Escherichia coli curved DNA binding protein A (CbpA) is a poorly characterised nucleoid associated factor and co-chaperone. It is expressed at high levels as cells enter stationary phase. Using genetics, biochemistry, and genomics, we have examined regulation of, and DNA binding by, CbpA. We show that Fis, the dominant growth-phase nucleoid protein, prevents CbpA expression in growing cells. Regulation by Fis involves an unusual “insulation” mechanism. Thus, Fis protects cbpA from the effects of a distal promoter, located in an adjacent gene. In stationary phase, when Fis levels are low, CbpA binds the E. coli chromosome with a preference for the intrinsically curved Ter macrodomain. Disruption of the cbpA gene prompts dramatic changes in DNA topology. Thus, our work identifies a novel role for Fis and incorporates CbpA into the growing network of factors that mediate bacterial chromosome structure.The  Escherichia coli, and other bacteria, radically re-model their chromosomes in response to environmental stress. This is most notable during periods of starvation, when the E. coli chromosome is super compacted. In dissecting the molecular mechanisms that control this phenomenon, we have found that regulatory cross-talk between DNA–organising proteins plays an essential role. Thus, the major DNA folding protein from growing E. coli inhibits production of the major chromosome organisers in starved cells. Our findings illustrate the highly dynamic nature of bacterial chromosomes. Thus, DNA topology, gene transcription, and chromosome folding proteins entwine to create a web of interactions that define the properties of the chromosome.Compaction of chromosomal DNA is a fundamental process that impacts on all aspects of cellular biology. However, our understanding of chromosome organisation in bacteria is poorly developed. Since bacteria are amongst the most abundant living organisms on the planet, this represents a startling gap in our knowledge. Despite our lack of understanding, it has long been known that  Escherichia coli, a particularly notable change in nucleoid structure occurs when cells are starved. Namely, the nucleoid adopts a super compact conformation that is believed to protect the genome. These changes in nucleoid structure coincide with changes in supercoiling Bacterial chromosomes are organised into a nucleoid by an integrated network of supercoiling, transcription and nucleoid associated DNA binding proteins E. coli cell extracts that preferentially bound curved DNA in vitrocbpA initiates from overlapping promoters referred to as P1 and P2 cbpA transcription is driven by the σ38 dependent P2 promoter with σ70 dependent P1 making only a small contribution E. coli, reaching 15,000 copies per cell after two days E. coli cells.The CbpA protein was first isolated as a factor present in crude cbpA regulatory region. When bound to these sites Fis prevents cbpA being transcribed from an aberrant promoter, located within the coding sequence of an adjacent gene. In starved cells, when cbpA transcription is induced by σ38, CbpA binds pervasively across the E. coli genome with a bias to the intrinsically curved Ter macrodomain. Disruption of the cbpA gene prompts dramatic changes in DNA supercoiling.In this work we sought to better understand the regulation and function of CbpA. We show that Fis plays a crucial role in preventing CbpA expression in growing cells. Hence, Fis binds to DNA target sites in the E. coli cells approach stationary phase they induce expression of two nucleoid proteins, Dps and CbpA. Previously, we found that Dps expression in growing E. coli cells is repressed by Fis cbpA regulatory region and part of the adjacent yccE gene, upstream of lacZ . For comparison, we also tested DNA fragments that drive expression of other nucleoid proteins and the nirB promoter, which has a high affinity for Fis cbpA regulatory region. Note that Fis forms three distinct complexes with the cbpA regulatory DNA, suggesting three separate Fis binding sites  and σ70 (lane 5) dependent DNA opening. As expected, our analysis identified DNA melting at the known P1 and P2 promoters . Thus, Fis only inhibited DNA untwisting at position −100 (open complex 4) which overlaps the Fis binding element.As a first step to understanding control of  in vivo , we werein vitro KMnO4 footprinting analysis we conducted in vivo mRNA primer extension experiments. This enabled us to identify cbpA transcript start sites, used in the presence and absence of Fis, in growing and stationary phase cells. The results of this analysis are shown in cbpA mRNA primer extension products. As expected these correspond to the P1 and P2 promoters  studies of Fis binding across the analysis . Examinaare high . Convers evident .cbpA Fis binding region to unrelated promoters. Furthermore, we expected that the Fis binding region would only inhibit promoters active during periods of growth, when Fis levels are high. Thus, we selected four promoter regions with different regulatory properties. Three of the promoters that we selected  are σ70 dependent promoters active in growing cells. Transcription from galP1 is stimulated by CRP, ynfE is FNR dependent and yeaR transcription requires NarL aer) is σ28 dependent and active during periods of starvation cbpA Fis binding element. When present the Fis binding region was placed between the promoter transcription start site and the lacZ start codon. The data show that the Fis binding element from the cbpA regulatory region repressed LacZ expression from all three “growth phase” promoters. Conversely, the aer promoter, which is active during periods of starvation, was not repressed (We reasoned that we should be able to “transplant” the repressive effect of the epressed .E. coli. To investigate DNA binding in vivo we utilised chromatin immunoprecipitation and DNA microarrays (ChIP-chip). In a preliminary analysis we compared CbpA binding in wild type E. coli BW27784 and the cbpM derivative MC108. The data show similar patterns of CbpA binding across the E. coli chromosome in both cases . Other plasmids in the sample were relaxed, as seen for starved cells (compare lanes 1–3). Plasmids isolated from the ΔdpsΔcbpA strain had similar topology to the plasmids isolated from the individual gene knockout strains (compare lanes 2–4). These effects were specific to plasmids isolated from starved cells; plasmid topoisomers obtained from growing cultures of each strain did not significantly differ  and by binding high affinity sites in the cbpA regulatory region (cbpA from σ70 dependent transcription). As cells divide, and Fis levels decrease, the different affinity of Fis for the dps and cbpA regulatory regions contributes to staged induction of dps and cbpA (2+ ions Taken together with previous work our results provide a detailed molecular explanation for the phenomenon of nucleoid reorganisation that occurs in starved y region  (to insuand cbpA . InterescbpA regulatory region sequences are numbered with respect to the P1 transcription start point (+1) and with upstream and downstream locations denoted by ‘−’ and ‘+’ prefixes respectively.Bacterial strains, plasmids and oligonucleotide sequences are listed in fis.Activities are shown in Miller units et al. cbpA regulatory DNA fragment cloned in pRW50. The 5′ end-labelled primer D49724, which anneals downstream of the HindIII site in pRW50 was used in all experiments. Primer extension products were analysed on denaturing 6% polyacrylamide gels, calibrated with arbitrary sequencing reactions, and visualized using a Fuji phosphor screen and Bio-Rad Molecular Imager FX.Transcript start sites were mapped by primer extension, as described in Lloyd et al. in vitro transcription experiments were performed as described et al.in vitro experiments are provided in the figure legends.CbpA and derivatives were all purified as described E. coli K-12 genome sequence . Topoisomers were separated on a 1% agarose gel containing 2.5 µg/ml chloroquine. Gels were run for 60 hours at 40 V in the dark. After washing with water for at least 2 hours the gel was stained with ethidium bromide for 2 hours and photographed under UV illumination.We monitored superhelicity of plasmid pJ204 in strain BW27784 and the ΔE. coli BW25113 were diluted 1∶100 into fresh LB media and grown at 37°C with aeration. Samples (1 ml) were taken at the indicated OD650 values and the cells harvested by centrifugation. Cells were re-suspended in Laemmli buffer so that the number of cells in each sample was equivalent. After boiling for 10 min cytoplasmic proteins were separated by SDS-PAGE and Fis was detected using Western immunoblotting as previously described Overnight cultures of Figure S1nirB DNA fragment was included as a positive control.Binding of Fis to DNA fragments carrying regulatory DNA sequences for genes encoding different nucleoid proteins. 175 nM, 350 nM or 700 nM Fis was incubated with different radiolabelled DNA fragments (∼20 nM) and the resulting protein-DNA complexes were separated by PAGE. Free DNA (F) and Fis-DNA complexes (C) are indicated. Promoters not known to contain Fis sites are shown in part A and panel B shows data for promoters with previously identified Fis binding sites. The (PDF)Click here for additional data file.Figure S2cbpA regulatory region DNA fragments. The figure shows DNA sequences for cbpA regulatory region DNA fragments. The P6, P4, P1 and P2 promoters and transcription start sites are highlighted in purple, orange, red and blue respectively. Coding DNA sequences for the yccE and cbpA genes are shown in upper case. The Fis binding site between position −108 and −94 with respect to the P1 promoter is underlined. Note that the −10 hexamers for the P1 and P2 promoters overlap by 1 base pair. Mutations in different promoter elements are highlighted by a star.