Chromatin proteins have expanded the mammalian synthetic biology toolbox by enabling control of active and silenced states at endogenous genes. Others have reported synthetic proteins that bind DNA and regulate genes by altering chromatin marks, such as histone modifications. Previously, we reported the first synthetic transcriptional activator, the “Polycomb-based transcription factor” (PcTF) that reads histone modifications through a protein–protein interaction between the polycomb chromodomain motif and trimethylated lysine 27 of histone H3 (H3K27me3). Here, we describe the genome-wide behavior of the polycomb-based transcription factor fusion protein. Transcriptome and chromatin profiling revealed several polycomb-based transcription factor-sensitive promoter regions marked by distal H3K27me3 and proximal fusion protein binding. These results illuminate a mechanism in which polycomb-based transcription factor interactions bridge epigenomic marks with the transcription initiation complex at target genes. In three cancer-derived human cell lines tested here, some target genes encode developmental regulators and tumor suppressors. Thus, the polycomb-based transcription factor represents a powerful new fusion protein-based method for cancer research and treatment where silencing marks are translated into direct gene activation. An engineered protein that manipulates DNA-protein packaging in cancer cells could offer a powerful new approach to treating tumors. Karmella Haynes and colleagues from Arizona State University in Tempe, USA, designed a protein that can recognize chemical tags on DNA-wound chromatin structures where gene activity is being silenced and then reactivate those epigenetically repressed genes. In previous work, Haynes’ team showed how this “Polycomb-based transcription factor” could stimulate gene expression at two silenced genes in one cancer cell line. Now, working in three different cancer cell types, the researchers demonstrate that treatment with this engineered protein increases the expression levels of many genes across the genome involved in developmental regulation and tumor suppression. If the engineered protein works specifically in cancer cells, while leaving healthy tissue alone, it could prove beneficial for patients. Then the effector proteins enhance or inhibit transcription initiation. Much of our current understanding about chromatin-mediated gene regulation comes from deconstructive methods such as genetic mutations and RNA interference  motif of CBX paralogs.10 We used this interaction to design a polycomb-based transcription factor (PcTF) that has VP64 (tetrameric VP16) and a visible red fluorescent tag (mCherry RFP) fused to the C terminus of the 60 amino acid PCD. The C-terminal VP64 domain allows PcTF to stimulate activation at repressed H3K27me3-associated genes . PcTF also activated endogenous silenced loci in an osteosarcoma cell line (U-2 OS).11 Low-resolution protein mapping experiments at four loci identified two promoter regions that were co-occupied by PcTF and H3K27me3. Further investigation is needed to understand and accurately predict targets of the PcTF transcription activator. Here, we report genome-wide analyses that substantially advance our understanding of PcTF function. PcTF-stimulated gene activation in three different cancer cell types identified a large cohort of universally upregulated genes with a signature H3K27me3 enrichment profile. PcTF is enriched at transcription start sites within the nucleosome-free region of promoters and this profile depends upon the methyl-histone-binding PCD domain. We present a model where PcTF bridges distal H3K27me3 with endogenous transcription factors near the transcription start site. These findings provide significant progress towards predicting targets of PcTF based on distributions of epigenomic marks.We constructed a synthetic transcription factor based on the natural effector protein CBX8. In many human cancers, lysine 27 on histone H3 (H3K27) becomes marked by trimethylation at abnormally high levels near tumor suppressor loci. CBX8-containing complexes accumulate at H3K27me3 and repress gene transcription.nes Fig. . In prev21 and protein mapping studies25 in human cancers and stem cells: ATF3, CADM1, CASZ1, CDH1, CDKN2A, CLU, HK2, HOXA6, HOXB4, IRF8, LAMB3, RUNX3, TRIM22, and WT1. We transfected U-2 OS, SK-N-SH, and K562 cells with a PcTF-expressing plasmid , neuroblastoma (SK-N-SH), and leukemia (K562). We selected a panel of 14 genes for which polycomb-mediated silencing is supported by genetic and pharmacologic disruption studiesmid Fig. . Primersmid Fig. . Next, wmid Fig. , Fig. S1CDKN2A was strongly upregulated by PcTF compared to ΔTF in U-2 OS cells.11 Here, our hydrolysis probe assay, which is more specific than the intercalating dye, shows modest upregulation (FC log2 = 0.68) of CDKN2A at the 24 h time point in U-2 OS and stronger upregulation in K562. CDKN2A is not upregulated in SK-N-SH. By including several more known polycomb targets, we have identified other genes that are specifically upregulated by PcTF in all three cell types: IRF8, CADM1, and RUNX3.In our previous work, we used an intercalating dye (SYBR green) for qRT-PCR and observed that CBX8, which matches the first ~200 bp of the PcTF open reading frame, indicated expression of the PcTF transgene  for control samples vs. fold change after PcTF expression showed that 13.4% of 23,245 annotated Refseq genes became up-regulated twofold or higher  from mock transfected (control) and PcTF-expressing cells (+PcTF) up to 96 h post-transfection. An increase in alignments to ene Fig. . Whole-ther Fig. . CASZ1 apes Fig. .Fig. 2GeMIR3658, which has 126 target genes (miRTarBase)27 and is upregulated in U-2 OS and SK-N-SH. MIR1248 has 154 target genes and is upregulated in SK-N-SH and K562. PcTF might also disrupt expression at the repressed genes by placing VP64 in a suboptimal position relative to the promoter.29We also observed down-regulation (fold-change ≤2) at 9.2% of the genes in U-2 OS. Fifty of these genes also became downregulated in SK-N-SH and K562. Repression is inconsistent with the transcriptional activation function of VP64. Based on the RNA-seq results alone, we speculated that the upregulated genes might be direct targets of PcTF . Regions of enrichment compared to non-immunoprecipitated bulk chromatin (input) were calculated using the Hotspot algorithm.30 We used shared and public data to determine H3K27me3 enrichment in SK-N-SH and K562 cells. CASZ1, IRF8, and CADM1 are marked by H3K27me3  and become upregulated upon PcTF expression in all three cell types. CLU and HOXA6 are enriched for H3K27me3 in two cell types, and only become up-regulated when H3K27me3 is present . These results are consistent with a mechanism where PcTF accumulates at H3K27me3 sites and induces gene transcription.To investigate