Epigenetic modifications of the herpesviral genome play a key role in the transcriptional control of latent and lytic genes during a productive viral lifecycle. In this study, we describe for the first time a comprehensive genome-wide ChIP-on-Chip analysis of the chromatin associated with the Kaposi's sarcoma-associated herpesvirus (KSHV) genome during latency and lytic reactivation. Depending on the gene expression class, different combinations of activating [acetylated H3 (AcH3) and H3K4me3] and repressive [H3K9me3 and H3K27me3] histone modifications are associated with the viral latent genome, which changes upon reactivation in a manner that is correlated with their expression. Specifically, both the activating marks co-localize on the KSHV latent genome, as do the repressive marks. However, the activating and repressive histone modifications are mutually exclusive of each other on the bulk of the latent KSHV genome. The genomic region encoding the IE genes ORF50 and ORF48 possesses the features of a bivalent chromatin structure characterized by the concomitant presence of the activating H3K4me3 and the repressive H3K27me3 marks during latency, which rapidly changes upon reactivation with increasing AcH3 and H3K4me3 marks and decreasing H3K27me3. Furthermore, EZH2, the H3K27me3 histone methyltransferase of the Polycomb group proteins (PcG), colocalizes with the H3K27me3 mark on the entire KSHV genome during latency, whereas RTA-mediated reactivation induces EZH2 dissociation from the genomic regions encoding IE and E genes concurrent with decreasing H3K27me3 level and increasing IE/E lytic gene expression. Moreover, either the inhibition of EZH2 expression by a small molecule inhibitor DZNep and RNAi knockdown, or the expression of H3K27me3-specific histone demethylases apparently induced the KSHV lytic gene expression cascade. These data indicate that histone modifications associated with the KSHV latent genome are involved in the regulation of latency and ultimately in the control of the temporal and sequential expression of the lytic gene cascade. In addition, the PcG proteins play a critical role in the control of KSHV latency by maintaining a reversible heterochromatin on the KSHV lytic genes. Thus, the regulation of the spatial and temporal association of the PcG proteins with the KSHV genome may be crucial for propagating the KSHV lifecycle. KSHV is a ubiquitous herpesvirus that establishes a life-long persistent infection in humans and is associated with Kaposi's sarcoma and several lymphoid malignancies. During latency, the KSHV genome persists as a multicopy circular DNA assembled into nucleosomal structures. While viral latency is characterized by restricted viral gene expression, reactivation induces the lytic replication program and the expression of viral genes in defined sequential and temporal order. Posttranslational modifications of the viral chromatin structure have been implicated to regulate viral gene expressions but the underlying gene regulatory mechanisms are still elusive. Here, we demonstrate that the latent and lytic chromatins of KSHV are associated with a distinctive pattern of activating and repressive histone modifications whose distribution changes upon reactivation in an organized manner in correlation with the temporally ordered expression of viral lytic genes. Furthermore, we demonstrate that the evolutionarily conserved Polycomb group proteins, that maintain the repression of genes involved in hematopoiesis, X-chromosome inactivation, cell proliferation and stem cell differentiation, also play a critical role in the regulation of KSHV latency by maintaining a repressive chromatin structure. Thus, the epigenetic program of KSHV is at the crux of restricting latent gene expression and the orderly expression of lytic genes. Chromatin is a highly dynamic structure of nucleosomes that are composed of DNA wrapped around the core histones . Over the past decade, several studies have demonstrated that histones are subject to various posttranslational modifications , which are capable of modulating chromatin structures to thereby influence gene expression Histone methylation status fluctuates in response to environmental and developmental conditions. A number of enzymes that add or remove methylation modifications have been discovered H3K27me3 is deposited by the evolutionary conserved 600-kDa Polycomb Repressive Complex 2 (PRC2), which consists of three Polycomb group (PcG) proteins  and the histone-binding proteins, RbAp48/46 Viruses replicating in the nucleus are also under the influence of the chromatin during different stages of their life cycles. Therefore, viruses have evolved various mechanisms to utilize or neutralize the impact of cellular chromatin factors, to ultimately control viral replication and gene expression A human tumour virus, called Kaposi's sarcoma-associated herpesvirus (KSHV) or human herpesvirus 8 (HHV8), has been consistently identified in Kaposi's sarcoma (KS) tumours, pleural effusion lymphoma (PEL), and Multicentric Castleman disease To elucidate the characteristics of the KSHV epigenome, we performed a high-resolution genome-wide analysis to map a set of activating and repressive histone H3 modifications on the entire KSHV genome during latency and reactivation. Based on their genome-wide profiles, we found that the KSHV genes are associated with a distinctive pattern of active and repressive histone modifications during latency, which ultimately changes upon reactivation. Importantly, the promoter regions of RTA and several E genes are associated with both H3K4me3 and H3K27me3 marks, suggesting that these promoters have a bivalent chromatin structure that maintains their repression during latency and is also poised for rapid activation upon stimulation. We also found that while the EZH2 histone methyltransferase is colocalized with H3K27me3 on the entire KSHV latent genome, it rapidly dissociates from the RTA promoter and other IE-E gene-rich genomic regions upon reactivation. This event ultimately results in reduced level of H3K27me3, which are concomitant with increasing levels of activating histone marks on the RTA promoter. Furthermore, treatment of latently KSHV-infected cells with a drug inhibiting the expression of PcG proteins, the small inhibitory RNA-mediated knockdown of EZH2 or the overexpression of H3K27me3 histone demethylases efficiently trigger the lytic reactivation of KSHV. These data collectively demonstrate that the Polycomb group proteins are involved in the maintenance of KSHV latency by preserving a reversible heterochromatin on the promoter regions of lytic genes such that they are silenced during latency but are poised for rapid activation upon reactivation.In this study we asked what histone modifications are associated with the KSHV genome during latency and how they change upon reactivation. For this, we used the well-characterized recombinant KSHV-positive primary effusion lymphoma cell line, TRExBCBL1-RTA, which expresses a Doxycycline (Dox) inducible myc/His-tagged RTA incorporated into the cellular genome In order to investigate the epigenome of KSHV during latency and lytic reactivation, we tested whether repressive histone modifications associated with lytic genes are responsible for maintaining the repression of lytic gene expression during latency and whether reactivation induces the deposition of activating histone modifications onto the viral genome for lytic gene expression. To uncover the global distribution of histone modifications on the KSHV chromatin during latency and reactivation, we mapped the genome-wide distribution of activating histone marks [acetyl-H3 (AcH3) and H3K4me3] and repressive marks (H3K9me3 and H3K27me3) on the KSHV genome , S5, S7.Because changes in H3 occupancy may affect the enrichment of histone modifications on the viral genome, we first investigated the global distribution of H3 on the viral genome during latency and upon reactivation. The histone H3 ChIP-on-chips revealed that histone H3 enrichment levels were comparable throughout the KSHV genome at 0 hpi and did not significantly change on most parts of the KSHV genome at 12 hpi , S3, S4.The RTA-induced initiation of KSHV lytic gene expression program results in the redistribution of histone modifications on viral genome. We calculated the 12hpi/input ratio to view the changes in histone modifications . Since HIn summary, these results demonstrate that the activating histone modifications of the latent KSHV genome are preferentially enriched in the constitutively active latency-associated genomic locus and in the early-lytic gene-containing genomic regions. In contrast, the H3K9me3 and H3K27me3 repressive histone marks are primarily enriched in the genomic regions that encode many late genes during latency (0 hpi), and they remain associated with these regions during the early phase (12 hpi) of reactivation as well.While only a few genes are expressed during latency, all viral genes are expressed upon lytic reactivation in a temporal and sequential order. This suggests that distinctive histone modifications may be associated with the different viral promoters to ultimately determine the timing and rate of viral gene expression. Thus, we attempted to delineate the characteristics of the chromatin structures associated with the regulatory regions of KSHV genes during latency and lytic reactivation , S6, S8.de novo expression of any viral proteins and can be rapidly induced upon reactivation of KSHV from latency. We found that although the IE genes  are silenced during latency, their 2-kb regulatory regions are enriched in the activating histone modifications H3K4me3 and AcH3, and this is further increased upon lytic reactivation at ORFs 48, 50 and K8 . Thus, this raises the possibility that the colocalization of H3K4me3 and H3K27me3 histone modifications at the same genomic region may be due to the mixture of latent and spontaneously reactivated KSHV genomes in the ChIPs assays. To determine whether H3K4m3 and H3K27me3 on the RTA promoter were simultaneously on the same KSHV genome and not independent histone marks on different viral genomes, sequential ChIP assay was applied . We perfExpression of IE genes is followed by the induction of E genes that encode a large variety of proteins for modulating immune responses, apoptosis or performing viral DNA synthesis. From the analysis of the TSS/promoter regions of E genes, three subgroups  of histone modification patterns emerged , S8. GroThe KSHV genes whose expression can be blocked by viral DNA replication inhibitors are classified as late genes. Because most of the late gene products are necessary for the assembly and egress of viral particles, their expression is required only after viral DNA synthesis. To achieve this temporal order of viral gene expression they should be completely silenced during the expression of IE and E genes. In agreement with this notion, we found that the promoter regions of most of the late genes were enriched in the repressive H3K27me3 and H3K9me3 histone modifications