Cse4 is lethal in cells lacking Psh1, the E3 ubiquitin ligase that targets CENP-ACse4 for degradation. To identify additional mechanisms that prevent CENP-ACse4 misincorporation and lethality, we analyzed the genome-wide mislocalization pattern of overexpressed CENP-ACse4 in the presence and absence of Psh1 by chromatin immunoprecipitation followed by high throughput sequencing. We found that ectopic CENP-ACse4 is enriched at promoters that contain histone H2A.ZHtz1 nucleosomes, but that H2A.ZHtz1 is not required for CENP-ACse4 mislocalization. Instead, the INO80 complex, which removes H2A.ZHtz1 from nucleosomes, promotes the ectopic deposition of CENP-ACse4. Transcriptional profiling revealed gene expression changes in the psh1Δ cells overexpressing CENP-ACse4. The down-regulated genes are enriched for CENP-ACse4 mislocalization to promoters, while the up-regulated genes correlate with those that are also transcriptionally up-regulated in an htz1Δ strain. Together, these data show that regulating centromeric nucleosome localization is not only critical for maintaining centromere function, but also for ensuring accurate promoter function and transcriptional regulation.The exclusive localization of the histone H3 variant CENP-A to centromeres is essential for accurate chromosome segregation. Ubiquitin-mediated proteolysis helps to ensure that CENP-A does not mislocalize to euchromatin, which can lead to genomic instability. Consistent with this, overexpression of the budding yeast CENP-A Cse4, which normally localizes exclusively to centromeres. Here, we investigated where CENP-ACse4 spreads in the genome when parts of its regulatory machinery are removed. We found that CENP-ACse4 becomes mislocalized to promoters, the region upstream of each gene that controls the activity of the gene. Consistent with this, the mislocalization of CENP-ACse4 to promoters leads to problems with gene activity. Our work shows that mislocalization of centromeric proteins can have effects beyond chromosome segregation defects, such as interfering with gene expression on chromosome arms.Chromosomes carry the genetic material in cells. When cells divide, each daughter cell must inherit a single copy of each chromosome. The centromere is the locus on each chromosome that ensures the equal distribution of chromosomes during cell division. One essential protein involved in this task is CENP-A The eukaryotic genome is packaged into chromatin, which consists of 147 bp repeating units of DNA wrapped around histone proteins to form nucleosomes . HoweverHtz1 (SGD ID: S000005372) histone variant is regulated by the SWR1 (SWR-C) and INO80 (INO80-C) chromatin remodeling complexes [Htz1 localizes to intergenic regions, specifically near transcription start sites (TSS) at the +1 and -1 nucleosomes surrounding nucleosome-depleted regions (NDRs) [Htz1 nucleosomes are correlated with high nucleosome turnover [Htz1 into nucleosomes by exchanging H2A/H2B dimers for H2A.ZHtz1/H2B dimers [Htz1 removal from nucleosomes by INO80-C is less well understood because it has two reported activities that both lead to H2A.ZHtz1 exchange, either by swapping H2A.ZHtz1/H2B dimers for H2A/H2B dimers [The genomic incorporation of the budding yeast H2A.Zs (NDRs) , 9–12. Iturnover , which iturnover –16. SWR-B dimers –19. In cB dimers  or by prThe localization of the CENP-A variant is regulated by the histone chaperone HJURP (Scm3 in budding yeast), which is targeted specifically to centromeres –25. CentCse4 (SGD ID: S000001532) is mediated by an E3 ubiquitin ligase called Psh1 (SGD ID: S000005415) [Cse4 is overexpressed in the absence of Psh1-mediated proteolysis [Cse4 in euchromatin. This also results in lethality, although the underlying cause has not been determined [Multiple mechanisms ensure that CENP-A does not mislocalize to euchromatin. A number of chromatin remodelers and histone chaperones are reported to help maintain centromeric chromatin or prevent CENP-A mislocalization, including Fun30, RSC, INO80-C, CAF-1, HIR, FACT, and RbAp48 and SWI/SNF –41. In a0005415) , 47. Wheteolysis , 46–48, termined , 46, 47.Htz1 is also a component of pericentromeric chromatin in budding yeast, where it localizes to nucleosomes flanking the CENP-ACse4 nucleosome and is important for chromosome segregation through unknown mechanisms [S. pombe have shown that CENP-ACnp1 tends to mislocalize to ectopic regions that are depleted of H2A.ZHtz1 [Similar to CENP-A, H2A.Z also contributes to chromosome segregation. In human cells, H2A.Z is found at pericentromeric regions, where it is incorporated at the inner centromere between the CENP-A nucleosome domains, and helps to establish centromeric heterochromatin , 50. Simchanisms , 52. Howchanisms . In cont2A.ZHtz1 .Cse4 mislocalizes to promoters that are enriched for NDRs flanked by H2A.ZHtz1, and this mislocalization is dramatically enhanced in cells that cannot degrade CENP-ACse4. This localization pattern appears to be due in part to co-opting of INO80-C to incorporate excess CENP-ACse4 into promoter nucleosomes that normally contain H2A.ZHtz1. Consistent with this, there was a significant correlation between transcripts that were misregulated in cells lacking H2A.ZHtz1 and those with high levels of CENP-ACse4 mislocalization. We also found that a subset of promoters that misincorporate CENP-ACse4 have decreased transcription, which may be the underlying cause of lethality. Together, these data suggest that it is essential that cells regulate CENP-ACse4 localization not only to ensure proper chromosome segregation, but also to protect cells from promoter nucleosome disruption and transcriptional misregulation.We set out to determine whether there are features of euchromatin that normally prevent budding yeast CENP-A misincorporation as well as to identify the functional consequences of CENP-A mislocalization to euchromatin. The identification of euchromatic sites that strongly misincorporate CENP-A may also shed light on the underlying cause of the lethality. To address these questions, we performed the first genome-wide analysis of CENP-A overexpression in the absence of ubiquitin-mediated degradation. We found that overexpressed CENP-ACse4 mislocalization in budding yeast, we performed ChIP-seq on endogenous and overexpressed CENP-ACse4 in the presence and absence of Psh1-mediated proteolysis. All strains contained a fully functional ectopic 3Flag-CSE4 gene integrated at the URA3 locus under the endogenous promoter and were deleted for the endogenous CSE4 gene. Cells overexpressing CENP-ACse4 contained an additional copy under the control of the GAL promoter (pGAL-3Flag-CSE4). As seen previously, CENP-ACse4 overexpression inhibited the growth of WT cells and resulted in lethality in psh1Δ cells , which cuts linker DNA between nucleosomes. The CENP-ACse4 nucleosomes were purified from the MNase-treated chromatin by immunoprecipitation of 3Flag-Cse4. The amount of CENP-ACse4 recovered in the ChIP samples reflected the starting levels in the chromatin  on chromosome arms that are enriched for mislocalized CENP-ACse4 and other kinetochore proteins [Cse4 peaks in the psh1Δ pGAL-3Flag-CSE4 strain (Cse4 was overexpressed to much higher levels in our study (150-fold compared to 3-fold), so the CLRs are a small fraction of the total peaks. Consistent with this, there was also enrichment in low confidence negative control regions (LCNCRs), indicating there is no preference for CLR localization. We also analyzed the AT content of the DNA bound by mislocalized CENP-ACse4, as this is a defining characteristic of centromeric DNA in budding yeast. As expected, CENP-ACse4 peaks were highly enriched for AT nucleotides in the WT strain. However there was only a moderate increase in AT% in the psh1Δ strain compared to the input nucleosomes, and almost no AT bias in the strains with overexpressed CENP-ACse4 (Cse4 is due to a more widespread effect than just centromere-like characteristics.We next asked whether the intergenic enrichment correlates with features known to be associated with centromeres. ChIP-seq of mildly overexpressed CENP-Aproteins . These C4 strain . HoweverNP-ACse4 . TogetheCse4 was specific to either promoters (defined as 500 bp upstream of the transcription start site (TSS)) or transcription terminators (defined as 500 bp downstream of the transcription termination site (TTS)) by calculating the number of peaks in these regions . In theectively . At the  of Psh1 .Cse4 in promoters and terminators is associated with the basal level of transcription in WT cells. We plotted CENP-ACse4 enrichment at the TSS and TTS of genes binned into quartiles by the published transcription levels in a WT strain, ranked from lowest transcription to highest transcription [Cse4 enrichment and the different transcription levels   . We ther at NDRs , as well at NDRs  and cent at NDRs , S5G Fig2A.ZHtz1 . The his2A.ZHtz1 . There wlization  TogetherHtz1 promotes CENP-ACse4 localization by performing ChIP on WT, psh1Δ, and psh1Δ htz1Δ cells overexpressing CENP-ACse4. htz1Δ cells are defective in induction from the GAL promoter [CSE4 levels. Overexpressed CENP-ACse4 bound to promoter regions in the psh1Δ htz1Δ double mutant, at levels similar to or even higher than the psh1Δ strain  is the Swi/Snf related ATPase in SWR-C that deposits H2A.ZHtz1 into nucleosomes [Cse4 in swr1Δ cells. We confirmed that H2A.ZHtz1 was reduced at a previously reported promoter nucleosome locus by ChIP-PCR  leads to a global alteration of H2A.ZHtz1 localization patterns genome-wide without affecting the overall levels of H2A.ZHtz1 incorporation in the genome [NHP10 (SGD ID: S000002160), a non-essential INO80-C subunit that facilitates binding to nucleosomes and DNA, but that does not affect catalytic activity in vitro [Cse4 levels, we performed chromatin fractionation in WT and nhp10Δ cells overexpressing CENP-ACse4. Similar to previously reported work, we did not detect a change in total H2A.ZHtz1 levels in the chromatin in the nhp10Δ strain  between the down-regulated genes and those with promoter CENP-ACse4 peaks  as the most significantly enriched transcription factor  in lactic acid media at 23°C and induced for 2 hours with 2% galactose. Yeast strains were constructed using standard genetic techniques. Epitope-tagged proteins were constructed using either a PCR integration technique [Microbial techniques and media were as described , 83. Forechnique  or by thCse4 levels were prepared as described [Protein extracts to check total CENP-Aescribed . Immunobescribed . For allescribed  for Psh1escribed  with thepGAL-3Flag-CSE4 strain. Note that the levels of H2A.ZHtz1 and H2B are somewhat variable between strains. This may be due to differential susceptibility of the cell wall to zymolyase digestion during the chromatin fractionation procedure, which seems to vary between strains. To control for this, we used H2B to determine the level of total chromatin in each condition.Chromatin fractionation assays were performed as described , followe3Flag-Cse4-containing nucleosomes were isolated by ChIP of 3Flag-Cse4 using monoclonal anti-Flag M2 antibodies . ChIPs were performed with Micrococcal nuclease -treated chromatin as described  with theS. cerevisiae reference genome version SacCer3 (Saccharomyces Genome Database (SGD)/UCSC) using the Burrows-Wheeler Aligner (BWA) [Dynamic Analysis of Nucleosome and Protein Occupancy by Sequencing, version 2 (DANPOS2) function Dpos with background subtraction [Cse4 nucleosome throughout the cell cycle [Raw reads  were mapped to the er (BWA) . The reser (BWA) . Mononucer (BWA) , 90–92. traction , and thell cycle , and smoll