further, we used data from chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) experiments to look for an epigenetic signature of PcTF-sensitive genes. We determined the enrichment of H3K27me3 at the 14 Polycomb targets Fig.  and near32 Genes with lower basal expression levels, prior to PcTF expression, show the highest levels of H3K27me3 enrichment, which is consistent with the role of H3K27me3 in gene silencing. Genes that became up-regulated in response to PcTF expression showed the highest H3K27me3 levels compared to non-responsive and down-regulated genes in K562 cells . In SK-N-SH and U-2 OS however, up-regulated genes showed the same or lower H3K27me3 levels as non-responsive genes. Therefore, PcTF-sensitivity may not be determined by the mere presence of H3K27 methylation within 5 kb of the transcription start site. Instead, positioning of H3K27me3 relative to the TSS might distinguish PcTF-sensitive genes.Overall, H3K27me3 enrichment was depleted near transcription start sites at silenced and active genes, indicated by a sharp valley at position zero in stratified TSS-plots Fig. . This prWe hypothesized that a specific H3K27me3 distribution pattern around the TSS determines which genes are activated by PcTF. For a subset of 194 commonly upregulated genes H3K27me3 is depleted at the TSS, which is surrounded by signal peaks at intervals of roughly 1 kb . The highest peaks for K562 were observed immediately downstream of the TSS and 3 kb upstream. These data suggest that PcTF-mediated activation of silenced genes may rely on H3K27me3 enrichment at positions distal to the TSS.11 Five of the eight genes that we identified as specifically PcTF-sensitive . IRF8, CLU, and WT1 expression decreased with increasing PcTF, suggesting that perhaps regulation of these three genes is complex and may involve endogenous factors that oppose activation. Overall, our dose-response experiment validated U2OS-PcTF as a robust system to further investigate PcTF-mediated gene regulation.In order to further investigate the mechanism of PcTF-mediated gene regulation, we used crosslinked chromatin immunoprecipitation  to measure the accumulation of PcTF and H3K27me3 at a single locus and throughout the genome. For these studies, we used an isogenic PcTF-expressing U-2 OS cell line. The cell line “U2OS-PcTF” carries a chromosomally-integrated, TetR-repressed gene that expresses myc-tagged PcTF when the cells are treated with doxycycline (dox) Fig. , as descive Fig.  showed aive Fig. . Frequenive Fig. . Express33 at a PcTF-sensitive locus in U2OS-PcTF cells that were formaldehyde-fixed at different time points after dox treatment. A time course flow cytometry analysis of RFP in U2OS-PcTF cells confirmed PcTF accumulation over time from 2 to 10 h . For ChIP analysis, we used CASZ1 as a model locus. This gene was consistently upregulated by PcTF in the three cells types tested here, responded to PcTF in a dose-dependent manner in U2OS-PcTF, and the locus is free of other overlapping or nearby genes that might complicate analysis. We analyzed five sites by quantitative PCR (qPCR) of DNA from IP-enriched chromatin. These sites were located at the TSS and along 10 kb of the upstream region ion Fig. . A GAPDHCASZ1 locus showed H3K27me3 enrichment ~ 2 kb upstream, and at lower levels at other sites, including the TSS , the TSS Fig. , Fig. S611 The binding profile of PCD showed enrichment across the 10 kb region upstream of CASZ1, which overlapped with H3K27me3 10 h. after induction , which indicated that PcTF and H3K27me3 distributions were not due to random crosslinking. We analyzed the distributions of PcTF and H3K27me3 ChIP at different genomic features and observed the greatest enrichment over background for intergenic regions . PcTF, but not H3K27me3, showed signal enrichment at promoters. These results indicate that PcTF and H3K27me3 co-occupy non-coding regions, and that PcTF is frequently enriched at promoter sites.To further investigate the mechanism of PcTF-mediated gene regulation, we used ChIP-seq to compare the distribution of PcTF and H3K27me3 throughout the genome and at TSS regions. A parental U-2 OS cell line  was used to observe unaltered H3K27me3 patterns in the absence of PcTF. ChIP-seq analysis of an isogenic, dox-inducible U2OS-ΔTF cell lineWe inspected promoter regions more closely by identifying PcTF and H3K27me3-enriched regions surrounding TSS’s. We identified 5535 total regions that were marked with PcTF or H3K27me3 or both. A TSS plot showed a peak of PcTF enrichment at the TSS over a 2 kb interval Fig. . RandomlIRF8, CASZ1, RUNX3, HOXB4, and WT1. We observed a significant (p < 0.05) increase in expression for two of these upregulated genes, IRF8 and CADM1 . We identified the closest element upstream or downstream of 23,245 TSS’s and calculated median distances between the midpoint of each element and the TSS , both up- and down-regulated genes were located near class 3 poised promoters, which are characterized by the presence of H3K27me3 and H3Kme4 and are thought to support gene state plasticity.36 Up- and down-regulated genes also showed greater proximity to class 12 repressed regions, which are typically enriched for Polycomb-associated marks such as H3K27me3. None of the chromatin classes we investigated showed a significant bias in TSS proximity specifically for the up-regulated genes. Our investigation of local chromatin features implicates poised promoters and Polycomb-repressed regions as factors for PcTF-sensitivity.Non-coding, intergenic regions play an important role in protein-mediated gene regulation. Many of these regions contain elements that show conservation of epigenetic marks across human cell types.TSS Fig. . Genes w37 suggesting a strong interaction with endogenous transcription factors. The C-terminal domain of PcTF includes VP64, which is composed of four copies of a core acidic transcription activation domain (TAD) from VP16.38 The TAD is derived from the H1 region of VP16, which has been shown to bind with high affinity (high nanomolar range) to the mediator complex subunit MED25.39 The VP16 TAD also interacts with MED17, several members of the transcription factor II (TFII) family, and the TATA promoter motif-binding protein TBP .45 Ectopic expression of IRF8 in renal cell carcinoma has been shown to decrease colony formation and migration.46 Overexpression of CADM1 reduced proliferation in cervical cancer cell lines C33A, HeLa, SiHa, and CaSki.47 It will eventually be important to directly compare the efficacy of PcTF to RNAi knockdown or chemical inhibition (e.g. UNC3866) of Polycomb proteins.48 This comparison will determine whether PcTF-mediated stimulation of the transcription activation complex at silenced genes is truly advantageous over the simpler delivery of small molecules that inhibit polycomb and generate indirect activation. Furthermore, work