(PDF)Click here for additional data file.Figure S3cbpA regulatory DNA (A) and a fragment carrying mutations in the putative Fis binding site (B). The DNA fragment was used present at a concentration of ∼20 nM.Effects of mutations in primary Fis binding sequence. (PDF)Click here for additional data file.Figure S4cbpA P4 promoter. Panel A shows different P4::lacZ fusions. The P4 promoter is illustrated by an orange arrow. The Fis binding element is shown as a yellow bar. In the P4 −10con construct the P4 promoter −10 hexamer has been improved from the wild type 5′-TAAAAT-3′ sequence to 5′-TATAAT-3′. Panel B shows LacZ activity values for each promoter::lacZ fusion in growing JCB387 cells. We confirmed that the P4 and P4 −10con promoters produced transcripts of the same length (i.e. that we had not inadvertently created a new promoter in P4 −10con) using in vitro transcription assays. Thus, DNA fragments carrying P4 or P4 −10con were cloned in plasmid pSR and used as templates for in vitro transcription. The repressive effect of Fis on P4 was also confirmed in these experiments (Panel C).Further characterisation of the (PDF)Click here for additional data file.Figure S5cbpA locus in vivo. The figure shows data from a ChIP-seq experiment to measure Fis binding across the E. coli chromosome (24) and an RNA-seq experiment using mRNA extracted from growing and stationary phase E. coli cells. The Fis binding profile is illustrated as a yellow line and reads mapping to different locations in the RNA-seq experiment are shown by arrows.Binding of Fis and transcription at the (PDF)Click here for additional data file.Figure S6E. coli+/−CbpM. Genome-wide view of CbpA binding in starved BW27784 (WT) and MC108 (ΔcbpM) cells. The figure shows ChIP-chip data for CbpA binding plotted against features of the E. coli genome in the form of a genome atlas. The data have been averaged across a 100,000 base pair window. The four chromosomal macrodomains (MD) are labelled.Chromosome-wide distribution of CbpA in starved (PDF)Click here for additional data file.Figure S7in vitro. A) Distribution of CbpA across the E. coli chromosome. Genome-wide view of CbpA binding in starved MC108 cells. The figure shows ChIP-chip data for CbpA binding plotted against features of the E. coli genome in the form of a genome atlas. The data have been averaged across a 100,000 base pair window. The four chromosomal macrodomains (MD) are labelled. Regions selected for in vitro binding assays are highlighted. B) CbpA binding to the glpX, yabN and paaA loci in vitro. The figure shows ethidium bromide gels on which DNA fragments corresponding to the different genomic loci have been run in the presence and absence of CbpA . Reactions contained 0.1 mM DNA.Binding of CbpA to different targets (PDF)Click here for additional data file.Table S1Strains, plasmids, and oligonucleotide sequences.(DOCX)Click here for additional data file.Table S2Raw array data.(ZIP)Click here for additional data file.Table S3Genome browser file.(ZIP)Click here for additional data file.Table S4E. coli genome file.(ZIP)Click here for additional data file.


The Paf complex of Opisthokonts and plants contains at least five subunits: Paf1, Cdc73, Rtf1, Ctr9, and Leo1. Mutations in, or loss of Paf complex subunits have been shown to cause defects in histone modification, mRNA polyadenylation, and transcription by RNA polymerase I and RNA polymerase II. We here investigated trypanosome CTR9, which is essential for trypanosome survival. The results of tandem affinity purification suggested that trypanosome CTR9 associates with homologues of Leo1 and Cdc73; genes encoding homologues of Rtf1 and Paf1 were not found. RNAi targeting CTR9 resulted in at least ten-fold decreases in 131 essential mRNAs: they included several that are required for gene expression and its control, such as those encoding subunits of RNA polymerases, exoribonucleases that target mRNA, RNA helicases and RNA-binding proteins. Simultaneously, some genes from regions subject to chromatin silencing were derepressed, possibly as a secondary effect of the loss of two proteins that are required for silencing, ISWI and NLP1. Saccharomyces cerevisiae, the Paf complex consists of five subunits: Paf1, Cdc73, Rtf1, Ctr9, and Leo1; the human complex also contains Ski8 PAF1 or CTR9 caused decreases in at least 300, and increases in over 900 mRNAs; over half of these were affected in similar ways by both deletions Arabidopisis, the complex is involved in transcriptional control of flowering The Paf complex has been implicated in several different aspects of gene expression within the nuclei of yeast and mammalian cells (Opisthokonts) and plants: defects have been shown to influence transcriptional elongation and termination, mRNA processing and export, and to result in epigenetic changes The complex has been shown to bind to RNA polymerase II; mutations in either Cdc73 or Rtf1 abolished the binding and resulted in reduced phosphorylation of the C-terminal domain or the largest polymerase II subunit in vitroEpigenetic changes associated with the Paf complex include histone methylation, ubiquitylation and acetylation. The Rtf1 component is implicated in histone ubiquitylation in vitroDefects in transcription elongation can affect choices between alternative polyadenylation and splice sites Very few studies have examined possible roles of the Paf1 complex in RNA polymerase I transcription. One found that Paf1 was required for histone K4 methylation on the rDNA trans splicing, and 3′ polyadenylation African trypanosomes multiply in the blood and tissue fluids of mammals (as the “bloodstream form”) and in Tsetse flies, where the form found in the midgut is called the “procyclic form”. Trypanosomes offer an interesting model for the study of RNA polymerase II transcription. Genes are arranged in polycistronic transcription units, which are transcribed from several start points Proteins involved in trypanosome polymerase II transcription apparatus differ in many ways from those of Opisthokonts. The largest subunit of RNA polymerase II lacks the characteristic heptapeptide repeats at the C-terminus, but that region is nevertheless serine-rich, phosphorylated, and required for transcription Trans splicing enables trypanosomes to produce protein-coding mRNAs using RNA polymerase I. The genes encoding the Variant Surface Glycoprotein (VSG) of the bloodstream forms, together with several associated genes, are transcribed by polymerase I from telomeric “expression sites”, while the EP and GPEET procyclins of the procyclic form, with a few downstream “procyclin associated genes” (PAG) are transcribed from chromosome-internal locations EP and GPEET procyclin genes is about ten times more active in procyclic forms than in bloodstream forms In this paper, we investigated the function of CTR9 in trypanosomes. We found evidence for interaction between trypanosome CTR9, CDC73, and a putative homologue of LEO1, and observed multiple effects on the transcriptome after CTR9 depletion.5′-GGCACCCTGCAGAATATTCT-3′ and 5′-CGGAGGTAAATTTGGCCGAG-3′. The resulting plasmid was linearized with Not I and transfected into bloodstream-form trypanosomes expressing the tet repressor (pHD 1313) and T7 RNA polymerase (pHD514). Transfectants were selected in 10 mg/ml hygromycin and cloned by limiting dilution. For the growth curve shown, RNAi was induced by adding tetracycline to the medium, at a concentration of 1 mg/ml. Samples were taken after every 24 hours of RNAi induction for three days. In later experiments, 100 ng/ml tetracycline was used, with similar effects on cell growth.For RNAi, a TbCTR9-specific DNA fragment was cloned into the vector p2T7TA blue Apa I and Hpa I sites (pHD2244), and expression induced using 100 ng/ml tetracycline. Expression of the C-terminally TAP-tagged version  was achieved similarly by cloning into pHD 918 to give pHD2245 For expression of C-terminally myc-tagged CTR9, the gene was amplified by standard PCR using the following oligonucleotides: 5′- GGGCCCATGCAATATATATCGGAACCC-3′ and 5′- GTTAACTATATTCCCATCATTAGCGGTGT-3′. The fragment was cloned into pHD1700 8 bloodstream cells expressing TAP or myc-tagged CTR9 were collected by centrifugation and analysed using NP-40 lysis and centrifugation To determine the subcellular location of CTR9, approximately 5×1010 cells collected. TAP purification was done as described by For tandem affinity purification, expression of TAP-tagged protein was induced for 24 h and about 1×10RNASeq analysis was done using poly(A)+ mRNA extracted 24 h after tetracycline addition; in the culture used, growth inhibition had not yet become apparent. RNASeq data for the CTR9 RNAi mutant were compared to those of 2 replicates of poly(A)+ mRNA from wild-type cells T. brucei. The tagged CTR9 was subjected to two steps of affinity purification and the proteins present in the entire preparation were identified by mass spectrometry. Four potential interaction partners were revealed , using the yeast, human or trypanosome sequences as queries. We used standard NCBI BLASTp settings