Genome instability plays a key role in multiple biological processes and diseases, including cancer. Genome-wide mapping of DNA double-strand breaks (DSBs) is important for understanding both chromosomal architecture and specific chromosomal regions at DSBs. We developed a method for precise genome-wide mapping of blunt-ended DSBs in human chromosomes, and observed non-random fragmentation and DSB hot spots. These hot spots are scattered along chromosomes and delimit protected 50–250 kb DNA domains. We found that about 30% of the domains (denoted forum domains) possess coordinately expressed genes and that PARP1 and HNRNPA2B1 specifically bind DNA sequences at the forum domain termini. Thus, our data suggest a novel type of gene regulation: a coordinated transcription or silencing of gene clusters delimited by DSB hot spots as well as PARP1 and HNRNPa2B1 binding sites. The study of chromosomal domains is hampered both by the complexity of chromosomal architecture and by the lack of experimental approaches for identification of structural and functional elements in chromosomes. The primary nucleosomal level in organization of chromosomes was detected by digestion of chromatin with exogenous nucleases. Many years ago, we detected 50–250 kb chromosomal domains that are released when genomic DNA is prepared under mild conditions, now known as forum domains. We assumed that this large-scale fragmentation of chromosomes by endogenous nucleases reflected some higher-order level of organization in chromosomes. However, both the existence of large 50–250 kb protected regions and short regions possessing hot spots of DNA breakage might reflect specific features in organization of chromosomes. Here, we used a procedure for genome-wide mapping of terminal regions of forum domains in human chromosomes. Our results show that hot spots of DNA breaks are not randomly distributed along chromosomes, that adjacent to these regions are binding sites of PARP1 and HNRNPA2B1, and that forum domains contain coordinately expressed gene clusters. DNA double-strand breaks (DSBs) in the human genome have been studied for many years, for two main reasons. First, DSBs, aberrant repair, and further chromosomal damage lead to massive genomic rearrangements Drosophila, plant, and yeast cells. The DNA domains within this 10% of chromosomal DNA were named “forum domains” Drosophila S2 cells and found that they are separated by hot spots of DSBs. Frequently, forum domain termini (FT), where individual DSBs reside, were found to correspond to regions of intercalary heterochromatin (I-HC) known as fragile and late replicating regions in Drosophila polytene chromosomes Drosophila chromosomes, the clusters of co-expressed genes Chromosomal integrity is challenged by both exogenous  and endogenous  influences DrosophilaThe non-random excision of forum domains from chromosomes and the correspondence of DSB hot spots to I-HC sites reminded us of Laird's old supposition that human fragile sites correspond to the I-HC detected in Here we report the results of genome-wide profiling of DSBs in human chromosomes from cultured HEK 293T cells. Our deep-sequencing data indicate a non-random distribution of DSBs that have blunt ends and that define mainly 50–250 kb forum domains that are protected from the breaks. Transcription profiling demonstrates that some domains contain coordinately expressed gene clusters, suggesting novel regulation of transcription inside large genomic regions delimited by hot spots of DSBs. A clue to a mechanism is provided by our finding that forum termini bind specifically the known transcriptional regulator PARP1, which is involved in chromatin organization and transcriptional regulation, and HNRNPa2B1, a nuclear RNA processing factor. Our data also suggest that the mapped breaks could correspond to fragile sites in human chromosomes, and indicate that our technique can be used to study fragile chromosomal sites in many types of human cells.To study hot spots of spontaneous DSBs causing forum domain excision, we isolated DNA samples in a solid phase, as described Drosophila genome FISH analysis  indicateFHIT and WWOX  have mapped DSB hot spots in HEK 293T cells. In the approximately 1500 kb fragile histidine triad gene, FHIT, eight FT were mapped in the gene itself and ten FT close to it. Monte-Carlo simulations indicated that the probability of the random occurrence of this number of FT consisting of overlapping reads is very low (p<10−7), both for FHIT itself and the whole region. Details are provided in WWOX gene, a target in FRA16D, seven FT corresponding to DSB hot spots were mapped    from the four FT analyzed was increased in the HU-treated cells . FT-4 waWWOX gene) were found to be undamaged. FT-4, which was slightly affected by replication stalling induced by HU, exhibited a high sensitivity to heat shock treatment. The results independently demonstrate a strong relationship between organization of transcription patterns and hot spots of DSBs.Profiling FT along chromosomes also revealed a correspondence between transcription patterns and hot spots of DSBs. To address the question of whether global gene expression changes could interfere with DNA breakage at FT regions, we analyzed the same FT set in heat shock-treated cells see . InteresWWOX gene and FT-4 are even more sensitive in fibroblasts than in HEK 293T cells, where they originally were detected. At the same time, FT-7 is absent in the HEF cells. Thus, not all FT retain their position in different cell types. The data suggest the functional significance of FT regions in differentiation.The observed relationships of FT regions with replication and transcription events suggest that at least some part of these hot spots of DSBs could be located in the same regions in different cell types due to stability of some higher-order chromosomal structures. The irrefragable answer could be found in the genome-wide profiling of FT in several cell types, which we currently are performing. In the present study, we used only quantitative PCR across several FT in one more cell line: human embryonic lung fibroblast (HEF) cells. in vitro binding experiments  using PARP1 and HNRNPA2B1 antibodies and quantitative PCR. In our eriments  we used eriments . The in cross FT . Alternacross FT . The resIntegrity and fragmentation are both natural states of chromosomal DNA. For example, extensive DNA fragmentation is observed in elongating spermatids during their normal chromatin remodeling The thousands of hot spots of spontaneous DSBs in human chromosomes described here also support this hypothesis. The RAFT procedure may provide a snapshot of preexisting transient physiological DSBs associated with different processes  in different genomic regions. Our PCR experiments across several FT independently support this notion. Transient DSB hot spots could appear upon the removal of DNA supercoiling from nucleosomes or the removal of torsion at the borders of different types of higher-order chromosomal structures required for the correct regulation of gene expression. Thus, the mapping of DSBs, hot spots, and protected regions (such as the 50–250 kb domains we have described) could contribute to our understanding of higher-order chromosomal structures.Drosophila, FT often correspond to the dispersed I-HC regions that are characterized by late replication and chromosomal breaks, and are involved in ectopic pairing Some mapped DSBs are in late replicated regions and in regions with a paucity of initiation sites, particular nucleotide content, or DNA secondary structures that may impair replication fork movement Drosophila, FT sites often correspond to the binding sites of 19–24 nt RNAs and Argonaute proteins http://www.ebi.ac.uk/Tools/emboss/align/index.html) and WebLogo (http://weblogo.berkeley.edu/logo.cgi) to find either the 12-nt topo II consensus sequence or to define any consensus sequence in a number of FT, using the sequences around the cut sites, but we did not observe any consensus. This may indicate that the topo II site (5′ ANCNT[A/G]T.NN[G/C]N[A/G] 3′) is actually more variable or that this enzyme is not involved in production of the majority of the mapped DSBs.We do not know what enzyme(s) is responsible for the chromosomal fragmentation. Topoisomerase I is required for CFS breaks, suggesting that polymerase–helicase uncoupling could be involved in the breakage DrosophilaDrosophila, about 20% of the forum domains were transcriptionally active and contained co-expressed genes, whereas the majority of domains contained silent gene clusters Initially, gene clusters exhibiting similar expression profiles shaping transcriptional territories were described in We speculate that small-RNA-directed DNA elimination in ciliates may lead to the selection of actively transcribed DNA domains that correspond to actively expressed forum domains, which accumulate in the macronucleus. In this way, widespread DNA breakage would lead to the sequestration of the active portion of the genome.The specific binding of PARP1 and HNRNPA2B1 proteins at FT sequences is very intriguing. PARP1 is a known regulator of chromatin structure and transcription HNRNPA2B1 belongs to the A/B subfamily of ubiquitously expressed heterogeneous nuclear ribonucleoproteins that bind to pre-mRNAs and are involved in the splicing and nucleocytoplasmic transport of mRNAs Our data suggest the presence of a novel type of gene regulation: a coordinated silencing or transcription of gene clusters within large chromosomal domains that are protected from fragmentation, delimited by non-randomly dispersed DSB hot spots and the binding sites of PARP1 and HNRNPA2B1. They highlight the relationship between human chromosomal fragile sites, chromosomal organization, and gene regulation, which may prove to be important for cancer genomics and gene therapy. The data strongly suggest a mechanistic link between DSBs, transcription patterns, and genomic instability. This outcome is in agreement with the recent conclusion that mapping of DSBs provides a novel tool to analyze genome architecture and that DSBs are distributed much more uniformly than was previously believed E. coli DNA polymerase I and then ligated with a molar excess of double-stranded biotinylated oligonucleotide  was labeled with Alexa Fluor 5 and Alexa Fluor 3, respectively, using a BioPrime total genomic labeling system (Invitrogen) according to the manufacturer's recommendations. G-banding was performed prior to FISH by use of a standard procedure Nuclear proteins from HEK293T cells were isolated as described 2, 0.2 mM EDTA, 1 mM DTT, and 30 µg of poly[d(I)/d(C)]. After pre-incubation, 0.4 µg of biotinylated RAFT preparation was added, and incubation at 20°C was carried out for 1 h with gentle mixing every 10 min. The bound proteins were selected on SA-PMP according to the manufacturer's recommendations.Initially, 150 µL of nuclear protein extract (150–300 µg of protein) was pre-exhausted for 10 min at 10°C in 400 µL of solution containing 20 mM HEPES, pH 7.6, 4% Ficoll, 5 mM MgClThe HEK 293T cell suspension was treated with 1% formaldehyde at 20°C for 10 min. The nuclei were washed and lysed, and chromatin was sheared to an