cycle . The resll cycle , 95. WigCse4, we analyzed the coverage relative to the centromere. Although CENP-ACse4 is constitutively localized to the centromere [Cse4 occupancy in each strain as any region where the CENP-ACse4 enrichment was above the threshold of the minimum average coverage at any centromere in the 3Flag-CSE4 strain using R Bioconductor packages Genomic Ranges, rtracklayer, and the UCSC SacCer3 reference genome [To identify genomic loci enriched for CENP-Antromere , its covntromere . We calle genome , 90–92 ae genome . rDNA Che genome . BrowserSaccharomyces Genome Database (SGD) annotations of the SacCer3 genome were used to call regions of centromeres, pericentromeres, telomeres, origins of replication, genes, and intergenic regions in that order, such that each base was assigned to only the first overlapping region type. To analyze the percentage of peaks from each strain in each genomic region, 1 bp regions at the center of each CENP-ACse4 peak were overlapped with each region so that each peak was counted only once using R Bioconductor packages Genomic Ranges, rtracklayer, and UCSC SacCer3 [Cse4 peaks that either did or did not overlap WT H2A.ZHtz1 peaks.Genomic regions were annotated using the following strategy:  SacCer3 , 90–92. Cse4 and H2A.ZHtz1 enrichment at the starts and ends of genes as well as centered on NDRs, origins of replication, or centromeres using the DANPOS2 profile function [Htz1 ChIP data is from [Htz1 coverage was calculated from the mapped reads with greater than 90% identity using the DANPOS2 function dpos with the default parameters [Cse4 or H2A.ZHtz1 coverage in 10 bp windows was calculated for 500 bp upstream and downstream of 3987 transcripts using custom gene files modified to use experimentally derived TSS data instead of open reading frame (ORF) start sites from Nagalakshmi et al, 2008 (GSE11209) [www.graphpad.com.We analyzed mean CENP-Afunction , 93. H2Arameters , 90–92. SE11209) . For theSE11209) , origin Cse4 peaks for each strain and reported H2A.ZHtz1 peaks (coarse grain nucleosome positions) from wild-type (WT) cells [Coverage data was visualized using IGV , 95. TheT) cells –92.RDS1 promoter are from [Cse4 ChIP, CENP-ACse4 was immunoprecipitated using anti-Cse4 (235N) antibodies [Cse4 expression induced from an inducible tetracycline repressed promoter [CEN3 [SAP4 promoter and the SLP1 promoter.ChIP was performed from 50 ml formaldehyde-crosslinked cultures as described . Chromatare from  and are tibodies  from strpromoter  after a er [CEN3 , the SAP® 2.0 Fluorometer .Total RNA was extracted from each sample using a hot acid phenol extraction protocol , followehttp://support.illumina.com/downloads/bcl2fastq_conversion_software_184.html). Reads of low quality were filtered prior to alignment to the reference genome (UCSC SacCer3 assembly) using TopHat v2.1.0[2 (ratio) | ≥ 0.585 (± 1.5-fold) with the FDR set to 5%. Normalized differential expression data are available as excel files (RNA-seq libraries were pooled (18-plex) and clustered onto a flow cell lane. Sequencing was performed using an Illumina HiSeq 2500 in “rapid run” mode employing a single-read, 50 base read length (SR50) sequencing strategy. Image analysis and base calling was performed using Illumina's Real Time Analysis v1.18 software, followed by 'demultiplexing' of indexed reads and generation of FASTQ files, using Illumina's bcl2fastq Conversion Software v1.8.4 . The number of significantly up or down regulated transcripts overlapped between the genotypes was compared using the hypergeometric distribution  using the total number of genes in the edgeR result files as the total population, using the GeneProf hypergeometric distribution calculator [The overlap between lists of genes with significantly changed transcription compared to WT yeast in lculator .S1 Fig3Flag-CSE4 (SBY10419), psh1Δ 3Flag-CSE4 (SBY10484), pGAL-3Flag-CSE4 (SBY10425) and psh1Δ pGAL-3Flag-CSE4 (SBY10483)) grown on the indicated media at the indicated temperatures. (B) Immunoblot of CENP-ACse4 and H2B levels in MNase-treated. chromatin (input) in the following strains: untagged WT (SBY3), 3Flag-CSE4 (SBY10419), psh1Δ 3Flag-CSE4 (SBY10484), pGAL-3Flag-CSE4 (SBY10425) and psh1Δ pGAL-3Flag-CSE4 (SBY10483). (C) Immunoblot of samples from the ChIP-seq experiment showing 3Flag-Cse4 in the insoluble pellet (Pellet), the MNase-treated input chromatin (Input), the unbound material (after anti-FLAG IP) (Unbound), and the anti-FLAG IP for the following strains: 3Flag-CSE4 (SBY10419), pGAL-3Flag-CSE4 (SBY10425), psh1Δ 3Flag-CSE4 (SBY10484), and psh1Δ pGAL-3Flag-CSE4 (SBY10483). Whole cell extract (WCE) from an untagged strain (SBY3) is also shown as a comparison. (D) Prepared Solexa libraries before sequencing for 3Flag-CSE4 (SBY10419), pGAL-3Flag-CSE4 (SBY10425), psh1Δ pGAL-3Flag-CSE4 (SBY10483) and psh1Δ 3Flag-CSE4 (SBY10484), Both Input (IN) and IP are shown for each strain.(A) 5-fold serial dilutions of ChIP-seq strains ((TIF)Click here for additional data file.S2 FigCse4 centromeric peaks at CEN4, the rDNA locus, SAP4 promoter, SLP1 promoter, and the UTH1 gene. % Input (means +/- 1 SEM) is shown for 2–4 biological replicates. Strains used were: untagged WT , 3Flag-CSE4 , psh1Δ 3Flag-CSE4 , pGAL-3Flag-CSE4  and psh1Δ pGAL-3Flag-CSE4 . (B) Fraction of total centromere like regions (CLRs) or low confidence negative control regions (LCNCRs) [Cse4 ChIP-seq peaks in each strain. (C-F) AT% per CENP-ACse4 ChIP or input nucleosome peak for the 3Flag-CSE4 strain (SBY10419), the psh1Δ 3Flag-CSE4 strain (SBY10484), the pGAL-3Flag-CSE4 strain (SBY10425), or the psh1Δ pGAL-3Flag-CSE4 strain (SBY10483).(A) ChIP-qPCR validation of CENP-A(LCNCRs)  that ove(TIF)Click here for additional data file.S3 FigCse4 ChIP compared to input nucleosome ChIP for 3Flag-CSE4 (SBY10419), psh1Δ 3Flag-CSE4 (SBY10484), or pGAL-3Flag-CSE4 (SBY10425) strains. Genes are binned by basal transcription level [(A-C) TSS and TTS profiles of CENP-Aon level .(TIF)Click here for additional data file.S4 FigCse4 ChIP for 3Flag-CSE4 (SBY10419), psh1Δ 3Flag-CSE4 (SBY10484), or pGAL-3Flag-CSE4 (SBY10425) strains. The 5’ and 3’ ends of genes are binned by direction of upstream or downstream transcription.(A-C) TSS and TTS profiles of CENP-A(TIF)Click here for additional data file.S5 FigCse4 ChIP coverage 500 bp upstream and downstream of all transcription start sites (TSS), for 3Flag-CSE4 (SBY10419), psh1Δ 3Flag-CSE4 (SBY10484), pGAL-3Flag-CSE4 (SBY10425), and psh1Δ pGAL-3Flag-CSE4 (SBY10483) strains separated by the presence of an annotated NDR [Cse4 ChIP coverage for the psh1Δ pGAL-3Flag-CSE4 (SBY10483) strain (left y axis) vs. mean H2A.ZHtz1 ChIP coverage [Cse4 ChIP coverage for 3Flag-CSE4 (SBY10419), psh1Δ 3Flag-CSE4 (SBY10484), pGAL-3Flag-CSE4 (SBY10425), psh1Δ pGAL-3Flag-CSE4 (SBY10483) strains at all origins. (G) Mean CENP-ACse4 ChIP coverage for 3Flag-CSE4 (SBY10419), psh1Δ 3Flag-CSE4 (SBY10484), pGAL-3Flag-CSE4 (SBY10425), and psh1Δ pGAL-3Flag-CSE4 (SBY10483) strains at all centromeres.(A-D) Mean CENP-Aated NDR  within t(TIF)Click here for additional data file.S6 FigCse4 (from psh1Δ pGAL-3Flag-CSE4 (SBY10483)) and H2A.ZHtz1 (from WT strain [Cse4 (from psh1Δ pGAL-3Flag-CSE4 SBY10483) and H2A.ZHtz1 , psh1Δ 3Flag-CSE4 , pGAL-3Flag-CSE4 , psh1Δ pGAL-3Flag-CSE4  strain and WT H2A.ZHtz1 coverage [Htz1 score distributions for each H2A.ZHtz1 nucleosome with (black) or without (blue) overlapping CENP-ACse4 peaks in the 3Flag-CSE4 (SBY10419), psh1Δ pGAL-3Flag-CSE4 (SBY10484), pGAL-3Flag-CSE4 (SBY10425) or psh1Δ pGAL-3Flag-CSE4 (SBY10483) strains. (E) The percentage of CENP-ACse4 peak centers separated by overlap with H2A.ZHtz1 peaks in each type of genomic region is graphed for the psh1Δ pGAL-3Flag-CSE4 strain (SBY10483). See Cse4 peaks in each genomic region.(A) Mean CENP-A(TIF)Click here for additional data file.S7 FigHtz1:H2B fold change vs. the pGAL-3Flag-CSE4 strain. Strains used: pGAL-3Flag-CSE4 3HA-HTZ1 (SBY12832), swr1Δ pGAL-3Flag-CSE4 3HA-HTZ1 (SBY12956), psh1Δ pGAL-3Flag-CSE4 3HA-HTZ1 (SBY12833), and swr1Δ psh1Δ pGAL-3Flag-CSE4 3HA-HTZ1 (SBY12924). n = 3. (B) Immunoblot of a chromatin fractionation experiment from strains as in (A). Pgk1 is a marker of the soluble fraction and H2B is a marker of the chromatin fraction. (C) Stability assay of pGAL-3Flag-CSE4 in WT (SBY12332), vs. swr1Δ (SBY12333) background. (-) samples were taken before adding 2% galactose to induce pGAL-3Flag-CSE4 overexpression. 0–60 minute timepoints were taken after adding 2% glucose and cyclohexamide (50ug/ml) to inhibit transcription and translation of pGAL-3Flag-CSE4. Pgk1 is shown as a loading control. (D) HA-Htz1 ChIP in untagged WT (SBY3), 3HA-HTZ1 3Flag-CSE4 (SBY12918), psh1Δ 3HA-HTZ1 3Flag-CSE4 (SBY13998), 3HA-HTZ1 pGAL-3Flag-CSE4 (SBY12832), psh1Δ 3HA-HTZ1 pGAL-3Flag-CSE4 (SBY12833) at the SAP4 and RDS1 promoters and within the ARE1 gene.(A) Means +/- 1 SEM from quantitative immunoblots of chromatin fractionations measuring H2A.Z(TIF)Click here for additional data file.S8 FigHtz1:H2B fold change vs. pGAL-3Flag-CSE4 strain. Strains used: pGAL-3Flag-CSE4 HA-HTZ1 (SBY12832), nhp10Δ pGAL-3Flag-CSE4 3HA-HTZ1 (SBY12930), psh1Δ pGAL-3Flag-CSE4 3HA-HTZ1 (SBY12833), and nhp10Δ psh1Δ pGAL-3Flag-CSE4 3HA-HTZ1 (SBY12959). n = 3. (B) Immunoblot of chromatin fractionation from the strains in (A). Pgk1 is a marker of the soluble fraction and H2B is a marker of the chromatin fraction. (C) 5-fold serial dilutions of WT (SBY3939), nhp10Δ (SBY11577), psh1Δ (SBY8336), psh1Δ nhp10Δ (SBY12346), pGAL-3Flag-CSE4 (SBY12349), nhp10Δ pGAL-3Flag-CSE4 (SBY12317), psh1Δ pGAL-3Flag-CSE4 (SBY12350), nhp10Δ psh1Δ pGAL-3Flag-CSE4 (SBY12348) strains on indicated media. (D) Immunoblot of CENP-ACse4 levels when induced with low levels of galactose (0.1%) in psh1Δ pGAL-3Flag-CSE4 3HA-HTZ1 (SBY12833) and nhp10Δ psh1Δ pGAL-3Flag-CSE4 3HA-HTZ1 (SBY12959) cells. Pgk1 is shown as a loading control. (E) Stability assay of pGAL-3Flag-CSE4 in wild-type (SBY12349) vs. nhp10Δ (SBY12317) background. (-) samples were taken before adding 2% galactose to induce pGAL-3Flag-CSE4 overexpression. 0–60 minute timepoints were taken after adding 2% glucose and cyclohexamide (50ug/ml) to stop transcription and translation of pGAL-3Flag-CSE4. Pgk1 is shown as a loading control.(A) Mean +/- 1 SEM from quantitative immunoblots of chromatin fractionations measuring H2A.Z(TIF)Click here for additional data file.S9 FigUTH1, a single copy gene. Graph shows the mean ratio +/- 1 SEM for 3 biological replicates. Strains used were: 3Flag-CSE4 (SBY10419), psh1Δ 3Flag-CSE4 (SBY10484), pGAL-3Flag-CSE4 (SBY10425), psh1Δ pGAL-3Flag-CSE4 (SBY10425). (B) rRNA expression analysis compared to ACT1 at t0 and t2 for strains used in the RNA-seq experiment (see psh1Δ pGAL-3Flag-CSE4. (D) Proportional Venn diagram of genes upregulated in psh1Δ pGAL-3Flag-CSE4 at t = 2 hours and genes with CENP-ACse4 peaks in their promoters in the psh1Δ pGAL-3Flag-CSE4 strain. p = 0.009 from a cumulative hypergeometric distribution test. (E) TSS and TTS profiles of CENP-ACse4 ChIP coverage in the psh1Δ pGAL-3Flag-CSE4 (SBY10483) strain at all genes (blue), upregulated differentially expressed (DE) genes in psh1Δ pGAL-3Flag-CSE4 only (red), upregulated DE genes in htz1Δ only (green), or upregulated DE genes in both psh1Δ pGAL-3Flag-CSE4 and htz1Δ (purple).(A) rDNA copy number ratio comparing the rDNA to ment see . Graph s(TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S5 Table(PDF)Click here for additional data file.S1 File(XLSX)Click here for additional data file.S2 File(XLSX)Click here for additional data file.S3 File(PDF)Click here for additional data file.