without filters, and matches with poor coverage were checked by reciprocal BLAST. Where results were negative, we increased the sensitivity by searching individual genomes for Ctr9, Cdc73 and Leo1, and for the Leo1 domain alone . ResultsCTR9, LEO1 and CDC73 were all found to be essential for normal growth of both bloodstream and procyclic trypanosomes CTR9 in bloodstream forms resulted in growth arrest after 1 day of tetracycline induction . If CTR9 were required for transcription processivity, we would expect that its depletion would result in preferential depletion of mRNAs that are towards the ends of transcription units. This was not seen - increases and decreases were scattered almost randomly through the transcription units . Table 3affected . Of the affected . This phCTR9 RNAi strain. About 37% of these sites were found in the database of predominant sites used that were determined previously on the basis of a collection of WT RNASeq data CTR9 knockdown does not significantly affect the use of splice acceptor sites.We also looked to see whether CTR9 depletion changed RNA processing patterns. Predominant splice acceptor sites were mapped as before EP .To verify the RNASeq results, we probed Northern blots. The increases in EP , GPEET aG2 mRNAs  were conG2 mRNAs . AttemptG2 mRNAs . Some CTEP and GPEET procyclin mRNAs seemed most likely to be a transcriptional effect, since according to the RNASeq results, the entire transcription units including the PAGs were affected: effects on RNA processing or degradation would be unlikely to affect each gene to such similar extents. To check this, however, we assessed the degradation rate of the EP procyclin mRNA. Results are shown in CTR9 RNAi, the onset of EP mRNA degradation was reproducibly delayed . Even a quick glance, however, was sufficient to explain why CTR9 RNAi was lethal: no fewer than 131 of the reduced mRNAs are essential for normal growth. Some examples of essential genes with at least 20-fold RNA decreases are shown in –10) of gene products in many categories related to nucleotide or nucleoside binding or hydrolysis, as well as motor activity; cytoskeletal categories were enriched with P values of less than 10–5 (not shown). Scrutiny of the list also suggested an explanation for deregulation of the procyclin loci: the mRNAs encoding ISWI (Tb927.2.1810) and NLP1 (Tb927.10.5450), were repressed 8-fold and 13-fold, respectively  were also at least 1.5-fold increased after RBP10 RNAi, and 8 after phloretin treatment. The CTR9 RNAi results also do not resemble those seen after a lethal knock-down of ZC3H11 . This suggests that the transcriptome changes after CTR9 RNAi were not simply caused by growth inhibition.Depletion of CTR9 caused many changes in the transcriptome. To find out whether these were simply a consequence of slowed growth, we compared the most up-and down-regulated genes with those previously detected, by microarray, after other growth-inhibitory treatments: inhibition of glucose transport using phloretin in vivo analysis of specific roles of CTR9 in polymerase II elongation. Next, the expression of NOT1 - a core component of the major deadenylation complex - and of XRNA, a 5′-3′ exoribonuclease that is important for degradation of relatively unstable mRNAs, was abolished. These two essential proteins are required for mRNA degradation. Transcripts encoding many RNA-binding proteins were also decreased; knock-on effects could have occurred if proteins that regulate mRNA degradation were lost while mRNA degradation pathways were still active. Finally, the increase that we observed in polymerase-I-transcribed mRNAs was most likely due to a reduction in chromatin silencing, consequent to the disappearance of ISWI and NLP1.By studying the time course of transcriptome changes after Paf1 depletion in yeast, Penheiter et al. C-terminally tagged CTR9 was found in both the cytoplasm and the nucleus. Although the tag might have influenced the result, the localisation is in accordance with the PSORTII prediction: the localization percentages of neighbours were 22% nuclear and 61% cytoplasmic for trypanosome CTR9. In contrast, yeast and mammalian proteins yielded 90%, and Drosophila 48%, nuclear scores. It is therefore possible that trypanosome CTR9 functions in both compartments, or is regulated by nucleo-cytoplasmic shuttling.Overall, our results suggest that trypanosome CTR9 may function as a complex with CDC73, LEO1, and perhaps other proteins. The complex components clearly have essential functions, most likely in gene expression in either the cytoplasm, the nucleus, or both, but the precise natures of these functions remain unclear.Figure S1Alignment of Ctr9 homologues, made using MegAlign (DNAStar). Residues that are identical to the majority consensus are highlighted in black, and residues that are within the consensus group (consensus #1) in grey. Groups are f-hydrophobic; p-polar, a-acidic, b-basic. Organisms are: Tb- T. brucei, At- Arabidopsis thaliana, NP_178674.6; Dd- Dictyostelium discoideum XP_642310; Eh- Entamoeba histolytica EAL45089; Hs- Homo sapiens NP_055448; Ng- Naegleria gruberi XP_002679971; Sc- Saccharomyces cerevisiae Ctr9; Sp- Schizosaccharomyces pombe CAA15833.(PDF)Click here for additional data file.Figure S2Alignment of Cdc73 homologues, made using MegAlign (DNAStar). Details as for T. brucei, Sc- Saccharomyces cerevisiae; Sp- Schizosaccharomyces pombe NP_595891.1; Hs- Homo sapiens NP_078805.3; Dd- Dictyostelium discoideum XP_644100.3; Ng- Naegleria gruberi XP_002683436.1; Pf- Plasmodium falciparum XP_001351765.1; Tt- Tetrahymena thermophila EAR84002.1; Pi- Phytophthora infestans XP_002903756.1; Eh- Entamoeba histolytica XP_648719.1; Tv- Trichomonas vaginalis XP_001310227.1; Tp- Thalassosira pseudonana XP_002287950.1.(PDF)Click here for additional data file.Figure S3Alignment of Leo1 homologues, made using MegAlign (DNAStar). Details as for T. brucei, At- Arabidopsis thaliana; Dd- Dictyostelium discoideum XP_637916.1; Tp- Thalassiosira pseudonana XP_002289477.1; Hs-Homo sapiens NP_620147.1; Sc- Saccharomyces cerevisiae Leo1; Ng- Naegleria gruberi XP_002677260.1; Tt- Tetrahymena thermophila EAR92336.1; Pi-Phytophthora infestans XP_002903832.1(PDF)Click here for additional data file.Figure S4TbCTR9-RPB1 co-immunoprecipitation. Method: Bloodstream-form trypanosomes cell lines expressing TAP-tagged TbCTR9 were used (lanes 5–8), with cells expressing TAP-tag alone as control (lanes 1–4). Protein expression was induced for 24 hrs. 1.5×108 cells were collected by centrifugation and washed once with 1×PBS. Cells were lysed in 500 µl breakage buffer as previously described 6 cells for lysate  and unbound  fractions; For wash 4  and the bead-bound fraction (lanes 4 and 5), the whole preparation was loaded. Results: A good signal of the bound CTR9-TAP protein was detected (lane 5) but there was no detectable RBP1 signal (lane 5). Similar results were obtained by immunoprecipiation of CTR9-myc (not shown).(PDF)Click here for additional data file.Table S1Effects of CTR9 on the transcriptome. The first Table is all genes, with ESAGs, VSGs and pseudogenes excluded. The second Table (sheet 2) includes only the “unique genes” of Siegel et al; these are single copy genes or, for repeated genes, a single representative of the gene family. Columns are as follows: A. TritrypDB accession number, v5; B. TritrypDB accession number, v5, but with additional zeros to give each gene ID within a chromosome the same number of digits. This enables a simple sort to arrange the genes in the order in which they appear on the chromosome. C. Annotation in TritrypDB; D. RNASeq results for poly(A)+RNA from cells after 1 day of RNAi targeting CTR9. The reads per kilobase per million reads for the ORF, divided by the ORF copy number . E. RNASeq results for poly(A)+ RNA from normal cells . F. RNASeq results (D) divided by (E). G. Half-life for total RNA . H. Developmental regulation, taken from Siegel et al., 2010. I. Position of the ORF near a start or stop site, as judged by the enrichment of modified histones as described in Siegel et al., 2009, and displayed on the TritrypDB web site. J. Details of the relative position of the ORF near a start or stop site: the number indicates whether the ORF was the first, second, third etc. relative to the transcription break. K. Nature of start/stop - convergent stops, divergent starts, or stops immediately followed by another start. L. Other notes - gene position near telomeres, pol I transcription, etc. M. Neighbouring locus, if transcribed by pol I or pol III. Only strongly-regulated genes are annotated. N. P-value for regulation (see tion see . Centrom(XLSX)Click here for additional data file.Table S2Oligonucleotides used to amplify cDNAs, with comments.(XLS)Click here for additional data file.Table S3Details of BLAST search results. Standard settings were used. Culumns C and D: For Arabidopsis thaliana, the single genome was searched. For all other genomes, the search was done using eleven genomes selected from the NCBI whole genomes database (http://www.ncbi.nlm.nih.gov/sutils/genom_table.cgi). The E values are therefore not comparable with those for Arabidopsis since a much larger database was searched. Columns E and F: Individual genes were blasted back against the S. cerevisiae or H. sapiens databases only. E values are given only if the relevant protein sequence was the best match.(XLSX)Click here for additional data file.