to determine the specificity of PcTF in cancer vs. healthy cells and to enhance efficient synthetic protein delivery will bring this promising new fusion protein-based technology closer to translation.Our work has significant implications for medicine. Currently, epigenetic engineering relies largely on small-molecule inhibitors that are intended to erase cancer-associated epigenetic silencing and indirectly activate therapeutic genes. These drugs have major shortcomings such as incomplete erasure of cancer-associated methyl-histone marks and DNA damage. Here, we used a synthetic chromatin protein, PcTF, to convert the methyl-histone signal into direct activation of therapeutic genes instead of inhibitors to erase cancer-associated marks. Genes that were consistently upregulated by PcTF in all three cell lines we tested have therapeutic potential. Work by others has shown that overexpression of the 11 The plasmid is constitutively expressed in the absence of a TetR protein . The full annotated sequences of PcTF  and ΔTF  are available online at Benchling—Hayneslab: Synthetic Chromatin Actuators (https://benchling.com/hayneslab/f/S0I0WLoRFK-synthetic-chromatin-actuators/).The doxycycline-inducible PcTF-expressing plasmid has been described.2 incubator. U2OS-stable cell lines were generated by previously published work.11 PcTF-expressing U-2 OS, K562, and SK-N-SH cells were generated by transfecting 5×105 cells in 6-well plates with DNA/Lipofectamine complexes: 2 μg of plasmid DNA, 7.5 μl of Lipofectamine LTX (Invitrogen), 2.5 PLUS reagent, 570 µl OptiMEM. Transfected cells were grown in pen/strep-free growth medium for 18 h. The transfection medium was replaced with fresh, pen/strep-supplemented medium and cells were grown for up to 48 h (K562) or up to 96 h (U-2 OS and SK-N-SH).Complete growth media contained 10% tetracycline-free fetal bovine serum and 1% penicillin and streptomycin (pen/strep). U-2 OS (ATCC HTB-96), U2OS-PcTF, U2OS-PCD, and U2OS-ΔTF cells were cultured in McCoy’s 5A. K562 (CCL-243), and SK-N-SH (ATCC HTB-11) cells were cultured in IMDM or EMEM, respectively. Cells were grown at 37 °C in a humidified CO6). Adherent cells (U-2 OS and SK-N-SH) were lysed directly in culture plates with 500 μl TRIzol. Suspended cells (K562) were collected into tubes, pelleted by centrifugation at 1000×g for 3 min at room temperature, separated from the supernatant, and lysed in 500 μl TRIzol. TRIzol cell lysates were extracted with 100 μl chloroform and centrifuged at 12,000×g for 15 min at 4 °C. RNA was column-purified from the aqueous phase (Qiagen RNeasy Mini kit 74104). SuperScript III (Invitrogen) was used to generate cDNA from 2 μg of RNA. Real-time quantitative PCR reactions (15 μl each) contained 1× LightCycler 480 Probes Master Mix (Roche), 2.25 pmol of primers ; cooling, ramp at 2.2 °C s−1–40 °C, hold 30 s. Crossing point (Cp) values, the first peak of the second derivative of fluorescence over cycle number, were calculated by the Roche LightCycler 480 software. Expression level was calculated as delta Cp, 2^[CpGAPDH–Cp experimental gene]. Fold change (log2) was determined as delta Cp transfected cells/delta Cp mock. Plasmid expression scaling for the values shown in Fig. 2) for mCherry (plasmid expression) for the 24-, 48-, and 72 h time points divided by the 24 h value. Values for target genes within each time point were multiplied by the appropriate quotient.Total messenger RNA was extracted from ~ 90% confluent cells ~1–2×10–2×106. ARNA-seq was performed using one sample per experimental condition for transiently transfected cells and three replicates for the U2OS-PcTF and U2OS-ΔTF cell lines. Total RNA was prepared as described for qRT-PCR. Overall, 50 ng of total RNA was used to prepare cDNA via single primer isothermal amplification using the Ovation RNA-Seq System (Nugen 7102-A01) and automated on the Apollo 324 liquid handler (Wafergen). cDNA was sheared to ~300 bp fragments using the Covaris M220 ultrasonicator. Libraries were generated using Kapa Biosystem’s library preparation kit (KK8201). In separate reactions, fragments from each replicate sample were end-repaired, A-tailed, and ligated to index and adapter fragments . The adapter-ligated molecules were cleaned using AMPure beads , and amplified with Kapa’s HIFI enzyme. The library was analyzed on an Agilent Bioanalyzer, and quantified by qPCR  before multiplex pooling and sequencing on a Hiseq 2000 platform (Illumina) at the ASU CLAS Genomics Core facility.49 with the hg19 reference genome (Feb. 2009 GRCh37). Enrichments normalized to input were calculated using the Hotspot algorithm (distribution version 4).31 ChIP-seq data for SK-N-SH was provided by B. Bernstein and K562 data was retrieved from the UCSC Genome Browser website.50 Galaxy 51 was used to identify overlaps with a minimum of 1 bp between gene intervals and ChIP-seq enrichment intervals. Raw sequence data (fastq) was analyzed with FastQC52 and processed to remove low-quality reads and adapters using TrimmomaticSE.53 RNA-seq alignments were carried out with de-multiplexed 50-bp single-end reads and the hg19 transcriptome. Within the public Galaxy online platform, splice junctions were mapped using Tophat  and Bowtie 2 and transcript abundance and differential expression were calculated using Cuffdiff54 and the Homo sapiens UCSC hg19 gene transfer format (gtf) file from Illumina iGenomes. Out-dated genes were identified by cross-referencing gene symbols with the NCBI database and removed from the Cuffdiff output in Galaxy, resulting in 23,245 genes. Gene comparisons and Venn diagrams were generated with Venny 2.1 (http://bioinfogp.cnb.csic.es/tools/venny/)55 and Biovenn (http://www.cmbi.ru.nl/cdd/biovenn/).56 Microsoft Excel was used to calculate distances between features  and to generate graphs and charts.ChIP-seq alignments were carried out using the Bowtie2 algorithmg for 5 min. Cell pellets were washed twice with 10 ml cold 1× PBS with Pierce Protease Inhibitors. Overall, 70 μl of cross-linked cells were resuspended in 112.5 μl of cell lysis buffer (10 mM Tris pH 8 (ThermoFisher), 10 mM NaCl, 0.2% IGEPAL (Sigma)) plus Protease Inhibitors and incubated on ice 10 min. Lysed cells were spun for 5 min at 400xg. Nuclei were resuspended and lysed in 1 ml of nuclei lysis buffer (1% sodium dodecyl sulfate (SDS) (Sigma), 10 mM ethylenediaminetetracacetic acid (EDTA) (Fisher Scientific), 50 mM Tris–HCl pH 8.1 (Sigma)) plus Protease Inhibitors and incubated on ice for 10 min. Lysed nuclei were diluted with 0.5 ml of ChIP dilution buffer (1% Triton X-100 (Santa Cruz Biotech), 2 mM EDTA, 150 mM NaCl (Sigma), 20 mM Tris–HCl, pH 8) and split into five 300 μl-aliquots. Samples were sheared with the Qsonica Q700A Sonicator with a 5.5″ Cup Horn. Sonicated chromatin samples were spun at 16,300xg for 10 min at 4 °C to remove impurities and frozen