Pseudomonas syringae pv. tomato DC3000. The alternative sigma factor HrpL directly regulates expression of T3SS genes via a promoter sequence, often designated as the “hrp promoter.” Although the HrpL regulon has been extensively investigated in DC3000, it is not known whether additional regulon members remain to be found. To systematically search for HrpL-regulated genes, we used chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-Seq) and bulk mRNA sequencing (RNA-Seq) to identify HrpL-binding sites and likely hrp promoters. The analysis recovered 73 sites of interest, including 20 sites that represent new hrp promoters. The new promoters lie upstream of a diverse set of genes encoding potential regulators, enzymes and hypothetical proteins. PSPTO_5633 is the only new HrpL regulon member that is potentially an effector and is now designated HopBM1. Deletions in several other new regulon members, including PSPTO_5633, PSPTO_0371, PSPTO_2130, PSPTO_2691, PSPTO_2696, PSPTO_3331, and PSPTO_5240, in either DC3000 or ΔhopQ1-1 backgrounds, do not affect the hypersensitive response or in planta growth of the resulting strains. Many new HrpL regulon members appear to be unrelated to the T3SS, and orthologs for some of these can be identified in numerous non-pathogenic bacteria. With the identification of 20 new hrp promoters, the list of HrpL regulon members is approaching saturation and most likely includes all DC3000 effectors.The type III secretion system (T3SS) is required for virulence in the gram-negative plant pathogen  Pseudomonas syringae pv. tomato DC3000 (DC3000), an important model pathogen in molecular plant pathology, causes bacterial speck disease in ArabidopsisNicotiana benthamiana (DC3000 mutants lacking virulence determinant HopQ1-1) hypersensitive response and pathogenicity (hrp) and hrpconserved (hrc) gene clusters hrpouter protein genes (hop) P. syringae strains were available. Experimental approaches included screening cosmid libraries for gain-of-function avirulence phenotypes hrpZ was recognized to support HrpL-dependent transcription hrp promoters associated with pathogenicity hrp promoter. Another high-throughput screen, also based on promoter trapping, identified 29 T3SS proteins in DC3000 hrpL mutant hrp promoters, and has been considered to be nearly complete or exhaustive P. syringae strains, resulting in the identification of 14 new potential regulon members in DC3000 Efforts to identify genes involved in virulence and pathogenicity were initiated well before the genome sequences of DC3000 and other in planta growth of D28E, a T3SS+ DC3000 derivative from which the 18 clustered effectors and 10 additional effectors were deleted, is significantly reduced relative to a T3SS– control strain hrp promoter, such as the one upstream of iaaLAlthough multiple approaches have been used to characterize the DC3000 HrpL regulon, several factors suggest that additional members may yet be found. First, inventory strategies have emphasized the identification and functional characterization of effectors, and thus non-effector genes that are important to bacterial growth and survival may have been overlooked. An example of a gene in this class is PSPTO_0834  hrp promoters and their activation by HrpL. The experiments identified most known members of the regulon  and promoter activity was tested using transcriptional fusions and quantitative reverse transcription-PCR (qRT-PCR). Computational analyses were used to search for additional members of the HrpL regulon in DC3000 and to investigate the conservation of HrpL regulon members and potential hrp promoters within the available Pseudomonadales genomes. This analysis revealed that some new HrpL regulon members are widely represented in both pathogenic and non-pathogenic bacteria. Finally, we conducted a translocation assay to demonstrate that one new HrpL regulon member, PSPTO_5633 (designated HopBM1), is translocated into plant cells in a T3SS-dependent manner.Here, we use chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-Seq), and a modified RNA-Seq protocol (incorporating the capture of mRNA 5′-ends), to provide evidence for direct binding of HrpL at hrp-minimal medium) Escherichia coli TOP10 (Invitrogen) was used as the host for sub-cloning and other plasmid manipulations. E. coli was grown at 37°C in Luria-Bertani (LB) DC3000 and its derivatives were grown at 28°C in MG medium hrp-inhibiting (KB medium) to hrp-inducing (MG supplemented with ferric citrate) conditions were carried out as follows. Colonies of hrpL-FLAG  and ΔhrpL were obtained from KB plates that had been incubated for 48 hours. Cells were re-suspended and grown overnight in 250 ml KB at 28°C with shaking at 250 rpm. Cultures were pelleted by centrifugation and washed in MG. Washed cells were re-suspended in MG and inoculated into bioreactors  containing 400 ml MG medium supplemented with ferric citrate (Sigma-Aldrich) to a final concentration of 50 µM as described previously Routine bacterial growth and medium shift experiments from Suicide vectors for gene deletions, single-crossover insertions or other purposes were introduced into DC3000 backgrounds using electroporation (hrpL) gene were amplified by primers oSWC04110/oSWC04112 and oSWC04114/oSWC04116  to confirm successful tagging.Regions flanking the PSPTO_1404 PSPTO_5633, PSPTO_0371, PSPTO_2130, PSPTO_2691, PSPTO_2696, PSPTO_3331, and PSPTO_5240. Regions flanking the gene of interest, designated flank A and flank B, were amplified from DC3000 genomic DNA by two primer pairs (see PSPTO_2691), and ligated with XbaI (or BamHI) digested pK18mobsacB. The resulting constructs were sequenced to confirm correct structure using primers M13F, M13R, and the two primers flanking the deletion region. The deletion constructs were introduced into DC3000, and ΔhopQ1-1 by electroporation. Merodiploid intermediates were selected for growth on medium containing kanamycin. Recombinants that had eliminated pK18mobsacB plasmid sequences were identified by sucrose counter-selection. Sucrose-resistant, Kan-sensitive colonies were screened by PCR using two primers flanking the deletion region. Mutants were confirmed by Sanger sequencing using four to six primers covering the manipulated region . The pBS181 plasmid was constructed by Gateway LR recombination (Invitrogen) between the pBS62 entry vector and the pBS180 destination vector. The pBS180 destination vector was constructed by replacing the lux operon of pBS59 iucD gene. The iucD open reading frame was PCR amplified from pENTR-Gus (Invitrogen) using oSWC750 and oSWC751, digested with HindIII and ligated with the 8.6 kb fragment of HindIII digested pBS59, generating pBS180. The hrpJ promoter region was PCR amplified from DC3000 genomic DNA using oSWC463 and oSWC464 and cloned in pENTR/D-topo (Invitrogen) to yield pBS62. The hrpJ promoter iucD fusion was then constructed by LR reactions between pBS62 and pBS180 to yield pBS181.The plasmid pBS181encodes a transcriptional fusion of the gfpmut2 gene was amplified from pTGS gfpmut2 start codon by 8 base pairs (bps). The gfpmut2 PCR product was digested with EcoRI and ligated to similarly digested pUCP24, yielding pBS12. pHL1 was constructed to function as a destination vector compatible with Gateway cloning. The Gateway cassette from plasmid pBS46 gfpmut2, and antibiotic resistance genes for tetracycline and kanamycin resistance. To test for the presence of hrp promoters, fragments of DNA (150 bps to 200 bps) containing candidate promoters were cloned from DC3000 genomic DNA using the Expand High Fidelity PCR system . The forward primers have four additional bases (CACC) for compatibility with TOPO cloning vectors. PCR fragments were cloned into pENTR/SD/D by directional TOPO cloning and were subsequently used to generate the gfp reporter constructs by LR reaction with the pHL1 destination vector  for 1 hour at room temperature. Proteins were detected after adding BCIP/NBT substrate (Sigma Aldrich) at room temperature.Proteins were resolved in a precast 4–20% polyacrylamide gel (Bio-Rad) and transferred to a PVDF membrane (Millipore) by electrophoresis. The membrane was then blocked in 5% non-fat milk for 2 hours at room temperature, and incubated with anti-FLAG M2 antibody (Sigma-Aldrich) for 1 hour at room temperature with gentle shaking. The membrane was washed three times in TBST buffer  and incubated with Alkaline Phosphatase conjugated 2g and washed twice in ice cold TBS. Washed pellets were stored at –70°C until processed. To lyse cells, 1 ml of CelLytic B (Sigma-Aldrich), supplemented with 10 µl LongLife Lysozyme  and 10 µl PMSF , was thoroughly mixed with thawed pellets by vortexing. Cells were incubated at 37°C for 10–15 minutes, and then disrupted by sonication . Continuous pulse power at 15% power was used to produce fragments of size around 300 bps. For each ChIP-Seq sample, 40 µl of ANTI-FLAG M2 Affinity Gel (Sigma-Aldrich) was pre-washed in cold TBS, added to the bacterial lysates, and incubated with gentle shaking at 4°C for 2 hours. Unbound DNA fragments were eluted using two washes with TBS. Note that hrpL-FLAG samples were prepared following the ChIP-exo protocol that includes specialized enzyme treatment and library preparation procedures , while the ΔhrpL samples were eluted, reverse cross-linked (below) and directly submitted for high-throughput sequencing without additional manipulation as detailed previously ChIP-exo and ChIP-Seq were performed as described hrpL-FLAG samples were treated using methods adopted from Rhee et al.For ChIP-exo, resin-bound DNA from the End polishing was accomplished using 4.5 U of T4 DNA polymerase, 200 µM dNTPs and 50 µg/ml BSA at 12°C for 30 minutes. This reaction generates blunt end DNA fragments.P2 adaptor , followed by two washes in ice cold TBS. DNA-protein complexes were eluted from resin using FLAG peptide (Sigma-Aldrich) at a final concentration of 150 ng/µl in 100 µl TBS, with slow shaking for 30 minutes. Supernatants were collected by centrifugation through the spin-X columns and saved as immunoprecipitated (IP) samples. To reverse formaldehyde crosslinking, IP samples were pre-incubated with 90 µl ChIP elution buffer  and 10 µl of protease (Sigma-Aldrich) (40 mg/ml in TBS) for 2 hours at 42°C, followed by 6 hours at 65°C. Qiagen PCR-purification spin columns were used to purify DNAs.To prepare ChIP-exo libraries for high-throughput sequencing, samples were incubated at 95°C for 5 minutes to denature double stranded DNAs. Primer P2  was added, allowed to anneal for two minutes at 30°C, and extended using 10 U of phi29 polymerase at 30°C for 20 minutes. The polymerase was inactivated by incubation at 65°C for 10 minutes. Blunt-end ligation of the P1 adaptor (10 uM) was accomplished using T4 DNA ligase 1000 U at 25°C for 60 min, followed by incubation at 65°C for 10 minutes to inactivate the ligase. DNA was