average length of 600 bp by sonication. X-ChIP was carried out using the OneDay ChIP kit , with 4 mg of antibodies against PARP1  or HNRNPA2B1 . The negative control was DNA precipitated using 4 mg of non-specific IgG from rabbit serum. The PCR primers corresponding to FT and non-FT regions are listed in http://bioinformatics.bc.edu/marthlab/PyroBayes). Elimination of primers sequences was performed by Perl script using BioPerl as interface to FASTA, with the assumption that a primer should be at either end of a read, but not further than 5 bp from it. All sequences shorter than 18 bp were removed from the dataset. The final mapping was performed using BWA (http://bio-bwa.sourceforge.net) and Samtools (http://samtools.sourceforge.net) with the Homo sapiens masked genome (assembly GRCh37p5/hg19) as the database (taken in the form of MFA files from ftp://ftp.ncbi.nih.gov/genomes/H_sapiens/Assembled_chromosomes/seq). The saturation curve indicating that practically all FT were defined is shown in Raw data in SFF format were obtained using a 454 Roche GS FLX Life Sciences pyrosequencing machine. Data were then decoded to FASTQ/FASTA format using PyroBayes (ftp://ftp.ncbi.nih.gov/genomes/H_sapiens/Assembled_chromosomes/gbs) by the Perl script with the BPLITE module as the interface for GBK/GBS files. Monte-Carlo simulations were performed using the following procedure. First, our investigations revealed that Perl's built-in random module (Math::Random) generates poor quality pseudorandom numbers. Therefore, we used the Mersenne Twister pseudorandom generator http://www.r-project.org). Because the standard interface sometimes cannot process the R output correctly, we reworked the processing procedure to meet our own needs. The details are described in All domain–gene comparisons and statistical evaluations were performed with each gene's database from the same GRCh37p5 genome build (taken in the form of GBS files from http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE35065). The reads are presented in .gff and .wig files, which are divided by chromosomes for convenience. The data in the .gff and .wig files are the same—only the format differs.The mapping result was deposited into the GEO database with the accession ID GSE35065  was used. The FT barcode is shown in red.(TIF)Click here for additional data file.Figure S2Overviews of chr13, chr14, chr15, chr17, chr18, chr19, chr20, chr21, and chr22. Integrated Genome Browser (Affymetrix) was used. The FT barcode is shown in red.(TIF)Click here for additional data file.Figure S3Overviews of chr2, chr6, chr7, and chrX. Integrated Genome Browser (Affymetrix) was used. The FT barcode is shown in red. The length and numbers of reads are shown above the barcode in chrX. The frequently and less frequently observed CFS detected in leukocytes are shown in red and in blue, respectively.(TIF)Click here for additional data file.Figure S4WWOX gene. This sequence escapes cloning in E. coli cells. The Z-DNA region (highlighted in yellow) was detected using ZHunt Online software (http://gac-web.cgrb.oregonstate.edu/zDNA/index). The region corresponding to FT reads is shown in blue (the Sau site is indicated in red). The underlined portion is a shorter amplified DNA fragment that also escapes cloning. Folding of the 1090 bp fragment was performed using the UNAFold Web Server http://mfold.rna.albany.edu/?q=mfold/DNA-Folding-Form).Z-DNA region and palindromes inside the 1090 bp sequence that possesses the mapped FT in the 3′ exon of the (PDF)Click here for additional data file.Figure S5WWOX gene. The results of four independent experiments are shown. Different forum domains (Forum) and control DNA (Intact) preparations, isolated as described in Quantitative real-time PCR across the FT in the 3′ exon in the (PDF)Click here for additional data file.Figure S6Expression levels inside forum domains in chr4, chr5, and chr6. The data for expression in HEK293T cells (wgEncodeEH002692_2) were used. The median values of transcription levels in coding regions  within a particular forum domain were used, and the result was plotted according to the position of the domain in its chromosome. The arrows indicate the position of the average expression level of forum domains in a particular chromosome. The value to the right of the arrow indicates the portion of forum domains in a chromosome that is more highly expressed.(PDF)Click here for additional data file.Figure S7Expression levels inside forum domains in chr7, chr8, and chr9. The data for expression in HEK293T cells (wgEncodeEH002692_2) were used. The median values of transcription levels in coding regions  within a particular forum domain were used, and the result was plotted according to the position of the domain in its chromosome. The arrows indicate the position of the average expression level of forum domains in a particular chromosome. The value to the right of the arrow indicates the portion of forum domains in a chromosome that is more highly expressed.(PDF)Click here for additional data file.Figure S8Expression levels inside forum domains in chr10, chr11, and chr12. The data for expression in HEK293T cells (wgEncodeEH002692_2) were used. The median values of transcription levels in coding regions  within a particular forum domain were used, and the result was plotted according to the position of the domain in its chromosome. The arrows indicate the position of the average expression level of forum domains in a particular chromosome. The value to the right of the arrow indicates the portion of forum domains in a chromosome that is more highly expressed.(PDF)Click here for additional data file.Figure S9Expression levels inside forum domains in chr13, chr14, and chr15. The data for expression in HEK293T cells (wgEncodeEH002692_2) were used. The median values of transcription levels in coding regions  within a particular forum domain were used, and the result was plotted according to the position of the domain in its chromosome. The arrows indicate the position of the average expression level of forum domains in a particular chromosome. The value to the right of the arrow indicates the portion of forum domains in a chromosome that is more highly expressed.(PDF)Click here for additional data file.Figure S10Expression levels inside forum domains in chr16, chr17, and chr18. The data for expression in HEK293T cells (wgEncodeEH002692_2) were used. The median values of transcription levels in coding regions  within a particular forum domain were used, and the result was plotted according to the position of the domain in its chromosome. The arrows indicate the position of the average expression level of forum domains in a particular chromosome. The value to the right of the arrow indicates the portion of forum domains in a chromosome that is more highly expressed.(PDF)Click here for additional data file.Figure S11Expression levels inside forum domains in chr19, chr20, and chr21. The data for expression in HEK293T cells (wgEncodeEH002692_2) were used. The median values of transcription levels in coding regions  within a particular forum domain were used, and the result was plotted according to the position of the domain in its chromosome. The arrows indicate the position of the average expression level of forum domains in a particular chromosome. The value to the right of the arrow indicates the portion of forum domains in a chromosome that is more highly expressed.(PDF)Click here for additional data file.Figure S12Expression levels inside forum domains in chr22 and chrX (A) and the average expression levels per forum domain in different chromosomes (B). The data for expression in HEK293T cells (wgEncodeEH002692_2) were used. The median values of transcription levels in coding regions  within a particular forum domain were used, and the result was plotted according to the position of the domain in its chromosome. The arrows indicate the position of the average expression level of forum domains in a particular chromosome. The value to the right of the arrow indicates the portion of forum domains in a chromosome that is more highly expressed.(PDF)Click here for additional data file.Figure S13HOXA gene cluster. The UCSC Genome Browser on Human Feb. 2009 (GRCh37/hg19) Assembly was used. UCSC genes, Human mRNAs from GenBank, the H3K37Ac mark from Encode, chromatin state segmentation by HMM from Encode/Broad, and some histone modifications by ChIP-Seq from Encode are indicated. The “RNA-seq” lane corresponds to expression of mRNAs in IMR90 cells (GEO accession number GSM438363). Forum domain containing actively transcribed genes are indicated by the red bracket. “HEK293T” lanes correspond to the expression of mRNA in HEK293T cells .Coordinated expression inside the 498 kb forum domain that possesses the (TIF)Click here for additional data file.Figure S14HOXC gene cluster (A) and inside the 673 kb forum domain that possesses the HOXD gene cluster (B). The UCSC Genome Browser on Human Feb. 2009 (GRCh37/hg19) Assembly was used. UCSC genes, Human mRNAs from GenBank, the H3K37Ac mark from Encode, chromatin state segmentation by HMM from Encode/Broad, and some histone modifications by ChIP-Seq from Encode are indicated. The “RNA-seq” lane corresponds to expression of mRNAs in IMR90 cells (GEO accession number GSM438363). Forum domains containing actively transcribed genes are indicated by the red bracket. “Domains” lanes indicate the forum domains containing the silent or weakly expressed genes (blue brackets). “HEK293T” lanes correspond to the expression of mRNA in HEK293T cells .Coordinated expression inside the 374 kb forum domain that possesses the (TIF)Click here for additional data file.Figure S15Saturation curve. % of FT plotted against decreasing % of reads reveals a plateau in the range between 90 and 100% of reads. The step was equal to 1% of reads in the range from 100% towards 70% and 5% down to 10% of reads. The curve indicates that practically all FT, corresponding to hot spots of DSBs, were defined.(PDF)Click here for additional data file.Table S1Primers used in quantitative PCR experiments.(DOC)Click here for additional data file.Table S2Primers used in the ChIP experiments shown in (DOC)Click here for additional data file.Table S3http://hgdownload.cse.ucsc.edu/admin/exe). The median values of transcription levels in coding regions  within a particular forum domain were used. The average expression level of forum domains in a particular chromosome was determined as a sum of expression data per chromosome divided into domains numbers. The values in the last four columns correspond to % of low expressing (below the average expression level in a particular chromosome) or silenced forum domains in a corresponding chromosome. Most domains in different cell lines are silent or expressed at very low levels.Percent of low expressing or silent forum domains in four cell lines. Names of cell lines and their corresponding accession numbers are indicated. The conversion of data for the second and third columns from hg18 to hg19 coordinates was performed using the LiftOver program ((DOC)Click here for additional data file.Text S1Extended Materials and Methods.(DOC)Click here for additional data file.