at −80 °C. To determine shearing efficiency, DNA from 100 μl of each sample was purified by incubation at 65 °C in 100 mM NaCl overnight, at 37 °C with 10 μg RNase A (Sigma) for 30 min, and at 62 °C with 10 μg Proteinase K (Qiagen) for 2 h. DNA fragment sizes of ~500 bp were confirmed via electrophoresis on a 1% agarose gel.Adherent U-2 OS cells were directly crosslinked in culture plates in 20 ml of 1% formaldehyde (Thermo Fisher Scientific)/1× PBS (Dulbecco’s) with gentle shaking for 10 min at room temperature. Cross-linking was stopped by adding 125 mM glycine, followed by 5 min gentle shaking. Quenched formaldehyde was aspirated and cells were washed twice for 5 min with gentle shaking at room temperature with 10 ml cold 1× PBS supplemented with Pierce Protease Inhibitors (Thermo Fisher Scientific). Cells were collected by scraping and spun at 200xOverall, 30 μg of each chromatin sample per immunoprecipitation was diluted to a final volume of 1 ml in ChIP dilution buffer. Magna ChIP Protein A + G Magnetic Beads (Millipore) were washed three times with PBS buffer + BSA (5 mg/ml) (Sigma). Chromatin samples were pre-cleared with 20 µl of washed beads and nutation for 3 h at 4 °C. Twenty percent of each pre-cleared sample was removed and set aside for input controls. Each chromatin sample was incubated at 4 °C for 12 h with nutation with each of the following antibodies: Anti-RNA PolII EMD Millipore 05-623, anti-H3K27me3 EMD Millipore 07-449, anti-H3K4me3 Abcam ab8580, anti-Myc (9B11) Mouse IgG Cell Signaling Tech 2276S, normal Mouse IgG Sigma 18765 (mock), and normal Rabbit IgG Cell Signaling Tech 2729S (mock). Magna ChIP Protein A + G beads were blocked by three washes in PBS buffer + BSA (5 mg/ml). Next, 20 μl of blocked beads were added to each antibody-chromatin and incubated for 3 h at 4 °C with nutation. Chromatin-antibody-bead complexes were washed twice for 10 min in RIPA buffer (50 mM HEPES pH 7.6 (Thermo Fisher Scientific), 1 mM EDTA, 0.7% sodium-deoxycholate (Sigma), 1% IGEPAL CA-630 (Sigma), 0.5 M LICl (Sigma)), twice for 10 min in a mild deteregent solution , and twice for 10 min in tris-EDTA pH 7.6 (Sigma). Elution of specifically bound chromatin was carried out in 100 μl of elution buffer , 0.1 M NaCl). Inputs were thawed and brought to 100 µl with elution buffer. Samples were incubated for 30 min with nutation at room temperature. DNA was purified as described above, cleaned with a QIAquick PCR Purification Kit (Qiagen), and eluted in 50 µl nuclease-free water.11 A ChIP-Seq DNA Sample Prep Kit (Illumina IP-102-1001) was used to prepare deep sequencing libraries from DNA that was purified from immunoprecipitated (IP) and non-IP (input) chromatin. End-repair was carried out at 20 °C for 30 min in the following 50 µl reaction: 30 µl ChIP DNA, 1× T4 DNA ligase buffer, 0.4 mM dNTP, 1 µl T4 DNA polymerase, 1 µl Klenow DNA polymerase, 1 µl T4 polynucleotide kinase. End-repair products were concentrated into a final volume of 34 µl Qiagen elution buffer (QIAquick PCR Purification kit 28104). 3′-end adenine base extension was carried out at 37 °C for 30 min in the following 50 µl reaction: 34 µl end-repair reaction product, 1× Klenow buffer, 0.2 mM dATP, 1 µl Klenow exonuclease. Base extension products were concentrated into a final volume of 10 µl H2O (Zymo Clean and Concentrator D4003). Adapter ligation was carried out at room temperature for 15 min in the following 30 µl reaction: 10 µl 3′-base extension reaction product, 1× ligase buffer, 1 µl adapter oligo mix, 4 µl DNA ligase. Ligation products were concentrated into a final volume of 10 µl H2O (Zymo Clean and Concentrator D4003). A total of 150–200 bp fragments from each ligation reaction were resolved and purified via gel electrophoresis and extraction (Zymoclean Gel DNA recovery kit D4001). DNA was back-eluted from the column twice with 10 µl H2O and brought to a final volume of 36 µl. Adapter-modified 150–200 bp fragments were enriched by the PCR in the following 50 µl reaction: 36 µl gel-purified DNA, 1× Phusion buffer, 0.3 mM dNTP, 1 µl PCR primer 1.1, 1 µl PCR primer 2.1, 0.5 µl Phusion polymerase. The cycling program was 98 °C/30 s, 18 cycles , 72 °C/5 min, 4 °C/∞. PCR products were concentrated into a final volume of 15 µl H2O (Zymo Clean and Concentrator D4003). Libraries were size-confirmed on an Agilent 2100 Bioanalyzer and subjected to deep sequencing with single-end 100 bp reads on an Illumina Hi-Seq SR flow cell platform.Chromatin immunoprecipitation was performed on U-2 OS Flp-In T-Rex cells, which carry a chromosomal insert of a TetR-repressible PcTF gene as previously described.−1–95 °C, hold 10 min; amplification, 45 cycles ; cooling, ramp at 2.2 °C s−1–40 °C, hold 30 s. To account for sampling 20% of the chromatin prep, input crossing point (Cp) values were adjusted by subtracting log2(20). % IP DNA was calculated as 100 × 2^[Cp input - Cp IP]. %Mock-IP DNA was calculated as 100 × 2^[Cp input - Cp mock] and subtracted from % IP DNA to determine % IP DNA enrichment relative to mock-IP.ChIP samples were analyzed using real-time quantitative PCR in 15 μl reactions containing 7.5 μl SYBR Green master mix, 2.25 pmol of primers, and 2 μl of IP, IgG-IP (mock), or input template DNA. The real time PCR program was run as follows: pre-incubation, ramp at 4.4 °C sSupplemental Figures and Tables


In contrast to unidirectional promoters wherein antisense transcription results in short transcripts which are rapidly degraded, bidirectional promoters produce mature transcripts in both sense and antisense orientation. To understand the molecular mechanism of how productive bidirectional transcription is regulated, we focused on delineating the chromatin signature of bidirectional promoters.We report generation and utility of a reporter system that enables simultaneous scoring of transcriptional activity in opposite directions. Testing of putative bidirectional promoters in this system demonstrates no measurable bias towards any one direction of transcription. We analyzed the NUP26L-PIH1D3 bidirectional gene pair during Retinoic acid mediated differentiation of embryonic carcinoma cells. In their native context, we observed that the chromatin landscape at and around the transcription regulatory region between the pair of bidirectional genes is modulated in concordance with transcriptional activity of each gene in the pair. We then extended this analysis to 974 bidirectional gene pairs in two different cell lines, H1 human embryonic stem cells and CD4 positive T cells using publicly available ChIP-Seq and RNA-Seq data. Bidirectional gene pairs were classified based on the intergenic distance separating the two TSS of the transcripts analyzed as well as the relative expression of each transcript in a bidirectional gene pair. We report that for the entire range of intergenic distance separating bidirectional