and depleted in the activating histone marks during latency and in the early phase of the reactivation (12 hpi) , group VIn agreement with the observed constitutive expression of the latent genes (LANA and vIRF3) during latency, their 2-kb TSS regions display enrichment only in H3K4me3 and AcH3 while lacking repressive histone modifications . InteresIn summary, we described the chromatin modification patterns of the TSS regions of the four expression classes  of KSHV genes during latency and lytic reactivation. Based on these data we propose that the diversity of the chromatin structure of the KSHV genome can reflect the various regulation mechanisms of viral gene expression, which allows a temporal and well-organized gene expression during reactivation.Because of the genome-wide distribution of H3K27me3 on the KSHV genome, we performed a ChIP-on-chip assay to determine whether EZH2, the H3K27me3 histone methyltransferase of the PcG proteins, associates with the KSHV genome. . ChIP-onin vitro, but its deletion also resulted in a dramatic decrease of the RTA-mediated autoactivation of its own promoter , has been shown to inhibit the expression of the Polycomb repressive complex 2 (PRC2) components , resulting in the suppression of H3K27me3 histone methylation and the upregulation of PcG target genes Besides the downregulation of EZH2 and SUZ12, DZNep also induced apoptosis of JSC-1 cells, detected by monitoring the cleavage of the apoptosis marker PARP  A, B. ToThe genome-wide transcriptional analysis of KSHV gene expression revealed that despite the differences in the features of their promoters, viral genes with similar functions display analogous expression patterns during the lytic replication cycle, implying the existence of a common regulatory mechanism for their gene expression KSHV genomic regions encoding a number of late genes are associated with repressive H3K9me3 and H3K27me3 modifications during both latency and the early phase of lytic reactivation, which is in accordance with the observed silencing of late genes. Strikingly, the high expression of late genes is observed at the time of the viral DNA replication concomitantly with the disassembly of viral chromatin , S2. Thede novo infection or rapidly express upon lytic reactivation Based on the histone modification patterns associated with the promoter regions of E genes, three distinctive groups could be observed , S8. TheHistone H3 ChIP-on-chip revealed that a small viral genomic region (between 15 and 30kb) mostly containing KSHV unique genes displayed a detectable decrease of H3 occupancy at 12hpi . Thus, tIn contrast to the genome-wide repressive role of H3K27me3, the effect of the heterochromatin histone mark H3K9me3 on viral gene expression seems to be limited: enrichment of the H3K9me3 is restricted to specific genomic regions  and the Genome-wide co-distribution of the EZH2 with the H3K27me3 repressive mark on lytic genes of KSHV and the EZH2 knockdown-mediated induction of lytic gene expression strongly implicate PcG proteins in the repression of lytic gene expression during latency , S7, S10Derepression of the PcG proteins-mediated silencing of gene expression have been linked to the recruitment of the H3K27me3 histone demethylases UTX and JMJD3 and the H3K4me3 histone methyltransferases MLL3 and MLL4 onto the target promoters, suggesting a tight cooperation between demethylation of the H3K27 and trimethylation of the H3K4 Collectively, our results indicate for the first time that histone modifications associated with the latent KSHV genome can be involved not only in the regulation of latency, but also in the control of the temporal and sequential order of the lytic gene expression cascade of KSHV upon reactivation. PcG proteins appear to play a key role in the maintenance of latency as well as in the inhibition of late gene expression during the early phase of lytic reactivation. Besides these beneficial roles of PcG proteins for the viral lifecycle, KSHV may adjust its viral genome through epigenetic modifications to tightly regulate viral expression to avoid promiscuous expression of lytic genes, which may trigger host immune system responses against KSHV infected cells.TRExBCBL1-RTA is a KSHV-positive cell line, which expresses a doxycycline-inducible Rta gene Expression plasmids HA-JMJD3 and HA-UTX were obtained from Kristian Helin (University of Copenhagen). HA-JMJD2A expression vector was gift from Yang Shi (Harvard University). HA-UTXm was generated by replacing the C terminus of the UTX in HA-UTX with PCR cloning to change histidine to alanine at amino acid 1146. pLuc3kb, pLuc1.9kb and pLuc0.9kb reporter plasmids were generated by inserting the Luciferase gene derived from pGL3-Basic and different sized fragments of the promoter region of KSHV Rta into pcDNA5/FRT. 293A and Vero.219 cells were transfected by Polyfect (Qiagen) according to the manufactrer's specifications. Luciferase assay was performed by using the Promega system. The luciferase activity values are the average of at least three independent experiments.Vero cells were transfected with plasmids expressing HA-tagged JMJD2A, JMJD3, UTX or UTXm respectively. 3 days post transfection, cells were fixed by 4% paraformaldehyde and then permeabilized by 0.2% Triton X100. 10% goat serum was used for blocking aspecific binding of antibodies followed by incubation of cells with antibodies against HA-tag, H3K9me3 and H3K27me3. After extensive washing with PBS, FITC- and TRITC-conjugated secondary antibodies were applied followed by Hoechst staining.Biotinylated DNA was made with biotin-conjugated primers and PCR amplification of sequences that are derived from the KSHV RTA promoter. KSHV (U75698.1 at GenBank) coordinates for fragment A and B are 69861–70500 and 70511–71130, respectively. The assay was performed essentially as described by Atanasiu et. al. The following antibodies were used in ChIPs and/or western blotting: rabbit anti-histone H3 (Abcam ab1791), rabbit anti-H3K27me3 (Millipore 07-448), rabbit anti-H3K9me3 (Millipore 07-442), rabbit anti-H3K4me3 (Millipore 04-745), rabbit anti-acetyl-histone H3 (AcH3) (Millipore 06-599), rabbit anti-RNA polymerase II (H-224)  (Santa Cruz sc-9001), mouse anti-RNA polymerase II (CTD4H8) (RNAPII Ser5) (Millipore 05-623), rabbit anti-CBF1 (Abcam ab25949), mouse anti-EZH2 (BD Biosciences 612666), rabbit anti-SUZ12 (Abcam ab12073). For western blotting the following antibodies were used: mouse anti-PARP (BD Biosciences 556494), mouse anti-actin (Abcam), anti-JMJD2A (Bethyl A300-861A), anti-JMJD3 (Abgent AP1022a), KSHV specific antibodies such as mouse anti-Rta gift from Koichi Yamanishi , rabbit anti-K8.1, mouse anti-K8 (Abcam ab36617), rabbit anti-K2 (ABI 13-214-050), rat anti-LANA (ABI 13-210-100), Epstein Barr Virus specific antibody such as mouse anti-Zebra (Zta) (Argene 11-007).7) were treated for 6, 12 and 24 hr with 1 ug/ml doxycycline. After fixation of the cells with 1% (v/v) formaldehyde for 10 min at RT, the cross-linking was stopped by adding glycine  for 5 min at RT. Cells were washed (3×) with cold PBS and then resuspended in Cell Lysis Buffer ) and incubated on ice for 10 min. After centrifugation , the pellet was resuspended in 1.8 ml of RIPA buffer , sonicated and centrifuged at 13000 rpm for 10 min at 4°C to remove cell debris. Aliquots of the supernatant  were stored at −80°C.TRExBCBL1-RTA cells (4×10For preparation of input DNA 20ul of chromatin was incubated in 100ul of TE buffer containing 50ug/ul RNase A for 30 min at 37°C. The samples were then adjusted to contain 0.5% SDS and 0.5mg/ml Proteinase K (Invitrogen) and incubated for 1 hr at 37°C. Formaldehyde crosslinks were reversed by adding sodium chloride  to the samples and incubated overnight at 65°C. DNA was extracted first by one volume of phenol/chloroform/isoamyl alcohol (25∶24∶1) saturated with 10 mM Tris, pH8.0 and 1 mM EDTA and then purified once by one volume of chloroform. DNA was precipitated by cold absolute ethanol, 10% (v/v) of 3 M sodium acetate, pH 5.2 and 1ul of 15 mg/ml Glycogen Blue (Ambion) at −80°C at least for 1 hr following by wash with 70% ethanol. Finally the input DNA was dried at RT and resuspended in 20ul of water.For ChIPs chromatin containing 10ug of DNA was first diluted in 500ul of RIPA buffer and precleared by Sepharose A beads. Immunoprecipitation was carried out with 1–2ug of antibodies overnight at 4°C. Next day to pull down the DNA/protein complexes, Protein-A/G agarose was added for 4 hr. Immunoprecipitation was washed sequentially with RIPA buffer once briefly and once for 10 min followed by washing with LiCl buffer  once for 10 min and with TE buffer two times for 10 min. The DNA/protein-Protein A/G agarose complex was resuspended in 100ul of TE buffer containing 50ug/ul RNase A and incubated for 30 min at 37°C. The Proteinase K treatment, crosslink reversal and DNA purification was done exactly as it was described at the preparation of input DNA. Both input and ChIP DNAs were measured by qPCR. Based on the standard curves for each primer pairs the enrichment of proteins and histone modifications on specific genomic regions were calculated as percentage of the immunoprecipitated DNA compared to input DNA. Each data points in ChIP figures were averages of at least three independent ChIPs using three independent chromatins.Sequential ChIP (seqChIP) was performed as follows. ChIPs were carried out as described above except that after the first ChIP (ChIP I.) the immunoprecipitated DNA/protein complexes were eluted from the Protein A/G agarose by 100ul of Elution buffer  by heating for 10–15 min at 65°C. 10% of the elution was saved for qPCR while the rest was diluted with RIPA buffer up to 1 ml and the second immunoprecipitation (ChIP II.) was performed exactly as described above for ChIP. The second ChIP was also measured by qPCR and the enrichment of the histone modifications on specific genomic regions was calculated as percentage of the immunoprecipitated DNA compared to the total amount of DNA eluted after the first ChIP. Each ChIP values were averages of three independent ChIPs using three independent chromatins.qPCR was performed using iQ SYBR Green Supermix (Bio-Rad) and CFX96 real-time PCR machine (Bio-Rad). PCR program was as follows: after an initial preincubation step at 95°C for 3 min, there were 40 cycles, each consisting of 95°C for 10sec, 64.5 or 59°C depending on the primers  for 20 sTotal cellular RNA was extracted with Tri reagent (Sigma) according to the manufacturer's instructions. 