MDM2 gene overexpression has been observed in several human tumors. Given that even modest changes in MDM2 levels might influence the p53 tumor suppressor signaling pathway, we postulated that sequence variation in the promoter region of MDM2 could lead to disregulated expression and variation in gene dosage. Two promoters have been reported for MDM2; an internal promoter (P2), which is located near the end of intron 1 and is p53-responsive, and an upstream constitutive promoter (P1), which is p53-independent. Both promoter regions contain DNA variants that could influence the expression levels of MDM2, including the well-studied single nucleotide polymorphism (SNP) SNP309, which is located in the promoter P2; i.e., upstream of exon 2. In this report, we screened the promoter P1 for DNA variants and assessed the functional impact of the corresponding SNPs. Using the dbSNP database and genotyping validation in individuals of European descent, we identified three common SNPs . Three major promoter haplotypes were inferred by using these three promoter SNPs together with rs2279744 (SNP309). Following subcloning into a gene reporter system, we found that two of the haplotypes significantly influenced MDM2 promoter activity in a haplotype-specific manner. Site-directed mutagenesis experiments indicated that the 40 bp insertion/deletion variation is causing the observed allelic promoter activity. This study suggests that part of the variability in the MDM2 expression levels could be explained by allelic p53-independent P1 promoter activity.Although mutations in the oncoprotein murine double minute 2 (MDM2) are rare,  MDM2ormation  and thers in p53 . BecauseMDM2 gene has a basal promoter (P1) and an alternative promoter (P2) starting in the intron 1 [MDM2 levels in stressed cells, whereas the promoter P1 functions mainly in a non-stressed environment [p53-responsive promoter of the MDM2 gene has been shown to increase the affinity of the transcriptional activator Sp1, resulting in higher levels of MDM2 mRNA and protein. This SNP has been shown to attenuate apoptotic activity and accelerate tumor formation [MDM2 promoters, we determined the SNP content and the haplotype structure of the constitutive P1 promoter. Here, we show that distinct P1 promoter haplotypes can influence the p53-independent promoter activity in an allele-specific manner.Functional sequence variants in promoter regions can lead to variable gene expression levels ,15; singintron 1 . The proironment ,24. The ormation ,25-27. Sormation -30; howeormation -33. In aMDM2 proximal promoter defined as 2.0 kb upstream of the transcription start site was done using the dbSNP database (build 128) [′AAAGCCCAAATTTCCTTGCT3′ (forward) and 5′CTCCATCTTTCCGACACACA3′ (reverse) primers, 2 mM MgCl2, 0.2 mM deoxynucleoside triphosphates (dNTPs), 1× Fast Start Taq DNA polymerase buffer and GC rich buffer, 2U Fast Start Taq DNA polymerase  and 15 ng of genomic DNA. The PCR program was 95°C for 3 min; 10 cycles with a denaturation at 95°C for 15 s; annealing at 55–50°C (each cycle decreases by 0.5°C) for 20 s and elongation at 68°C for 2 min; followed by 25 cycles at 50°C for annealing. The amplicons were dot-blotted in duplicate on a nylon membrane and were hybridized with allele-specific oligonucleotides (ASOs) as previously described [′TTTCCTTTCTGGTAGGCTGG3′ (forward) and 5′CACCTACTTTCCCACAGAGA3′ (reverse) primers, 1.5 mM MgCl2, 0.2 mM dNTPs, 1× Fast Start Taq DNA polymerase buffer and GC rich buffer, 1U Fast Start Taq DNA polymerase , and 15 ng of genomic DNA. The PCR program was 95°C for 3 min; 32 cycles with a denaturation at 95°C for 30 s; annealing at 52°C for 30 s; and elongation at 72°C for 20 s. Hardy-Weinberg equilibrium was tested with a χ2 test for goodness of fit. Haplotypes were generated by PHASE software  [The initial search for promoter SNPs (pSNPs) in ild 128) . Seven Sescribed . OligonuWA, USA) .The two major promoter haplotypes (approximately 2.0 kb region) were amplified from genomic DNA of known homozygous individuals and cloned individually in the promoterless pGL3basic Firefly luciferase vector  using the Gateway Technology . Specific mutations were introduced by site-directed mutagenesis  according to the manufacturer's instructions. Clones chosen for transfection were sequenced to confirm the presence of the SNPs and then purified using the Qiagen plasmid mini kit  prior to transfection.Renilla luciferase cytomegalovirus (CMV) immediate early enhancer/promoter region (pRL-CMV) (ratio 100:1) were co-transfected to control transfection efficiency. The pGL3basic promoterless plasmid (Promega) was used as a negative control and the pGL3SV40 plasmid (Promega) was used as a positive control. The transfected cells were plated in 96-well plates with approximately 6 × 104 cells per well. The cells were harvested 24 h following transfection, and luciferase reporter gene activity was measured with dual-luciferase reporter assay system (Promega) in a SpectraMax 190 luminometer according to the manufacturer's protocol . Firefly luciferase activities of the allelic constructs were normalized using the Renilla luciferase pRL-CMV activity. The results were expressed as the ratio of Firefly luciferase activity divided by the pRL-CMV internal control activity and expressed as relative luciferase (means ± standard deviation) of four replicates. Three independent experiments were carried out for each cell line. Statistical analyses were performed using unpaired Student's t test to determine p values. Global p value is calculated with Fischer's inverse Chi-squared test [The resulting constructs were used to transiently transfect three cell lines  using lipofectamine reagent according to the manufacturer's protocol (Invitrogen). Constructs (99 ng) and SV40-driven (1 ng) red test .http://www.genomatix.de) was used to determine the presence of putative binding sites for known transcription factors. The predicted gain or loss of putative transcription factor binding site (TFBS) due to a given SNP was determined by the optimized matrix threshold as defined in the MatInspector program.MatInspector program from Genomatix Software GmbH . By genotyping a panel of 91 unrelated Western Europeans, we found four pSNPs  to be polymorphic. For rs2279744 the observed minor allele frequency of 35% was similar to the one previously reported for Caucasians [D′ and R2; these values between rs1144944 and rs937282 are 0.977 and 0.934, respectively, and 0.968 and 0.471 between these two SNPs and rs2279744. This indicates that rs2279744 (SNP309) is tightly linked with the P1 promoter's variants. The search for SNPs in the constitutive P1 promoter of ucasians . Among tMDM2. The 309 G allele was only present in 1.0% of individuals carrying haplotypes other than H1A (data not shown); therefore, European individuals carrying the allele G of this SNP are more likely to have the high P1 promoter activity haplotype because of the linkage disequilibrium.To assess the functional impact of the major promoter haplotypes 1 and 2, we subcloned the promoter haplotypes in the promoterless pGL3 basic Firefly luciferase reporter vector and we carried out transient transfection experiments for each haplotype-specific constructs in three cell lines . In an attempt to identify the cis-acting elements responsible for the observed changes in MDM2 P1 promoter activity, we modified the allele combination in both haplotypes using site-directed mutagenesis . Previous work has shown a correlation of rs937282 with allelic differences in promoter activity, with the allele −182 G having high promoter activity [At the promoter level, regulation of  element ,47. We sactivity . HoweverIn vitro studies have shown that the allele SNP309G increased the affinity of Sp1 transcription activator for a putative binding site and increase the steady-state levels of MDM2, which in turn reduced the basal p53 levels [MDM2 expression in certain tissues [Most previous studies have been focused on the impact of rs2279744 (SNP309 (T > G)), which is located in the p53-dependent promoter P2. 3 levels ,49. Alth3 levels ,50. A cl3 levels . The exp3 levels . A recen3 levels . FinallyMDM2. This linkage disequilibrium could also contribute to cancer associations with SNP309 suggesting that haplotype constructions of MDM2 pSNPs would add force to these association studies. In this report, we showed that SNP309G was associated with the high P1 promoter activity haplotype. We believe that looking at the impact of haplotypes rather than individual SNPs on promoter activity is a more suitable approach because it takes into account the putative interaction between SNPs. In conclusion, this study revealed differential constitutive P1 promoter activities, at least in vitro. This observation implies that individuals who carry distinct p53-independent P1 promoter haplotypes might have a modified risk for cancer development. Association studies in large patient cohorts will helps us to further determine the importance of these haplotypes in cancer.These conflicting rather than conclusive results might be explained by several reasons, including linkage disequilibrium between SNP309 and another, yet unknown, functional SNP in The authors declare that they have no competing interests.MEL carried out most molecular genetics experiments and drafted the manuscript. MO and ML participated in some molecular studies. MEL, MO, EK, and DS contributed to the interpretation of the data. MEL, DS and EK conceived the study, and participated in its design and coordination. All authors read and approved the final manuscript.