Streptococcus suis is a major bacterial pathogen of young pigs causing worldwide economic problems for the pig industry. S. suis is also an emerging pathogen of humans. Colonization of porcine oropharynx by S. suis is considered to be a high risk factor for invasive disease. In the oropharyngeal cavity, where glucose is rapidly absorbed but dietary α-glucans persist, there is a profound effect of carbohydrate availability on the expression of virulence genes. Nineteen predicted or confirmed S. suis virulence genes that promote adhesion to and invasion of epithelial cells were expressed at higher levels when S. suis was supplied with the α-glucan starch/pullulan compared to glucose as the single carbon source. Additionally the production of suilysin, a toxin that damages epithelial cells, was increased more than ten-fold when glucose levels were low and S. suis was growing on pullulan. Based on biochemical, bioinformatics and in vitro and in vivo gene expression studies, we developed a biological model that postulates the effect of carbon catabolite repression on expression of virulence genes in the mucosa, organs and blood. This research increases our understanding of S. suis virulence mechanisms and has important implications for the design of future control strategies including the development of anti-infective strategies by modulating animal feed composition. Streptococcus suis is a major bacterial pathogen of young pigs and a worldwide economic problem for the pig industry. Furthermore, S. suis is emerging as a zoonotic pathogen associated with meningitis and septicaemia in humans S. suis across the mucosal epithelium in the upper respiratory tract S. suis are unknown and probably depend on the environmental conditions and the bacterial genotype including presence of virulence genes. In vitro studies on adhesion and invasion by S. suis have often been performed in medium containing glucose as a carbon source which does not accurately reflect the situation in vivo. In the oropharyngeal cavity including the saliva, glucose may be present but concentrations usually diminish rapidly (within 30 min) after ingestion S. suis sly gene, may release, through its cytotoxic effect on epithelial cells, cellular glycogen S. suis virulence factors such as suilysin. Streptococcal pathogens contain genes required for efficient utilization of α-glucans including amylases and/or pullulanases which cleave α-1,4 and α-1,6 glycosidic bonds in starch or glycogen S. suis serotype 2 (apuA-SSU1849) was necessary to support bacterial proliferation on the α-glucan starch/pullulan  as a single carbon source and to promote adhesion of S. suis to porcine tracheal epithelial cells Currently, little is known about the expression and regulation of S. suis the expression of carbohydrate metabolic enzymes are under the control of the global transcriptional regulator, catabolite control protein A (CcpA) that mediates carbon catabolite control (CCC) in presence of a preferred sugar, usually glucose. CcpA can repress or activate transcription by binding to cis-acting catabolite response element (cre) sites in the gene promoter regions As in all Gram-positive bacteria, in S. suis, we compared the genome-wide effects of growth on glucose or the α-glucan starch/pullulan on S. suis metabolism and virulence gene expression. We first constructed a metabolic map for S. suis and used it to analyze the key metabolic pathways altered by growth on glucose or pullulan. In addition to genes that play roles in (carbohydrate) metabolic pathways, 19 virulence genes were differentially expressed, seven of which were strongly induced (ratios>10) by growth in pullulan compared to glucose and nine were shown to contain a consensus cre site in their promoter sequences. The regulation of apuA, the virulence factor most strongly induced in pullulan compared to glucose, was investigated in detail using qPCR analysis of gene transcripts in bacteria grown in different carbon sources, promoter mapping and binding studies with CcpA and a newly identified transcriptional regulator, ApuR. The biological consequences of carbohydrate metabolism and virulence gene expression were also assessed in an in vitro porcine tracheal cell model using qPCR, hemolytic assays and adhesion and invasion assays. Based on these findings we propose a model for the transcriptional regulation of production of virulence factors during different stages of infection dependent on a CcpA-mediated, carbon catabolite control-dependent mechanism. To verify the predictions of this model, the in vivo expression of apuA and sly was measured for S. suis serotype 2 recovered from the blood and organs of pigs infected under controlled conditions.Given the relative abundance of dietary complex carbohydrates such as α-glucans and low level of glucose in the porcine upper oropharyngeal niche occupied by S. suis serotype 2 strain S10 S. suis S10 is more than 99% identical to the genome of S. suis 2 strain P1/7, a sequenced reference strain of which the genome had been annotated previously (NCBI Genome and NCBI BioProject http://www.ncbi.nlm.nih.gov/genome/?term=Streptococcus%20suis) S. suis was grown in Todd-Hewitt broth (THB) (Difco) or on Columbia agar plates with 6% sheep blood (Oxoid) at 37°C under 5% CO2 for 18 hr. A complex medium (CM) (S. suis only grows to high density in CM when exogenous carbohydrates are added 600 using a SpectraMax M5 reader (Molecular Devices LLC).The virulent ium (CM)  was prepS. suis S10 was grown to exponential (e) and early stationary (s) phase as indicated in 260/A280 ratio (NanoDrop 8000 UV-Vis Spectrophotometer). For qPCR, cDNA was synthesized using SuperScript VILO (Invitrogen). Primers were designed using Oligo Program version 6  . Two replicates of all samples and primer pairs were included and the experiment was performed in triplicate. Non-template controls were included for each gene in each run.For RNA extraction,  Norway) . Quantitene proS  [26]. ThS. suis oligoarray (8×15 K) containing in situ synthesized 60-mers was produced by Agilent Technologies , based on the genome sequence of S. suis P1/7 S. suis samples was labeled using the Cyanine 5 (Cy5) labeling reaction . Co-hybridization with labeled cDNA probes was performed on these oligonucleotide arrays at 42°C for 16 h in Slidehyb#1 . The data were normalized using Lowess normalization http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE40658. Details of the custom-made 60-mer oligonucleotide array design  are deposited in the ArrayExpress database (http://www.ebi.ac.uk/arrayexpress/) under accession number A-MEXP-1671.An S. suis bacteria grown in CM supplemented with pullulan (Pul) or glucose (Glc) and harvested at early exponential (e) or early stationary (s) phase was cross-compared in different combinations . Overlapping and unique differentials were visualized using Venn diagrams at http://bioinfogp.cnb.csic.es/tools/venny/index.html.Differential gene expression of S. suis P1/7 genome, Gene Ontology  and KEGG pathway annotations were obtained using the BLAST2GO software (www.blast2go.org) For all genes and proteins identified in the S. suis genes and proteins according to standard Gene Ontology (GO) (www.geneontology.org) nomenclature. BLAST2GO uses the integrated Gossip package BLAST2GO was used to annotate all known http://meme.sdsc.edu/meme/meme.html) software suite (version 4.1.0) was used for the identification of motifs OM1 and OM2 MdxE and MalE genes, with well-characterized cre sites, from Bacillus and Listeria . A range of motif widths (15 nt in length) and zero or one motif per sequence were specified in our queries and FIMO, part of MEME, was used to search for this motif in the genome of S. suis P1/7. The algorithm in MAST, also part of the MEME suite, calculates position scores for the motif at each possible position within a sequence P value below 10−4 were considered to identify putative CcpA binding sites. In silico searches and comparisons of predicted cre sites within the S. suis P1/7 genome sequence and reconstruction of cre locations in the predicted operons were conducted using the corresponding databases provided by the MicrobesOnline database web server (http://microbesonline.org) and RegPrecise database The MEME .The 5′-rapid amplification of cDNA ends (RACE) system (Invitrogen) was used to determine the transcription start site of the mer ASP1 . A homopS. suis 2 S10 genomic DNA using GoTaq (Promega) with primers ApuR_F/R and CcpA_F/R respectively (The genes encoding the regulators ApuR (amino acids 2 to 312) and CcpA (amino acids 2 to 333) were amplified by PCR from ectively . The recectively  [19]. FrS. suis P apuA promoter sequence (cre) were synthesized , PCR-amplified with labelled Pr2F/R primers and incubated with the proteins. Free and bound DNAs were separated on 5% Tris-Borate-EDTA (TBE) native gels for 30 min at 10 cm/v. The gels were visualized using a LI-COR Odyssey Imager and scanned at 800 nm wavelengths.Infrared electrophoretic mobility shift assays (EMSA) were performed utilizing three pairs of fluorescent InfraRed-dye 800 (IRdye 800) labelled primer on the 5′ ends . The IRdye-primers used to PCR-amplify three DNA fragments of ca. 120 bp (Pr1-Pr3) contained overlapping regions to cover the full length of the sequence . The IRdS. suis bacteria were grown in CM+Glc and CM+Pul at three different growth stages: lag (OD600 0.1–0.2), exponential (OD600 0.2–0.5) and stationary (OD600 0.5–0.7). The supernatant was collected from 1 ml for each culture by centrifugation at 12000 g for 1 min. Serial twofold dilutions (150 µl) of test samples were prepared in polystyrene deep-well titer plates (Beckman) with 