1ug of total RNA was treated by DNase I (Sigma), reverse transcribed by iScript cDNA Synthesis kit (Bio-Rad) and the cDNA was measured by either conventional PCR or qPCR. The relative quantification of gene expression was calculated with the ddCt method, where actin mRNA was used for normalization. RT-PCR graphs were made based on the average of at least two independent experiments.The shRNA constructs were prepared by using the pLKO.1 lentiviral vector. The target sequences are listed in The 15 bp-tiling KSHV microarray contains both KSHV and human probes and was manufactured by Agilent Technologies. The probes were spotted on 8×15K array format. The majority of the oligonucleotides are overlapping 60-mer probes covering the entire KSHV genome (U75698 and U75699 at GenBank). In addition, probes specific for the gene regulatory regions of 71 human genes were also included in the microarray. The human probes are derived from the Agilent Human CoC 2×244k microarray design. For ChIP-on-chip 30 ug of chromatin and 5–6 ug of antibodies were used per ChIP. The ChIP and DNA purification was performed exactly as described above. ChIP DNA was amplified by Complete Whole Genome Amplification (WGA2) kit (Sigma-Aldrich) and purified with QIAquick PCR Purification kit (Qiagen) according to the instructions of the manufacturers. Labeling, hybridisation and scanning of the microarrays were done at the Functional Genomics Core, Microarray Service at City of Hope . Probe signals were extracted using the Agilent Feature Extraction software (version 10.5.1.1). Each ChIP-on-chip experiment was performed two times and the average of the biological replicates is shown in 2 (Cy5/Cy3) of the mean signals of the probes were calculated, and imported into R software (version 2.9.0) (http://www.r-project.org). Using the Bioconductor package “aroma.light,” lowess normalization as implemented by the function “normalizeLoess,” was used to correct for die bias. The log2 ratios for samples at 0hpi and 12hpi were scaled to have the same median absolute deviation (MAD) using the function “normalizeBetweenArrays,” from the “limma” Bioconductor package Log2 ratios for each overlapping probe were averaged and a score was assigned to each base pair (bp). A moving average was calculated across the genome by sliding a 150-bp window stepwise by 1 bp unidirectionally. When calculating the moving average, the window is shifted past regions where there are missing probes so that regions with missing probes do not have any weight. Regions where no probes exist were assigned an enrichment value of “0.” Log2 ratios were converted to linear scale, and tracks in BED format were created for each sample. Tracks were then imported into Genetrix software (Epicenter Software) for visualization.To visualize enrichment across the KSHV genome, the normalized log2 ratios were calculated as described above. Log2 ratios were averaged for each 50 bp nonoverlapping window upstream and downstream of the TSS within the promoter for each gene. The TSS was assigned a single score. The resulting matrix was applied to perform hierarchical clustering with Cluster 3.0 (http://bonsai.ims.u-tokyo.ac.jp/~mdehoon/software/cluster/software.htm). The results of clustering were then imported into Java TreeView for visualization (version 1.1.4r2) For the KSHV promoter array analysis promoter regions were defined as 1000 bp upstream and downstream of the translational start sites (TSS) of each viral gene. The coordinates of ORFs are listed in While the 0 hpi-ChIPs were compared to the 0 hpi-DNA inputs in ChIP-on-chips the 12 hpi-ChIP-on-chips were analysed in two different ways. (1) 12 hpi-ChIPs were compared to 0 hpi-ChIPs , S8, S10Figure S1(A) TRExBCBL1-RTA cells were treated by 1 ug/ml of doxycycline for 6, 12 and 24 hours and subject to immunoblot analysis with the indicated antibodies. myc/His-Rta was detected by anti-myc antibody. Actin was used to monitor protein amounts in the cell lysates. (B) An aliquot of cells used in (A) was used for purification of total RNA to measure the indicated viral mRNAs. (C) Using the same number of cells, the copy number of KSHV genome in Dox-induced  TRExBCBL1-RTA cells relative to that in non-induced (0 hpi) cells were determined by qPCR using primers specific for the indicated genomic regions. The amount of viral DNA was normalized for the cellular DNA input.RTA-mediated reactivation of KSHV. (0.35 MB TIF)Click here for additional data file.Figure S2(A) The schematic map shows the KSHV genome and arrows indicate the KSHV genomic regions tested in ChIPs. RTA and LANA promoters are shown in more details. 1 to 16 represents genomic regions spanning the RTA promoter and the intron of RTA (+0.8 kb) relative to the translational start site of RTA. CBF1-binding sites are highlighted in blue. (B) Non-induced and Dox-induced TRExBCBL1-RTA cells were used to perform ChIPs for hisone H3 on different viral gene promoters or (C) on cellular promoters. (D) Non-induced and Dox-induced TRExBCBL1-RTA cells were tested in immunoblot for histone H3 and indicated histone modifications.Dissociation of histone H3 from the replicating KSHV genome. (0.59 MB TIF)Click here for additional data file.Figure S3A) ChIP-on-chip for histone H3. (B) Histone modifications on the KSHV genome during latency. (C) Changes of histone modification on the KSHV genome upon lytic reactivation.Genome-wide mapping of histone modifications on the KSHV genome during latency and reactivation. It shows one of the biological replicates of the ChIP-on-chip experiments (Chromatin A). Details are described in (0.88 MB TIF)Click here for additional data file.Figure S4A) ChIP-on-chip for histone H3. (B) Histone modifications on the KSHV genome during latency. (C) Changes of histone modification on the KSHV genome upon lytic reactivation.Genome-wide mapping of histone modifications on the KSHV genome during latency and reactivation. It shows one of the biological replicates of the ChIP-on-chip experiments (Chromatin B). Details are described in (0.88 MB TIF)Click here for additional data file.Figure S5Normalization of the ChIP-on-chip data by histone H3 at 0 hpi. The ChIP-on-chip experiments were performed as described in (0.67 MB TIF)Click here for additional data file.Figure S6A) The 0 hpi-ChIP-on-chip dataset used in the hierarchical clustering without normalization for histone H3. The clustering was taken from B) The 0 hpi-ChIP-on-chip dataset used in the hierarchical clustering was normalized for changes in histone H3.Comparison of the hierarchical clustering of histone modifications associated with the regulatory regions of viral genes with and without H3 normalization. The clustering was performed as described in (2.96 MB TIF)Click here for additional data file.Figure S7A) Histone H3 ChIP-on-chip is the same as in B) Histone modification ChIP-on-chips were performed using non-induced TRExBCBL1-Rta cells. (C) ChIP-on-chips were performed with TRExBCBL1-Rta cells induced by doxycycline for 12 hours. Changes of the distribution of histone modifications during reactivation (12 hpi) are shown as the normalized Cy5/Cy3 ratio of 12 hpi-ChIP DNA over 0 hpi-ChIP DNA. Red line indicates that the Cy5 (ChIP at 12 hpi)/Cy3 (ChIP at 0 hpi) ratio equals one showing that there is no change in the level of histone modifications between 0hpi and 12hpi. Numbers in the left upper corners show the maximum values of Cy5/Cy3. Missing probes in specific genomic regions are shown below the genome scale (**). The alternating dark and light blue squares atop display the viral ORFs where the white triangle indicates ORFs that are expressed from the reverse DNA strand. The hpi stands for hours post-induction.Genome-wide mapping of histone modifications on the KSHV genome during latency and reactivation. Each ChIP-on-chip experiment is an average of two biological replicates. ((0.85 MB TIF)Click here for additional data file.Figure S8Hierarchical clustering of histone modifications associated with the regulatory regions of viral genes. Based on their expression patterns the viral genes were grouped as latent, IE, E and L genes and hierarchical clustering was performed within the groups. Details are described in (2.64 MB TIF)Click here for additional data file.Figure S9IE genes are repressed during latency but rapidly induced upon reactivation. Total RNAs were purified from non-induced (0 hpi) and induced  TRExBCBL1-RTA cells followed by RT-PCR using specific primers for the indicated IE transcripts. RT+: cDNA synthesis was performed with reverse transcriptase, RT−: cDNA synthesis reaction did not include reverse transcriptase.(0.25 MB TIF)Click here for additional data file.Figure S10Colocalization of EZH2 and H3K27me3 on the regulatory region of KSHV genes. Hierarchical clustering of EZH2 and H3K27me3 associated with the regulatory region of KSHV genes is performed as described in (1.34 MB TIF)Click here for additional data file.Figure S11(A) Schematic diagrams of deletion mutants of the RTA promoter fused to the luciferase reporter gene. The size of the tested promoter regions relative to the translational start site of RTA is indicated on the left. (B) The deletion mutants were transfected into 293A cells in the absence or presence of RTA and assayed for luciferase activity. Data represent the average of three independent experiments. (C) Nuclear extract of BCBL1 was subject to DNA affinity purification with DNA fragments derived from the RTA promoter region. Fragment A includes the 3 CBF1 binding sites at −1.4 kb relative to the translational start site of RTA. Fragment B is derived from the adjacent promoter region of Fragment A that does not contain any CBF1 binding sites. Immunoblot was performed with an anti-CBF-1 specific antibody.Binding of CBF1 to the RTA promoter is essential for RTA-mediated activation of its own promoter. (0.13 MB TIF)Click here for additional data file.Figure S12(A) Overexpressed HMTs were detected by immunoblotting with HA-, JMJD2A- and JMJD3-specific antibodies. (B and C) HA-tagged JMJD2A, JMJD3, UTX and UTXm were transfected into Vero cells, which were subject to immunofluorescent analysis at 3 days post-transfection. The expression of enzymatically active JMJD2A suppressed the steady-state level of H3K9me3, whereas JMJD3 and UTX decreased H3K27me3 in transfected cells.Overexpression of histone demethylases in Vero cells. (1.27 MB TIF)Click here for additional data file.Figure S13(A) JSC-1 cells were treated with 5 uM DZNep for 1, 2 and 3 days (dpt) and then harvested for immunoblot analysis with an anti-PARP antibody. (B) JSC-1 cells were treated with different concentrations of DZNep for 3 days and then subject to immunoblot analysis with the indicated antibodies. (C) JSC-1 cells were treated with 200 nM of staurosporine (STS) for 1, 2 and 3 days followed by immunoblot analysis for the indicated cellular proteins and histone modifications . (D) A portion of control and 3dpt JSC-1cells used in (C) was used for RNA purification and the levels of the indicated KSHV mRNA were measured by RT-qPCR.Induction of apoptosis is not sufficient to induce KSHV reactivation. (0.55 MB TIF)Click here for additional data file.Table S1Primer sequences(0.06 MB DOC)Click here for additional data file.Table S2Genomic coordinates of the open reading frames of KSHV. IE = immediate early, E = early, L = late. Start codon coordinates indicate the first nucleotide of the KSHV genes and the stop codon coordinates indicate the last nucleotide in the stop codon of the KSHV genes. Coordinates are based on U75698.1 at GenBank.(0.05 MB DOC)Click here for additional data file.