MYCN is a transcription factor that is expressed during the development of the neural crest and its dysregulation plays a major role in the pathogenesis of pediatric cancers such as neuroblastoma, medulloblastoma and rhabdomyosarcoma. MeCP2 is a CpG methyl binding protein which has been associated with a number of cancers and developmental disorders, particularly Rett syndrome.Using an integrative global genomics approach involving chromatin immunoprecipitation applied to microarrays, we have determined that MYCN and MeCP2 co-localize to gene promoter regions, as well as inter/intragenic sites, within the neuroblastoma genome (MYCN amplified Kelly cells) at high frequency . Intriguingly, the frequency of co-localization was significantly less at promoter regions exhibiting substantial hypermethylation (8.7%), as determined by methylated DNA immunoprecipitation (MeDIP) applied to the same microarrays. Co-immunoprecipitation of MYCN using an anti-MeCP2 antibody indicated that a MYCN/MeCP2 interaction occurs at protein level. mRNA expression profiling revealed that the median expression of genes with promoters bound by MYCN was significantly higher than for genes bound by MeCP2, and that genes bound by both proteins had intermediate expression. Pathway analysis was carried out for genes bound by MYCN, MeCP2 or MYCN/MeCP2, revealing higher order functions.ALK, AURKA and BDNF.Our results indicate that MYCN and MeCP2 protein interact and co-localize to similar genomic sites at very high frequency, and that the patterns of binding of these proteins can be associated with significant differences in transcriptional activity. Although it is not yet known if this interaction contributes to neuroblastoma disease pathogenesis, it is intriguing that the interaction occurs at the promoter regions of several genes important for the development of neuroblastoma, including  MYCN is a member of the MYC family of basic helix-loop-helix (bHLH) transcription factors which regulate a diverse range of cellular processes including proliferation, differentiation and apoptosis MYCN occurs in multiple pediatric cancers, and for neuroblastoma it is the most important genetic prognostic indicator of poor clinical outcome CATGTG motif than for CACGTG, which is significantly associated with c-MYC binding sites MYC family members heterodimerize with MAX at DNA target sequences known as E-boxes, recruiting histone acetyltransferases (HAT) and activating gene expression Genome-wide analysis of MYCN binding has also established a more global role for MYCN as a mediator of chromatin structure et al.However, this classic model of MeCP2 as a transcriptional repressor has been called into question, as methylated and imprinted genes remain silent in MeCP2-deficient mice In this study, we have carried out genome-wide analysis of MYCN and MeCP2 binding sites in combination with methylation analysis, and have characterized a novel co-occupancy of these proteins at promoter regions. Gene expression analysis of bound promoters reveals differential expression levels of genes bound individually by, or in combination with MYCN and MeCP2.Taken together, our results suggest that the majority of hypermethylated MYCN sites are also bound by the methyl binding protein MeCP2, that a greater number of MYCN/MeCP2 positive sites occur outside of hypermethylated loci and points to a role for MeCP2 in the modulation of gene expression in MYCN amplified tumors.MYCN amplified neuroblastoma cell line Kelly was immunoprecipitated with an anti-MeCP2 antibody and then hybridized to the NimbleGen HG18 two-array promoter set and to a custom designed tiling array representing 528 miRNA loci, as described previously We previously reported that MYCN co-localizes to regions of hypermethylated DNA in neuroblastoma cell lines at a significantly higher than expected frequency SST, MEF2C, GPRIN1 and SGK  software. The top 5 enrichment categories for genes uniquely bound by MYCN included cellular movement, cell death, gene expression, cell cycle and cell signaling, all terms which might be expected based on numerous prior studies of MYCN . AlthougCDC7, CDC23, CKS1B, NDC80 and NUF2 whereas those uniquely bound by MeCP2 were often involved with cell cycle arrest or senescence. This observation is consistent with our finding that genes uniquely bound by MYCN are generally more over-expressed than genes uniquely bound by MeCP2. Further analysis of the broad functional category of cell death revealed that genes co-bound by MYCN/MeCP2 were enriched for the subcategories of “apoptosis of neuroblastoma cell lines” and “survival of neuroblastoma cell lines”. These subcategories included many genes known to be important in neuroblastoma pathogenesis, including ALK, AURKA and BDNF , with no clear preference for any particular E-box variant. This was also the case for regions only occupied by MeCP2 (1.2-fold).10 ; To investigate other potential transcription factors that might be associated with MYCN/MeCP2 co-binding sites, we determined if other transcription factor binding motifs were significantly over-represented. We then cross referenced these significance values with the mRNA expression for these genes to establish which transcription factors were expressed in Kelly cells. The mRNA expression for each transcription factor was plotted against the significance of its motif enrichment [−loget al.et al.et al.MYCN amplified cell line expressing high levels of the protein.Using ChIP-chip methods we have identified a novel pattern of high frequency co-localization of the MYCN transcription factor and the MeCP2 methyl binding protein to genomic sites in neuroblastoma Kelly cells. Through a co-immunoprecipitation experiment we also demonstrate that a protein-protein interaction has occurred, but whether both, or only one protein, is binding to DNA is uncertain. The interaction of MeCP2 with another transcription factor, CREB1, has also been documented by Chahrour et alMeCP2-null mice and from transgenic mice which over-express MeCP2. In contrast to the classical model, they noted that gain of MeCP2 caused significantly more activation than repression, while loss of MeCP2 caused an increase in repression and a decrease in activation. Sequential ChIP analysis of chromatin immunoprecipitated with CREB1 and MeCP2 followed by luciferase reporter and gene expression assays confirmed that the two factors associated with the promoter regions of specific genes (including Sst and Mef2c) and activated transcription. Our own analysis of gene expression indicates that many genes with MeCP2 bound promoters are transcriptionally active. However, our results also reveal the influence of MYCN, which is expressed at high levels in Kelly. As expected from a transcriptional activator, the group of genes bound by MYCN but not MeCP2 had significantly higher levels of expression  than those bound by MeCP2 alone. Groups of genes bound in combination with MYCN and MeCP2 had median expression that was intermediate to those bound by only MYCN or MeCP2. These results suggest that both proteins can act as modulators of transcription.Chahrour et al. revealed that the majority of MeCP2 binding sites were unmethylated, occurred outside of CpG islands, and that downstream genes were actively expressed Previous ChIP-chip analysis of MeCP2 binding sites within the neuroblastoma cell line SH-SY5Y by Yasui Dlx5/Dlx6 locus using 3C combined ChIP with an anti-MeCP2 antibody, demonstrated the MeCP2 was required for the formation of a silencing chromatin loop, and that Mecp2-deficient mice had increased expression of Dlx5 and Dlx6 as a result of aberrant loop formation. The influence of MeCP2 in the formation of chromatin loops does not seem to be limited to transcriptional silencing, however, as gene expression and ChIP-chip analysis of several genes, including JUNB, within the NB cell line SH-SY5Y, indicate that these genes are modulated by distal and proximal MeCP2 binding sites JUNB. Our own ChIP-chip analysis of JUNB in the Kelly cell line reveals several putative overlapping MYCN and MeCP2 binding sites around the promoter region of JUNB which are similar to those previously described in SH-SY5Y. The mechanism that modulates chromatin looping remains poorly understood, but it is thought that changes in chromatin flexibility are necessary to allow loop formation. It has been suggested that chromatin flexibility is regulated by histone acetylation et al. revealed that over 90% of the identified genomic euchromatic marks, acetylation of histone H3K9 and trimethylation of H3K4, were dependent upon MYCN expression An intriguing possibility is that MYCN and MeCP2 can co-operate to regulate gene expression by altering higher-order chromatin structure. Several recent studies using chromosome conformation capture (3C) techniques have shown that distally located cis-regulatory elements can be brought into proximity to the promoter of active genes, indicating that a chromatin loop forms to allow the adjacent association of both elements. A study of the imprinted et al.The MYC family of transcription factors have been previously associated with specific DNA binding sequence motifs known as E-boxes. We have previously shown a preference of certain E-box motifs  in the MYCN amplified state. An E-box analysis of hypermethylated regions which were MeCP2 positive and MYCN positive, revealed enrichment for both CATGTG and CACCTG motifs. Interestingly, the classic E-box motif CACGTG was highly enriched (4.3 fold) where MeCP2 was bound to hypermethylated regions in the absence of MYCN. This is consistent with the report by Perini et al ALK, AURKA, and BDNF.In conclusion, our results indicate that MYCN and MeCP2 protein interact and co-localize to similar genomic sites at very high frequency. Overall, genes with MYCN and MeCP2 bound promoters have a median expression that is intermediate between genes that are uniquely bound by either MYCN or MeCP2. Our results also support the concept that MeCP2 has a repressive effect on transcriptional activity, except