purified using Agencourt AMPure magnetic beads and amplified by PCR using DNA polymerase Phusion in 18 PCR cycles. Finally, samples were purified using 80 µl Agencourt AMPure magnetic beads (Beckman Coulter Genomics), following the manufacturer’s instructions, and eluted to a final volume of 30 µl.Total RNA was prepared using an RNeasy Kit (Qiagen) following the manufacturer’s instructions, using the optional on-column DNaseI digestion. RNA was treated twice with DNase I (Ambion) to remove residual DNA and then cleaned and concentrated using RNA cleanup and concentrator-5 (Zymo Research). Integrity of the RNA was assessed using the Agilent Bioanalyzer .4 to prevent circularization before ligation of an RNA oligonucleotide  to RNA 5′-ends. The ligation reaction was incubated overnight at 16°C. Products were recovered using RNA cleanup and concentrator-5 (Zymo research) and eluted in 10 µl H2O.Ribo-Zero rRNA Removal Kit (Epicenter) was first used to remove ribosomal RNA (rRNA). RNAs were then treated with Terminator 5′-Phosphate-Dependent Exonuclease (Epicentre), as described previously Stranded RNA-Seq libraries were prepared using the ScriptSeq v2 RNA-Seq Library Preparation Kit (Epicentre) and 500 pg to 50 ng rRNA-depleted RNAs following the manufacturer’s protocol. Briefly, RNAs were fragmented and cDNA synthesis was carried out using random-sequence primers containing a tagging sequence at their 5′-ends. The 3′-ends were tagged using the Terminal-Tagging Oligo. Di-tagged cDNAs were purified using magnetic beads and subjected to a limited-cycle PCR as recommended (10–15 cycles). Samples were indexed for multiplexing using the appropriate primers. Libraries were purified using the AMPure XP system (Beckman Coulter) in 20 µl total volume. Library quality was assessed using the Agilent Bioanalyzer. Sequencing was performed on the Illumina HiSEQ2000 by the Cornell DNA Sequencing Core Facility.For RNA-Seq data, reads bearing the unique 18 nucleotide sequence (tag) at the 5′ end were first identified and separated from the set of all RNA-Seq reads. The tag was then removed from each read, leaving the nucleotide sequences derived from the original RNAs. Thereafter, the de-tagged RNA-Seq reads (5′ capture data), the untagged RNA-Seq reads and reads obtained from the ChIP-exo procedure (ChIP-Seq data) were handled in the same way. Quality scores for the sequence reads were accessed using FastQC Reads that aligned perfectly to a single location were retained and all others were discarded. The “sinister profiles” are histograms representing the number of trimmed reads whose 5′-ends uniquely map to each position Enriched regions (those overrepresented in the ChIP-Seq data set) were identified using Genetrack ΔhrpL mutant was also computed to present enrichment attributable to unspecific binding.The normalized number of sequence reads associated with each site enriched by ChIP-Seq was also computed. The Un-normalized ‘peak’ height is an average of reads on both chromosomal strands in a window of 30 bps centered at ChIP-Seq ‘peak’. The background height was average of reads on both chromosomal strands in 2 windows of 30 bps that are 400 bps upstream and downstream of the ChIP-Seq ‘peak’. This background height was used to normalize the corresponding un-normalized ‘peak’ height. The normalized value on Sequences of length 40–50 bps (FASTA format) upstream from captured 5′ transcription start sites were used as input to MEME -dna -mod anr –nmotifs 20 -minw 14 -maxw 35 -maxsize 150000The MEME package also generates sequence logos for each sequence pattern detected .hrp promoters as a training set  ning set . DNA seqrom NCBI . Prodigapromoter . The sam signals . Custom gyrA and gap1, were the internal and negative controls, respectively, for all tested regions. To test for transcript abundance, regions downstream of mapped transcriptional start sites were amplified. Transcript levels for each region  were calculated relative to the level for the housekeeping gene gyrA.cDNA synthesis was accomplished using qScript cDNA Synthesis  and random primers as part of the kit. qPCR steps were performed using iQ SYBR Green Supermix (Bio-Rad). Primer pairs  were selected to amplify a region of approximately 100 bps (Beacon Designer). For evaluating enrichment of ChIP-Seq binding sites, the primers amplified within the putative enriched regions. Enrichment (fold change) was calculated for DNA recovered at each tested binding site in the immune-precipitated (IP) sample compared to that in the lysate sample. The housekeeping genes, Solanum lycopersicum or Nicotiana benthamiana plants were germinated and grown in a greenhouse with approximate 16/8 hr. light/dark cycles. Four to five week old tomato plants or two to three week old N. benthamiana plants were inoculated with a 3×104 CFU/ml bacterial suspension using blunt syringe infiltration. Bacteria were recovered from plants by sampling leaf tissue at the site of infection using a #2 disk punch  at 2 days post infection (dpi), 4 dpi and 6 dpi. Leaf disks were homogenized by mechanical disruption in 700 µl of 10 mM MgCl2. Serial dilutions of the tissue homogenate were plated on KB agar supplemented with rifampicin and the number of colony forming units per milligram leaf tissue was calculated.7 CFU/ml for DC3000 derivatives unless otherwise noted. HR was observed and documented by photography after 2 days.Inoculation was performed as described above for virulence assays except the inoculum concentration was 3×102, and adjusted to an OD600 of 0.05 (∼5×107 CFU/ml). Bacterial suspensions were inoculated into N. benthamiana leaves using a blunt tip 1 ml syringe and plants were placed on the lab bench. After 6 hours, 2 leaf discs per sample were excised using a 1 cm-diameter cork borer and frozen in liquid nitrogen along with 2 copper-coated beads . The leaf discs were finely ground by vigorous vortexing and resuspended in 300 µl of 0.1 M HCl. The leaf extract was centrifuged at 6500 rpm for 10 minutes, and the supernatant was transferred to a fresh tube. A 10-fold dilution was prepared in 0.1 M HCl and used for the Cya assay since it was observed that diluting the extract resulted in greater sensitivity. Total pmol cAMP in each sample was determined using the Direct cAMP ELISA kit (Enzo Life Sciences) following the manufacturer’s instructions. Three plants were inoculated in each experiment and the samples were analyzed in 2–5 independent experiments.Translocation assays were performed as described previously hrpL locus at its native position in DC3000 chromosome  is required for DC3000 virulence. To perform a global inventory of genomic sites likely to bind this sigma factor, we tagged HrpL with a FLAG epitope at the C-terminus by modifying the romosome  and perfromosome  and estaromosome . Resultsimepoint .et al.hrpL strain were prepared using a conventional ChIP-Seq protocol HrpL-FLAG sample to determine levels attributable to non-specific binding.Using the high-resolution ChIP-exo procedure described by Rhee hrp promoters , particularly in the –35 region (hrpL-FLAG strain (ranging from 4.7 for P_922939 to 502.0 for P_5384493) are typically much larger than the corresponding values from the ΔhrpL strain . The resemblance of the candidate promoters to the canonical hrp promoter consensus sequence, together with the evidence that HrpL binds at their genomic locations suggests that they are genuine HrpL-responsive promoters.Because of the high resolution afforded by ChIP-exo, we were able to align enriched sequences and identify by inspection a conserved motif that resembles the accepted sequence for the HrpL-responsive promoter, GGAAC(–35)-N5 region . The reg5 region . All enrActive promoters should be associated with nearby transcription start sites (TSSs). Using high-throughput sequencing methods, we mapped TSSs by modifying a stranded RNA-Seq protocol to enrich for primary mRNA transcripts  hrpL-FLAG sample and 1472 sequences from the ΔhrpL sample (overlapping sequences within a sample were merged) and used the sequences as input to MEME hrpL-FLAG sequences (20- TANaaT-3′) 15-GcCGAta-3′) hrp promoter  10-TGC-3′) ΔhrpL samples, motif 1 (RpoD) and motif 2 (RpoF). The putative RpoF promoters derived in both cases are upstream of genes likely to be regulated by this sigma factor, such as fliC (PSPTO_1949), fliD (PSPTO_1951), flgK (PSPTO_1944), cheY-2 (PSPTO_1980) and cheA-2 (PSPTO_1982) . As expected, the HrpL motif was identified in the hrpL-FLAG but not the ΔhrpL samples.To identify conserved motifs associated with TSS, the 1500 tagged ends with the most abundant reads were selected for analysis. We extracted 50 nucleotides directly upstream from each captured 5′-end, resulting in 1451 sequences derived from the equences . These iet al.hrp promoter) using the similar MEME parameters but different DC3000 culture conditions and sample preparation methods. However, in agreement with that report, we also observed that captured ends tend to occur in tightly spaced clusters . Although these promoters may simply be inactive, it is also possible that the TSSs are undetectable because they are rapidly degraded in the cell or during sample processing. Previous experimental evidence suggests that all four of these hrp promoters are genuine hrp promoter candidate, between PSPTO_3948 and PSPTO_3949, is approximately 30 bps distant from the closest TSS. Validation tests suggest that this promoter supports HrpL-dependent transcription (below). In summary, all 20 candidate hrp promoters are associated with regions enriched by ChIP-Seq and a plausible hrp promoter motif; most are positioned close to captured 5′-ends (between 0–7 bps). It is therefore likely that these represent bona fide HrpL-dependent promoters.The overall number of detected promoter motifs contrasts with results reported by Filiatrault hrp promoters discussed above were analyzed further using ChIP-qPCR, reporter fusions and qRT-PCR to confirm HrpL binding, promoter function and HrpL-dependent transcription.The twenty Cells for ChIP-qPCR were cultured and collected independently from those used for ChIP-Seq. Analysis of the DNA isolated by immunoprecipitation confirmed enrichment at 19 out of 20 targets . The exchrp-inducing medium compared to KB . A hrp promoter motif at this location was identified in an earlier global transcriptional map A plasmid-based reporter trap assay was designed to test promoter activity at defined chromosomal regions and to isolate promoter activity from potentially confounding effects such as read-through from transcription initiated further upstream. Genomic fragments of 100 to 200 nucleotides containing candidate promoters were used to construct transcriptional fusions with a green fluorescent protein (GFP) reporter. Although the basal expression of each promoter was different on KB, all twenty regions showed strong induction in ed to KB . P_39290hrp