WT1). To rapidly identify coordinately expressed prostate cancer growth control genes that may be regulated by WT1, we used an in silico approach.Gene expression analyses have led to a better understanding of growth control of prostate cancer cells. We and others have identified the presence of several zinc finger transcription factors in the neoplastic prostate, suggesting a potential role for these genes in the regulation of the prostate cancer transcriptome. One of the transcription factors (TFs) identified in the prostate cancer epithelial cells was the Wilms tumor gene  gene commonly known as the prostate specific antigen (PSA) gene. This analysis located several potential WT1 TFBS in the PSA gene promoter and led to the rapid identification of a novel putative binding site confirmed in vivo by ChIP. Conversely for two prostate growth control genes, androgen receptor (AR) and vascular endothelial growth factor (VEGF), known to be transcriptionally regulated by WT1, regulatory sequence conservation was observed and TF binding in vivo was confirmed by ChIP.Evolutionary conserved transcription factor binding sites (TFBS) recognized by WT1, EGR1, SP1, SP2, AP2 and GATA1 were identified in the promoters of 24 differentially expressed prostate cancer genes from eight mammalian species. To test the relationship between sequence conservation and function, chromatin of LNCaP prostate cancer and kidney 293 cells were tested for TF binding using chromatin immunoprecipitation (ChIP). Multiple putative TFBS in gene promoters of placental mammals were found to be shared with those in human gene promoters and some were conserved between genomes that diverged about 170 million years ago , therefore implicating these sites as candidate binding sites. Among those genes coordinately expressed with Overall, this targeted approach rapidly identified important candidate WT1-binding elements in genes coordinately expressed with WT1 in prostate cancer cells, thus enabling a more focused functional analysis of the most likely target genes in prostate cancer progression. Identifying these genes will help to better understand how gene regulation is altered in these tumor cells. In the United States, prostate cancer is the most common form of cancer in men and is the second most deadly cancer in men killing more than 27,000 annually . Nearly WT1) and the early growth response gene (EGR1), zinc finger transcription factors that bind at G-rich promoters of genes that regulate growth. In fact, the WT1 TF binds at several G-rich sites (GNGNGGGNG), including the EGR1 consensus binding site GCGGGGGCG [Global gene expression analyses have led to a better understanding of growth control of prostate cancer cells -5. OngoiCGGGGGCG -9. Both CGGGGGCG -12. WT1 CGGGGGCG .AR) and vascular endothelial growth factor (VEGF) gene promoters [in silico analyses were based on 24 genes expressed in prostate cancer epithelium that were likely to influence growth of prostate cancer cells. Putative TFBS were computationally predicted; however, the identification of functional TFBS is a challenge and requires an alternative approach. Availability of complete genomic sequence from multiple species allows identification of evolutionary conserved elements, e.g. cis-regulatory elements. Functionally important elements are likely to experience purifying selection pressure [While identifying prostate growth control pathways potentially regulated by WT1, we have focused our studies on candidate genes belonging to known growth regulatory pathways. We have previously described WT1 regulation of the androgen receptor .Importantly, for the ls Table . The EGRGATA2, WT1, and NPY genes showed 25% to 100% conservation between human and other primates. Conservation of AP2 sites was the strongest in the NPY gene promoter as these sites are also conserved between human and opossum  that are conserved among several mammalian genomes  was conserved between all primates, rodents, and opossum, thus, depicting millions of years of conservation of this particular site , an important diagnostic marker. Sequence alignment of the KLK3 promoter revealed three WT1 sites and two SP1 sites, with two-thirds of the WT1 and one-half of the SP1 sites conserved between human and other primates (Table in vivo. PCR primers were designed to flank the region where adjacent conserved WT1 (human 1332–1352) and the SP1 sites (human 1404–1418) were identified  in hormone responsive LNCaP prostate cancer cells  and SP1 (human 1721–1735) site and a WT1 site (human 1755–1771) were conserved between primates and dogs, but not in rodents  has been successful in identifying functionally important regions, including those regions that regulate gene expression ,27-34. Hin silico TFBS predictions, to identify those sites most likely to regulate expression of target genes that influence growth of prostate cancer cells. Regulatory regions with functional importance can be expected to exhibit sequence conservation due to selection. Thus, predicted TFBS that are located in the orthologous positions in multiple genomes are likely to be truly functional. Our identification of evolutionary conserved WT1 and SP1 binding sites in the PSA promoter indeed supports this notion  is a homolog of WT1 [EGR1 has been implicated as a cancer suppressor gene and activates genes required for differentiation [EGR1 promoter in vitro [EGR1 target genes, such as the N-myc downstream regulated gene 1 (NDRG1), an α/β hydrolase. In many cancer cell lines it has been shown to be up-regulated by both hypoxia and hormone treatment suggesting that it could be linked to androgen induced differentiation and signaling in the prostate [The early growth response 1 gene .While analyzing the homologous sequences of the different gene promoters, numerous overlapping TFBS were found, suggesting competition for binding and differential regulation of these gene promoters. Several studies have shown that EGR1 and SP1 TFBS often overlap ,64,65. WTGFβ1) and EGR1 promoters, while EGR1 functions as an activator [Additionally, WT1 and EGR1 compete for binding at shared TFBS. WT1 recognizes and binds to EGR1 sites on the promoters of many different genes ,9,66-68.ctivator ). For exPSA promoter where an overlapping EGR1/SP1 site is 50 base pairs downstream of a WT1 site and in the VEGF promoter where an EGR1/SP1 overlapping site is 20 base pairs away from a WT1 site. Such sites can facilitate synergistic interactions or may be required for inducible expression, as described for AR and GATA2 interactions in the PSA promoter [VEGF promoter an SP1 site adjacent to a non-canonical estrogen receptor (ER) TFBS contributes to hormone induction of VEGF expression [IGF1R) promoter [Combinations of adjacent and overlapping EGR1, WT1 and SP1 TFBS conserved between multiple species were found in multiple gene promoters. Adjacent sites were found in the promoter . Additiopression . Similarpromoter . These cPSA promoter was verified in vivo by ChIP assays. Similarly, a transcriptionally active WT1 binding site in the VEGF promoter was confirmed by ChIP and found to be in a region conserved amongst primates. Thus, these genes could be part of a novel network of regulatory pathways initiated by WT1 and have important implications in the progression of prostate cancer.Genes coordinately expressed in prostate cancer epithelial cells have conserved regulatory elements and an abundance of overlapping zinc finger TFBS. Potential WT1 gene targets were identified based on TFBS sequence conservation, and the significance of the WT1 TFBS in the Homo sapiens) genome, the NCBI m36 assembly of mouse (Mus musculus) genome, the Pan Tro 2.1 assembly of chimp (Pan troglodytes) genome, a whole genome shotgun (WGS) preliminary assembly Btau_3.1 of cow (Bos Taurus) genome, a WGS assembly Can Fam2.0 of dog (Canis familiaris) genome, a WGS preliminary assembly Mmul_1 of rhesus monkey (Macaca mulatta) genome, the Mon Dom5 assembly of opossum (Monodelphis domestica) genome, and the RGSC3.4 assembly of rat (Rattus noregicus) genome. Since major regulatory elements are located within several hundred base pairs of transcription start sites [EGR1 promoter this extended beyond 1.5 kb, so was assigned a negative number. The genome viewer and annotation program Artemis was used to ensure the correct context of genomic sequences [For each of the 24 prostate cancer growth regulatory genes differentially expressed, the complete or draft genomes of eight different mammalian species were downloaded from the Ensembl Genome Browser ,72. The rt sites , 1.5 kb equences . In eachAR and VEGF promoter sequences containing the functional WT1 TFBS for the human AR and VEGF promoters were obtained from Ensembl . For alignment analyses of known functional sites [al sites ,15, an oTFBS of WT1, EGR1, SP1, SP2, AP2 and GATA1 were predicted for each gene by the program MatInspector  that utiRegions that are conserved in multiple genomes are often found to correspond to functionally important ones . HoweverWt1 gene (lacking both KTS insertion and exon 5) fused to GFP coding region were obtained from Dr. A. Ward [LNCaP prostate cancer cells (ATCC-CRL 1740) and human embryonic kidney 293 cells ATCC-CRL 1573) were cultured in RPMI or DEM/F12  media, respectively, as described [73 were c A. Ward . All DNATaq polymerase  and the following set of primers: VEGF primers (F) 5'TTCCTAGCAAAGAGGGAACG3' and (R) 5'ACCAAGGTTCACAGCCTGAA3'; AR primers (F) 5'TATCTGCTGGCTTGGTCATGGCTTG3' and (R) 5'CTGCTTCCTGAATAGCTCCTGCTT3'; and PSA primers (F) 5'TCTGCCTTTGTCCCCTAGAT3' and (R) 5'AACCTTCATTCCCCAGGACT3'. Following an initial 10 min denaturation at 95°C, DNA was amplified by 32 cycles of: 1) 20 sec denaturation at 95°C, 2) 30 sec annealing at either 58°C (for VEGF primers) or 59°C (for AR and PSA primers) and 3) 30 sec extension at 72°C; amplification was completed with a 2 min final extension at 72°C. PCR products were fractionated on 1% agarose gel, and ethidium bromide stained DNA was visualized by a gel doc system . Specificity controls are shown in Additional file The Farnham ChIP protocol  was usedWT1: Wilms tumor 1; TFBS: transcription factor binding sites; ChIP: chromatin immunoprecipitation; AR: androgen receptor; VEGF: vascular endothelial growth factor; PSA: prostate specific antigen; KLK3: kallikrein-related peptidase 3; PCR: polymerase chain reaction; GFP: green fluorescent protein.TFs: transcription factors; in silico analysis, and drafted the text. ST performed in silico analysis. ABazarov performed in silico analysis. ABrett performed ChIP analysis and contributed to the text. GF planned functional assays, guided student research, and drafted the text. HP planned in silico analyses, guided student research, and drafted the text.KE performed ChIP analysis, Evolutionary conserved TFBS in promoters of 24 genes expressed in prostate cancer epithelium. This table lists evolutionary conserved transcription factor binding sites in promoters of 24 genes expressed in prostate cancer.Click here for fileWT1 bound the proximal, but not the distal, region of the amphiregulin (AREG) gene promoter in chromatin of LNCaP cells. This specificity control illustrates WT1 binding to the proximal region (known to bind WT1), but not the distal region of the AREG promoter in LNCaP chromatin.Click here for file


