor superfamily that regulates several important cellular processes including hepatic glucose and alcohol metabolism. However, mechanistic understanding of transcriptional regulation of the ERRγ gene remains to be elucidated. Here, we report that activating transcription factor 6α (ATF6α), an endoplasmic reticulum (ER)-membrane–bound basic leucine zipper (bZip) transcription factor, directly regulates ERRγ gene expression in response to ER stress. ATF6α binds to ATF6α responsive element in the ERRγ promoter. The transcriptional coactivator peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α) is required for this transactivation. Chromatin immunoprecipitation (ChIP) assay confirmed the binding of both ATF6α and PGC1α on the ERRγ promoter. ChIP assay demonstrated histone H3 and H4 acetylation occurs at the ATF6α and PGC1α binding site. Of interest, ERRγ along with PGC1α induce ATF6α gene transcription upon ER stress. ERRγ binds to an ERRγ responsive element in the ATF6α promoter. ChIP assay confirmed that both ERRγ and PGC1α bind to a site in the ATF6α promoter that exhibits histone H3 and H4 acetylation. Overall, for the first time our data show a novel pathway of cross talk between nuclear receptors and ER-membrane–bound transcription factors and suggest a positive feed-forward loop regulates ERRγ and ATF6α gene transcription. Estrogen-related receptors (ERRs) are members of the NR3B subfamily of nuclear receptors, which include ERRα, ERRβ and ERRγ. These orphan nuclear receptors regulate transcription via estrogen response elements and the closely related ERR response elements (ERREs) but do not bind endogenous estrogen . The ERRRecently, numerous studies demonstrate the importance of ER stress in the pathogenesis of various liver diseases, including chronic viral hepatitis, insulin resistance, nonalcoholic fatty liver disease, ischemia-reperfusion injury, genetic disorders of protein misfolding and alcoholic liver disease . The ER PGC-1α, a member of a small family of coactivators, was identified using yeast two-hybrid assays for peroxisome proliferator-activated receptor γ (PPARγ)-interacting proteins  and is iPrevious reports suggest different nuclear receptors and many transcription factors cross talk to regulate mammalian gene expression in response to ER stress. ER stress–induced activation of ATF6α decreases insulin gene expression via upregulation of orphan nuclear receptor small heterodimer partner (SHP) . One reptrans-activate each other. Moreover, our study reveals ERRγ is induced earlier that ATF6α in response to ER stress. Together, we present a novel mechanistic pathway that would encourage further study to elucidate the relation between nuclear receptors and ER stress.Here, we examined the mechanism of cross talk between a nuclear receptor, ERRγ and an ER-membrane–bound bZIP transcription factor, ATF6α. In response to ER stress, expression of both transcription factors increases significantly through reciprocal activation. ATF6α directly binds to the ERRγ promoter, and ERRγ directly binds to the ATF6α promoter. Of most interest, PGC1α acts as a coactivator in both cases. The physical interaction of PGC1α with both ERRγ and ATF6α increases significantly upon ER stress that identifies the importance of PGC1α in this cross talk. We observed a significant increase in histone H3 and H4 acetylation in both promoters upon induction of ER stress by Tm treatment. Knockdown of either factor significantly decreases the expression of the other, suggesting their ability to FRONTIER), anti-ATF6 (Imgenex), anti-PGC1α (Santa Cruz), anti-acetyl-histone H3 , anti-acetyl-histone H4 , anti-GRP78 (abcam), anti-PDK4 (abcam), anti-CHOP (Santa cruz), anti-XBP1 (Santa cruz) and anti-ATF4 (Santa cruz). The primary antibodies were used at a dilution ranging from 1:200 to 1:1000 for western blot analysis, and at a dilution of 1:200 for immunoprecipitation.Tm and Brefeldin A were obtained from Sigma-Aldrich; Thapsigargin was obtained from Sigma, and GSK5182 was synthesized as described previously . Antibodsupplemental table. All plasmids were confirmed via DNA sequence analysis. For ectopic expression of the genes, adenoviral delivery was used. Adenoviruses (Ad) encoding GFP only (Ad–GFP), Ad–ERRγ, Ad–shERRγ, Ad–ATF6α, Ad–PGC1α and Ad–shPGC1α