promoters in their native genomic context. Relative transcript abundance for candidates was computed by comparing relative transcript levels at 1.5 hours after shifting bacterial cells from KB to MG (iron supplemented) medium, normalized to mRNA levels for gyrA, a housekeeping gene  showed the largest induction in this experiment (>1000x), with four others showing levels ≥100x. P_2012122 (upstream of PSPTO_1843) shows only small differences in mRNA levels between the two backgrounds. P_4199618, upstream of PSPTO_3721, showed no significant induction using qRT-PCR but did exhibit HrpL-dependent behavior in the reporter trap assay. This promoter may depend on other factors for transcription or may ordinarily function at a very low level of activity.A qRT-PCR assay was used to assess HrpL-dependent transcription in regions downstream from candidate ing gene . Most prperiment . When thhrp promoter candidate is in an intragenic region downstream of PSPTO_4750  but is oriented in the antisense direction. As shown in hrp-inducing compared to KB rich medium.P_5384493 was verified in all experiments. This hrp promoters, we scanned the DC3000 genome using a hidden Markov model trained using previously annotated and new hrp promoter sequences . As a further precaution, the E-value threshold for a match was reset to 0.01, an even more relaxed level that predicts 424 hrp promoters in the DC3000 genome. Among matches that are unassociated with confirmed promoters, five are in regions where ChIP-Seq shows weak enrichment (hrp promoter-like sequence upstream of PSPTO_0816 (Type IV pilus biogenesis protein) was examined more closely. This candidate shows no significant enrichment using ChIP-qPCR, and does not demonstrate HrpL-dependent transcription using qRT-PCR (data not shown). However, a transcriptional promoter fusion involving this region exhibits a 10.9 fold induction in hrp-inducing medium . In order to determine whether the new genes contribute to pathogenicity, we constructed deletion mutants (in WT DC3000 and ΔhopQ1-1 backgrounds) for seven candidates whose annotated functions were suggestive of plant association and examined them for an altered virulence phenotype in N. benthamiana . No phenotypic differences were observed for in planta growth, virulence, or HR for any strain (data not shown). The result for PSPTO_2130 is consistent with another analysis reported for this gene The annotated functions of the genes associated with the new P. syringae pv. maculicola, Erwinia tracheiphila, Citrobacter rodentium, Burkholderia phymatum, Xanthomonas campestris and Yersinia mollaretii), which is frequently the case for an effector. However, the TEREE  score for this gene is –2 hrp promoter motif, aligned with the ChIP-Seq, RNA-Seq and 5′-capture profiles in this genomic region.Although PSPTO_5633 has no annotated function, it shares high sequence similarity with hypothetical proteins in some pathogenic bacterial species . The level of cAMP observed using a T2SS–strain was indistinguishable from that seen with DC3000  is defined by NCBI in their taxonomy database (http://www.ncbi.nlm.nih.gov/taxonomy). We identified orthologs to the DC3000 genes immediately downstream from the 73 hrp promoters and then examined the DNA sequences upstream from them for patterns matching the hrp promoter (P. syringae group genomes contain HrpL orthologs. hrp promoter motifs are observed upstream of most other regulon orthologs (bright red squares) including those corresponding to the new regulon members (shown with names on a green background). Genes for core T3SS functions are widely shared across the group, whereas orthologs for the DC3000 effector genes are not as conserved, reflecting effector diversity. About half of the new regulon members are as conserved as the core T3SS genes. Others (such as PSPTO_5633 and PSPTO_3481) exhibit a different pattern and are found only in DC3000 and a few other P. syringae genomes , most of which are neither plant pathogens nor contain HrpL orthologs , classified by Ferreira et al.hrp promoter was also predicted within PSPTO_1369 (shcN) upstream of PSPTO_1370 (virulence factor HopN1) hrp promoter upstream of PSPTO_1369, where HrpL binding is very clearly observed  in an earlier 5′-capture experiment All previously reported observed . Finallyhrp promoters that had been noted previously but described as weakly supported. A putative promoter upstream of PSPTO_1645, reported by Ferreira et al.hrp promoter associated with it. However, we detected HrpL binding, a captured TSS, and a hrp promoter motif upstream from this gene. PSPTO_2691 was also recently identified as a HrpL regulon member by Mucyn et al.hrp promoter upstream of the hopAT1 pseudogene (PSPTO_5618) was noted in We found evidence for three et al.et al., are not in common. A likely explanation for the disagreement is that Mucyn et al. optimized their experiments to identify genes that respond to HrpL either directly or indirectly, and employed a cloned hrpL gene under the control of an arabinose-inducible promoter on a multi-copy plasmid. Some genes identified in this fashion would be expected to have no associated HrpL-binding activity or hrp promoter motifs in their upstream regions. This appears to be true for the 12 candidates reported by Mucyn et al. that are not in agreement  is secreted by the T1SS and degrades flagellin monomers et al. In aprI is downstream from a functional hrp promoter and is therefore a member of the HrpL regulon. The identity of the promoter is strongly supported by several lines of evidence . It is possible that the target for AprI in the periplasm is another protease unrelated to AprA. However, deletions in aprI in either DC3000 or ΔQ1-1 backgrounds exhibit no growth or virulence phenotypes in planta, leaving the role of AprI a mystery. AprI orthologs are widespread in the P. syringe group . We suggest however that evidence for HrpL binding, the presence of a motif, and demonstrated transcriptional activity together best define the regulon in practical terms. The remaining challenge is to determine what roles, if any, the new regulon members play in the process of pathogenesis.Figure S1Likely promoter motifs recovered by MEME using 5′-end capture data from hrpL-FLAG and ΔhrpL cells.(TIF)Click here for additional data file.Figure S2qRT-PCR analysis showing HrpL-dependent transcription downstream from 38 known HrpL regulon members. Relative transcript change was compared between DC3000 and ΔhrpL strains. Relative induction of each gene was normalized to the housekeeping gene gap1. No transcription induction was observed in KB, while significant induction was seen after medium shift to MG supplemented with iron  after 1.5 hr. Values are averages of three replicates with standard deviations.(TIF)Click here for additional data file.Figure S3ChIP-Seq and RNA-Seq data for selected HrpL regulon members. The red line represents mapped reads corresponding to the positive strand and the green line shows reads mapped to the negative strand. Genome annotation is shown below profiles. The sequence containing the hrp promoter motif is represented by green boxes. The 5′ capture profile from Filliatrault et al.(TIF)Click here for additional data file.Figure S4Ortholog inventory of HrpL regulon in P. syringae group. Green represents newly found members; black represents previously annotated regulon members. The values shown represent counts of orthologs of HrpL regulon members across 121 species.(TIF)Click here for additional data file.Table S1All primers used in this study.(XLSX)Click here for additional data file.Table S2hrp promoter motif sequences included in the HMM training set.(DOCX)Click here for additional data file.Table S3Orthologs and hrp promoter motif scores for HrpL regulon members in 1060 Pseudomonadales genomes. A blank (white) cell indicates that no ortholog was detected. “ =  = ” indicates that an orthologous gene was identified but no upstream sequence could be extracted (due to incomplete sequence information and segmentation in draft genomes). For cases in which orthologs were detected and upstream sequences recovered, the color represents the –logarithm (base 10) of the HMM E-value for the best motif matching the hrp promoter model in the upstream sequence. A continuous color scheme is used where blue represents a poor match , dark red indicates an intermediate match , and bright red indicates a good match . Most verified HrpL promoters in DC3000 match with values above 3. The leftmost gene column represents orthologs for the HrpL sigma factor, PSPTO_1404. In DC3000, this sigma factor is transcribed from a RpoN-responsive promoter hrp promoters are shown in columns, as they appear in CEL, hrp/hrc cluster, followed by type III effectors, chaperones and helpers, and non-type III function genes. Newly found members are in green background. 3-color scale is used: Color: Blue …… dark red …… light red Value: 0…….…………2………….…….5.(XLSX)Click here for additional data file.Table S4Additional hrp promoter-like sequences with limited experimental support.(DOCX)Click here for additional data file.Table S5Data for all hrp promoters.(DOCX)Click here for additional data file.Dataset S1S1.1 to S1.6. Artemis-loadable profiles for the DC3000 chromosome, plasmid A and Plasmid B. These can be used to visualize results for ChIP-exo of hrpL-FLAG, t = 1.5 hours (dataset S1.1 to S1.3); ChIP-Seq of ΔhrpL, t = 1.5 hours (dataset S1.4 to S1.6), respectively.(ZIP)Click here for additional data file.Dataset S2S2.1 to S2.6. Artemis-loadable profiles for the DC3000 chromosome, plasmid A and Plasmid B. These can be used to visualize results for RNA-Seq of hrpL-FLAG, t = 1.5 hours (dataset S2.1 to S2.3); ChIP-Seq of ΔhrpL, t = 1.5 hours (dataset S2.4 to S2.6), respectively.(ZIP)Click here for additional data file.Dataset S3S3.1 to S3.6. Artemis-loadable profiles for the DC3000 chromosome, plasmid A and Plasmid B. These can be used to visualize results for 5′ capture (TSSs) of hrpL-FLAG, t = 1.5 hours (dataset S3.1 to S3.3); ChIP-Seq of ΔhrpL, t = 1.5 hours (dataset S3.4 to S3.6), respectively.(ZIP)Click here for additional data file.Dataset S4S4.1, S4.2. gff files containing positions of 1500 top peaks in 5′ capture data of hrpL-FLAG and ΔhrpL samples.(ZIP)Click here for additional data file.Dataset S5gff file containing new hrp promoters in DC3000.(ZIP)Click here for additional data file.Dataset S6gff file containing results obtained from the HMM scan (p = 0.001) for hrp promoter motifs in the DC3000 genome.(ZIP)Click here for additional data file.Dataset S7MEME analysis of sequences upstream from captured 5′-ends using RNA-Seq data from hrpL-FLAG cells at t = 1.5 hours.(ZIP)Click here for additional data file.Dataset S8MEME analysis of sequences upstream from captured 5′-ends using RNA-Seq data from ΔhrpL cells at t = 1.5 hours.(ZIP)Click here for additional data file.