C. elegans is a powerful system for comparative molecular analyses of environmental stress response mechanisms. Infection of worms with bacterial and fungal pathogens causes the activation of well-characterized innate immune transcriptional programs in pathogen-exposed hypodermal and intestinal tissues. However, the pathophysiological events that drive such transcriptional responses are not understood. Here, we show that infection-activated transcriptional responses are, in large part, recapitulated by either physiological or genetic activation of the osmotic stress response. Microarray profiling of wild type worms exposed to non-lethal hypertonicity identified a suite of genes that were also regulated by infection. Expression profiles of five different osmotic stress resistant (osr) mutants under isotonic conditions reiterated the wild type transcriptional response to osmotic stress and also showed substantial similarity to infection-induced gene expression under isotonic conditions. Computational, transgenic, and functional approaches revealed that two GATA transcription factors previously implicated in infection-induced transcriptional responses, elt-2 and elt-3, are also essential for coordinated tissue-specific activation of osmosensitive gene expression and promote survival under osmotically stressful conditions. Together, our data suggest infection and osmotic adaptation share previously unappreciated transcriptional similarities which might be controlled via regulation of tissue-specific GATA transcription factors.The soil-dwelling nematode  The innate immune system is an evolutionarily ancient response to pathogen infection C. elegans has proven to be a powerful model system for genetic and genomic analysis of innate immunity C. elegans can be infected through both the intestine and hypodermis by several bacterial and fungal pathogens, including the human opportunistic pathogen Pseudomonas aeruginosaC. elegans by most bacterial pathogens does not involve cell invasion, replication, and lysis C. elegans are still not clear. Nonetheless, genetic screens have led to the identification of a conserved ASK/p38 MAP kinase signaling pathway that is activated by infection and required for proper infection inducible gene expression, toxin defenses, and normal lifespan The nematode C. elegans, the transcription factors functioning downstream of p38 MAPKs to execute infection regulated gene expression programs are less well defined. Recent work C. elegans GATA-type transcription factors, elt-2 and elt-3, may promote pathogen resistance and pathogen-inducible gene expression in the intestine and hypodermis, respectively. Drosophila and humans also play functionally important roles in pathogen resistance Drosophila, GATAs function together with rel-type transcription factors, such as Dif, Dorsal, and Relish to promote immune responsive gene expression C. elegans genome does not express known homologs of rel-type transcription factors, suggesting that GATA factors may function as key integrators of cellular stressors and immune responses. Currently, it is unclear whether C. elegans GATA factors are involved in the response to specific pathogen signals or to more general pathogen-evoked disruptions in host cell homeostatic processes.In contrast to the clear roles for p38 MAP kinase signaling pathways in the regulation of innate immune responses in C. elegans, mimics previously described transcriptional responses to bacterial and fungal infection. Transcriptional profiling of five different osmotic stress resistant (osr) mutants reiterated the wild type response to osmotic stress and also exhibited a substantial overlap with numerous infection models, suggesting that the overlap is genetically regulated. Using bioinformatics, transgenic promoter analysis, and loss-of-function studies, we found that two GATA factors, elt-2 and elt-3, are required to mediate osmosensitive gene expression in the intestine and hypodermis, respectively. We also find that simultaneous inhibition of both elt-2 and elt-3 suppresses physiological and genetic resistance of animals to osmotic stress. These data add to our understanding of the mechanisms of infection by suggesting that osmotic dyshomeostasis may represent a significant pathophysiological consequence of infection in C. elegans. Furthermore, our findings further implicate GATA factors as key post-developmental integrators of diverse stress responses in C. elegans. Given the highly conserved links between innate immunity and osmoregulation in mammals, understanding the molecular mechanisms of osmosensing and osmosensitive gene expression in C. elegans may also shed new light on mechanisms of pathogen-induced cellular toxicity.In this work, we show that osmotic stress, a natural environmental stress for C. elegans is exposed to unstable osmotic environments and must adapt to these environments to survive. Previously, we showed that upregulation of a glycerol biosynthetic enzyme resulted in osmotically induced glycerol accumulation, which is required for proper adaptation to hyperosmotic environments gpdh-1, without causing significant lethality As a soil-dwelling organism, Osmotically Regulated Genes (ORGs). We validated our microarray data by performing real-time quantitative PCR on a subset of ORGs from independently derived biological samples. In all cases, the qPCR data correlated well with microarray results . However, of these 78 genes, only 42.3% (33/78) showed concordance in the direction of transcriptional regulation. Furthermore, the makeup of the Cd2+ and osmotic stress responses were functionally distinct. For example, Cd2+ induced the expression of 14 heat shock proteins, whereas osmotic stress failed to induce any HSPs and repressed the expression of three HSPs. In addition to these differences between ORGs and Cd2+ responsive genes, ORGs did not exhibit significant overlap with transcriptional responses to either ER stress or hypoxia, two additional stressors that are also thought to cause tissue damage Using stringent statistical criteria see ‘’, we ide results . Since oosr) mutants grown under isotonic conditions. osr mutants exhibit many of the physiological characteristic of wild type animals adapted to hyperosmotic stress, such as high gpdh-1 expression, glycerol accumulation, and resistance to normally lethal levels of hypertonicity osr mutants mis-express these phenotypes under isotonic conditions, suggesting that osr mutants encode genes that are negative regulators of osmosensitive signaling pathways and that loss of function mutations in osr genes cause constitutive activation of these pathways. The osr mutants osm-7 and osm-11 encode homologous secreted proteins that may also function as Notch ligands dpy-9 and dpy-10 encode extracellular matrix collagens, and osm-8 encodes a secreted mucin-like protein . osr mutants exhibit no obvious signs of cellular or tissue damage under isotonic conditions osr mutants are likely responsive to osmosensitive signaling pathways rather than secondarily responsive to tissue damage.To further determine whether activation of ORGs was responsive to tissue damage or to genetically regulated activation of osmosensitive signaling pathways, we performed microarray analysis of five osmotic stress resistant  were also regulated by all five osr mutants under isotonic conditions . Taken together, these data suggest that the osr mutants genetically activate a transcriptional program that is also physiologically activated by hyperosmotic stress in wild type C. elegans.Using stringent statistical criteria see ‘’, we fouype ORGs , S5, S6. mM NaCl , suggestnditions , which i dataset . Genes sosr mutants, we noticed an enrichment for several genes and gene classes previously associated with microbial infection in C. elegans. These included neuropeptide like proteins (nlp), caenicin (cnc), and c-type lectin (clec) gene classes . This suggested that, like other complex physiological processes such as ageing in vivo osmotic stress resistance. To identify transcription factors that might regulate osmotic stress responses in vivo, we first performed an unbiased search of the promoters (1Kb) of all upregulated ORGs and their C. briggsae orthologs for transcription factor binding sites using all binding motifs described in the TRANSFAC motif database  gpdh-1 promoter for all three predicted GATA binding sites  exposure to 200 mM NaCl  also suppressed the genetic activation of intestinal, but not hypodermal, gpdh-1p::GFP expression in the osr mutant osm-7 (elt-2(RNAi) on osmotic stress responses was not simply due to a general defect in intestinal gene expression, since elt-2(RNAi) exhibited normal expression of an intestinal ER-stress reporter (hsp-4p::GFP) (data not shown).The above data suggested that one or more GATA factors may play functionally important roles in the regulation of osmosensitive gene expression and pression . elt-2, elt-3(gk121), and double mutant elt-2(RNAi); elt-3(gk121) animals following exposure to hyperosmotic stress. Each of the single elt mutants survived a hyperosmotic challenge as well as wild type animals  mutants was not significantly suppressed in either elt-2(RNAi) or elt-3(gk121) backgrounds (osm-8(n1604); elt-2(RNAi); elt-3(gk121) triple mutants exhibited a significant reduction in chronic osmotic stress resistance  by which activation of osmotic stress responses mimic infection is currently unclear but one intriguing mechanism may relate to infection-induced disruptions in osmotic homeostasis. For example, many pathogens, via their production of pore-forming toxins and Type III secretion systems, create ion and water permeable pores in the plasma membrane C. elegans to a purified pore-forming toxin C. elegans are reduced following infection with toxin-compromised pathogens.Previous studies of infection in Drosophila, salt stress induces a broad transcriptional response that includes the induction of numerous infection regulated genes C. elegans, these data suggest the intriguing hypothesis that cellular osmotic stress may be an evolutionarily conserved and physiologically significant activator of immune responses. Interestingly, the C. elegans genome does not contain rel-type transcription factors similar to NFAT5/TonEBP, suggesting that the functional links between osmotic stress and immunity may have preceded the evolution of this family of transcription factors. C. elegans should provide an outstanding model system in which to better explore the functional, molecular, and evolutionary relationships between immunity and osmotic stress.Functional links between infection and osmotic stress have also been observed in other systems. For example, in mammals, T-cell activation in the thymus occurs in hypertonic environments, the maintenance of which requires the rel-type transcription factor NFAT5/TonEBP C. elegans does not express a homolog of this or any other rel factor. Therefore, the transcription factor(s) controlling osmosensitive