10 mM Tris-buffered saline . Subsequently, 150 µl of a 2% washed horse erythrocyte suspension in 10 mM Tris-buffered saline containing 0.5% BSA was added to each well. After the wells were sealed, the plates were incubated on a Coulter mixer for 2 h at 37°C. Unlysed erythrocytes were sedimented by centrifugation (1500 g for 10 min), 150 µl portions of the supernatant were transferred to a polystyrene flat-bottom microtiter plate and measured at 540 nm with a microELISA (enzyme-linked immunosorbent assay) reader . A 100% lysis reference sample was obtained by lysing cells with 1% Triton-X and the background lysis was subtracted before calculation of hemolytic activity.The hemolytic activity was assayed as previously described S. suis was added to the cell culture at an multiplicity of infection (m.o.i.) of ∼50 and incubated for 2 h at 37°C with 5% CO2 to allow cellular invasion by the bacteria. The monolayers were then washed three times with PBS; 1 ml of cell culture medium containing 100 µl/ml gentamycin and 5 µg/ml penicillin G was added to each well, and the plates were incubated for 2 h at 37°C, 5% CO2 to kill extracellular and surface-adherent bacteria. The monolayers were washed three times with PBS, and cells were disrupted by the addition of 800 µl of ice-cold Milli-Q water and repeated up-and-down pipetting to release intracellular bacteria. To enumerate the viable bacteria, serial dilutions of each cell lysate were plated in triplicate on Columbia sheep blood agar plates and incubated at 37°C for 24 h. The rate of invasion was expressed as percentage of initial inoculum that was recovered per well. Two independent assays were performed in triplicate.Culturing of Newborn pig tracheal cells (NPTr) S. suis-specific symptoms occurred. Caesarean-derived, colostrum-deprived (CDCD) piglets were infected i.v. with 1×106 CFU of S735-pCOM1-orf2 in vitro qPCR assays but was selected because it was known to be effectively recovered from the blood and organs in experimental pig infections. Genes encoding apuA and sly are both present in the genome of this strain (not shown). Animals were euthanized when specific S. suis symptoms  were observed, or at the end of the experiment (6 days post infection). After animals were euthanized; organs and specific sites of S. suis infection were examined macroscopically and bacteriologically. Tissue samples were collected during post-mortem observation and snap-frozen in liquid nitrogen.The animal experiment described in this paper was approved by the ethical committee of the Central Veterinary Institute of Wageningen UR, Lelystad, The Netherlands under proposal number 2010113c in accordance with Dutch legislation  and the general principles governing the use of animals in experiments of the European Communities (Directive 86/609/EEC). To reduce unnecessary suffering of animals, humane end points were used: piglets were euthanized as soon as in vivo samples were thawed on ice, resuspended in 600 µl of Trizol  and subjected to 40 seconds of 6.5 m s−2 in the Fastprep-24  to disrupt bacteria. The mixture was extracted with 120 µl of chloroform, mixed for 15 seconds, incubated for 3 min at RT and centrifuged for 15 min at 20.000 x g. Supernatant was removed and extracted with 1 volume of chloroform. RNA in the supernatant was precipitated with 1 volume of isopropanol. After incubation for 30 min at RT or 16 h at −20°C, RNA was collected by centrifugation, and washed with 70% ethanol. The pellet was resuspended in water. Subsequently, RNA was purified as described above. To remove eukaryotic RNA all samples were treated using the MicrobEnrich kit . Bacterial-enriched RNA was amplified using the Ovation PicoSl WTA system v2 . cDNA was purified using MinElute spin columns  and diluted 25 times for qPCR analysis. Primers were designed using PrimerExpress software  . In each run a standard curve was incorporated consisting of seven 10-fold dilutions of a vector containing the cloned target of the PCR. In this way, both target genes and the reference genes could be related to a standard line. For each reaction, negative water controls were included. Analysis was performed using the ABI7500 Software (Applied Biosystems).Blood collected in PAXgene tubes was treated as recommended by the manufacturer . Samples taken from heart, brain and joints consisted of a mixed pellet of porcine cells combined with bacteria. All pellets from CA, USA) . Each rein vitro studies were analyzed using GraphPad Prism version 5.0 software . All qPCR experiments and hemolytic activity assay were reproduced at least two times in triplicates and, where indicated, representative experiments are shown. Two-way ANOVA tests were carried out using Bonferroni's post hoc test. The adhesion and invasion assays were performed at least two times using triplicate samples. All numerical data presented here are expressed as means ± standard error of the mean (SEM). Statistical significance was determined using a two-tailed unpaired Student's t test. Differences were considered significant at p<0.05. Statistical significance was indicated as follows: * p<0.05; ** p<0.01; *** p<0.001.The results obtained in the S. suis gene expression we compared genome-wide transcriptomic data from exponential (e) and stationary phase (s) cultures of S. suis in complex media (CM) supplemented with 1% of starch/pullulan  (Pul) or glucose (Glc) as carbon sources  during exponential growth and 1015 (51% of annotated genes) during early stationary growth . In totaS. suis P1/7 genes using BLAST2GO (see Methods)  functional gene annotations of all differentially expressed Methods) . SimilarMethods) . The higMethods) . Other eS. suis in presence of these different carbon sources leads to changes of 35–50% of the S. suis transcriptome, affecting not only carbohydrate metabolism but also basal metabolic and stress survival pathways. To summarize these broad changes in global transcriptomes, we generated a visual representation of the main carbohydrate pathway genes for six growth conditions comparisons according to their expression ratios (see Methods)  .S. suis grown in starch/pullulan vs glucose revealed that growth in pullulan induced expression of the maltodextrins utilization gene cluster that includes apuA and the corresponding phosphotransferase systems (PTS) and ATP-binding cassette (ABC) transporters. The expression of the glycolysis pathway genes did not change significantly during growth in pullulan compared to glucose, suggesting that activity of this pathway was not altered during growth in both carbon sources. Rather, it appeared that excess intracellular glucose was converted in glycogen as energy reserves (glycogen biosynthesis pathways) or had entered into hexose-pentose carbohydrate pathways as an alternative to glycolysis (sugar interconversion and Leloir pathways)  . The bioS. suis virulence genes. Seven genes possibly involved in the invasion of mucosal tissues or avoidance of host defenses by streptococcal pathogens were highly upregulated (expression ratio>10) in starch/pullulan compared to glucose (S. suis P1/7 for catabolite-responsive element (cre) sites, short DNA regions that can be bound by CcpA, using the consensus B. subtilis cre sequence We analyzed the transcriptome data to see whether the differential fermentation of starch/pullulan and glucose affected expression of known or predicted  glucose , Text S2cre sites located upstream of the start codon of predicted proteins (P) or in proximity of the gene transcription start site (G) were predicted to control expression of 172 genes through interaction with CcpA  or in an S. suis serotype 2 ΔccpA mutant compared to wild-type 38 potential ith CcpA . Of the ith CcpA . In Tablcre sites were commonly associated with the predicted promoters of carbohydrate PTS and ABC transporters (17%) and enzymes for carbohydrate metabolism (25%). Additionally, cre sites were identified in the promoters of regulators (10%) and 9 (8%) out of the 19 virulence genes (47%) that were differentially expressed in pullulan compared to glucose, including sly and apuA  and maltotriose   of apuA expression was observed 30 min after addition of maltotriose  B. subtilis 168; 49% identity) Listeria monocytogenes; 47% identity) Enterococcus faecium E1162; 49% identity) We hypothesized that the induction of rminator . Homologrminator  which coapuA is considered a relevant virulence factor and its regulation by different carbohydrates could be of biologic relevance, we decided to investigate the binding of CcpA and ApuR to operator sites in the apuA promoter. To elucidate the apuA promoter regulatory modules we determined the transcription start site, conducted homology searches for binding motifs of known regulators in silico, and performed in vitro promoter binding assays with purified transcription factors (TFs). The transcriptional start site of the apuA transcript was determined experimentally by 5′-RACE to be 31 nt upstream of the start codon. The consensus −35 element (5′-TTGCAA-3′) for RpoD (sigma 70) and the −10 element (5′-TATATA-3′) required for interaction with the RNA polymerase and transcription initiation were found near the expected positions upstream of the transcription start site  that were also listed in the RegPrecise database mdxR-like operator motif, designated here as OM1, was found 13 bp upstream of the −35 element; a second operator motif predicted cre site designated OM2/cre, located at position −30 nt, overlapping the conserved −35 element; this overlap could interfere with binding of the RNA polymerase . A single rApuR-DNA complex was observed with promoter fragments Pr2 and Pr3 suggesting the binding motif