Cd14 expression was associated with this background specific susceptibility to intestinal inflammation. Polymorphisms of the Cd14 promoter were found to be likely causative for strain specific expression, and Cd14-knockout mice revealed a protective role of this gene-product in experimental IBD. In this study, luciferase reporter assays confirmed an increased activity of the C3Bir derived Cd14 promoter compared to the one of B6. Promoter truncation experiments and site-directed mutagenesis in both strains resulted in reduced Cd14 promoter activity and confirmed that a central AP1 and the proximal SP1 transcription factor binding sites mediated the basal activity of the Cd14 promoter in the mouse. Moreover, a T to C exchange at position -259 replaced putative STAT1 and CDX1 sites in the Cd14 promoter from B6 by a SP2 site in C3Bir. Ablation of the Sp2 site through truncation was associated with a decreased promoter activity. Site-directed mutagenesis also demonstrated that the inactivation of SP2 led to a substantial loss of promoter activity in C3Bir. Performing electrophoretic mobility shift and supershift assays demonstrated interaction of SP2 with its potential binding site. In addition, retroviral—mediated overexpression of the SP2 transcription factor in primary bone marrow macrophages derived from C3Bir mice caused a significant increase in Cd14 transcription. These data characterized SP2 as important factor responsible for higher Cd14 expression and reduced IBD susceptibility mediated by the C3Bir allele.Genetic analysis in the IL10-deficient mouse model revealed a modifier locus of experimental inflammatory bowel disease (IBD) on chromosome 18, with the allele of the strain C3H/HeJBir (C3Bir) conferring resistance and the allele of C57BL/6J (B6) conferring susceptibility. Differential  CD14 was identified as a marker and modifier gene in human IBD   and beta actin with F(ab’) 2 rabbit anti mouse IgG [HRP]  as secondary antibodies, luminescence of the different bands was activated by using Roti-Lumin 1 and 2 solution (Carl Roth). Detection of the bands was performed by x-ray film The two cell lines were tested for their basal Cd14 full-length promoters of C3Bir respectively B6 were served as templates .Cd14 expression, the full-length promoter constructs of both strains were truncated to map regions essential for transcription regulation.To figure out which binding sites were associated with the strain specific Cd14 to -722/+199 increased promoter activity significantly sixfold to more than 3 x 105 associated with the loss of OCT1 binding sites. Therefore, the distal part of the promoter contained a stimulating element. Further truncation to -844/+199 did not change the reporter expression -like 2) in rat alveolar macrophages after transient tranfection in vitro increased the Cd14 protein level while the expression of an anti-NRF2 siRNA accomplished the opposite effect [Truncation experiment revealed an inhibitory element in the distal -1067 to -977 region of the B6 allele containing an OCT1 binding site that was replaced by a CUX1 binding motif in the C3Bir allele through a T to C exchange at position -1041. Mutagenesis of this OCT1 consensus sequence demonstrated clearly that the OCT1 site has activating functions. The stimulating effects of OCT1 were already shown on B cells and in the expression of onocytes ,41. There effect . NRF2 sue effect . Moreovee effect  suggestiCd14 promoter region from the B6 allele containing the STAT1/CDX1 transcription factor binding site between -298 and -181 as well as after the mutagenesis of these binding motifs the reporter expression was diminished emphasizing the stimulatory function of STAT1 or CDX1. The transcription CDX1 was characterized as an intestine-specific homeoprotein but in contrast to the hypersensitivity to DSS-induced intestinal inflammation after the deletion of the closely related factor CDX2 the ablation of CDX1 has no effect of colitis susceptibility [Cd14 expression was mediated by STAT1 in human moncytic cells [Following elimination of the tibility , 46. On ic cells  indicatiCd14 allele derived from C3Bir the STAT1/CDX1 consensus sequences were disrupted creating a new SP2 binding motif introduced through a T to C substitution at position -244. We showed that the depletion of the SP2 element from the promoter by truncation and mutation led to a significant transcriptional down-regulation. Moreover, EMSA and supershift experiments proofed the protein-DNA interaction between SP2 and its C3Bir specific binding site [CD14 in monocytes and hepatocytes [Cd14 expression compared to untreated cells. Also Bösinger and colleagues found that the retroviral infection with HIV correlated with a lower expression of Cd14 and TLR4 [Cd14 transcription compared to wild type BMMs. Concerning the low binding affinity of SP2 to DNA [Cd14 promoter allele of C3Bir. While the SP1 and SP3 bind the same target gene sequences mediated by conserved carboxy-terminal zinc finger domains, SP2 binds in a complex with the transcription factor Nf-y to a trimeric histone-fold CCAAT box motif to SP2 specific target genes [Cd14 as a major target of SP2 [Cd14 was characterized as positively regulated target gene of SP2 in C3Bir mice. SP2 may contribute to the differential expression level between B6 and C3Bir mice and was identified as innovative target for the therapy of inflammatory bowel diseases supporting the upregulation of the protective CD14 protein.In the ing site . Moreoveatocytes . The retand TLR4 . Compens2 to DNA  these reet genes . Genome t of SP2 , 50,51, t of SP2 . TherefrS1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


Chromatin immunoprecipitation (ChIP) is widely used to identify chromosomal binding sites. Chromatin proteins are cross-linked to their target sequences in living cells. The purified chromatin is sheared and the relevant protein is enriched by immunoprecipitation with specific antibodies. The co-purifying genomic DNA is then determined by massive parallel sequencing (ChIP-seq).Drosophila embryos. Employing several polyclonal and monoclonal antibodies directed against their signature subunits, ACF1 and RSF-1, robust profiles were obtained indicating that both remodelers co-occupied a large set of active promoters.We applied ChIP-seq to map the chromosomal binding sites for two ISWI-containing nucleosome remodeling factors, ACF and RSF, in Further validation included controls using chromatin of mutant embryos that do not express ACF1 or RSF-1. Surprisingly, the ChIP-seq profiles were unchanged, suggesting that they were not due to specific immunoprecipitation. Conservative analysis lists about 3000 chromosomal loci, mostly active promoters that are prone to non-specific enrichment in ChIP and appear as ‘Phantom Peaks’. These peaks are not obtained with pre-immune serum and are not prominent in input chromatin.Mining the modENCODE ChIP-seq profiles identifies potential Phantom Peaks in many profiles of epigenetic regulators. These profiles and other ChIP-seq data featuring prominent Phantom Peaks must be validated with chromatin from cells in which the protein of interest has been depleted. The validity, quality and robustness of ChIP-seq experiments depends on several variables, such as the specificity and avidity of the antibodies used, the fractional occupancy of chromatin loci by the protein of interest, the nature of its chromatin interaction and the likelihood that this interaction will be trapped by chemical crosslinking. Furthermore, native binding profiles may be distorted by biases introduced by experimental procedures, including the immunoprecipitation process, the shearing of chromatin, DNA library preparation, sequencing and the final bioinformatics analysis . To avoiDrosophila RSF, ACF and CHRAC (and their orthologous complexes in yeast) are best known for their role in regenerating the integrity of the nucleosomal arrays (nucleosome ‘spacing’) after inevitable disruptions of the fiber during utilization of the genetic information  and 3602rsf-1 (We used 0–12 h old Oregon-R wild-type (WT) embryos for chromatin immunoprecipitation (ChIP) assays. The mutant alleles sf-13602  were gen9 nuclei/ml.For nuclei isolation, embryos were slowly thawed and dounced using a glass homogenizer  with 20 strokes each of the A and B pestles in ice-cold NX-I buffer . The lysate was filtered through Miracloth and nuclei were pelleted at 3500 rpm, 10 min, 4°C. The pellet was washed in NX-I buffer. Finally, the nuclei were resuspended in RIPA  and washed three times. Nuclei were then counted and frozen at −80°C in aliquots of ∼108/ml by dilution in RIPA and sheared with a Covaris S220 system  at 110 Watts, 20% duty factor and 200 cycles per burst for 25 min. Chromatin was pre-cleared using protein a A+G bead (1:1) mix for 1h at 4°C. Immunoprecipitations were set up overnight at 4°C with 200 μl chromatin and 4 μl of the respective antibody adjusted to 500 μl with RIPA. The antibody amounts had been titrated by following ChIP-qPCR enrichment along several candidate regions obtained from modENCODE ISWI profiles . RIPA-equilibrated protein A+G (1:1) mix was then added to precipitate the immune-complexes for 3 h at 4°C. For the rat monoclonal antibody (3F1), the chromatin immunoprecipitation was performed using pre-sorbed protein G beads, with an excess of antibody, for 3 h at 4°C. Beads were washed subsequently five times for 10 min each in 1 ml RIPA buffer. Residual RNA was digested by RNase A  at 37°C for 20 min. Subsequent protein digestion  and reversal of cross-linking were performed together at 68°C for 2 h. DNA was purified using GenElute™ PCR Clean-Up Kit .For shearing and ChIP, thawed nuclei were adjusted to ∼2×10ChIP DNA was quantified using the Qubit® dsDNA HS Assay Kit  and sequencing libraries were prepared using the MicroPlex Library Preparation kit  starting from 2 ng DNA, whenever possible. PCR amplification was monitored by quantifying amplified libraries (maximum 19 cycles). The libraries were sequenced on a HighSeq 1500 (Illumina) instrument to yield roughly 15–25 Mio reads of 50 bp, single end sequences per sample. Raw, de-multiplexed sequence data files are available at GEO (GSE67323).Drosophila embryos of 7, rsf-13602acf1 and WT genotypes were used for nuclei preparation as described earlier in the method section. Nuclear extract was prepared using nuclear complex co-IP kit  according to manufacturer's guidelines. Immunofluorescence microscopy analyses were carried as described  1065–1463, while the rat monoclonal antibody 3F1 was raised against full length ACF1 but recognizes an epitope in the N-terminal half (data not shown). The rabbit polyclonal antibody was raised against RSF-1 aa 2049–2390. For Western blotting, an overnight collection of 100–300 mg escribed  using a Drosophila melanogaster genome assembly Dmel3 v5.75 using Bowtie v1.1.1 with unique mapping criteria of ‘m-1’. The quality of the raw reads was assessed using FASTQC v0.10.1. Background-subtracted tag densities were obtained using the SPP package . The deletions are documented by the absence of sequencing reads for the corresponding genomic region in input chromatin of the 7acf1 mutant sample and absence of traces obtained from RNA polymerase II ChIP-seq enrichments over the gene body in the 3602rsf-1 background . The absence of ACF1 or RSF-1 expression in mutant embryos was confirmed on Western blots and by IFM . The ACF1 monoclonal antibody 3F1 and the polyclonal Rb1 are highly specific, whereas the Rb2 shows minor cross-reactivity with some extract proteins . The RSF-1 antibody, which allows detection of the antigen in Drosophila embryos by IFM, does not yield signal in RSF-1-deficient embryos, demonstrating its specificity .For ChIP we made use of two different rabbit polyclonal antibodies  raised against the C-terminal region of ACF1, a rat monoclonal antibody (3F1) that detects the N-terminus of ACF1 and a rabbit polyclonal antibody raised against a central fragment of RSF-1. The specificities of the antibodies were assessed by Western blotting and immunofluorescence microscopy (IFM) of mutant embryos. eletions . The acfFormaldehyde-fixed chromatin from embryos collected during a 12 h time window (0–12 h after egg laying) was sheared with adaptive focused acoustics (Covaris) to 200 base pair (bp) mean fragments and subjected to ChIP-seq analysis . When tested on candidate genomic loci obtained from modENCODE ISWI profiles  all antibodies show enrichment of