were described elsewhere  according the manufacturer’s instructions as described elsewhere . Brieflyn = 5) were maintained ad libitum for desired experimental period and sacrificed. Liver tissue samples were used for Co-IP assay. For western blot analysis, cell lysates were prepared and analyzed as previously described (Coimmunoprecipitation (Co-IP) and western blot analyses were performed as described previously . For Co-escribed .Confocal microscopy was performed as described elsewhere . In brieKnockdown of PGC1α was performed using the pSuper vector system ,51. AML1Total RNA was isolated using the TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. The mRNAs of ATF6α and PGC1α were analyzed by RT-PCR or quantitative real-time RT-PCR (qPCR) as indicated. DNA samples from total RNA reverse transcription or from chromatin immunoprecipitation (ChIP) assays served as the templates for qPCR, which were performed with TOPreal SYBR Green PCR Kit (Enzynomics) and the Step One Plus real-time PCR system (Applied Bioscience) in triplicate. mRNA expression levels were normalized to those of β-actin (ACTB). The RT–PCR and qPCR primer sequences are available on request.Formaldehyde cross-linking of cells, ChIPs and real-time PCR analyses were performed as described elsewhere . After sad libitum. Tm  was administered by Intraperitoneal injection into C57BL/6J mice (n = 5 per group) for 0–12 h for time-course study, or Ad-GFP or Ad-ERRγ were injected via tail vein into male C57BL/6J mice (n = 5 per group). Where indicated, GSK5182 was administered first  by intraperitoneal injection, and after 30 min, Tm  was administered by intraperitoneal injection into C57BL/6J mice (n = 5 per group). All experiments were conducted as previously described  were maintained on a 12-h/12-h light/dark cycle and fed t test. Differences were considered statistically significant at P < 0.05.Data are expressed as means ± SEM. Statistical analysis was performed using the two-tailed Student in vivo, mice were injected with Tm. A significant increase in ERRγ mRNA  and protein . All these results indicated that ER stress induced ERRγ gene expression was mediated through ATF6α. To test this hypothesis, endogenous ATF6α was knocked down by Ad-shATF6α in presence of Tm in AML12 cells. As expected, the Tm mediated increase in ERRγ protein level was significantly decreased in response to ATF6α knock down . To identify the DNA sequence conferring ATF6α-mediated ER stress effect on the ERRγ promoter, a series of deletion constructs was analyzed. Deletion of the ERRγ promoter sequence from 1.5 to 1.253 kb drastically decreased the promoter activity conferred by ATF6α, suggesting that the region from 1.5 to 1.253 bp conferred the activation of ERRγ promoter (TGACGTG(G/A)  . We aligsfection C. Next, sfection D. To furpromoter E, thereftylation , to detetylation F. Tm treSupplementary Figure S3A). Because Tm induces transcription of ATF6α . As we noticed an early gene induction of ERRγ compared with ATF6α upon ER stress . To further confirm this, GSK5182, an inverse agonist of ERRγ . To identify the DNA sequence conferring ERRγ-mediated ER stress effect on the ATF6α promoter, we used a series of deletion constructs of ATF6α promoter for transient transfection assay. Deletion of the ATF6α promoter sequence from 2.6 to 2.3 kb drastically decreased the promoter activity conferred by ERRγ, suggesting that the region from 2.6 to 2.3 kb conferred the activation of the ATF6α promoter  through the National Research Foundation of Korea funded by the Ministry of Education, Science, and Technology Grant [20100019512 to H.S.C.], [2012M3A9C3048686 and 2011-0011433 to S.H.B] and the Korea Research Institute of Bioscience and Biotechnology (KRIBB) Research Initiative Program (to C.H.L.). Portions of this work were supported by National Institutes of Health (NIH) grants . Funding for open access charge: National Creative Research Initiatives Grants from the Korean Ministry of Education, Science, and Technology and the Future-based Technology Development Program (BIO Fields) through the National Research Foundation of Korea funded by the Ministry of Education, Science, and Technology Grants, KRIBB Research Initiative Program grants, and NIH grants.Conflict of interest statement. None declared.