for ApuR lies within the 64 bp overlapping region (cre operator binding motifs. The non-specific competitor DNA fragment had no effect on Pr2 complex formation whereas the specific competitor (Pr2) substantially reduced rApuR binding . The adherence of S. suis was significantly increased  after growth in 1% pullulan  in glucose. Invasiveness of S. suis was nearly 9-fold higher when grown in pullulan (0.40%±0.01) (P<0.01)  , correspsly was strongly induced in pullulan compared to glucose, both in exponential and stationary phase (+18.1/+17.9 ratio in pullulan vs glucose). The presence of a cre site in the sly promoter region suggested that the induction of sly during growth in pullulan was due to relief from CcpA-mediated carbon catabolite repression rather than a specific induction by starch/pullulan. As suilysin has been proposed to compromise the integrity of the host epithelium and facilitate entry into the body we measured erythrocyte hemolytic activity (HA) of culture supernatants of S. suis grown in glucose or pullulan. HA was significantly higher in supernatants of S. suis grown in pullulan compared to glucose over a wide range (ca. 0.5) of OD600 values . Pullulan, a starch, was chosen since we had previously found that growth of S. suis in presence of this carbohydrate induces expression of apuA, a gene known to be involved in carbohydrate metabolism Asymptomatic carriage of apuAA switch from growth in glucose to starch/pullulan resulted in a change of ca. 50% of its transcriptome, involving multiple GO function categories . As antiS. suis genes might be regulated by CCC we searched for conserved cre sites in the genome of S. suis P1/7 and identified 172 genes, some of which were organized in operons  S. suis serotype 2 ΔccpA mutant with the corresponding wild-type strain In other streptococcal pathogens CcpA and CCC have been shown to play a role in virulence gene expression as well as colonization  operons . Of thesapuA encoding a cell-surface amylopullulanase required for growth on starch or glycogen that also contributes to adherence to porcine epithelial cells in vitro and thus may be relevant to mucosal colonization apuA was highly induced during growth in starch/pullulan or maltotriose, the most abundant degradation product of pullulan, and repressed during growth in glucose  . The mos glucose  and 4. Ag motifs  and EMSAor, ApuR . The simtotriose . In contapuA, eighteen other predicted or known virulence genes were differentially regulated in pullulan vs glucose, seven of which were highly upregulated in pullulan compared to glucose  S. suis sly mutant, lacking of the hemolytic characteristics, was nearly as invasive as the wild-type parental strain in a pig infection studies after bacteria intravenous injection. The authors of the study by Allen et al (2001) et al (2001) sly was present in a significantly higher number of isolates from pigs with meningitis, septicemia, and arthritis than isolates from pigs with pneumonia. Conversely, sly is also significantly overrepresented in asymptomatic carriage strains of pigs sly does not exclusively correlate with invasive isolates. In addition to suilysin, it is likely that other virulence factors are relevant for the invasion of connective tissue, such as the hyaluronidase and heparinase II/III-like proteins. Note that the function of these putative virulence proteins still has to be demonstrated. In contrast to our results, sly expression was not increased in a recent study which compared gene expression of a wild-type strain with a ΔccpA mutant grown to stationary phase sly is controlled by a CcpA-independent carbon catabolite repression mechanism although a cre site is present in the sly promoter region.In addition to  glucose . One of  glucose ; its inc glucose . SuilysiS. suis in the oropharyngeal cavity. Additionally relief from CCR leads to higher expression of other virulence genes involved in mucosal infection  would repress expression of apuA and sly by CCR  than in the blood (from 4.4 to 6.6 mM) S. suis  in synovial fluid We propose that at mucosal surfaces the high ratios of starch to glucose promotes production of extracellular ApuA to support growth of nfection . Indeed,y by CCR . Indeed,e organs  supporti S. suis . AdditioapuA and other virulence genes. We propose a schematic model of how carbohydrate content of different tissues could modulate S. suis metabolism at the different stages of infection Click here for additional data file.Figure S2S. suis genes with the annotation “carbohydrate metabolism” that were differentially upregulated in starch/pullulan vs glucose. A. early exponential (e) and B. early stationary phase (s) Enrichment analysis performed using BLAST2GO .GO term distribution of (TIF)Click here for additional data file.Figure S3S. suis pathogenesis and expression ratios are shown for exponential (white bar) and early stationary (black bar) growth phases. Envelope: cps2E*-SS0519 putative galactosyl transferase; wchF*-SSU0520 putative rhamnosyl transferase; cps2C-SSU0517 tyrosine-protein kinase; cpss_lpl-SSU1123 putative glycosyltransferase; cpss_lpl2-SSU1124 putative rhamnosyl transferase pgdA-SSU1448 peptidoglycan GlcNAc deacetylase, dltA-SSU0596 D-alanine-poly(phosphoribitol) ligase subunit1; Envelope/Adhesion neuB-SSU0535 putative N-acetylneuraminic acid synthase; neuC-SSU0536 putative UDP-N acetylglucosamine 2-epimerase; neuA-SSU0538 N-acylneuraminate cytidylyltransferase; Adhesion: apuA*-SSU1849 amylopullulanase; sadP-SSU0253 putative surface-anchored protein receptor; gnd-SSU1541 6-phosphogluconate dehydrogenase; Adhesion/Invasion: srtA-SSU0925 sortase; fbpS-SSU1311 fibronectin-fibrinogen binding protein; gapdH-SSU0153 glyceraldehyde-3-phosphate dehydrogenase; eno-SSU1320 enolase; pepD-SSU1215 putative surface-anchored dipeptidase; dpp IV-SSU0187 Xaa-Pro dipeptidyl-peptidase; Invasion: sly* SSU1231 suilysin (haemolysin); hepII/III*-SSU1048 heparinase II/III-like protein; hyl*-SSU1050 hyaluronidase precursor; ssnA*-SSU1760 surface-anchored DNA nuclease; arcB*-SSU0580 arginine deaminase; igaP-SSU1773 putative surface-anchored serine protease; sspA-SSU0757 cell envelope proteinase; ofs-SSU1474 serum opacity factor; luxS-SSU0376 S-ribosyl homocysteinase; Marker: mrp-SSU0706 muramidase-released protein precursor; ef-SSU0171 putative surface-anchored protein. * Indicates the presence of a predicted cre in the virulence gene promoter region.Putative and characterized virulence gene expression ratios in pullulan vs. glucose. The genes are grouped according to their predicted or described function in (TIF)Click here for additional data file.Figure S4S. suis apuR gene (black arrows). The annotations of the genes downstream of apuR are also indicated and colored to show functional relatedness. Gene names are indicated above the arrows.Gene homologues in Gram-positive bacteria that share ≥45% of protein identity with the (TIF)Click here for additional data file.Figure S5A. Coomassie stained SDS-PAGE gel (12%), showing purified fraction His-ApuR at the expected size of 38 kDa and B. Western Blot of the same gel with a monoclonal His-tag antibody protein. C. Coomassie stained SDS-PAGE gel (12%) of purified His-CcpA at expected size of 40 kDa.SDS polyacrylamide electrophoresis of purified transcriptional regulators. (TIF)Click here for additional data file.Figure S6A. Schematic representation of the apuA promoter (PapuA) and fluorescently labeled PCR amplified DNA fragments  used for EMSAs. B. to F. DNA amplicons and DNA/rApuR protein complexes visualized in native 5% acrylamide gels using the Odyssey Imager. In these EMSA assays the concentration of each DNA amplicon was around 6 ng (∼50 nM) B. EMSA of 100 nM rApuR binding to Pr1, Pr2 and Pr3. C. Increasing DNA/rApuR complex formation in presence of 4 ng Pr2 DNA amplicon and an increasing amount of rApuR  D. Competitive EMSA using increasing concentrations of non-fluorescent non-specific competitor DNA (lacking OM1 binding motifs). E. Competitive EMSA using increasing concentrations of non-fluorescent Pr2 as a specific competitor. The amounts of competitor DNA added are indicated (25–150 nM) F. Identification of specific ApuR binding sites in PapuA. PR2: native promoter region fragment 2. ΔOM1 and ΔOM2/cre are synthetic DNA fragments of PapuA that lack the predicted binding sites. + rApuR recombinant present - rApuR recombinant absent. G. to J. DNA amplicons and DNA/rCcpA protein complexes visualized in native 5% acrylamide gels using the Odyssey Imager. G. DNA/rCcpA complexes in the presence of increasing amounts of rCcpA as indicated. H. Competitive EMSA using increasing concentrations of non-fluorescent specific competitor I. Competitive EMSA using increasing concentrations of non-fluorescent non-specific competitor DNA (lacking OM2/cre binding motifs) J. DNA/rCcpA complex formation with fluorescent Pr2 and two synthetic promoter Pr2 fragments lacking either OM2/cre or OM1.EMSA with purified ApuA and CcpA. (TIF)Click here for additional data file.Table S1Oligonucleotide primers used in this study.(DOCX)Click here for additional data file.Table S2S. suis genes differentially regulated during growth in pullulan (Pul) vs glucose (Glc) in the exponential (e) or stationary (s) phase.Microarray data (xl file): (XLSX)Click here for additional data file.Table S3cre-site prediction in the genome of S. suis P1/7.(DOCX)Click here for additional data file.Table S4cre like motifs identified in S. suis 2 P1/7 and other Gram positive bacteria.Motif OM1-OM2/(DOCX)Click here for additional data file.Text S1Material and methods.(DOCX)Click here for additional data file.Text S2Results .(DOCX)Click here for additional data file.