genes, the expression profile of such genes (symmetric or asymmetric) matches the histone modification profile of marks associated with active transcription initiation and elongation.We demonstrate unique distribution of histone modification marks that correlate robustly with the transcription status of genes regulated by bidirectional promoters. These findings strongly imply that occurrence of these marks might signal the transcription machinery to drive maturation of antisense transcription from the bidirectional promoters.The online version of this article (10.1186/s12864-018-4697-7) contains supplementary material, which is available to authorized users. Saccharomyces cerevisiae revealed that cryptic transcripts arise from divergent transcription from well-defined gene promoters, which suggest that bidirectional transcription is an inherent property of eukaryotic promoters [In the human genome a substantial portion of genes 10%) are arranged in a head-to-head manner, referred to as bidirectional genes 0% are ar, 3. It hromoters . These cromoters . A few sromoters –12. Howeromoters –15. Histromoters . HistoneTo monitor bidirectional transcription, we designed an experimental system with two reporter genes arranged in a head to head orientation without any promoter element in between them as described recently . We then+ T cells. We categorized the bidirectional genes based on the intergenic distance that separates them, in terms of number of nucleosomes that can be accommodated between the two TSS. Such analysis revealed that histone modifications that mark actively transcribing promoters are coincident with transcriptional status of each gene in a bidirectional gene pair.To verify whether the observed bimodal pattern holds true across most, if not all, bidirectional promoters, we further analyzed publicly available datasets for histone modifications associated with active transcription initiation and successful transcription elongation. Histone modification profiles from two cell types were analyzed to confirm whether these profiles were cell type specific or not. We analyzed Histone 3 Lysine K tri-methylation (H3K4me3), Histone 3 lysine 27 acetylation (H3K27ac) near the TSS of 974 bidirectional gene pairs, and Histone 3 lysine 36, lysine 79 methylation  in the gene bodies of the 974 pairs in H1 human embryonic stem (ES) cells and human CD4For pairs wherein the expression is asymmetric, i.e., each gene in a pair is expressed at significantly altered level relative to its partner, a significant bias in H3K4me3 enrichment that mirrors the transcription profile was observed in both the datasets tested. Interestingly, H3K27ac does not show this correlation in both cell types and the correlation is markedly higher in H1 ES cells. In the case of symmetrically expressed bidirectional gene pairs, the histone enrichment profile for H3K4me3 is bimodal and this is never observed in any of the unidirectional genes that were used as a control set of genes. Thus, the bimodal distribution of histone modification marks that strongly correlates with the transcription status of genes presumably constitutes a chromatin signature unique to bidirectional promoters.Normal rabbit IgG (12–370) and normal mouse IgG (12–371) were purchased from Millipore/Upstate. H3K79me3 , H3K36me3 , H3K27me1 (17–643), H3K4me3 (07–473), Acetyl-Histone H4 (17–630) and H3K9ac (17–658) antibodies were procured from Millipore. All of the above antibodies were used for ChIP analysis at 5 μg antibody per 30 μg chromatin. Oct-3/4 (sc-8628) and Sox2  antibodies were procured from Santa Cruz Biotechnology. Anti-Nanog (AF1997) was purchased from R&D Systems. Anti-H3 (ab1791) was procured from Abcam. All of the above antibodies were used for immunoblot analysis at 1:1000 dilution.2 atmosphere. For transfections, HEK-293 T cells were grown up to 60% confluency in 6 well culture plates at 37 °C in DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS) (Invitrogen) and penicillin/streptomycin, under 5% CO2 atmosphere. Cells were transfected using Lipofectamine 2000 as per manufacturer’s instructions (Invitrogen). The medium was supplemented with 10% FBS 6 h post-transfection. The cells were allowed to grow for 48 h post-transfection before being imaged by confocal microscopy and flow cytometry analysis. For flow cytometry, cells were washed twice with ice cold PBS, followed by fixing with 3.7% paraformaldehyde. Cells were then acquired using FACS CANTO II flow cytometer (Becton Dickinson).HEK-293  and Jurkat  were obtained from the ECACC (European Collection of Authenticated Cell Cultures) repository. HEK293 cells and Jurkat cells were grown in DMEM and RPMI 1640 respectively. For both cells media was supplemented with 10% fetal bovine serum and Penicillin/Streptomycin, at 37 °C under 5% CO-(ΔΔCt).Total RNA was isolated using TRIzol (Invitrogen). Five hundred ng of total RNA was reverse transcribed using ImProm-II™ Reverse Transcription System (Promega). Quantitative real-time PCRs were performed using SYBR green PCR master mix (Applied Biosystems), with annealing and extension of primers at 60 °C. Fold changes were calculated using the formula; Fold change = 2ChIP assay was performed as described . For quaThe recovery is the % . Relative occupancy was calculated as a ratio of specific signal over background:Relative occupancy is then used as a measure of the protein association with a specific locus.6 cells were seeded in each 100 mm dish. Cells were grown for 24 h prior to addition of RA. Next day, RA was added to a final concentration of 13.7 μM and cells were maintained in RA upto 7 days, with media replacement (containing freshly thawed RA) every day. After 7 days of RA treatment, cells were harvested for ChIP, RNA and protein.All-trans-retinoic acid (RA)-induced differentiation of N2D1 cells was performed in 100 mm culture dish. RA was reconstituted at a concentration of 5 mg/ml in DMSO (Sigma) and stored in dark at − 80 °C. For differentiation experiments, NT2D1 cells were harvested using 0.05% Trypsin and resuspended in fresh media. Cells were counted and 2 × 10ENCODE data for H1 ES cells were as follows:SRR018455 for H3K4me3, SRR029348 for H3K27ac, SRR067953 for H3K79me1, and SRR018454 for H3K36me3 ChIP-Seq respectively.ENCODE data for CD4 T cells were as follows:SRR787517 for H3K4Me3 and SRR980415 for H3K27Ac ChIP-Seq respectively.The RNA-Seq datasets were SRX007165 for H1 ES cells and SRR643766 for CD4 T cells respectively.‘—no-unal’ option was used to write only the aligned reads to the output file. RSEM [For RNA-Seq, the raw reads were aligned to the hg19 reference assembly using HISAT2 with default options for alignment and the TPM was used as opposed to the usual Fragments Per Kb Per Million (FPKM) because TPM is a more robust measure of quantitating transcripts even in absence of multiple samples. RSEM abundance is calculated for each transcript in each sample independently. Therefore this abrogates the need of replicates for assigning a significance value to the estimate of transcript abundance. The estimated transcript abundances were stored as text files with ENST IDs that served as unique identification markers for each transcript isoform.