gene expression in C. elegans have remained mysterious. Our data reveal an important role for two GATA-type transcription factors, elt-2 and elt-3, in this process. Both GATA factors are also required for other developmental and non-developmental processes elt-2 and elt-3 to determine their specific responses. For example, while elt-2 is required for both osmosensitive and infection-inducible gene expression, the sek-1/pmk-1 p38 MAP kinase signaling pathway is only required for pathogen-induced transcriptional responses and not osmosensitive gene expression elt-2 and studies of another developmentally required transcription factor, skn-1, which is required for oxidative stress responses in the C. elegans intestine. Multiple signaling pathways converge on skn-1, which integrates these signals to coordinate appropriate responses to ageing, insulin signaling, caloric restriction, and oxidative stressors elt-2, like skn-1, exhibits direct regulation of localization, DNA binding, and/or activity in response to osmotic stress and/or infection.In animals, the molecular mechanisms regulating osmosensing and osmosensitive gene expression are not well understood. As mentioned above, the rel factor NFAT5 is essential for osmosensitive gene expression in mammals, yet C. elegans. Tissue-specific GATA-type transcription factors elt-2 and elt-3 play a major role in coordinating both of these gene expression programs and are required for the response to osmotic stress. Our studies functionally link cellular osmotic stress responses with innate immunity and suggest that osmotic dyshomeostasis might represent a new and evolutionarily significant paradigm for the physiological basis of host cell pathogen sensing.In conclusion, our findings show that both osmotic stress and pathogen infection regulate similar transcriptional targets in E. coli strain OP50. NGM media was made hypertonic by the addition of NaCl to the indicated concentration. The following strains were used: LGI: kbIs5; LGII: dpy-10(e128), osm-8(n1518); LGIII: osm-7(n1515); LGIV: dpy-9(e12); LGX: osm-11(n1604), elt-3(gk121). Unless otherwise noted, all strains were grown at 20°C. The wild type N2 strain was obtained from the Caenorhabditis elegans Genetic Stock Center.All strains were maintained using standard culture methods and fed with the gpdh-1::GFP reporter constructs were created using PCR or site directed mutagenesis of the construct described in myo3p::dsRed2 plasmid and 30 ng/µl of the relevant gpdh-1 construct using standard microinjection methods Hypochlorite synchronized L1 stage wild type animals were grown to the young adult stage on standard NGM plates. Worm were washed off plates using isotonic M9 solution (∼170 mOsm) and placed onto NGM plates containing 200 mM NaCl for 15 minutes, 1 hour, or 6 hours. For the steady state samples, synchronized L1 stage animals were placed onto NGM plates containing 200 mM NaCl and allowed to grow to the young adult stage. For all samples, 1000 adult worms were collected into 1.5 ml tubes using gating criteria on the COPAS Biosort that excluded L4 and younger animals. Immediately following sorting, worms were pelleted at 2,000 RPM and the supernatant was aspirated, leaving ∼100 µl on top of the worm pellet. 400 µl of Trizol was added and the solution was vortexed for two minutes. Worms were stored at -80° until RNA isolation. For each timepoint, five individual replicates were performed. The best three preparations  were used for RNA labeling and hybridization. The remaining two preparations were used for qPCR validation of microarray results.C. elegans GeneChips. All protocols were conducted as described in the Affymetrix GeneChip Expression Analysis Technical Manual. Briefly, 2 µg of total RNA was converted to first-strand cDNA using Superscript II reverse transcriptase primed by a poly(T) oligomer that incorporated the T7 promoter. Second-strand cDNA synthesis was followed by in vitro transcription for linear amplification of each transcript and incorporation of biotinylated CTP and UTP. The cRNA products were fragmented to 200 nucleotides or less, heated at 99°C for 5 min and hybridized for 16 h at 45°C. The microarrays were then washed at low (6X SSPE) and high  stringency and stained with streptavidin-phycoerythrin. Fluorescence was amplified by adding biotinylated anti-streptavidin and an additional aliquot of streptavidin-phycoerythrin stain. A confocal scanner was used to collect fluorescence signal at 3 um resolution after excitation at 570 nm. The average signal from two sequential scans was calculated for each microarray feature.Total RNA was extracted using a combined Trizol (Invitrogen)/RNAeasy  column method. Purified total RNA was submitted to the University of Pennsylvania Microarray Core facility for RNA quality control, labeling, and hybridization to Affymetrix Affymetrix .cel files for all arrays were uploaded into the Partek Genomics Suite and intensity values were normalized using the GC-RMA algorithm. Using Affymetrix present/absent calls, 13,866/22,625 probes were ‘present’ in at least 3/30 datasets, and these probes were used for all subsequent statistical analysis. Data across the wild type series was analyzed using the Significance analysis of Microarrays (SAM) algorithm cpt-6, which encodes a carnitine palmitoyl transferase whose expression levels are unchanged by hypertonicity. Initial test showed that data normalization using probes against clh-3 and lam-3, whose expression was also unchanged by hypertonicity, showed similar results to those achieved with cpt-6 (data not shown).cDNA was reverse synthesized from 1 µg total RNA  and qPCR was performed using an ABI7300 thermocycler and TaqMan probe sets . Each reaction was performed in 20 µl reactions in technical triplicate or quadruplicate from 2–3 biological replicates. Data were normalized to expression levels of Survival assay were performed by placing 10–20 young adult animals onto OP50-seeded NGM plates containing the indicated amount of NaCl. Animals were scored for survival after 24 hours. Worms were considered dead when they failed to respond to harsh plate tapping and light touch with a platinum wire. Animals that died as the result of internally hatched progeny (“bag-of-worms” phenotype) were censored from the analysis. Each NaCl concentration was repeated in quadruplicate and each assay was repeated 2–3 times. Figures illustrate the data from one representative experiment.elt-2 RNAi experiments, L1 stage animals were fed control RNAi bacteria (HT115 containing the pPD129.36 empty vector (EV) plasmid) for 24 hours at 16°, washed from plates, and distributed to EV or elt-2(RNAi) plates for 72 hours at 16° prior to either survival assays or COPAS measurements. Using these conditions, we were able to circumvent the developmental arrest phenotype of elt-2(RNAi) and still achieve ∼90% knockdown of elt-2 mRNA, as assessed by qPCR (data not shown). For survival assays, EV(RNAi) and elt-2(RNAi) animals were transferred to NGM RNAi plates containing the indicated levels of NaCl and survival was measured as described above. For COPAS assays, kbIs5 animals were washed onto new RNAi plates containing the indicated RNAi clone and either 50 mM or 200 mM NaCl. At the indicated timepoints, animals were analyzed on a COPAS Biosort for time-of-flight (TOF) and GFP expression. Raw data were filtered to select adult animals (TOF>300) and the GFP data was normalized to the TOF measurement (GFP/TOF).For the C. elegans ORG dataset (foreground dataset) and their C. briggsae orthologs were searched for the presence of a transcription factor binding motif using the 811 motifs or Positional weight Matrices (PWM) in TRANSFAC 10.2 (www.gene-regulaiton.com). We also performed the search using 8451/8956 C. elegans/C. briggsae ortholog pairs that exhibited detectable expression in our microarray data (background dataset) The motif scan was done using a previously described tool, PWM_SCAN th percentile of range of scores achievable by a PWM. For a motif, given the number of matches in the foreground dataset (ORGs), the p-value corresponding to the motif occurrence was estimated based on 1000 random samplings of the background dataset and computing the fraction of times that the motif occurence equaled or exceeded its occurrences in the foreground dataset. Only motifs with p-values<0.05 in both the C. elegans and C. briggsae datasets were considered significant.1 kb of promoter sequence (upstream of the start ATG) from each gene in Groups 2–5 of the Data are presented as means ± S.E. Survival and qPCR gene expression data were analyzed using either the Student's T-test or one-way ANOVA, as implemented in Graphpad Prism . Enrichment scores and enrichment significance were calculated as previously described Figure S1qPCR validation of hypertonic stress microarray data. cDNA prepared from two independent biological replicates was used as the template for quantitative RT-PCR see . Data sh(0.47 MB TIF)Click here for additional data file.Figure S2ORGs do not exhibit significant overlap with other stress-induced transcriptional responses. ORGs were compared to previously published microarray studies of the endoplasmic reticulum unfolded protein response (UPR) and the response to hypoxia (0.38 MB TIF)Click here for additional data file.Figure S3Pearson Correlation analysis between WT ORGs and osr mutants. The pairwise Pearson Correlation coefficient was determined between each dataset. Each experimental replicate is shown under the indicated genotype.(0.77 MB TIF)Click here for additional data file.Figure S4Truncation analysis of the gpdh-1 promoter. Transgenic animals containing the indicated lengths of the gpdh-1 promoter (from start ATG) were fused to GFP using a PCR fusion method [52]. GFP expression in transgenic animals was quantified on a COPAS Biosort following exposure to either 50 mM NaCl or 200 mM NaCl. N>150 animals for each measurement.(0.42 MB TIF)Click here for additional data file.Table S1osr mutants (Excel file).Osmotically regulated genes in wild type following 15 minute, 1 hour, 6 hour, and 1 generation of exposure to 200 mM NaCl and their regulation in five (0.10 MB XLS)Click here for additional data file.Table S2osm-7(n1515) mutants and their regulation in four other osr mutants (Excel file).Genes regulated in (0.06 MB XLS)Click here for additional data file.Table S3osm-8(n1518) mutants and their regulation in four other osr mutants (Excel file).Genes regulated in (0.23 MB XLS)Click here for additional data file.Table S4osm-11(n1604) mutants and their regulation in four other osr mutants (Excel file).Genes regulated in (0.09 MB XLS)Click here for additional data file.Table S5dpy-9(e12) mutants and their regulation in four other osr mutants (Excel file).Genes regulated in (0.59 MB XLS)Click here for additional data file.Table S6dpy-10(e120) mutants and their regulation in four other osr mutants (Excel file).Genes regulated in (0.70 MB XLS)Click here for additional data file.Table S7Overlap between ORGs and genes regulated by eight different models of pathogen infection (Excel file).(0.02 MB XLS)Click here for additional data file.