Plasmodium falciparum genome is equipped with several subtelomeric gene families that are implicated in parasite virulence and immune evasion. Members of these families are uniformly positioned within heterochromatic domains and are thus subject to variegated expression. The best-studied example is that of the var family encoding the major parasite virulence factor P. falciparum erythrocyte membrane protein 1 (PfEMP1). PfEMP1 undergoes antigenic variation through switches in mutually exclusive var gene transcription. var promoters function as crucial regulatory elements in the underlying epigenetic control strategy. Here, we analysed promoters of upsA, upsB and upsC var, rifA1-type rif, stevor, phist and pfmc-2tm genes and investigated their role in endogenous gene transcription by comparative genome-wide expression profiling of transgenic parasite lines. We find that the three major var promoter types are functionally equal and play an essential role in singular gene choice. Unlike var promoters, promoters of non-var families are not silenced by default, and transcription of non-var families is not subject to the same mode of mutually exclusive transcription as has been observed for var genes. Our findings identified a differential logic in the regulation of var and other subtelomeric virulence gene families, which will have important implications for our understanding and future analyses of phenotypic variation in malaria parasites.The  Plasmodium falciparum evade antibody-mediated host immunity by altering parasite-encoded antigens exposed on the surface of infected red blood cells (iRBCs) through antigenic variation  (stevor (subtelomeric variable open reading frame)    , stevor rif gene family is the largest in P. falciparum , upsB-type var (PFL0005w), upsC-type var (PFL1960w), rifA1 (PF13_0004), stevor (PFL2610w), phistb (PFL2540w), pfmc-2tm  and cam (PF14_0323) (control). Note that the rifA1 and upsA promoters are present on the sense and antisense strand, respectively, of the 2.8 kb intergenic sequence separating these head-to-head oriented genes. All sequences were cloned into the context of the parental reporter vector pBcam by replacement of the cam promoter (bsd) resistance cassette was used to obtain transfectants carrying stable episomes. Importantly, this initial selection is independent of the activity of the test promoters driving transcription of the downstream drug-selectable reporter hdhfr-gfp (human dihydrofolate reductase fused to green fluorescent protein). All constructs carry a var gene intron element downstream of the hdhfr-gfp cassette for consistency. We chose this approach, rather than cloning family-specific introns, since the role of the var intron in silencing and mutual exclusion is exerted by the intron's own promoter activity  and is thus sequence-independent  carried an activated upsA promoter and was used in all subsequent experiments. Noteworthy, populations transfected with pBupsA were also successfully selected on WR, and reciprocal drug swaps had no effect on parasite growth suggesting that both activities of this bi-directional promoter were active simultaneously (data not shown). Southern blot analysis revealed that plasmids were maintained episomally in all lines except 3D7/stevor where integration into the endogenous stevor locus occurred .The BSD-selected reporter lines 3D7/upsA†, 3D7/upsB, 3D7/upsC, 3D7/rifA1, 3D7/stevor, 3D7/phist, 3D7/pfmc-2tm and the control line 3D7/cam exhibited similar plasmid copy numbers ranging from two to eight copies per parasite . After We pBupsA . In thisper se cause alterations in mRNA profiles. Indeed, we found 127 genes either up- or downregulated by more than twofold in at least one time point in all transfected lines compared with the untransfected control . Interestingly, this set contained several genes coding for proteins involved in DNA/RNA metabolism, protein translation and export, and stress response. These changes are compatible with possible responses to plasmid maintenance and replication as well as to the mode of BSD action, which inhibits protein synthesis ; TP2: 14–22 hpi; TP3: 22–30 hpi; TP4: 30–38 hpi] during the intra-erythrocytic developmental cycle (IDC) from 3D7 wild-type parasites and each of the WR-selected transfected lines (except for 3D7/upsA where a BSD-selected population was used). This sampling strategy was designed to include the temporal peaks of transcription for each endogenous gene family . Within var, stevor, pfmc-2tm and cam promoters demonstrating that the episomal sequences contained sufficient information to recapitulate the temporal expression of endogenous gene families. Intriguingly, the rifA1 promoter correlated with endogenous var rather than rif transcription implying that this bi-directional rif promoter displays a var-specific activation pattern. This interesting observation may indicate differential regulation of this particular subset of rif genes compared with other members of the family. For the phistb promoter we observed divergence in early schizonts where episomal promoter activity increased and endogenous phist transcription decreased. The reason for this discrepancy is unclear but may be due to lack of the full complement of regulatory cis-acting sequences on the 2.5 kb upstream region cloned into pBphistb, or different mRNA stability properties of the reporter compared with endogenous phist transcripts.As an important consideration for comparative analyses, we tested whether the stage-specific activities of the episomal promoters matched the temporal transcription profile of the cognate endogenous gene families. To do this, we compared reporter gene mRNA abundance profiles in selected lines to the average relative expression of the corresponding gene family members . This analysis revealed high correlations for the var and subtelomeric non-var families show the same behaviour. The cam promoter that is not epigenetically regulated and the upsB and upsC var promoters served as important negative and positive controls for silencing respectively. As expected, quantitative reverse transcription PCR (qRT-PCR) analysis revealed high levels of induction for the upsB promoter (18.2-fold ± 1.0 s.d.) and upsC promoter (9.4-fold ± 3.7 s.d.) relative to the negative control promoter cam (2.1-fold ± 0.4 s.d.)  displayed weaker but still elevated levels of induction compared with the control, which may be indicative for silencing of both activities of this bi-directional promoter in a considerable fraction of the unselected populations. In contrast, we found no evidence for alternative states of activity for the stevor, phistb and pfmc-2tm promoters. These results therefore suggest for the first time that promoters of subtelomeric non-var families may be unable to recruit transcriptional silencing autonomously. This is in distinction to var promoters that are characterized by alternative states of activity and reversible responsiveness to epigenetic control mechanisms  or an active (activated) state . By quan.4 s.d.)  and S4. var transcription  in all lines investigated, underscoring the specificity of the changes observed for TP1 and TP2.Activation of upsB and upsC promoters leads to downregulation of endogenous cription . Here, wvar expression levels at TP1. With the exception of 3D7/stevor, average var transcription was reduced in all transfected lines compared with 3D7. Importantly, however, var downregulation was much less pronounced in 3D7/phist, 3D7/pfmc-2tm and 3D7/cam compared with the lines carrying activated var promoters. Compared with 3D7/cam, significant downregulation occurred specifically only in 3D7/upsA, 3D7/upsB, 3D7/upsC and 3D7/rifA1 parasites. This is also illustrated by comparing the relative expression levels for each individual var gene . This is particularly interesting given that all hyp4 paralogues are located in head-to-tail orientation directly upstream of a pfmc-2tm locus. These observations suggest that expression of both families is co-regulated and that their exported gene products may cooperate in the same functional pathway in the iRBC. As a first step towards testing this hypothesis we generated a double transgenic cell line simultaneously expressing C-terminally tagged PFMC-2TM-GFP and HYP4-CherryFP and demonstrate that indeed HYP4 colocalizes with PFMC-2TM to MCs .Next, we were interested in asking if any of the recently described and uncharacterized var gene activation involves a single regulatory pathway, or if distinct transcriptional regulators mediate subtype-specific activation of var genes. To do this, we interrogated our datasets to identify genes with differential expression in the 3D7/upsA, 3D7/upsB and 3D7/upsC transfectants. This comparative approach identified only minor transcriptional differences between these cell lines. We pinpointed a total of only 20 transcripts with greater than threefold changes in relative expression in TP1 and TP2 in any one compared with both other cell lines. Eight, three and nine genes were differentially expressed in 3D7/upsA, 3D7/upsB and 3D7/upsC respectively . Nearly all of these changes affected heterochromatic genes that are expected to undergo variegated expression. Interestingly, one of the genes upregulated in 3D7/upsA encodes the ApiAP2 factor PfSIP2 that has been implicated in silencing of subtelomeric upsB-type var genes  and 3′ UTR (hrp2 versus PbDT terminator) sequences. These differences may cause differential regulation at the level of mRNA stability, which in turn would affect the abundance of steady-state transcripts. Second, it is impossible to control or assess the proportion of parasites carrying active promoters prior to selection; differences in these proportions are probably reflected in the different default activity levels in 3D7/upsA†, 3D7/upsB and 3D7/upsC, which were lowest for upsB and highest for upsC. These uncertainties notwithstanding, activation of episomal upsA promoters led to a specific downregulation of endogenous var transcription as previously reported for upsB and upsC. We therefore believe that although the active upsA promoter was placed in a different plasmid context it still functioned as a regulatory entity equivalent to upsB and upsC promoters. Hence, despite considerable sequence variation between different var promoter types mutually exclusive activation of all var genes appears to be governed by a common regulatory mechanism. This assumption is also supported by our failure to identify distinct signatures of transcripts coding for potential transcriptional regulators in 3D7/upsA, 3D7/upsB and 3D7/upsC parasites.The three