Geminiviridae, genus Begomovirus) are major limiting factors for the production of numerous dicotyledonous crops throughout the warmer regions of the world. In the Old World a small number of begomoviruses have genomes consisting of two components whereas the majority have single-component genomes. Most of the monopartite begomoviruses associate with satellite DNA molecules, the most important of which are the betasatellites. Cotton leaf curl disease (CLCuD) is one of the major problems for cotton production on the Indian sub-continent. Across Pakistan, CLCuD is currently associated with a single begomovirus  and the cotton-specific betasatellite Cotton leaf curl Multan betasatellite (CLCuMuB), both of which have recombinant origins. Surprisingly, CLCuBuV lacks C2, one of the genes present in all previously characterized begomoviruses. Virus-specific transcripts have only been mapped for few begomoviruses, including one monopartite begomovirus that does not associate with betasatellites. Similarly, the transcripts of only two betasatellites have been mapped so far. The study described has investigated whether the recombination/mutation events involved in the evolution of CLCuBuV and its associated CLCuMuB have affected their transcription strategies.Whitefly-transmitted geminiviruses  from infected Overall, the transcription of CLCuBuV and the recombinant CLCuMuB is equivalent to earlier mapped begomoviruses/betasatellites. The recombination events that featured in the origins of these components had no detectable effects on transcription. The transcripts spanning the mutated C2 gene showed no evidence for involvement of splicing in restoring the ability to express intact C2 protein. Geminiviridae family have circular single-stranded (ss)DNA genomes and classify into four genera according to their host range, insect vector and genome organization), that also interferes with host cell-cycle and is the only virus encoded protein required for virus replication, the C2 protein  that is involved in up-regulating late, virion-sense encoded genes (in some cases) as well as host genes and is a RNA silencing suppressor (accession number AM421522) and associated betasatellite, CLCuMuB-[PK:Veh:06] (AM774307) were used to infect Total RNA was isolated from plants using Trizol reagent (Gibco-BRL) as described by the manufacturer. The extracted RNA was dissolved in diethylpyrocarbonate-treated water and stored at −80°C.32P]dCTP using a Megaprime labeling kit (Amersham). Hybridization was performed at 45°C for 16 h. Following stringent washing, radioactive signals were detected using a storage phosphor screen and the images were acquired after 3 h exposure using a Typhoon phosphoimager (Amersham). A betasatellite specific DNA probe was derived from the complete coding region of the βC1 gene, labelled with a DIG PCR labelling kit and exposed to X-ray film after treatment with CDP-Star (Roche).Total RNA 10 μg) was electrophoresed on 1.2% agarose MOPS gels, blotted onto nylon membranes  and UV cross-linked. DNA fragments for virus complementary-sense (coordinates 1059–66) and virion-sense (coordinates 124–1059) probes were PCR amplified using specific primers CF/CR and VF/VR (Table0 μg was ′ RLM-RACE PCR was performed using an RLM-RACE Kit (Ambion) according to the manufacturer’s instructions. Total RNA was treated with alkaline phosphatase to remove the 5′ phosphate group of non-capped mRNAs, followed by treatment with tobacco acid pyrophosphatase (TAP) to decap mRNAs. TAP treated RNA was ligated to a synthetic 5′ RNA adapter (Table′ termini of virus and betasatellite specific transcripts.5er Table using T4er Table to ampli′ RLM-RACE PCR was performed according to the manufacturer’s instructions (Ambion). Total RNA (1 μg) was reverse transcribed with an oligo (dT) adapter primer  according to the manufacturer’s instructions, and were cloned into the pTZ57R/T vector (Fermentas). PCR products (without cloning), as well as cloned products, were sequenced . Sequences were analyzed using the Lasergene software package (DNASTAR Inc.).N. benthamiana plants are shown in FigureN. benthamiana tissue to either probe. In addition to the single long transcript on the blot probed with the virion-sense probe, a smear was observed at the bottom of the blots. This most likely represents degradation products of the major transcript  and BuC2F (inner) were used in combination with 5′ RACE outer and inner primers respectively for the 3′ end of ~1.7 kb transcripts. Similarly the outer primer BuC2F and inner primer REn-′3B were used for the 3′ end of small transcript of ~0.7 kb. Sequencing results showed that all the complementary-sense transcripts have a single major transcription termination site mapping at coordinate 1059  of 21nt  and BC2bb (inner) were used in combination with 3′ RACE outer and inner primers respectively ), none of CLCuMuB,30, have′ ends and a common 3′ end. The polyadenylation sites are arranged such that complementary- and virion-sense RNAs overlap at their 3′ endsaAUGGCu)[C2 and C3 AUG initiation codons are in a more favourable context (AAAAAAUGca and cAACcAUGGa). Thus in contrast to TGMV, CLCuBuV may translate both C2 and REn from both the size classes of C2/C3 transcripts.The analysis detected no RNAs that span only the C2 and C3. For TGMpression. For CLCaAUGGCu), whereasC1 and (truncated) C2 terminator codons and the start of C3 suggests that it may involve reinitiation or internal initiation. For TGMV the translation of C3 from the short