RNA Polymerase II (RNAP II) is recruited to core promoters by the pre-initiation complex (PIC) of general transcription factors. Within the PIC, transcription factor for RNA polymerase IIB (TFIIB) determines the start site of transcription. TFIIB binding has not been localized, genome-wide, in metazoans. Serial analysis of chromatin occupancy (SACO) is an unbiased methodology used to empirically identify transcription factor binding regions. In this report, we use TFIIB and SACO to localize TFIIB binding regions across the rat genome.A sample of the TFIIB SACO library was sequenced and 12,968 TFIIB genomic signature tags (GSTs) were assigned to the rat genome. GSTs are 20–22 base pair fragments that are derived from TFIIB bound chromatin. TFIIB localized to both non-protein coding and protein-coding loci. For 21% of the 1783 protein-coding genes in this sample of the SACO library, TFIIB binding mapped near the characterized 5' promoter that is upstream of the transcription start site (TSS). However, internal TFIIB binding positions were identified in 57% of the 1783 protein-coding genes. Internal positions are defined as those within an inclusive region greater than 2.5 kb downstream from the 5' TSS and 2.5 kb upstream from the transcription stop. We demonstrate that both TFIIB and TFIID  bound to internal regions using chromatin immunoprecipitation (ChIP). The 5' cap of transcripts associated with internal TFIIB binding positions were identified using a cap-trapping assay. The 5' TSSs for internal transcripts were confirmed by primer extension. Additionally, an analysis of the functional annotation of mouse 3 (FANTOM3) databases indicates that internally initiated transcripts identified by TFIIB SACO in rat are conserved in mouse.Our findings that TFIIB binding is not restricted to the 5' upstream region indicates that the propensity for PIC to contribute to transcript diversity is far greater than previously appreciated. The core promoter is the major regulatory element responsible for determining transcriptional output. The core promoter spans a region of 40–50 bases and encompasses the transcript start site . The corRecently, the isolation and analysis of the mouse transcriptome by the functional annotation of mouse 3 (FANTOM3) consortium indicates that most protein-coding genes produce multiple transcripts . ImportaIn this report, we use serial analysis of chromatin occupancy (SACO) to identify TFIIB binding regions in the rat genome. SACO allows an unbiased and genome-wide interrogation of transcription factor binding regions ,16. In tnude mice [insulin and cFos [Fcgr2b and muscle specific, myocardin, Mycd)  Figure . TFIIB bs Figure . ChIP as6 GSTs indicative of putative TFIIB binding regions. A portion of the library corresponding to 19,204 GSTs was sequenced. Of these, 12,968 (68%) could be assigned to a unique position in the rat genome, similar to the fraction of unique GSTs in previously characterized SACO libraries [The TFIIB SACO library contains approximately 10ibraries ,16. We mgamma-aminobutyric acid A receptor subfamily (Gabrs). We then localized all TFIIB GSTs in the library relative to RefSeq gene boundaries. TFIIB GSTs were classified as 5' promoter associated if they were found in an inclusive region 2.5 kb outside and 2.5 kb inside the transcript start site (TSS), 3' promoter associated if they were within a region 2.5 kb outside and 2.5 kb inside the 3' end, and internal if they were greater than 2.5 kb inside either the 5' or 3' ends of gene boundaries. The 2.5 kb limit was chosen based upon agarose gel determination of the uppermost size of the chromatin fragments included in library construction , mammalian core promoters less frequently contain TATA boxes, have a lower frequency of pairing TATA with Inr elements, and many promoters, including those within CpG islands, appear to lack all three of these core elements [u is upstream of the TATA box and BREd is downstream. It is known that both the BREu and the BREd modulate promoter activity but that this effect is dependent on the specific composition of elements present within a core promoter [In the last few years, we have begun to understand the complexity and diversity of core promoters that reside in the mammalian genome . This iselements . Two TFIpromoter . This copromoter . Using apromoter  or this The most significant finding of our study is that TFIIB binding localizes to positions other than the 5' promoter of protein-coding genes. While the strong co-occurrence of TFIIB GSTs with transcript start sites identified by the FANTOM3 group suggest that these alternative positions identify promoters, TFIIB has been shown to have additional roles outside of transcript start site selection. Singh and Hampsey recently reported that TFIIB binding to terminator sequences located downstream of protein coding regions . InsteadThe results presented here provide the first genome-wide mapping of TFIIB binding in a metazoan. Because TFIIB is required for RNAP II dependent transcription, our SACO screen provides an unbiased localization of promoter elements. Our finding that TFIIB occupation of internal regions is common within genes suggests that the full-length protein-coding transcripts may in fact represent a fraction of the genetic output from these loci. Identification of evolutionarily conserved internal promoters suggests that adjacent transcripts may be subjected to regulation that is independent of the 5' untranslated region and promoter elements. Clearly we are only at the very beginnings with our understanding of the transcriptome and its regulation in higher eukaryotes.2 and were 70–75% confluent at the time of harvesting.Rin-m cells (ATCC #CRL-2057), passage 5–10, were grown in RPMI 1640 (Invitrogen) supplemented with 10% FBS (Hyclone), 100 units/ml penicillin, 100 units/ml streptomycin, and 5 mM L-glutamine. Cells were maintained at 37°C and 5% CO6 cells and were conducted as reported [Antibodies used for ChIP included: 3 μg TFIIB c18 , 3 μg RNAP II , 10 μl TFIID , and 3 μg Gal4 . According to the manufacturer the TFIID antibody is directed against TBP. ChIP assays contained 5 × 10reported ,27. Briereported . PrimersFor a complete protocol for constructing a SACO library see ,21. The Sequencing was performed at High-Throughput Sequencing Solutions .6 Rin-m cells were lysed using a Qiashredder column (Qiagen) and RNA was isolated using the RNeasy kit (Qiagen). Genomic DNA was removed using the DNA free kit (Ambion). First strand cDNA was synthesized from 1 μg of DNA free RNA using a random hexamer primer (Invitrogen) and Superscript III Reverse Transcriptase (Invitrogen). Reactions lacking reverse transcriptase were analyzed to survey the presence of genomic DNA. Real-time PCR was conducted with 50 ng of cDNA and the assay was capable of detecting 10–50 copies of target cDNA.5 × 10A modification of the first choice RNA mediated rapid amplification of cDNA ends kit  was used to identify 5' capped transcripts associated with TFIIB GSTs as reported ,21. TotaCBP were end-labeled with 32P-γ-ATP (Perkin Elmer) using T4 polynucleotide kinase (NEB). Unincorporated 32P-γ-ATP was removed using a NucAway spin column (Ambion) and specific activity of the probes was determined using a 2200CA liquid scintillation analyzer (Packard). Total RNA was isolated using TRIzol (Invitrogen) and 10 μg was annealed to the radiolabeled primer at 58°C for one hour. cDNA was extended using AMV reverse transcriptase (Promega). Products were resolved on a 7 M urea/1× TBE /8% polyacrylamide denaturing gel using a sequencing gel apparatus . An end-labeled Φ× HinfI digested ladder was included for size reference.Primer extension was performed with the primer extension/AMV reverse transcriptase system (Promega). Primers were designed approximately 50 bp downstream of the 5' capped nucleotide of Initial processing and placement of the GSTs followed the pipeline established for our previous SACO libraries ,16. ChroGSY and SM conceived the experimental design. GSY constructed the SACO library, performed the chromatin immunoprecipitations, cap-trapping, and primer extensions. RC assisted with validation of the SACO library. VR and SM positioned TFIIB GSTs relative to individual chromosomes and to RefSeq gene annotation. VR and SM also performed the analysis with the mouse TSS data deposited by the FANTOM3 consortium. GSY and SM wrote the manuscript. All authors gave final approval of the version to be published.Distribution of TFIIB GSTs across rat chromosomes. A list of mapped GSTs, corresponding chromosomes, and number of GSTs identified per megabase (Mb) of DNA.Click here for fileLocation of TFIIB GSTs and associated RefSeq genes on chomosome 10. Gray bars on the chromosomes are the TFIIB GSTs within 2.5 kb of a RefSeq gene (indicated above the chromosome).Click here for fileSonicated chromatin input for SACO library construction. Ethidium bromide stained 1% agarose gel of sonicated chromatin used to generate the TFIIB SACO library. L: 1 kb DNA ladder (Invitrogen). The sizes of some ladder fragments are indicated in nucleotide bases.Click here for file


Altogether, our results provide the molecular explanation to the B12/23 constraint and also uncover a ‘Dβ1 23RSS-mediated’ restriction operating beyond chromatin accessibility, which directs Dβ1 ordered rearrangements.T cell receptor (TCR) β gene assembly by V(D)J recombination proceeds via successive Dβ-to-Jβ and Vβ-to-DJβ rearrangements. This two-step process is enforced by a constraint, termed beyond (B)12/23, which prohibits direct Vβ-to-Jβ rearrangements. However the B12/23 restriction does not explain the order of TCRβ assembly for which the regulation remains an unresolved issue. The initiation of V(D)J recombination consists of the introduction of single-strand DNA nicks at recombination signal sequences (RSSs) containing a 12 base-pairs spacer. An RSS containing a 23 base-pairs spacer is then captured to form a 12/23 RSSs synapse leading to coupled DNA cleavage. Herein, we probed RSS nicks at the TCRβ locus and found that nicks were only detectable at Dβ-associated RSSs. This pattern implies that Dβ 12RSS and, unexpectedly, Dβ 23RSS initiate V(D)J recombination and capture their respective Vβ or Jβ RSS partner. Using both  Immunoglobulin (Ig) and T-cell receptor (TCR) genes are assembled from separate variable (V), diversity (D) and joining (J) gene segments via a series of site-specific events of DNA rearrangement, termed V(D)J recombination. This process requires the binding of the lymphocyte-specific recombination activating gene 1 and 2 (RAG1/2) protein complex to recombination signal sequences (RSSs) flanking the rearranging sides of individual V, D and J gene segments The molecular mechanism of V(D)J recombination has been described in great detail i) cell lineage specificity (with for example TCR gene rearrangement occurring in T lymphocytes only); (ii) developmental specificity (with for example TCRβ gene rearrangement occurring prior to that of TCRα); and, at some loci, (iii) allele specificity . By and large, these controls are thought to involve lineage- and developmentally-regulated changes in chromatin structure that precisely modulate the accessibility of individual Ig/TCR gene loci and/or segments, with their associated RSSs, to the unique RAG1/2 recombinase A tight regulation of V(D)J recombination ensures proper lymphocyte development and eludes lymphoid malignancy-causing chromosomal translocations in vivo and in vitro, we provide evidence that, at the TCRβ locus, Dβ-flanking 23- and 12RSSs constitute primary anchoring sites for PC formation for D-to-J and V-to-DJ rearrangements respectively. Most importantly, we found that the Dβ1 23RSS also prohibits RAG1/2-mediated nicking at the adjoining 5′Dβ1 12RSS. These data elucidate the mechanism of B12/23 and reveal a role for the Dβ1 23RSS in imposing ordered (‘D-J prior to V-DJ’) rearrangement at the Dβ1 locus.Beyond the chromatin barrier, individual 12- and 23RSS-flanked gene segments can still display high disparity in recombination frequency, mainly due to nucleotide variations in their RSSs and/or adjacent coding flanks The oligo-capture assay, initially described by Curry et al. −CD8− double-negative (DN) thymocytes of a WT mouse was oligo-captured using heptamer-specific oligonucleotides, T4 DNA ligase and proper restriction enzymes. Next, the digested DNAs were fractionated using