when it is interacting with MYCN and potentially other transcriptional activators. Currently, it is not known if this interaction contributes towards neuroblastoma disease pathogenesis or represents an important and normal process occurring in the development of the neural crest. Further studies are warranted to address these questions, particularly since the interaction of these two proteins occurs at the promoters of several genes that are important for the development of neuroblastoma, including Kelly cells were obtained from the European Collection of Animal Cell Cultures  and were cultured in RPMI-1640 supplemented with 2 mM glutamine, 10% fetal bovine serum and penicillin/streptomycin. The Kelly neuroblastic cell line, derived from the metastatic tissue of a female neuroblastoma patient, contains an amplification of the MYCN oncogene and expresses elevated levels of MYCN mRNA and protein et al.2 ratios and the NimbleScan peak finding function. An in-built peak analysis algorithm detected significantly enriched regions that had at least 4 probes above a threshold value of log 2.0. These enriched regions were then identified as “peaks” and a false discovery rate (FDR) score was assigned. Enriched sites with a false discovery rate (FDR) of less than 0.1, which were shared across duplicate ChIP reactions were filtered using an in-house Java application and selected for further analysis. Raw log2 ratio data for array experiments are available at www.ebi.ac.uk/arrayexpress (Accession E-MTAB-621).ChiP-chip analysis was performed as previously described by Murphy et al.H19 locus was used as the control and fold enrichment of this locus was determined relative to the unmethylated H3B locus 2 ratio data was calculated and a one-sided Kolmogorov-Smirnov (KS) test using a sliding window of 750 bp was used to determine whether the probes were drawn from a significantly more positive distribution of intensity log-ratios than those on the rest of the microarray. Each probe was assigned a –log10p-value from the windowed KS test and hypermethylated sites of enrichment were detected by searching for at least two probes with a minimum –log10 p-value of two. Peaks within 500 bp of each other were merged and SignalMap version 1.9 was used to visual the resulting data files.The protocol used was as previously described by Weber , Carlsbad, CA), 10 µg of total RNA was used to synthesize double-stranded cDNA. The Cy3 fluorophore  was used to label the cDNA and 4 µg of this labeled cDNA was hybridized to the homo sapiens 4×72 K Gene Expression Array . Arrays were scanned using GenePix 6.0 software on the Axon 4000B microarray scanner. Data analysis was performed using the expression robust multi-array module of Nimblescan Version 2.4. The raw data from the gene expression array experiments are available at www.ebi.ac.uk/arrayexpress (Accession E-MEXP-3121).Total RNA was extracted from Kelly cells using the RNeasy Mini Kit  and the complete removal of DNA was ensured via an on-column digestion of DNA using the RNase-free DNase set . The integrity of the RNA was confirmed using the Experion RNA StdSens Analysis Kit . Using the Superscript Double-Stranded cDNA Synthesis Kit from Invitrogen , CDC25A (Hs00947994_m1), MeCP2 (Hs00172845_m1), MYCN (Hs00232074_m1), c-MYC (Hs00153408_m1), TP53 (Hs99999147_a1), TERT (Hs99999022_m1) and β-Actin (HS99999903_m1) were obtained from Applied Biosystems and were used to validate the gene expression array results. Analysis was performed using the 7900HT Fast Realtime System . The results of the gene expression array were correlated with the Ct values of gene expression probes using Spearman's rank correlation coefficient.Taqman gene expression assays for 6 Kelly cells were harvested from a T75 cell culture flask and nuclei were isolated by re-suspending the cells in a complete hypotonic buffer for 15 min on ice. After the addition of detergent, the cells were centrifuged at 14,000 x g in a pre-chilled microcentrifuge and the nuclear fraction was retained. The nuclei were enzymatically sheared by incubation at 37°C for 10 min after re-suspension in complete digestion buffer containing an enzymatic shearing cocktail. The reactions were stopped with the addition of 0.5 M EDTA and, after centrifugation, the supernatant containing the digested nuclei was retained. The nuclear extract (500 µg) was incubated with 5 µg of rabbit polyclonal MeCP2 antibody  or 5 µg of normal rabbit IgG  on a rotating platform for 4 h at 4°C. Protein G magnetic beads were added to the reactions and the incubation was continued using the same conditions for a further hour. After incubation, the protein beads were precipitated using a magnet and after washing, the protein complexes were eluted by re-suspending the beads in 20 µl of Laemmli buffer and incubating the samples at 100° for three minutes. Each sample (10 µl) was loaded onto a 10% SDS-PAGE gel and resolved by electrophoresis.The protein was transferred to a nitrocellulose membrane which was then blocked overnight at 4°C with a 5% solution of BSA. Blots were probed with a 1 in 500 dilution of B8.4.B  antibody, followed by a 1 in 500 dilution of Clean-Blot IP Detection Reagent (HRP) .Co-immunoprecipitations were performed using a modified version of the Universal Magnetic Co-IP Kit protocol . Briefly, 5×10http://www.ingenuity.com). To identify the top functions of lists bound by MYCN and MeCP2, the biological functional analysis was performed on all datasets separately. The results of this analysis were compared across datasets, using Fisher's Exact test, biological functions were deemed to be significant if P<0.05.Lists of genes which were identified as bound uniquely by, or in combination with, MYCN and MeCP2 using an in-house Java based software program. These lists were then imported into Ingenuity Pathway Analysis (IPA) version 8.8 (http://string-db.org) DNA sequence data for the processed ChIP regions for each cell line were retrieved from the UCSC database. Phylogenetically conserved sequence between Human (HG18) and Mouse was selected for motif analysis. The occurrence of DNA binding motifs was assessed in relation to their background frequencies within the sequence tiled on the promoter array. Significance for over- or under-representation was assessed using P-values based on Chi-square test. Transcription factor motif enrichment was assessed by examining occurrence of 482 transcription factor binding motifs (from 358 transcription factors), represented by their position-weight-matrices (PWM), in the conserved DNA sequences from the various peak datasets. We then calculated over-representation of each motif compared to our promoter array background and generated a significance value from the Chi-squared test. The String database was used to retrieve information on known and predicted proteins interactions  and the custom tiled array (C). Correlation scores above R = 0.85 were observed between replicate experiments confirming that the experiments reproducibly detected MeCP2 binding sites. (D) SYBR Green qPCR validation of positive MeCP2 binding sites. Fold enrichment of positive MeCP2 target sites is displayed. Experiments were carried out in duplicate using the standard delta delta Ct method. Results are plotted relative to a region negative to MeCP2 Binding (H3B) which is set to 1.Pair-wise comparisons of log(TIF)Click here for additional data file.Figure S2SST, (B) GPRIN1, (C) MEF2C (D) SGK (E) SNRPN and (F) BDNF. The base pair position of the promoters is indicated by the scale across the top of the panels. The fluorescent intensity of the probes across replicates is expressed as log2 ratios and is represented as the tracks with green bars. Statistically significant MeCP2 binding sites are represented as red bars. The position of the genes and the tiled region on the array are indicated by the two lower tracks.Identification of MeCP2 binding within the promoter regions of previously published target genes (A) (TIF)Click here for additional data file.Figure S3Pie charts representing the percentage of MeCP2 sites which are unique to the MeCP2 dataset and which overlap sites enriched for MYCN binding and regions of hypermethylation.(TIF)Click here for additional data file.Figure S4CDC7, CDC25A, MeCP2, MYCN, MYC, P53, TERT and β-Actin. The Ct values for these probes were correlated with the expression values obtained for each of these genes on the array platform using Spearman's Rank Correlation Test. Gene expression validation of nimblegens 4-plex 72K arrays. Taqman gene expression probes were selected for (TIF)Click here for additional data file.Figure S5ALK (B) promoter region of BDNF (C) promoter region of AURKA. Data from replicate MYCN and MeCP2 ChIP-chip experiments is presented. Red bars represented regions with statistically significant over-representation of the respective proteins.Co-localization of MYCN and MeCP2 at the promoters of neuroblastoma relevant genes. (A) promoter region of (TIF)Click here for additional data file.Figure S6Assessment of motif enrichment at unique and commonly bound MYCN and MeCP2 sites in intergenic regions identified from the miRNA custom tiling array data set. Here we illustrate the frequency, relative to background, of the various classes of canonical E-boxes (CANNTG) in non-methylated intergenic sites bound by MYCN alone (a), MeCP2 alone (c) and both MYCN and MeCP2 (b); and methylated intergenic sites bound by MYCN alone (d), MeCP2 alone (f) and both MYCN and MeCP2(e). We also include the putative MeCP2 binding motif proposed by Klose et al (37). Motifs with 1.5-fold change over background and P<0.05 are highlighted.(TIF)Click here for additional data file.Figure S7H19 gene promoter relative to the non-methylated H3B promoter following MeDIP from Kelly cells.qPCR showing enrichment of the imprinted (TIF)Click here for additional data file.Table S1Comparison of MeCP2 ChIP-chip results with Yasuai et al 2008.(XLSX)Click here for additional data file.Table S2PCR Primers.(XLSX)Click here for additional data file.