the corresponding amplicons by quantitative PCR . The antibodies were titrated in the dilution range of 1:62.5–1:1000 and a 1:125 dilution (4 μl of rabbit antiserum) was selected for the immunoprecipitation. This antibody concentration is comparable to our previous ChIP studies where we monitored JIL-1 (5 μl antiserum), MSL1 (5 μl), MSL2 (1 μl), MSL3 (4 μl), MLE (4 μl) and MOF (2.5 μl) and obtained meaningful profiles of excellent quality. The RSF-1 antibody works under similar concentration range in both ChIP and IFM (1:150 dilution) .The smoothened, background-subtracted sequence tag density profiles are all highly similar and reveal series of prominent peaks, albeit with variable intensities between different antibodies    indicatiWe derived conservative sets of peaks for ACF1 and RSF-1 by employing the HOMER software . The pro7acf1 chromatin profiles . Likewise, the avid RSF-1 antibody retrieves 3030 common regions from chromatin of WT and 3602rsf-1 embryos . To summarize, Phantom Peaks are not a result of a particular FA fixation protocol.Conceivably, over-fixation of tissue using higher concentrations of formaldehyde (FA) might be a source of artifacts. Our ChIP-seq profiles were derived from 3.7% FA fixed tissues. We previously optimized and used this setting to obtain genome-wide localization of MSL1 , which iDe novo DNA sequence motif analysis using HOMER reveals promoter motifs that are enriched in up to 40% of the peaks , identifying promoters as major contributors Figure . Furthers Figure . For sevt Figure ] that art Figure . We furtt Figure  and showt Figure  35)..De novo Drosophila transcription factors (a modENCODE and BDTNP initiative). The analyses identified 1962 HOT genomic regions that are bound by more than 8 and up to 24 different transcription factors. These sites of clustered ChIP signals collectively constitute roughly 3% of the genome. Not all HOT sites have known functions in transcription control and it has been speculated that some may constitute storage hubs for transcription factors. We observed that about one third of the Phantom Peaks overlap with annotated HOT regions , data not shown  of the peaks overlapping with Phantom Peaks  to fragments of mean sizes of roughly 200 bp. This method is increasingly used for ChIP –42 as itot shown ]. To expot shown . This chP-value <0.05), histone deacetylases HDAC1 and HDAC6  and insulator proteins such as BEAF-32, CTCF and CP190 . These overlap percentages probably underestimate the numbers of shared peaks considering the fact that not all potentially enriched regions are called as peaks by the software in both Phantom Peak and modENCODE cases. Similarly processed tracks for weakly overlapping factors such as Prd and Su(H)  and representatives of the high overlapping factors show similar regions of enrichments of the ChIP signals along the Phantom Peaks in the latter cases regulators such as Sin3A, Fer-3, Chriz and Hr78 . Comparison of antibody-generated modENCODE profiles and DamID profiles with the Phantom Peaks set revealed similarities and differences. The overlap of Sin3A-DamID and BEAF32-DamID with the Phantom Peaks are strong and therefore many sites may indeed be functional. By contrast, DamID profiles of the insulators CTCF and of Su(var)3–9 show poor overlap with Phantom Peaks , raising doubts about the reliability of the corresponding ChIP-seq profiles. Interestingly, the DamID profile for ISWI (a core component of ACF and RSF complexes) shows poor overlap with Phantom Peaks . We further observe that most of the Phantom Peaks are localized within the chromatin derived from housekeeping gene .DamID offers an antibody-free approach to map chromatin binding sites. In this case, the protein of interest is expressed as in fusion with quencing . Compariquencing ,28 usingquencing . The peabona fide binding sites for the remodelers. This additional specificity control is not commonly included in ChIP-seq analyses because suitable mutants are sometimes not available and knockdown strategies are often too inefficient to remove the antigen completely.ChIP-seq profiles are commonly assumed valid if the data have been generated in concert with the modENCODE guidelines that were drafted to assure a good quality of data entered in public repositories. These guidelines demand that researchers validate the specificity of their antibodies by Western blotting and immunofluorescence microscopy (if possible) and that two independent antibodies against a chromatin protein yield consistent results . Our ChIAlthough the enrichment of Phantom Peaks is non-specific from the antibody point of view, the genomic sites that are retrieved are by far not random. The majority of Phantom Peaks maps to promoters of actively transcribed genes that are characterized by open chromatin and are bound by multiple transcription factors and the transcription machinery. A large fraction of Phantom Peaks also coincides with previously mapped HOT regions, where between 8 and 24 transcription factors have been found co-localizing.We speculate that the enrichment of these sites is brought about by a combination of poor ChIP specificity and interaction-prone (‘sticky’) surfaces at the Phantom Peak locations. Phantom Peaks are unlike to be retrieved if the specific ChIP reaction is efficient, like in our earlier experiments on the MSL proteins . In the Why should regions of high transcription factor occupancy and recruitment of the RNA polymerase machinery be prone to non-specifically interact with antibodies? We speculate that many transcription factors and components of the transcription machinery contain ‘unstructured’ protein surfaces or molten-globule conformations, sometimes of low sequence complexity or highly charged, that are poised to interact with close-by targets and to acquire a specific structure only through this interaction –52. For bona fide binding sites and Phantom Peaks, with varying contributions of each group. Undoubtedly many published interactions at Phantom Peak sites will prove correct—after all, many of these are highly active promoters, where numerous regulators are known to accumulate. However, in cases where no consensus binding sequences for a sequence-specific factor can be found at the mapped site (as is the case for many insulator proteins), or the functional significance of presumed interactions is unclear (as in the case of HDAC complexes at highly active promoters), or when the massive enrichment of many factors at some sites does not make functional sense (as in the case of HOT regions), caution should be taken.We found that a considerable number of modENCODE ChIP-seq profiles display enrichments that coincide with Phantom Peaks. It is currently not possible to evaluate whether these signals reflect true chromatin binding events or other examples of Phantom Peak manifestation. Conceivably, many profiles may be composites of Drosophila. False positive signals that are obvious from irrelevant antibodies (e.g. GFP) or pre-immune sera can be reduced by appropriate normalization procedures (Our study echoes related observations in other systems. Hyper-ChIPable regions at accessible genomic regions have previously been described in bacteria, yeast and mammals that can be retrieved using antisera directed against non-chromatin proteins –57 demonocedures ,54. Crosocedures , or emplocedures ,59. ThesDrosophila genomics community will profit from a listing of such dubious sites, just as the proteomics community has profited from publication of a listing of the cRAP protein sequences of common contaminants in mass spectrometric analyses (Concerning some shortcomings of antibodies, such as relaxed specificity and cross-reactivity, the increasing awareness in the research community has led to initiatives toward increased standardization and quality control measures . For antanalyses .


Drosophila chromatin with high and low concentrations of MNase to reveal two distinct nucleosome types: MNase-sensitive and MNase-resistant. MNase-resistant nucleosomes assemble on sequences depleted of A/T and enriched in G/C-containing dinucleotides, whereas MNase-sensitive nucleosomes form on A/T-rich sequences found at transcription start and termination sites, enhancers and DNase I hypersensitive sites. Estimates of nucleosome formation energies indicate that MNase-sensitive nucleosomes tend to be less stable than MNase-resistant ones. Strikingly, a decrease in cell growth temperature of about 10°C makes MNase-sensitive nucleosomes less accessible, suggesting that observed variations in MNase sensitivity are related to either thermal fluctuations of chromatin fibers or the activity of enzymatic machinery. In the vicinity of active genes and DNase I hypersensitive sites nucleosomes are organized into periodic arrays, likely due to ‘phasing’ off potential barriers formed by DNA-bound factors or by nucleosomes anchored to their positions through external interactions. The latter idea is substantiated by our biophysical model of nucleosome positioning and energetics, which predicts that nucleosomes immediately downstream of transcription start sites are anchored and recapitulates nucleosome phasing at active genes significantly better than sequence-dependent models.Nucleosomal DNA is thought to be generally inaccessible to DNA-binding factors, such as micrococcal nuclease (MNase). Here, we digest  Eukaryotic DNA is packaged in the nucleus into a compact nucleoprotein complex called chromatin. The fundamental unit of chromatin is a nucleosome comprising 147 base pairs (bp) of DNA wrapped around a histone octamer core in ≈1.67 left-handed superhelical turns ,3. The ‘in vivo and in vitro nucleosome occupancy profiles (in vivo by ATP-dependent chromatin remodeling enzymes (remodelers)—abundant nuclear factors which effect nucleosome sliding and unfolding using energy of ATP hydrolysis  and fission (S. pombe) yeast, nucleosome positions can be determined with nucleotide precision using a recently developed chemical cleavage approach (CC-seq) , which preferentially digests linker DNA, followed by paired-end sequencing of undigested DNA fragments (MNase-seq) . Alignme(CC-seq) –40. Repl(CC-seq) ,41. The Drosophila genome by chromatin digestion with two different concentrations of MNase. We used a high concentration of MNase (MNaseHIGH) for complete digestion of chromatin to mononucleosomes, and a low concentration of MNase (MNaseLOW) for partial digestion. This procedure produced two distinct maps representing MNase-resistant and MNase-sensitive nucleosomes, respectively. Sequences occupied by MNase-resistant and MNase-sensitive nucleosomes differ significantly with respect to their dinucleotide composition. MNase-resistant nucleosomes are assembled on sequences depleted of A/T and enriched in G/C-containing dinucleotides. In contrast, MNase-sensitive nucleosomes are found on A/T-rich sequences. Furthermore, nucleosome occupancy profiles of MNase-resistant, but not MNase-sensitive, nucleosomes can be partially predicted using sequence-dependent models  on grape juice plates supplied with yeast paste. S2 cells were grown in Schneider's media (Invitrogen) supplied with 10% Fetal Calf Serum (Life Technologies) at 27°C, unless specified otherwise.Drosophila embryos and S2 cells was performed as described in Supplemental Methods. Libraries for Illumina sequencing of nucleosomal DNA were prepared with Illumina or SureSelect kits and paired-end sequenced (2 × 25 bp or 2 × 50 bp) on Illumina HiSeq 2000. To assess the temperature effects on nucleosome sensitivity/accessibility to MNase, S2 cells were incubated overnight (≈16 h) at 18°C and fixed with formaldehyde prior to MNase digestion. All MNase reactions were performed at 25°C for 10 min.MNase digestion of formaldehyde cross-linked chromatin of HIGH-ChIP-seq assays were done as described in Supplemental Methods. In brief, following the complete MNase digestion of embryonic chromatin, nucleosomal fragments were precipitated with ChIP-grade antibodies against histone H3  or H2B (MNase) or H2B , and seqLOW-ChIP-seq, embryonic chromatin was partially digested and fractionated on the 5–30% sucrose gradient. Fractions corresponding to mono-nucleosomes were ChIPed with anti-H3 and anti-H2B antibodies and paired-end sequenced on Illumina HiSeq 2000 . RNA-seq for Drosophila S2 cells was performed according to the standard Illumina protocols by ServiceXS (the Netherlands).For MNasehttp://bowtie-bio.sourceforge.net/bowtie2/index.shtml) against