‘macs2 bdgcmp’ was then used to generate an output file that contained the fold enrichment of that particular histone mark at each location in the genome. The resultant output file was used to plot the enrichment of histone modifications at the desired locations in the genome.Histone modifications present in both the data sets were analyzed for comparison across cell types. The raw reads were aligned to the hg19 reference genome assemble using bowtie2 with default options. The alignments for input control and the immunoprecipitated (ChIPed) sample were used to calculate enrichment of histone modifications at all locations in the genome. ‘macs2 callpeak’ was used to identify ‘peaks’ of histone modification enrichment and the output was stored as bedgraph files. A ‘q’ value of 0.05 was used to retain peaks that showed a significant enrichment over Input. The list of bidirectional genes was obtained from  and eachGenomic coordinates for transcription start sites for each partner in a gene pair were used to calculate the mid point between the two TSS for a bidirectional gene pair. If the distance between the two TSSs was an odd number, 1 was added to the total distance before dividing by 2 to find the midpoint between the two TSS. Using the midpoint co-ordinates for each gene pair as a starting point, a 4 Kb window was calculated. These co-ordinates were used for calculating the enrichment of histone modifications. A BED file that contained genomic co-ordinates that lie within each 4 Kb window was calculated using the ‘bedtools chop -stagger’ tool. This generates overlapping genomic co-ordinates that are separated by a specific length and staggered by a specific length, each can be specified independently. The sliding windows used for this analysis were 170 bases in length with a stagger of 75 bases. The tool ‘bedmap -mean’ was used to map the signal of histone enrichment over each interval in the BED file. This plots the mean signal enrichment over input for each histone modification.Genes were classified as symmetric or asymmetric as described above. For each bidirectional gene pair, the midpoint between the two TSS was considered as a reference point for generation of 4 Kb windows as explained above. For each gene pair, histone modifications were quantified on each side of the reference point with overlapping reads discarded from this analysis. In this manner, enrichments of histone modifications were calculated independently for each gene within a bidirectional pair. The cumulative enrichment was calculated using the bedgraph files generated by MACS2 during peak calling. The tool bedmap -sum was used to calculate the cumulative enrichment of signal over input only in cases where the q value of peak calling was lower than 0.05. For asymmetrically expressed gene pairs, the cumulative enrichment of each histone modification for the higher expressing partner gene and lower expressing partner gene from a bidirectional pair was compared with the unpaired t - test. For symmetrically expressed gene pairs, distribution of histone modification enrichment calculated for the ‘upstream’ partner (i.e. upstream of the midpoint) and downstream partner gene (i.e. downstream of the midpoint) was compared with the unpaired t-test. All of the statistical tests were performed using ‘R’ and the resulting data was plotted as violin plots with the ‘p’ value from the t test included.For each histone modification, the enrichment for that modification in each interval in the 4 Kb window was summed over each interval across all bidirectional gene pairs in one gene category. The resultant data was divided by the highest value of enrichment to scale all the values between 0 and 1. This step yielded a normalized profile of histone modifications for each modification in each cell type. Since the size of each window is identical, the windows can be overlaid over each other while keeping the relative locations of the TSS and the intergenic region constant across all bidirectional gene pairs. These are not absolute values and cannot be compared across samples or categories. The plots denote the profile of histone modifications for each set of bidirectional gene pairs with respect to the locations of the transcription start sites for each gene in a pair.To study bidirectional transcription in vivo we designed a vector system comprising two reporter proteins, namely eGFP and mCherry. These reporter genes are arranged in head-to head orientation and the expression of these reporters can be driven by a common promoter element placed in between the two transcription start sites Fig. . To valiAfter validating the pDR vector we cloned 8 putative bidirectional promoter regions from previously reported bidirectional gene pairs in the pDR vector in both sense and antisense orientations and transfected them into 293 T cells. Microscopy analysis revealed that all 8 clones express both the reporter proteins in both sense Fig.  and antiFor BDPs S2, S3, S4, S6, S8, analysis of the RNA-Seq data revealed a bias at the endogenous loci that was skewed in favor of transcription from the promoter oriented towards GFP in the clones tested. This bias resulted in transcription that differed by at least one order of magnitude between the two promoters. In such cases there is a skew towards the GFP signal. We surmise that these sequences are inherently skewed by a margin that is extremely large and this is evident in the bias observed in the flow cytometry data. Nevertheless, in case of promoters where the bias is lower or negligible the intergenic region exhibits unbiased expression of both reporters.Recently, polyadenylation sites found in the antisense direction to the unidirectional promoters have been shown to induce the decay of transcripts upstream to the promoter , 23. Sin+ T cells). The bidirectional gene pairs chosen for this analysis were previously reported [II [The bidirectional gene pair NFYA and OARD1 was assessed for histone modifications exhibited enrichment of histone marks that are in agreement with the transcription status of each gene within the pair. Thus, chromatin modifications appear as strong candidate for transcription regulation of bidirectional promoters. To demonstrate this further, we analyzed bidirectional gene pairs that were transcribed in an asymmetric fashion (one gene among the pair is transcribed at a much higher level than the other) as opposed to those whose expression profiles were symmetrical (both genes in a pair are either transcribed or silent) in two different cell types. To assess whether the correlation between transcription status and promoter associated histone modifications, namely H3K4me3 and H3K27ac, persist on a genome-wide scale when considering multiple bidirectional gene pairs, we analyzed 974 bidirectional gene pairs in two different cell types, namely the H1 human embryonic stem cells (H1 ES) and CD4 positive naive T cells isolated from peripheral blood  in the expression levels within a bidirectional gene pair were classified as asymmetrically expressed genes. Gene pairs wherein both genes were not expressed to detectable levels (Transcript per million < 1) were considered to be silent but were classified as symmetrically expressed genes. This classification was performed independently for each cell type. Thus, the genes that were asymmetric in H1ES cells can be re-classified as being symmetric in CD4 T cells or vice versa.