main dhfr-gfp mRNA levels in unselected 3D7/upsA† parasites were higher than those in most other WR-selected lines  demonstrates that the refractoriness of 3D7/upsA† to WR challenge was not due to irreversible silencing. One possibility is that the upsA 5′ untranslated region (5′ UTR) mediates inefficient translation, which in turn may result in insufficient hDHFR concentrations to support WR resistance. Interestingly, the 5′ UTR of var2csa has been implicated in translational repression  and PfSIR2B in silencing of var gene subgroups and neighbouring upsA var and rifA1 genes  of neighbouring upsA var and rifA1 genes .Analysis of the head-to-head stevor, phistb and pfmc-2tm promoters showed no evidence for reversible states of activity indicating that these promoters are not directly targeted by the silencing machinery. Both the stevor and pfmc-2tm promoters were properly controlled in terms of temporal activity during the IDC. The phistb promoter was active coincident with endogenous phist transcription in ring stages and trophozoites, but remained active in early schizonts when endogenous phist transcription was reduced. If this discrepancy is due to the absence of critical cis-acting elements on the 2.5 kb episomal promoter, or to differences in mRNA stability between the episomal and endogenous transcripts is unclear. However, in light of the substantial activity displayed by the phistb promoter we envisage it was still controlled by the same transcription factors that also mediate endogenous phist transcription in ring stages and trophozoites. Importantly, for all three promoters we observed no specific downregulation in transcription of the endogenous rif, stevor, phistb and pfmc-2tm families. In these parasites the corresponding plasmid-encoded and endogenous promoters were active simultaneously, rather than in a mutually exclusive manner. In summary, these results demonstrate that transcriptional regulation of non-var families is controlled by mechanisms different from those governing mutually exclusive var transcription. They furthermore suggest that transcription of non-var families is not mutually exclusive, which is consistent with previous reports showing that more than one RIFIN or STEVOR variant can be expressed concurrently in a single parasite  covering the complete intra-erythrocytic developmental cycle was used as reference sample. Total RNA was prepared by disrupting 1.5 ml of RBC pellet (> 3% parasitaemia) with 10 ml of TriReagent (Sigma-Aldrich) following manufacturer's instructions. cDNA was generated from total RNA, and the test and reference samples were labelled as previously described . All samples were tested negative for contaminating gDNA by quantitative PCR (qPCR). RNA was reverse transcribed using the RETROscript Kit® (Ambion). gDNA was extracted from the same parasite samples by addition of 3% SDS/0.27 M Na-acetate to the parasite pellet followed by phenol/chloroform extraction and precipitation. gDNA was resuspended in ddH2O and diluted 1:100 for qPCR analysis.Parasites were released from iRBCs in 0.15% saponin. Total RNA was isolated using TriReagent (Sigma-Aldrich) and further purified using the RNeasy Plus Mini Kit (Qiagen) for removal of gDNA. Residual gDNA was digested with TURBO DNA-dhfr-gfp by the value obtained for the PFL0900c (arginyl-tRNA synthetase) locus  for 3D7/upsA, 3D7/upsB, 3D7/upsC, 3D7/rifA1, 3D7/phist and 3D7/cam, and at 22–30 hpi (TP3) for 3D7/stevor and 3D7/pfmc-2tm respectively. qRT-PCR experiments were performed on three independent biological samples. Plasmid copy numbers were determined by qPCR on gDNA and calculated by dividing the absolute copy numbers obtained for hg and washed twice in 1 ml of RPMI-HEPES. The pellet was resuspended in 400 µl of RPMI-HEPES containing 2.5 µg ml−1 DAPI and incubated for 10 min at 37°C. Three microlitres of packed RBCs were mixed with 2 µl of Vectashield (Vector Laboratories) and immediately analysed on a Leica DM 5000B microscope. Images were taken at 100× magnification with a Leica DFC 300 FX camera and acquired via the Leica IM 1000 software, and processed using Adobe Photoshop CS2.Five hundred microlitres of culture  was pelleted for 1 min at 200 32P-dATP-labelled hdhfr.gDNA was subjected to Southern analysis using standard procedures. gDNA was digested with EcoRI/PvuII and separated on 0.7% agarose gels. Southern blots were probed with random-primed


MCK), are differentially expressed in fast- and slow-twitch muscle fibers, but the fiber type-specific regulatory mechanisms are not well understood.Hundreds of genes, including muscle creatine kinase  containing a paired E-box/myocyte enhancer factor 2 (MEF2) regulatory motif resides within MR1. The SIE's transcriptional activity equals that of the extensively characterized 206-bp MCK 5'-enhancer, but the MCK-SIE is flanked by regions that can repress its activity via the individual and combined effects of about 15 different but highly conserved 9- to 24-bp sequences. ChIP and ChIP-Seq analyses indicate that the SIE and the MCK 5'-enhancer are occupied by MyoD, myogenin and MEF2. Many other E-boxes located within or immediately adjacent to intron 1 are not occupied by MyoD or myogenin. Transgenic analysis of a 6.5-kb MCK genomic fragment containing the 5'-enhancer and proximal promoter plus the 3.2-kb intron 1, with and without MR1, indicates that MR1 is critical for MCK expression in slow- and intermediate-twitch muscle fibers , but is not required for expression in fast-twitch muscle fibers (types IIb and IId).Modulatory region 1 (MR1) is a 1-kb regulatory region within MCK expression in slow- and intermediate-twitch muscle fibers and that MR1's positive transcriptional activity depends on a paired E-box MEF2 site motif within a SIE. This is the first study to delineate the DNA controls for MCK expression in different skeletal muscle fiber types.In this study, we discovered that MR1 is critical for  MCK) is among the most abundant transcripts in striated muscle 2 TG[C/T]2 N[C/G] [MCK-SIE control elements may not be functionally important. The MEF2 motif in all six species conforms fully to the MEF2 consensus sequence ([G/T][C/T]TA[A/T]3 ATA[A/G][A/C/T])   and 10 t[A/C/T]) . In addi[A/C/T]) . The cluenhancer ,46.MCK gene expression, the MR1 region was deleted from the entire 6.5-kb MCK sequence , and the effect of the deletion was examined in differentiated skeletal myocytes (MM14). To gauge the relative change in transcriptional activity caused by the loss of MR1, we compared 6.5MCKΔMR1-CAT to a construct that contains a deletion of the well-characterized MCK 5'-enhancer . Expression from each test plasmid was normalized to the activity of a muscle-specific MCK enhancer-driven alkaline phosphatase (AP) reference construct.To address the function of MR1 in MCK gene construct (P < 0.01) .Deletion of MR1 results in an approximately fivefold lower transcriptional activity in differentiated MM14 cultures than that produced by the entire 6.5-kb MCK-SIE is critical for MCK gene transcription, it was deleted from the 6.5MCK-CAT construct and the resulting 6.5MCKΔSIE-CAT was tested in differentiated skeletal muscle cultures , demonstrating that, in the context of the 6.5-kb MCK genomic sequence, the MCK-SIE is likely responsible for much of the positive transcriptional activity of MR1.To determine whether the MCK proximal promoter , a greater than 10-fold increase in transcription (P < 0.01) relative to the MR1-PP-CAT construct was observed. In fact, the MCK-SIE synergizes with the proximal promoter, as does the 5'-enhancer  gene. This sequence is roughly 40-kb 3' of the MCK-SIE on mouse chromosome 19 and is within a 1-kb region that entirely lacks the core E-box binding motif CAnnTG; thus it should serve as a truly negative control for MyoD and myogenin occupancy of the MCK-SIE. The second "negative" control primer pair spans the exon 1/intron 1 boundary and amplifies a 217-bp region located 690 bp upstream of the MCK-SIE, 242 bp downstream of the active promoter E-box and 1,149 bp downstream of the active MCK 5'-enhancer right E-box  should be significantly greater than those at any of the negative control regions.Three "negative" control primers were used to rule out the possibility of cross-enrichment from factors binding to non-s Figure . The firx Figure . The moux Figure . It contMCK-SIE region. In contrast, neither antibody enriched the exon 2 and Mark4 genomic regions significantly above nonspecific IgG. This demonstrates that MyoD and myogenin bind neither to nonconserved, and presumably nonfunctional, E-box motifs in the regions surrounding the MCK-SIE, nor to chromatin regions that lack E-boxes. There is a slight enrichment at the exon 1/intron 1 boundary. However, this could be caused by cross-enrichment due to MyoD and myogenin occupancy of the nearby and functional proximal promoter E-box . In. InMCK iMCK-SIE's high activity is largely due to the paired E-box and MEF2 motifs, since their mutation or deletion caused a significant decrease in transcription, while mutations affecting the AP-1/MAF half-site motifs did not . On the basis of our mutagenesis studies, the MCK-SIE 3'-E-box appears to be the more active site, since its deletion caused a greater reduction of transcriptional activity  are present in myocyte cultures , but MEFentially , and theand MHox ,55,68-72s Figure , whereasMCK-SIE. These MR1 regions can repress the MCK-SIE's activity via the combined or individual effects of at least 15 highly conserved 9- to 24-bp sequences , while expression levels in fast-twitch fibers (type IIb and type IId) remained high  transcription factor demonstrated high-level transgene expression in the soleus (slow- and intermediate-twitch muscle fibers) as well as in EDL (fast-twitch muscle fibers) ), Felis catus (cat [GenBank: AC135221.3AC135221.3]), Canis familiaris (dog [GenBank: AC137538.2]), Bos taurus (bovine [GenBank: AC137535.2]), Sus scrofa (pig [GenBank: AC139878.2]) and Mus musculus (mouse GenBank: [AC118017.15]) were obtained from compiled genomic sequences in the Entrez Genome Project database and subjected to sequence alignment using ClustalW [http://www.gene-regulation.com/cgi-bin/pub/programs/match/bin/match.cgi) , FelMCK gene  [MCK-CAT [MCKΔMR1-CAT construct was generated from the 6.5MCK-CAT construct by introducing ClaI restriction sites 5' and 3' of MR1  using the QuikChange Site-Directed Mutagenesis Kit , according to the manufacturer's directions. MR1 was then deleted by digestion of the plasmid with ClaI and religation of the remaining vector. The 6.5MCKΔEnh-CAT construct was generated by site-directed mutagenesis