BDGP5/dm3 D. melanogaster genome.Sequence reads were aligned using Bowtie2 v 2.1 . The MNaseR software (https://www.r-project.org) function fft was used to compute the discrete Fourier transform of nucleosome dyad arrays, followed by the calculation of absolute magnitudes with the mod function. For NRLs of 100–225 bp in length, the resulting magnitudes were linearly interpolated with the step of 0.01 bp using the approx function, spline smoothed with the smooth.spline function (the smoothing parameter was set to 0.8) and scaled to the maximum magnitude.Nucleosome repeat lengths (NRLs) were estimated by the Fourier analysis of normalized nucleosome dyad profiles. Specifically, the in vivo, such as chromatin remodeler activity and temperature.Nucleosomal DNA is not permanently wrapped around histone octamer cores—rather, nucleosomes undergo partial unwrapping at the entry and exit sites due to thermal fluctuations and the action of ATP-dependent chromatin-remodeling enzymes. Moreover, extra DNA may be pulled into the nucleosome and form a loop. Propagation of such DNA loops along the histone octamer surface, accompanied by breaking and re-forming of histone–DNA contacts, may mediate nucleosome translocation . FinallyDrosophila embryos with gradually increasing amounts of MNase . The magnitude of these trends is again smaller than the difference between MNaseHIGH and MNaseLOW chromatin digestions.We have mapped sequence reads obtained in MNaseS Figure . In addiDrosophila embryos was digested as before, using high and low concentrations of MNase. Mono-nucleosomes obtained by the MNaseHIGH digestion were precipitated using anti-H2B or anti-H3 antibodies, and the resulting DNA fragments were paired-end sequenced (MNaseHIGH-ChIP-seq). Mono- and oligonucleosomes obtained by low-MNase digestion were first separated according to their molecular weight by sucrose gradient fractionation. The resulting fractions were analyzed on an agarose gel and a fraction containing mononucleosomes was immunoprecipitated with anti-H2B or anti-H3 antibodies, followed by paired-end sequencing (MNaseLOW-ChIP-seq). For each antibody and MNase concentration, two independent biological replicates were analyzed. At high MNase concentration, the distribution of fragment lengths is consistent between replicates and does not depend on the antibody used . However, at low MNase concentration both H3 replicates are enriched in short fragments . This is consistent with the presence of partially disassembled nucleosomes, which retained the core H3-H4 tetramer but lost the outer H2A-H2B dimers.It is conceivable that a fraction of the DNA fragments identified by MNase digestion correspond to DNA-bound proteins other than histones, especially in promoters. To alleviate this concern, we have carried out a set of MNase-ChIP-seq experiments . Briefly, chromatin of HIGH and MNaseLOW data sets, we have mapped MNaseLOW-ChIP-seq and MNaseHIGH-ChIP-seq to the Drosophila genome, creating nucleosome occupancy and dyad density profiles. Since there is enrichment for short fragments with anti-H3 antibodies, we have divided fragment lengths from H3 experiments into short and long subclasses, with the long subclass window sizes consistent with MNase-seq . For ChIP-seq maps, which employed anti-H2B antibodies, we use only the long-fragment subclass. A table of linear correlation coefficients  shows that MNaseHIGH and MNaseLOW nucleosome occupancy profiles are highly correlated with their ChIP-seq counterparts, regardless of the antibody used. The correlations are strongest when the same fragment lengths are considered and become much weaker when occupancies of short and long fragments are compared. Nevertheless, correlations within MNaseHIGH and MNaseLOW groups tend to be much higher than the correlations between any two data sets produced using different levels of MNase. We conclude that DNA fragments isolated via MNaseHIGH-seq and MNaseLOW-seq come predominantly from nucleosomes. Further, treatments with high and low MNase concentrations isolate distinct subpopulations of MNase-resistant and MNase-sensitive nucleosomes.As with the MNaseLOW 1n), and shows that gene promoters are not nucleosome-depleted—rather, they are occupied by MNase-sensitive nucleosomes, a significant fraction of which are partially assembled or partially unwrapped. The enrichment of short-length nucleosomal fragments immediately upstream of the TSS is also visible in the heat maps . Interestingly, the difference between short and long DNA fragments in the MNaseLOW-ChIP-seq H3 experiment is much less pronounced around TTS, although both are enriched compared to the MNaseLOW-ChIP-seq H2B nucleosome map.After complete digestion, the average dyad density around TSS and TTS is consistent across all data sets . In contrast, after partial digestion there is marked enrichment of short DNA fragments obtained by ChIP-seq with anti-H3 antibodies . This enrichment is comparable in magnitude to that observed in the MNase-seq experiment (MNaseDrosophila Schneider 2 (S2) cells. Unlike a mixture of cells extracted from 0 to 12 h Drosophila embryos, S2 cells represent a homogeneous cell type; we expect their gene expression states to be similar across the population. Besides, nucleosome unwrapping and partial disassembly, nucleosome remodeling and chromatin fiber packaging (the primary factors that affect DNA accessibility to MNase) are not limited to embryonic chromatin. Supplementary Figure S4A demonstrates our procedure for extracting nucleosomal DNA from Drosophila S2 cells. As with embryos, we have used an agarose gel to analyze DNA fragments obtained at low and high MNase concentrations. In the MNaseHIGH-seq experiment, we have collected and sequenced mononucleosome-size (1n) fragments, which represent MNase-resistant nucleosomes. The distribution of DNA fragment lengths from paired-end sequencing  is similar to that observed with the embryos . In particular, it exhibits oscillatory substructure indicative of stepwise nucleosome unwrapping and disassembly. In the MNaseLOW-seq experiment, both mono- and di-nucleosome-sized fragments  were isolated and sequenced using paired-end reads . The distribution of 1n fragment lengths  is again similar to that observed in embryonic chromatin , while the lengths of 2n fragments are concentrated in the 300–400 bp range . As with embryos, mononucleosome fragments represent MNase-sensitive nucleosomes, while di-nucleosome fragments should be enriched in MNase-resistant nucleosomes. Indeed, as with embryos the di-nucleosome gel band progressively disappears as MNase concentration is increased, showing that more and more fragments are digested to the mononucleosome level.Next, we have mapped nucleosomes at high and low MNase concentrations in Drosophila genome. Midpoints of 1n fragments were identified with nucleosome dyad positions. For 2n fragments, the dyads were assumed to be located at one-fourth and three-fourth positions with respect to the start of the fragment. MNaseHIGH and MNaseLOW 1n nucleosome occupancy profiles are reasonably well correlated with their embryonic counterparts, demonstrating that both studies yield similar subsets of MNase-resistant and MNase-sensitive nucleosomes . Furthermore, whereas in S2 cells the MNaseHIGH 1n nucleosome occupancy is only weakly correlated with MNaseLOW 1n nucleosome occupancy (Pearson's correlation coefficient r = 0.39), the correlation with the MNaseLOW 2n occupancy profile is much stronger (r = 0.69). This observation confirms our view of distinct MNase-resistant and MNase-sensitive nucleosome subsets, and suggests that a combined MNaseLOW 1n+2n nucleosome map provides a more comprehensive representation of relative occupancies of all nucleosomes present in Drosophila S2 cells.We have mapped both 1n and 2n nucleosome fragments to the Drosophila embryos, MNase-resistant nucleosomes are depleted in promoters and around TTS  to 18°C  than with its high-temperature counterpart (r = 0.33 with MNaseHIGH 1n) . This observation is consistent with the fact that at 18°C MNase-resistant nucleosomes, many of which are MNase-sensitive at 27°C, are enriched in promoters and around TTS compared to MNase-resistant nucleosomes at 27°C , it appears that the marked increase in nucleosome sensitivity to MNase with temperature is mediated primarily through changes in compactness and density of chromatin fibers. Indeed, genome-wide Fourier analysis demonstrates that the average linker length becomes shorter as the temperature is lowered to 18°C, indicating more compact nucleosome placement genome-wide and, to a smaller extent, in the vicinity of active genes . Thus nucleosome sensitivity to MNase digestion may be partially mediated by DNA sequence.We have studied whether MNase-sensitive and MNase-resistant nucleosomes are associated with distinct sequence motifs (such as mono- and di-nucleotide distributions) that might partially explain their genome-wide positioning and varying sensitivity to MNase. We find that in S2 cells mononucleosomal sequences obtained by the MNased Figure ,C. In cos Figure ,D, consis Figure ,53. Intes Figure . Consistt Figure , while fn Figure . The dift Figure , showingLOW 1n, MNaseHIGH 1n and MNaseLOW 1n+2n nucleosome occupancy profiles , we classify genomic regions into MNase-resistant/MNase-sensitive and, separately, into open/closed categories  occupancy are classified as MNase-resistant or MNase-sensitive, respectively. Likewise, regions in which MNaseLOW 1n nucleosome occupancy is higher or lower than the total occupancy correspond to ‘open’ or ‘closed’ chromatin. Indeed, in regions of closed chromatin relatively few mononucleosome-size fragments were obtained at low MNase concentration, and vice versa. The notions of MNase sensitivity and chromatin accessibility are closely related: MNase-resistant chromatin is predominantly closed, while MNase-sensitive chromatin is predominantly open . Strikingly, this bimodality is also reflected in the nucleosome organization in the vicinity of coding regions: nucleosomes corresponding to active genes form well-ordered arrays, whereas nucleosomes in and around silent genes are virtually unordered and uniformly distributed . The boundary between active and silent genes  separates these two distinct types of nucleosome organization. This bimodality is also observed in embryonic gene expression levels and, correspondingly, in embryonic chromatin organization , likely through interactions with chromatin remodelers, components of transcriptional machinery and regulatory factors. One simple mechanism of nucleosome organization is ‘phasing’ off a barrier created by a DNA-bound factor with sufficiently high binding affinity, so that it is not displaced through thermodynamic competition with nucleosomes . AnotherHIGH 1n) nucleosomes are depleted inside DHS  nucleosomes , which correspond to regions of open chromatin enriched in DNA-bound factors Figure . MoreoveS Figure ,B, likels Figure . The phaLOW 1n+2n data set in which nucleosome formation energy is a function of the mono- and dinucleotide content at each nucleosomal site. Our biophysical model relies on a closed-form solution, which allows us to infer nucleosome formation energies directly from the observed dyad density profile . Briefly, the model exploits the analogy between nucleosome arrays and a one-dimensional fluid of particles with steric exclusion, subject to an arbitrary external field , we find that MNase-resistant nucleosomes are indeed more stable Figure . Note thn Figure .Nucleosomes are dynamic structures—they can be unfolded, relocated or partially unwrapped, with the help of chromatin remodeling enzymes or by thermal fluctuations alone ,61–64. IDrosophila embryos and S2 cells , with MNase-sensitive and MNase-resistant nucleosomes exhibiting unique sequence signatures . Histone exchange rates and sensitivity to MNAse at the +1 nucleosome correlate with RNA Pol II occupancy in the vicinity of TSS. Thus it appears that the presence of RNA polymerase both stabilizes the +1 nucleosome, precisely defining its position to a degree not achievable with DNA sequence alone, and at the same time increases its histone exchange rates. The latter requires an active remodeling mechanism since thermally-activated histone exchange should be impeded if the nucleosome is stabilized through interactions with external factors such as Pol II.MNase-seq sequencing data have been deposited to NCBI GEO under accession number GSE69336.