With the advent of high throughput and high resolution genome-wide protein-DNA detection assays, the inter-relationships between chromatin and the transcription machinery are now becoming clearer. Here I will discuss our recent findings using MNase ChIP-seq to map nucleosome positions, and an ultra-high resolution mapping technique called ChIP-exo that we recently developed . What is apparent from these studies is the following: 1) transcription factors bind to many more locations in the genome than previously appreciated. 2) transcription complexes form at the interface between nucleosomes and nucleosome-free promoter regions. 3) Chromatin remodeling complexes target specific nucleosome positions, working in concert to organize nucleosomes at the beginning and end of genes. Many remodeler subunits interact asymmetrically with the nucleosome core across the genome, which may be important for the directional passage of RNA polymerase II.


Schizosaccharomyces pombe has well characterized regions of heterochromatin and has proven to be a powerful model for elucidation of epigenetic silencing mechanisms. Research in S. pombe led to the breakthrough discovery that epigenetic silencing is not solely a chromatin-driven transcriptional repression and that RNA interference of nascent transcripts can guide epigenetic silencing and associated histone modifications. Over the last 10 years, an eloquent integration of genetic and biochemical studies have greatly propelled our understanding of major players and effector complexes for regulation of RNAi-mediated epigenetic silencing in S. pombe. Here, we review recent research related to regulation of the epigenetic state in S. pombe heterochromatin, focusing specifically on the mechanisms by which transcription and RNA processing interact with the chromatin modification machinery to maintain the epigenetically silent state.Post-translational modifications of histones are critical not only for local regulation of gene expression, but also for higher-order structure of the chromosome and genome organization in general. These modifications enable a preset state to be maintained over subsequent generations and thus provide an epigenetic level of regulation. Heterochromatic regions of the genome are epigenetically regulated to maintain a “silent state” and protein coding genes inserted into these regions are subject to the same epigenetic silencing. The fission yeast  The organization of chromosomal DNA within the nucleus is achieved through direct interaction with a wide range of proteins. The basic unit of the DNA-protein complex, known as chromatin, is the nucleosome that contains approximately 147 bp of DNA wrapped around a core of eight histone proteins: two copies each of the H2A, H2B, H3 and H4 histone proteins. Post-translational modification of core histone proteins and further interactions with nuclear proteins regulates the nature of chromatin and its subsequent activity. In general, the majority of active protein-coding genes are located within euchromatin, regions of chromatin in which the nucleosome particles have an “open” packaging structure amenable to access by regulatory proteins and the transcriptional machinery. In contrast, inactive genes and structural regions of the genome, such as the centromeres and telomeres, are packaged with the nucleosome in a more tight “closed” state known as heterochromatin. The post-translational modifications of chromatin that define its state enable a preset gene activity to be maintained even in the absence of a regulatory signal. This is also a heritable state that can be transferred to subsequent generations without requiring change of DNA sequence, and is thus referred to as epigenetic regulation.The N-terminal regions of histone proteins, commonly called the “histone tails” as they extend from the nucleosomes, are highly conserved among eukaryotic organisms and represent the target site for most common epigenetic modifications. The major post-translation modifications involved in epigenetic regulation are found in the histone H3 tails, particularly at the lysine 4 (H3K4) and lysine 9 (H3K9) residues. The chromatin at actively transcribed genes and associated euchromatic regions is marked by methylation of lysine 4 (H3K4me) and acetylation of lysine 9 (H3K9ac). The opposite is found in heterochromatin, where methylation of lysine 9 (H3K9me) and subsequent coating by Heterochromatin Protein 1 (HP1) proteins is a hallmark of epigenetically silent chromatin . It is iAlthough heterochromatic regions of the genome contain few protein-coding genes, the mechanisms for epigenetic silencing are directly relevant to that occurring at protein-coding genes and for the control of mobile transposon elements that can strongly influence neighboring genes. Epigenetically silent heterochromatin also serves an essential function in chromosome dynamics, supporting kinetochore formation to ensure correct chromosome segregation and for maintenance of telomere integrity at chromosome ends. Mutations that disrupt any aspect of epigenetic silencing, such as those that impair H3K9me or reduce HP1 association with chromatin, often display phenotypes of lagging chromosomes and missegregation, and in severe cases cell lethality  flanked by two inverted inner repeats (imr) that share exact identity, which is further flanked by the pericentromeric outer repeat region (otr) that contains multiple copies of dg and dh repeat sequences (cnt and imr) is unique to each chromosome whereas the pericentromeric repeats of each chromosome otr are similar and vary in number depending on the chromosome  region that is another well studied region of heterochromatin. The mating type region contains an upstream active mat1 locus and two downstream donor loci, mat2 and mat3, within a silent heterochromatin domain. Switching of the upstream mat1 gene with one of two downstream donor loci determines the mating type of the cell. Correct regulation of heterochromatin silencing is essential for efficient mating type switching. This region long served as a target site for heterochromatin studies in S. pombe  is a PTGS regulation that either represses translation of target transcripts or results in their cleavage and rapid degradation. The RNAi process is mediated by small RNAs, microRNAs or short interfering RNAs (siRNAs), that have complementary sequence to the target transcript and determine the site of action for the RNA machinery . Small RArgonaute genes were suggested to have an important role in cell differentiation in plants (S. pombe due to the fact that its genome only contained a single copy of Argonaute (ago1+). Deletion of ago1+ and the related single Dicer (dcr1+) and RdRP (rdp1+) genes led to the surprising finding that these core components of the RNAi machinery were required for maintaining the silent state of pericentromeric regions  complex is a second Ago1-containing complex that is proposed to mediate the loading of Ago1 with mature single-stranded siRNA for its activity within the RITS complex   (The RITS complex interacts with nascent transcripts from the pericentromeric regions, and the activity of RITS in processing these transcripts is thought to be guided by complementary single-stranded siRNAs loaded into Ago1 (In the next step of the loop, RITS interacts with both RDRC and CLRC through its Ago1 subunit (The dsRNA products provided by RDRC activity then proceed through the loop to function as substrates for Dcr1 and are processed into siRNAs. The mechanism by which dsRNA is released from RDRC and interacts with Dcr1 remains unclear, although it may have parallels to that for transcription as suggested by the presence of polyA polymerase subunit in RDRC and contribution of splicing components (It is important to emphasize that the complexes described here should not be considered as fixed, stable complexes. One example of this is the ability of CLRC subunits Rik1 and Dos1 to associate and interact with chromatin even in the absence of Clr4 (The concept of the self-enforcing loop is based on the interdependent relationship between Clr4 activity and the RNAi pathway. Clr4 is required for the accumulation of high siRNA levels and the mutual interaction between RNAi complexes . On the de novo formation of heterochromatin that leads into the self-enforcing loop is one aspect that requires further investigation. Low levels of H3K9me persist at the centromeric repeat regions in RNAi-deficient cells (S. pombe (de novo heterochromatin assembly. However, another report argues against an upstream role for siRNAs. Using a genetic approach to transiently deplete CLRC or RNAi components, it was shown that Ago1 was not required to initiate de novo H3K9me at centromeres by Clr4 (The process for S. pombe . Based oSimilar to the fact that epigenetic silencing complexes are thought to be highly dynamic, consideration also needs to be made of the dynamic nature of chromatin and how RNAi-mediated epigenetic silencing interacts with other molecular processes that occur along the chromosome. One chromatin remodeling process expected to conflict with the self-enforcing loop is that of DNA replication. Centromeric transcripts and siRNAs accumulate predominantly during S-phase, and it is at this point that H3K9me needs to be transferred or spread along the newly synthesized DNA (Rik1 and Dos2 have been identified as part of a second complex separate from CLRC, which contains the transcriptional activator Mms19 and DNA polymerase subunit Cdc20 , promoteOur understanding of the major players and effector complexes for RNAi-mediated epigenetic silencing and heterochromatin assembly has advanced considerably over the last 10 years. However, as new components and functions are discovered, it becomes apparent that that there are still many questions that remain unanswered. Examples of this are Ago1 and Rik1, which are two core proteins that are located in multiple complexes. The identification of ARC provided a concept for siRNA loading of Ago1 in RITS, but it remains to be determined how siRNA transfers between the two complexes, whether the same Ago1 molecule shuttles between the two complexes, and the extent that separate functions for Ago1 are controlled by its other subunits. Rik1 was one of the first genes to be implicated in heterochromatin formation and is now known to form two distinct complexes. These complexes explain the critical role for Rik1 and how multiple processes of transcription, chromatin modification and replication are intertwined. However, the nature of Rik1 contribution to these complexes still requires clarification and the dynamics behind subunit interactions remain vague. The HP1 protein Swi6 gained prominence as a signal for completion of typical heterochromatin formation irrespective of RNAi involvement and its role was thought to be clear. However, Swi6 has now been shown to participate in the initiation of replication within pericentromeric repeats . Much reA central question for future research to understand heterochromatin formation is what exactly defines the region to be targeted for epigenetic silencing. It has been eloquently demonstrated that tethering of Clr4 is sufficient to trigger formation of heterochromatin, and importantly, that the RNAi pathway is dispensable for assembling of this silent heterochromatin into a functional centromere , whereas