to delete the MCK 5'-enhancer .A 6.5-kb construct of the mouse  +3,230)  was clon[MCK-CAT . The 6.5SphI (5') and BstI (3'). The MR1 amplicon was cloned upstream of the proximal promoter by replacing the 5'-enhancer in the e-358-CAT reporter construct [SphI and BstI. The mouse MCK PP region used in these studies extends from -358 to +7. All other deletions and mutations described in this study were generated using the QuikChange Site-Directed Mutagenesis Kit.The MR1 region was polymerase chain reaction-amplified from the existing 6.5-kb construct with primers containing the restriction sites onstruct  using Sp5 log phase cells/dish and were allowed to proliferate under growth conditions  added at approximately 12-hour intervals) for about 24 hours. Myoblasts were cotransfected using a standard calcium phosphate method [5 cells with test constructs driving the expression of the CAT reporter gene and an AP reference plasmid, which contains the 5'-enhancer placed 5' of the basal promoter sequence (-80 to +7). Transfected MM14 cultures were induced to differentiate four hours after beginning the transfection by aspirating the growth medium, rinsing once with saline G, incubating for 2 minutes at room temperature in 15% glycerol 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-buffered saline, rinsing again with saline G and then adding 10 mL of differentiation medium  [MCK enhancer-AP reference plasmid is expressed only in differentiated muscle cells, it provides a control for plate-to-plate variability in transfection efficiency and extent of muscle differentiation in skeletal myocyte cultures.MM14 skeletal myoblasts were cultured as described previously . Collagee method  at aboutinsulin) . Relativinsulin) . Since t5 log phase MM14 cells/dish and grown to near confluence (about 4 × 106 cells/dish), then allowed to differentiate in proliferation medium without additional bFGF for four to six days prior to harvesting. All cultures contained more than 90% terminally differentiated myocytes as assessed by immunostaining a parallel culture with the myosin-specific antibody MF-20. This procedure produced more than 7 × 106 differentiated myonuclei per 100-mm dish. Cells were sonicated with 16 rounds of 15-second pulses with 45 seconds of rest between pulses  on a Model 100 Sonic Dismembrator  at the highest setting. Antibodies used for immunoprecipitation described in this study were as follows: anti-myogenin (M-225) sc-576 X, anti-MyoD (M-318) sc-760 X, anti-MEF2A (C-21) sc-313 and normal rabbit IgG sc-2027 . The primers used in ChIP analyses were MCK 5'-enhancer: 183 bp forward: 5'-GCCCATGTAAGGAGGCAAGGCC-3', reverse: 5'-CACCAGGGACAGGGTTATTTTTAGAGC-3', MCK exon 1/intron 1 boundary: 217 bp forward: 5'-GGGTCACCACCACCTCCACAG-3', reverse: 5'-GCCTTGCAAGGAGGGGACACTTG-3', MCK-SIE: 168 bp, forward: 5'-CTTGAGGCCCAGAGCCTGGCTG-3', reverse: 5'-GAGACCCAAAGCCCTTGAAGCTGCTAC-3', MCK exon 2: 207 bp, forward: 5'-GTCCCAAAGGCCGCCACCATG-3', reverse: 5'-GGGTTGTCCACCCCAGTCTGG-3' Mark4 gene region: 205 bp, forward: 5'-GGATGCCATGCCTGGTGGCCAT-3', reverse: 5'-GCCATGCAGCTTTCACGCAGAGG-3'.ChIP assays were performed using a modification of the Fast-ChIP method as described previously ,80 with MCK-SIE): AGGAGCATCTAAAAATAGCCACAAAG, MEF2 (MCK-SIE)-M1: AGGAGCATCCGAAAACGGCCACAAAG and MEF2 (MCK-SIE)-M2: AGGAGCATCATAAAAATGCCACAAAG.EMSA was carried out as previously described . Nuclearhttp://www.bdbiosciences.com/home.jsp) and IgG normal rabbit sc-2027 .Antibodies used for EMSA were anti-MEF2A  (Santa Cruz Biotechnology), anti-TEF-1  in 20% fetal bovine serum (FBS)/Dulbecco's modified Eagle's medium (DMEM)  and 0.025% glutaraldehyde  for 10 minutes. A total of 2 × 107 cells were fragmented to about 100 to 300 bp using 30-second, 12-W cycles on a Misonix 3000 sonicator http://www.fishersci.com/ecomm/servlet/fsproductdetail?aid = 2819374&storeId = 10652 for a total sonication time of 15 minutes. The sheared chromatin was immunoprecipitated using 100 μL of sheep anti-mouse IgG M280 beads (Invitrogen) and 5 μg of MEF2 monoclonal antibody (clone B4) from Santa Cruz Biotechnology or 200 μL of sheep anti-rabbit IgG M280 beads and 10 μg of MEF2 polyclonal antibody (clone H300) from Santa Cruz Biotechnology. Illumina libraries for sequencing were made using their ChIP-Seq library kit  as described by the manufacturer, except that a 10-cycle amplification was performed before gel selection according to the method of Johnson et al. (library 2) [ChIP assays for MEF2 ChIP-Seq were performed according to the protocol described by Johnson et al.  with thebrary 2) . Librarybrary 2) . Mapped brary 2)  and werebrary 2) .MCK gene sequence and the sequence with MR1 deleted were cloned upstream of the β-gal reporter gene to generate the 6.5MCK-β-gal and 6.5MCKΔMR1-β-gal constructs, respectively. DNA for microinjection was prepared by enzymatic digestion to linearize the plasmids and gel-purified by freeze-and-squeeze columns . Transgenic mice were produced using eggs from C57BL/6J × C3H crosses through the University of Washington Transgenic Resource Program. Founders were crossed to C57BL/6J to generate F1s. Lines of mice analyzed were either F1s or the progeny of F1s (N2 or N3) that were back-crossed with C57B/6J.The 6.5-kb http://www.leica-microsystems.com/, mounted onto glass slides at room temperature and then stored at -80°C.Adult mice (1+ months) were killed according to methods approved by the University of Washington Institutional Animal Care and Use Committee. TA and soleus muscles were dissected and mounted in a 2:1 mixture of optimal cutting temperature compound and 10% gum tragacanth in cryomold cassettes. Cassettes were then frozen in liquid nitrogen-cooled isopentane. Tissues contained in blocks were cryosectioned at a thickness of 6 μM at -25°C using a Leica cryostat 2 , 0.01% sodium oxycholate and 0.02% Nonidet P-40 and stained in a standard X-gal reagent solution [http://www.sigmaaldrich.com). Images were obtained using a Zeiss Axiovert 200 microscope http://www.zeiss.com/micro with a Zeiss AxioCam MRm camera (Zeiss), and acquired using AxioVision software (Zeiss). Images were then uniformly false-colored using Adobe PhotoShop version 7 software .Slides were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 15 minutes at 4°C and washed in 100 mM sodium phosphate (pH 7.3), 2 mM MgClsolution  for 4 hohttp://www.hyclone.com/) supplemented with 10% FBS ) and penicillin-streptomycin (Sigma). Cultures were then switched to serum-free medium and incubated for two or three days. The medium was collected, centrifuged and filter-sterilized  and monoclonal antibodies were concentrated by HiTrap column chromatography . High-protein concentration fractions as determined by the Bradford method [http://www.piercenet.com/), and then stored at -20°C. Slides were treated with blocking buffer  and incubated with about 10 μg/mL BA-D5, SC-71 and BF-F3 for 1 hour, washed three times for five minutes in blocking buffer and incubated with goat anti-mouse secondary antibodies IgG2b Alexa Fluor 350, IgG1 Alexa Fluor 594 and IgM Alexa Fluor 488 (Invitrogen) for 30 minutes. Slides were washed as before, rinsed in PBS and mounted in gelvatol. Images were acquired as described above.Monoclonal antibodies specific for myosin isoforms MYHC1, MYHC2A and MYHC2B were produced from cultures of hybridoma lines BA-D5, SC-71 and BF-F3, respectively . These ad method  were pooBRG1: Brahma-related gene 1; CAT: chloramphenicol acetyl transferase; ChIP: chromatin immunoprecipitation; ERANGE: Enhanced Read Analysis of Gene Expression; KLF3: Krupple-like factor 3; Mark4: MAP/microtubule affinity-regulating kinase 4 gene; MAZ: Myc-associated zinc finger protein; MEF2: myocyte enhancer factor 2; MCK and MCK: muscle creatine kinase gene and protein; MCK, MCK-SIE: MCK small intronic enhancer; MR1: modulatory region 1; MYHC: myosin heavy chain; Oct-1: octamer-binding protein; TA: tibialis anterior muscle.AP: alkaline phosphatase; bFGF: basic fibroblast growth factor; β-gal: β-galactosidase; The authors declare that they have no competing interests.PWLT carried out the sequence alignments; made the test gene constructs; carried out the transfection assays, the ChIP analyses, the immunohistochemistry and immunofluorescence assays; and drafted parts of the manuscript describing Hauschka Lab data. KIFA conceived of the redesign of the ChIP-Seq fixation, performed and analyzed MEF2 ChIP-Seq and drafted portions of the manuscript describing Wold Lab data. CLH carried out the EMSA study and helped to draft the manuscript. CLS participated in the immunohistochemistry and immunofluorescence assays. APM participated in the transfection analyses. DLH carried out the whole muscle extract transgene expression assays. JCA and REW prepared and labeled the MYHC monoclonal antibodies and participated in the immunohistochemistry assays. BJW conceived of the global ChIP-Seq analysis of multiple myogenic transcription factors, participated in the design and coordination of the MEF2 ChIP-Seq studies and drafted parts of the manuscript describing the Wold Lab data. Together with PWLT, SDH conceived of the overall study, participated in its design and coordination between the two laboratories and played a major role in writing the manuscript. All authors read and approved the final manuscript.Figure S1. A six-species sequence alignment of modulatory region 1 (MR1), which demonstrates the conserved nineteen subregions throughout the region. The MR1 sequences of six mammalian species  were aligned to reveal sequence conservation. Bases that are fully conserved between the six species are highlighted in black, while those conserved in three to five species are highlighted in gray. Gaps in the sequence alignment are represented as hyphens. The 5' and 3' flanks of MR1, as defined in this study, are marked with red right-angled arrows. Nineteen conserved subregions  were tested for transcriptional activity  Conserved regions within MR1  were deleted from MR1-proximal promoter-chloramphenicol acetyl transferase (MR1-PP-CAT) and tested for transcriptional activity in skeletal myocytes (gray bars). (B) MM14 cells were transiently transfected with constructs containing each of the 19 different conserved motif deletions, and cells were harvested as described in the Figure MCK 5'-enhancer alkaline phosphatase (AP) reference plasmid and compared to the intact MR1-PP-CAT (black bar) and to the PP-CAT (white bar). Expression levels of MR1-PP-CAT were scaled to equal 1.0. Asterisks indicate constructs that did not result in a statistically significant change in transcriptional activity.Click here for file