transcript may occur by leaky scanning, since the C3 initiation codon occurs before the C2 terminator codon[C2 termination codon occurring before the C3 initiation codon and there is an ORF , which could possibly be expressed by leaky scanning through the C2 initiation codon. Translation of the C* ORF might adversely affect C3 expression. However, further studies will be required to investigate these possibilities.The mechanism by which REn is expressed is unclear. The gap between both the or codon. HoweverIt remains unclear why geminiviruses would express REn from multiple transcripts. A possible explanation is that this protein may be required in larger amounts than possible from a single transcript or its expression requires more subtle control. The protein may be required at several stages of the virus infection cycle and thus may be expressed at different times from different mRNAs.′ ends identified represent heterogeneous initiation sites of a bicistronic mRNA encoding both C2 and C3. The predicted sizes of RNAs initiating at the 5′ and 3′ends mapped here is consistent with the complementary-sense RNAs detected in infected tissue. Identified transcription start sites are located downstream of putative TATA boxes at optimal distances of 20 to 35 nt. These results suggest that CLCuBuV uses a bicistronic transcription strategy, in common with other geminiviruses, to translate C2 and REn from a single transcript[C3 gene-specific primers did not reveal any major transcription start between the C2 and C3 start codons, or splicing to remove the upstream C2 start codon.The transcript 5anscript. This isC1, truncated C2 and C3 genes  of the C4 gene and would thus appear not to direct translation of the C4  complementary-sense transcripts could potentially allow translation of the  kb compl′ UTR of 143 nt that spans the origin of replication and promoter elements far from the complementary-sense genes. A similar transcript has previously been observed for TGMV[′ UTRs are 20–40 nt long and the promoter elements are located at an optimal distance from transcription initiation sites as reported previously for others geminiviruses and thus represent the authentic 5′ ends for these transcripts[′ UTRs, these elements are far removed from the transcription initiation sites. It has been recognized that translation initiation efficiency from the start codon (AUG) depends on its position and sequence context within, and also on its distance from, the 5′ UTR[′ UTRs may be associated with a decrease in translation initiation efficiency and vice versa[For the long complementary-sense transcripts TATA and CAAT boxes are located at the requisite distances of approximately 30bp from the transcription initiation sites  tail of the mRNA is added 21 nt downstream of canonical AATAAA polyadenylation signal. This site is located at an optimal distance downstream of a consensus polyadenylation termination signal suggesting that it represents the authentic 3′ end for these transcripts. Polyadenylation signals normally occur 10–30 nt upstream of the polyadenalytion site[′ UTR for virion-sense transcripts determined in this study are short, being 21 nt. The poly (A) tail is added 22 nt downstream of a polyadenylation signal. A similar arrangement has been observed for other geminiviruses; MYMV and SpCTV[The RACE clone sequences for the complementary-sense showed a single predominant 3ion site. The 3′ nd SpCTV,31.′ and 3′ ends at coordinates 563 and 190, respectively  and for the non-recombinant CLCuMuB [βC1 transcripts, RNA was extracted from transgenic plants harbouring dimeric betasatellite constructs – such plants would not be expected to contain episomally replicating satellite. The transcripts here are from a bona fide begomovirus-betasatellite infection. However, Saunders et al.[′ termini to the presence of a cryptic polyadenylation in the sequence of AYVB. No such cryptic polyadenylation signals  are present in the recombinant CLCuMuB.The single transcript identified for CLCuMuB maps with 5for AYVB. The crifor AYVB. The diffor AYVB (multiplerminus), may be s et al. attributet al.[Sequencing of clones of the short transcript spanning the C2 mutation in CLCuBuV did not show any splicing, or other editing event, which might restore expression of a full-length C2 protein, supporting the conclusion of Amrao et al. that an et al.[The transcription of CLCuBuV and the recombinant CLCuMuB is equivalent to earlier begomoviruses/betasatellites that were transcription mapped. The recombinations/mutations that led to their appearance caused no detectable differences at the transcription level. Nevertheless, the study provides some avenues of investigation to follow-up in the efforts to determine the mechanism of resistance breaking in cotton by CLCuBuV and its betasatellite. These include the possible differences in the C4 protein, or expression thereof, and possible effects on REn expression, in addition to the hypothesis put forward by Amrao et al. that theet al.. These pThe authors declare that they have no competing interests.FA performed the experiments and prepared the first draft of the manuscript. MS provided overall directions regarding the designing of all experiments, writing and supervised the work. FV supervised the hybridization experiments during the short-term visit of FA in Basel and discussed the different results with FA. RWB was involved in critical review of the work and in writing the manuscript. The final manuscript was read and approved by all authors.