+ T cells  regulates expression from either TSS. On the other hand, the dual reporter system pDR  mediated differentiation of NT2D1 human embryonic carcinoma cells. These cells behave similar to embryonic stem cells since they express all pluripotency factors and can be differentiated into specific cell types upon providing specific signals . We diffThe regulation of bidirectional transcription is not clearly understood and this study aims at answering this question from an epigenetic perspective. A luciferase reporter based system was typically used to characterize bidirectional promoters . HoweverPrevious studies have already established that bidirectional promoters exhibit specific enrichment of active transcription associated chromatin signatures . The occAs a category of transcriptionally distinct gene sets, the symmetric and asymmetric transcription units show a distinct difference from each other. It would be difficult to extend this analysis quantitatively to each particular gene pair since quantitation of histone modifications from ChIP-Seq data is not possible at the same resolution as quantitation of transcripts from RNA-Seq. As such, individual gene pairs cannot be tested for a quantum of difference in the extent of histone modification that correlates directly to a difference in transcript abundance, as the accuracy of one estimation is lower than the other. However, if analyzed as a group then the differences between gene sets become apparent. Since the promoter element between a bidirectional gene pair has been previously reported to a single unit, whose disruption leads to loss of expression from both transcripts from the pair , asymmetA mechanism of transcription using transcription factors (TFs) as the primary driving force for transcription regulation is not sufficient to explain these stark differences between histone modification enrichment patterns. Transcription factors have specific sites that they recognize, however, the sites that have been reported to enriched on bidirectional promoters are for proteins such as SP1, YY1 etc. , which aIt is equally probable that a stark difference a difference in transcription activity would result into a difference in the histone occupancy profiles at the endogenous loci in the cell.Either way, correlation between the histone modification profile and transcriptional profile is evident. Thus, it is possible that these modifications help in preserving transcriptional status of the gene pair that overrides the inherent tendency of bidirectional promoters to transcribe in both orientations. We confirmed a functional connection between histone marks associated with elongation for at least one gene pair. We used RA mediated differentiation of the human embryonic carcinoma NT2D1 cells as model system. Based on a 10-fold differential expression for the NUP62CL-PIH1D3 upon RA mediated differentiation, we selected this gene pair for ChIP analysis of transcription elongation marks H3K79me3, H3K36me3 and H3K27me1. Such stark difference in expression for the gene pair in the two states of differentiation rendered it an attractive candidate for assessment of histone marks that are dependent on active transcription. We analyzed the enrichment of these elongation marks in 2 Kb upstream and downstream regions from TSS of NUP62CL-PIH1D3 gene pair including the intergenic region. Interestingly, all three histone marks exhibited significant enrichment downstream to TSS on both gene bodies, which explains the significance of bimodal distribution of elongation marks in productive bidirectional transcription from sense and antisense orientations. This is the first instance where a direct correlation of histone modification marks in regulating the transcriptional state of the bidirectional genes driven by the bidirectional promoters has been proposed. The results presented here identify an epigenetic histone modification signature of bidirectional promoters that sets them apart from all other transcribing loci in the genome  The strategy for introducing eGFP and mCherry under a common regulatory DNA element is shown. This vector construct is designed to provide a quantitative readout in live cells based on strand-specific promoter activity. Intense red and green colors indicate direction of promoter activity. Three constructs are shown in the scheme, in middle is pDR vector with no promoter element, Right side shows construct with CMV promoter antisense to mCherry, left side shows construct with CMV promoter antisense to eGFP. (B) Clones from Fig. Additional file 2:Figure S2. Characterization of the bidirectional gene pair NFYA-OARD1. (A) The figure shows relative expression of NFYA-OARD1 in Jurkat cells as measured by quantitative RT-PCR analysis. The expression levels are normalized to GAPDH (B) Fluorescence images of the NFYA-OARD1 intergenic region when cloned into the pDR vector. The orientation of each clone with respect to NFYA is depicted below each set of images. Scale bar denotes 400 μm. (C) Flow cytometry plots of cells transfected with NFYA-OARD1 cloned into pDR vector in which NFYA is in sense orientation to mCherry. As a control NFYA cloned into eGFP-N1 vector was used wherein NFYA drives the expression of GFP. The axes denoting mCherry and eGFP are depicted adjacent to the plots. (PDF 1434 kb)Additional file 3:Figure S3. H3K4me3 distribution on bidirectional gene with different intergenic distances in H1 ES cells. The figure shows enrichment of H3K4me3 at the bidirectional genes pairs with intergenic distance upto 1000 bp. Intergenic distance is represented as the number of nucleosomes that could potentially be accommodated. Data are shown for the gene pairs which have intergenic region that could contain 2 to 6 nucleosomes assuming 170 bp length for wrapping around each octamer and inclusive of the 20 bp linker. Cumulative expression is calculated by summation of fold enrichment signal at every location in a 4 KB window for each category and dividing by the