We are studying a 181-bp PRE from the Drosophilaengrailed gene. This PRE causes pairing-sensitive silencing of mini-white in transgenes. Here we show that the 181-bp PRE also represses mini-white expression in flies with only one copy of the transgene. To isolate mutations that alter the activity of the 181-bp PRE, we screened for dominant suppressors of PRE-mediated mini-white repression. Dominant suppressors of mini-white repression were rare; we recovered only nine mutations out of 68,274 progeny screened. Two of the nine mutations isolated are due to the same single amino acid change in the transcriptional activator Woc (without children). Reversion experiments show that these are dominant gain-of-function mutations in woc. We suggest that Woc can interfere with the activity of the PRE. Our data have implications for how Polycomb group proteins act to either partially repress or completely silence their target genes.Polycomb group response elements (PRE) are  Drosophila as genes necessary to maintain the silencing of homeotic genes, it is now evident that PcG proteins have many other targets (reviewed in Polycomb group genes (PcG) encode proteins that mediate transcriptional repression. First identified in Drosophila, PcG proteins are associated with Polycomb group response elements (PRE), DNA elements that recruit PcG proteins to the DNA .In engrailed and invected PREs, pairing-sensitive silencing was observed at a frequency of 21–62% of insertion sites  it represses inappropriate expression in both en- and Ubx-reporter genes in embryos , which encodes a transcriptional activator. Our data suggest that there is a competition between transcriptional activators and PcG repression and that certain types of activators may be better able to overcome PcG repression.We conducted a genetic screen for dominant suppressors of pairing-sensitive silencing by a transgene that contained the 181-bp For EMS mutagenesis, adult males were fed EMS as described , and thewoc transcription unit was sequenced.DNA was isolated from homozygous or hemizygous mutant adults or larvae, and the entire en PRE was amplified with the primers GCGGAATTCGAGATGGCATGTGGCTCT and GCGGAATTCGCATGCTGGAGCTGTCAG, cut with EcoRI, and cloned into EcoRI cut, phosphatased EK710, which contains loxP sites on both sides of the EcoRI site . A fragment of DNA containing the 181-bp PRE and flanking loxP sites was cut with NotI and cloned into NotI cut CaSpeR4. The resulting clone was sequenced to determine the orientation of the insert.The 181-bp P[L181PRE] was injected into homozygous Df(1)w67c23, y embryos using standard techniques. Some lines were generated by P-element mobilization by crossing to a strain with the endogenous transposase insertion +∆2P99B . P[L] derivative lines lacking the en181bp-PRE were obtained by crossing males with the P[L181PRE] insertion to virgin females that carried a constitutively active Cre recombinase transgene  w67c23, y flies. Two individual +w male progeny were selected from each insertion line and crossed to the appropriate balancer chromosome. P[L] lines were established, and the deletion of the en181bp-PRE was confirmed by PCR with primers flanking the loxP sites.ew]/Sco) . Progeny1118w, (2) 1118; P[181PRE]8-10Cw, (3) 1118; wocD1w, and (4) 1118;P[181PRE]8-10C; wocD1w. Total RNA from 3rd instar larvae, 1-day-old pupae, or adult fly heads was prepared  en181bp-mediated mini-white repression. We used the line P[181PRE]8-10C, which contains a P-construct with the 181-bp PRE of en DNA cloned into pCaSpeR  caused a darkening of the P[181PRE]8-10C heterozygous eye color. As none did, we suspected that both mutations are gain-of-function alleles that produce proteins with altered activities. If so, then a mutation that inactivates the mutant protein should revert the dominant suppression of the P[181PRE]8-10C eye color. Therefore, we tried to revert both mutations.We mapped the mutations on chromosome 3 using the markers woc.We used both X-rays and EMS to generate revertants. We recovered three X-ray-induced revertants  and four EMS-induced revertants (from 5500 progeny). The revertants are lethal over deficiencies for the region 96F1–98A5. By crossing to overlapping deficiencies and lethals in the region, we found that all of the revertants are lethal or semilethal mutations in the gene woc gene from our original suppressor mutation chromosomes showed that both of these mutations are due to the same single amino acid change in a position evolutionarily conserved throughout the Drosophila lineage, as well as in most insects (D1woc and D2woc (for Dominant1woc and Dominant2woc), and we named the revertant alleles based on the allele reverted and the mutagen used  generated from D1woc by EMS (E) mutagenesis). The wocD1/+ and wocD2/+ flies have no phenotypic defects. The D1woc and D2woc homozygotes survive, are fertile, and also show no phenotypic defects.Sequencing of the  insects . This amD1woc and D2woc, as well as an additional lesion in the woc transcription unit . Because Dwoc dominantly alters the eye color of P[181PRE]8-10C heterozygotes, we looked at whether Dwoc could dominantly alter the eye color of flies heterozygous for P[L181PRE] insertions that show mini-white repression. For 14 out of 15 P[L181PRE] lines tested, Dwoc does not alter the eye color. However, in P[L181PRE]-8A, the eye color was slightly darker in a Dwoc mutant (data not shown). P[L181PRE]-8A is inserted just upstream of the PcG-regulated gene CycA (at 3L:11826614). To determine whether the effect on the eye color of P[L181PRE]-8A flies was due to the PRE, we examined the eye color of P[L]-8A flies in which the 181-bp PRE had been removed. We found that Dwoc had no effect on the eye color of flies that lacked the PRE. This shows that, at least at this chromosomal location, the change in eye color mediated by Dwoc is dependent on the PRE. However, as Dwoc does not influence the eye color of most P[L181PRE] lines, we believe that Dwoc is not working on the PRE directly but on sequences flanking the P[181PRE] insertion sites.We next examined whether GstS1 and CG30456 transcripts were altered in D1woc mutants, both in the presence and in the absence of the P[181PRE]8-10C insertion. We examined RNA levels at three developmental stages: 3rd instar larvae, 1-day-old pupae, and adult heads. We saw no significant differences in the expression levels of GstS1 and CG30456 between D1woc and wild-type 3rd instar larvae or one-day old pupae (data not shown). However, we saw a twofold increase in the level of GstS1-RNA in the adult heads of homozygous D1woc mutants compared to wild-type . Finally, we tested whether Dwoc altered the transcription level of CycA in adult heads. We saw no significant differences in CycA levels between wild-type and D1woc; P[181PRE] heads (data not shown).We examined whether the levels of ild-type . Flies wP[181PRE]8-10C effect the expression levels of GstS1 or CG30465 in fly heads by qRT-PCR; we saw no effect on transcript levels of either gene (data not shown). Thus, a change in transcription level of a GstS1 is not required for suppression of the pairing-sensitive silencing of P[181PRE]8-10C. Finally, we examined the effects of three the other suppressor mutants on the eye color of P[L181PRE] at multiple insertion sites. Like Dwoc, none of the other suppressor mutations altered the eye color of P[L181PRE] at the insertion sites.We tested whether four of our other dominant suppressors of pairing-sensitive silencing of loxP sites and cloned it upstream of the mini-white reporter in pCaSpeR (P[L181PRE]), in the same orientation and position as in the construct P[181PRE] . All lines were treated with Cre recombinase to excise the 181-bp PRE, yielding P[L]. None of the lines without the PRE showed pairing-sensitive silencing. In 7 of the 15 pairing-sensitive lines, the eye color of heterozygous flies became darker upon removal of the PRE, showing that some repression of mini-white expression occurred even in the heterozygotes  and examined whether the insertion occurred in or near a transcription unit  HOX gene . The 181-bp PRE is known to bind PcG proteins, and a binding site for the DNA-binding PcG group protein Pho is required for 181-bp–mediated mini-white repression  and transcriptional activation of GstS1 . Second, changes in the chromatin environment, here caused by a gain-of-function mutation in the transcriptional activator Woc, can inactivate a PRE. The dependence of PRE activity in transgenes on chromosomal environment has long been recognized and is dramatically demonstrated in a recent report showing the effect of chromosome environment on the activities of the Abd-B Fab-7 PRE and the vg PRE  and one decapentaplegic (dpp) enhancer]. At that time, neither vg nor dpp was thought to be regulated by PcG proteins. However, since then, a vg PRE and a dpp PRE have been identified  to ccurring . Therefo