streptavidin-conjugated magnetic beads and the captured DNAs tested for the presence of TCRβ sequences of interest using PCR and Southern blotting (−/−) thymocytes. We also assessed background level from DNA samples treated in parallel but omitting the heptamer oligonucleotide. Finally, each captured DNA at Jβ, Dβ or Vβ gene segments were compared with that at a Cβ2 gene fragment lacking RSS sequences.We used the oligo-capture approach to probe RSS nicks associated with rearranging TCRβ gene segments in early developing T lymphocytes. Briefly, genomic DNA from cell-sorted CD4vs. Dβ differential recovery since when focusing on the Vβ8.1/8.2/8.3 segments  representing ∼20% of total Vβ rearrangements in vitro. The results indicate that the pCACAGTG-biotin heptamer can capture an RSS carrying a RAG1/2-mediated nick (According to the previous study suggesting that the 12RSS initiates V(D)J recombination and captures the 23RSS, we expected to observe some nicks at Jβ 12RSS. However, we didn't detect any oligo-captured Jβ1 or Jβ2 DNA. Of note, a greater number of Jβ gene segments cannot explain the difference between the amounts of Dβ versus Jβ capture since we investigated all segments together within each Jβ1 or Jβ2 genomic cluster (see legend to −/−) thymocytes in which Dβ-Jβ clusters display a heterochromatin structure To verify that RAG1/2 cleavage activity is primarily dependant on RSS accessibility, we used DNA from TCRβ enhancer-deleted  of V(D)J recombination and especially the aptitude of RSSs to form a functional RAG∶RSS single complex, visualized by the production of single-strand nicks. To do so, the incubation with the RAG1/2 extract was limited to 5 min, and the two 38 and 27 nucleotides (nt) fragments corresponding to respectively RAG1/2-mediated 12- and 23RSS nicks, were monitored  or quite low (∼1.5% for 3′Dβ1-Jβ2.5 substrates). This outstanding asymmetry is consistent with a model of PC nucleation whereby the RAG1/2 proteins first react with the Dβ1 23RSS before the capture of a free Jβ 12RSS. Nicking profiles of Dβ2-Jβ2 and Dβ1-Jβ substrates are qualitatively similar. However, Dβ1 and Dβ2 23RSS yielded different amount of single strand nicks, respectively ∼30% and 4% of input material , leading to a defect of Vβ/Jβ PC formation.Altogether, our in vivo oligo-capture assay at the Dβ1 locus, since (conversely to Dβ2 locus) nicks at Dβ1 12- and 23RSSs can be distinguished due to their divergent heptamers. In our previous oligo-capture assay  requires the previous elimination of the Dβ1 23RSS. To test if the inhibition of RAG1/2 cleavage activity on the Dβ1 12RSS is mediated by the neighboring Dβ1 23RSS and not by the chromatin structure, we performed in vitro cleavage assays. We first carried out in vitro nicking assays using Dβ-based substrates. As previously shown, nicking at a single RSS can occur in presence of Mg2+ ions in the buffer 14), in which the Dβ1 23RSS is replaced by the Vβ14 23RSS (gel 4). On the contrary, preferential cleavage fell on the 12RSS. These data therefore suggest a regulatory function unique to the Dβ1 23RSS which, in the germline situation, might anchor RAG1/2 catalytic activity at the expense of the neighboring Dβ1 12RSS.cis-rearrangement, we next performed in vitro RSS coupled-cleavage assays using various forms of recombination substrates (1) and a related substrate carrying the Vβ14 23RSS at the 3′side of Dβ1 gene segment (pVDv). Conversely, cleavage was severely reduced when using a substrate carrying the Dβ1 23RSS (pVD1). Additional experiments demonstrated coupled cleavage within Dβ1/Jβ1-containing substrates whether the Dβ1 12RSS was present or not , arguing that this site has no detrimental effect on PC nucleation involving the downstream 3′Dβ1-/Jβ1-associated RSSs.To further assess the possibility that the Dβ1 23RSS impairs Vβ-to-5′Dβ1 in vitro data using non chromatinized templates demonstrate that RAG1/2 catalysis preferentially targets the Dβ1 23RSS instead of Dβ1 12RSS. Since Dβ1 23RSS mediates the inhibition of the adjacent Dβ1 12RSS nicking, the nucleation of Dβ/Vβ synaptic complex formation is impeded. Thereby, this ‘Dβ1 23RSS-mediated restriction’ provides a potential mechanism to direct ordered rearrangements (‘D-J prior V-DJ’) at the Dβ1 locus.Overall, our wt) minilocus system. This system has been shown to recapitulate the main features of endogenous TCRβ gene assembly, including B12/23 restriction and ordered TCRβ assembly  In vitro results, using D1V14 and pVDv substrates, have shown that Dβ1 12RSS cleavage is not impaired when the Vβ14 23RSS (instead of the Dβ1 23RSS) lies at the 3′side of Dβ1  type, display broad disparities in their overall ability to undertake the first catalytic step of V(D)J recombination, RAG1/2-mediated nicking. Within the limits of sensitivity of single strand nick assays, these range from a relatively high potential (Dβ1 23RSS) to lower aptitude  to near ineffectiveness . The proficiency of the Dβ1 23RSS to undergo RAG1/2-mediated nicking activity is coupled with an inhibition of that at the 5′ adjoining 12RSS. These data have a number of implications for the biology of V(D)J recombination and the control of TCRβ gene assembly. Notably, the emerging picture that nicks preferentially accumulate at Dβ segments strengthens the model that recombination synapsis proceeds via the capture of a free RSS by a RAG1/2-loaded partner in vitro, with complete absence at the endogenous TCRβ locus in WT DN cells. These data strongly argue that one aspect of the B12/23 constraint results from the inability of Vβ 23- and, especially, Jβ 12RSSs to initiate PC assembly, and therefore to form a synapse together. Our data do not establish where RAG1/2 proteins bind; therefore they don't discriminate between two possible hypotheses to explain the scarce nicking at Vβ and Jβ RSSs: these RSSs are poor substrates for either RAG binding or for the RAG nicking reaction per se. The first hypothesis is not supported by previous EMSA studies showing that RAG binding to Dβ1 12RSS and to Jβ 12RSSs was equivalent 2+ buffer, thus it cannot be excluded that DNA binding properties of RAG1/2 proteins in Ca2+ and Mg2+ buffers differ slightly. Also, if we considered that some additional proteins could be involved in RAG binding to RSS We observed an ineffective RAG1/2-mediated nicking of Vβ/Jβ substrates i.e., avoid PC nucleation at Jβ RSSs). In contrast heptamers and nonamers of Dβ RSSs are close to the consensus sequences. Additionally, Dβ RSSs display high conservation across distant species  AP-1 binding site), affect both nicking and coupled cleavages of 3′Dβ1-Jβ1.1 substrates and also partially abolish the ‘Dβ1 23RSS-mediated restriction’ J recombination, thus if the heptamer is flanked by a “bad” coding sequence (such as T or A stretch) the recombination efficiency may decrease Our genome is scattered with sequences akin to RSS , but surprisingly, these cryptic RSSs are rarely mis-targeted by the recombinase  and the germline promoter pDβ1, controlling the initiation of V(D)J recombination −/− thymocytes confirming that RSS accessibility is a prerequisite for RAG1/2 cleavage activity. Therefore chromatin structure and epigenetic marks, by modulating appropriately RSS accessibility (or inaccessibility) of the various TCRβ gene segments, could be sufficient for a tight regulation of V(D)J recombination. Nonetheless, mechanisms distinct from RSS accessibility exist to ensure B12/23 restriction +CD8+ T-cells undergoing V(D)J recombination, Vβ gene segments upstream of a functional VDJβ1 rearrangement are maintained in an active chromatin environment but were still restricted from further rearrangement despite the proximity of Dβ2 gene Similarly to other DNA transactions, V(D)J recombination is prominently regulated by chromatin structure and modifications −/−) −/−) WTDMF mice were housed under specific pathogen-free conditions, and handled in accordance with French and European directives.The D10 cell line −/− or Eβ−/− thymocytes was prepared as previously described Total thymocytes were incubated 1 h at 37°C in the presence of rabbit complement  and rat IgM anti-mouse-CD4 (RL172.4) and -CD8α (3.155) antibodies. Living cells were collected on a ficoll gradiant . Cell preparations were >95% DN as determined by flow cytometric analysis. Genomic DNA from purified DN WT, RAG12 and 1 U Taq DNA polymerase (Invitrogen). Amplified DNAs were separated through a 1% agarose gel, transferred onto a Biodyne B membrane, and hybridized using a 5′ end 32P radio-labeled specific probe , individually produced by PCR amplification using a plasmid from group II as template and oligonucleotide primers #181 and #318 .Substrates for DNA cleavage were constructed using PCR amplified fragments from various genomic DNA regions within the mouse TCRβ locus and standard molecular cloning procedures. PCR amplifications  were performed using Platinum® Taq DNA Polymerase High Fidelity (Invitrogen) and appropriate oligonucleotide primers Table S4The RAG1/2-containing extract was prepared from heat-shocked D10 cells according to a published protocol 2, 1 mM DTT) supplemented with the RAG1/2 extract (20–30 µg), 1.5 mM rATP, and proper recombination substrate (0.3 pmol). To increase cleavage efficiency, 6 µg of a nuclear extract prepared from mouse WT thymocytes were also added. Negative controls were carried out using similar conditions without addition of the RAG1/2 extract. After phenol extraction and ethanol precipitation, the DNA samples were electrophoresed through a 1% agarose gel and analyzed by Southern blot using a Biodyne B transfer membrane . Membranes were hybridized with TCRβ-specific, radio-labeled probes A (5′-GAGAAGAGTAGAGGACTGTGGGCCTTGG-3′) or B (5′-GACTTGAATCATGTTGTTTTCC-3′). For RAG1/2-mediated nicking assays, the substrate was first digested by restriction enzymes AccI and NcoI. The resulting 700 bp fragment was gel purified  and radio-labeled at 5′ ends using T4 polynucleotide kinase (Invitrogen) and γ32P-[ATP] . The labeled substrate (∼0.1 pmol) was used for DNA cleavage as described above, except that incubation was for 5 min. The DNA samples were then deproteinized and further digested by restriction enzymes EcoO109I/XbaI (that cut within RSS-intervening sequences) to ensure proper quantification of nicked vs. intact RSSs. Formamide loading buffer was added to the digests and samples were heated at 95°C then separated by 15% polyacrylamide gel electrophoresis (PAGE) under denaturing conditions (7 M urea). Nicking assays of the 5′Dβ1, 3′Dβ1, Dβ1 or D1V14 amplified products used similar conditions except that the deproteinized samples were electrophoresed directly without further restriction enzyme treatment.RAG1/2-mediated coupled cleavage was performed for 3 h at 30°C in a final volume of 25 µL of cleavage reaction buffer Click here for additional data file.Figure S2Pairwise modulation of RAG1/2-mediated coupled cleavage.(0.20 MB PDF)Click here for additional data file.Figure S3RAG1/2-mediated coupled cleavage of pTCRβWT and pTCRβDMF substrates;(0.12 MB PDF)Click here for additional data file.Figure S4Phylogenetic profiles of Dβ and Jβ RSSs(0.15 MB PDF)Click here for additional data file.Figure S5Effect of Dβ1 23RSS spacer mutations on RAG1/2-mediated cleavages(0.16 MB PDF)Click here for additional data file.Figure S6Cloning strategy to construct recombination substrates(0.02 MB PDF)Click here for additional data file.Figure S7Cloning strategy to construct additional recombination substrates(0.03 MB PDF)Click here for additional data file.Table S15′-phosphorylated 3′-biotinylated oligonucleotides (7-mers) and restriction enzymes used in the oligo-capture assays to displace and ligate the nicked strand and to restrict the genomic DNA before purification on streptavidin-conjugated magnetic beads.(0.05 MB PDF)Click here for additional data file.Table S2DNA sequences of the RSSs  used in this study.(0.01 MB PDF)Click here for additional data file.Table S3Oligonucleotide primers and hybridization probes used in the oligo-capture assays for PCR amplification and Southern blotting identification of the captured DNAs(0.05 MB PDF)Click here for additional data file.Table S4Oligonucleotides used in the construction of the various DNA cleavage substrates analyzed in this study.(0.03 MB PDF)Click here for additional data file.Text S1Consideration of an Alternative capture model.(0.07 MB PDF)Click here for additional data file.