highest value of signal in the respective category as described in ‘Additional file 4:Figure S4. H3K4me3 distribution on bidirectional gene with different intergenic region in CD4+ T cells. The figure shows enrichment of H3K4me3 at the bidirectional genes pairs with intergenic distance up to 1000 bp. Intergenic distance is represented as the number of nucleosomes that could potentially be accommodated. Data are shown for the gene pairs which have intergenic region that could contain 2 to 6 nucleosomes assuming 170 bp length for wrapping around each octamer and inclusive of the 20 bp linker. Cumulative expression is calculated by summation of fold enrichment signal at every location in a 4 Kb window for each category and dividing by the highest value of signal in the respective category as described in ‘Additional file 5:Figure S5. H3K27ac distribution on bidirectional gene with different intergenic region in H1 ES cells. The figure shows enrichment of H3K27ac at the bidirectional genes pairs with intergenic distance up to 1000 bp. Intergenic distance is represented as the number of nucleosomes that could potentially be accommodated. Data are shown for the gene pairs which have intergenic region that could contain 2 to 6 nucleosomes assuming 170 bp length for wrapping around each octamer and inclusive of the 20 bp linker. Cumulative expression is calculated by summation of fold enrichment signal at every location in a 4 Kb window for each category and dividing by the highest value of signal in the respective category as described in ‘Additional file 6:Figure S6. H3K27ac distribution on bidirectional gene with different intergenic region in CD4 T cells. The figure shows enrichment of H3K27ac at the bidirectional genes pairs with intergenic distance up to 1000 bp. Intergenic distance is represented as the number of nucleosomes that could potentially be accommodated. Data are shown for the gene pairs which have intergenic region that could contain 2 to 6 nucleosomes assuming 170 bp length for wrapping around each octamer and inclusive of 20 bp linker. Cumulative expression is calculated by summation of fold enrichment signal at every location in a 4 Kb window for each category and dividing by the highest value of signal in the respective category as described in ‘Additional file 7:Figure S7. H3K79me1 distribution on bidirectional gene with different intergenic region in H1 ES cells. The figure shows enrichment of H3K79me1 at the bidirectional genes pairs with intergenic distance up to 1000 bp. Intergenic distance is represented as the number of nucleosomes that could potentially be accommodated. Data are shown for the gene pairs which have intergenic region that could contain 2 to 6 nucleosomes assuming 170 bp length for wrapping around each octamer and inclusive of 20 bp linker. Cumulative expression is calculated by summation of fold enrichment signal at every location in a 4 KB window for each category and dividing by the highest value of signal in the respective category as described in ‘Additional file 8:Figure S8. H3K36me3 distribution on bidirectional gene with different intergenic region in H1 ES cells. The figure shows enrichment of H3K79me1 at the bidirectional genes pairs with intergenic distance up to 1000 bp. Intergenic distance is represented as the number of nucleosomes that could potentially be accommodated. Data are shown for the gene pairs which have intergenic region that could contain 2 to 6 nucleosomes assuming 170 bp length for wrapping around each octamer and inclusive of 20 bp linker. Cumulative expression is calculated by summation of fold enrichment signal at every location in a 4 KB window for each category and dividing by the highest value of signal in the respective category as described in ‘Additional file 9:Expression profile 10 bidirectional gene pairs upon RA mediated differentiation of NT2D-1 cells. These gene pairs were tested for expression differences with real-time PCR upon RA mediated differentiation as described in


ChIP-seq performed on lymphoblastoid cell lines (LCLs), expressing epitope-tagged EBNA3A, EBNA3B or EBNA3C from EBV-recombinants, revealed important principles of EBNA3 binding to chromatin. When combined with global chromatin looping data, EBNA3-bound loci were found to have a singular character, each directly associating with either EBNA3-repressed or EBNA3-activated genes, but not with both. EBNA3A and EBNA3C showed significant association with repressed and activated genes. Significant direct association for EBNA3B loci could only be shown with EBNA3B-repressed genes. A comparison of EBNA3 binding sites with known transcription factor binding sites in LCL GM12878 revealed substantial co-localization of EBNA3s with RUNX3—a protein induced by EBV during B cell transformation. The beta-subunit of core binding factor (CBFβ), that heterodimerizes with RUNX3, could co-immunoprecipitate robustly EBNA3B and EBNA3C, but only weakly EBNA3A. Depletion of either RUNX3 or CBFβ with lentivirus-delivered shRNA impaired epitope-tagged EBNA3B and EBNA3C binding at multiple regulated gene loci, indicating a requirement for CBF heterodimers in EBNA3 recruitment during target-gene regulation. ShRNA-mediated depletion of CBFβ in an EBNA3C-conditional LCL confirmed the role of CBF in the regulation of EBNA3C-induced and -repressed genes. These results reveal an important role for RUNX3/CBF during B cell transformation and EBV latency that was hitherto unexplored. In vitro, infected mature B cells give rise to lymphoblastoid cell lines (LCLs) that carry the EBV genome as an extra-chromosomal episome and remain in the latency III state expressing all latency associated EBV genes and resembling cycling, antigen-activated B blasts. The ease of obtaining continuously proliferating LCLs from virtually any genetic background has led to LCLs being used in diverse studies with extensive data being generated on genome-wide transcription factor localization, global chromatin dynamics and analyses of the global epigenetic landscape (see below).Despite being associated with various cancers—including several B cell lymphomas—Epstein-Barr Virus (EBV) infects, persistently and asymptomatically, >90% of the human population ,2. The EBIM/BCL2L11  tag  was fuse methods ,40, geneThe recombinant BAC was transfected into HEK293 cells, the integrity of the EBV genome was tested by episome rescue and restriction enzyme digestions analysed by pulsed-field gel electrophoresis. Virus was produced and its titer assessed as described previously (6 on the previous day). Virus-containing medium was collected 48 h post-transfection (∼4 ml in each case).Double-stranded DNA for the stem sequence of each shRNA was created by the annealing of single stranded oligos  and clon6 cells in 6ml of medium 15 min before infections. Cells were then pelleted by centrifugation, re-suspended in 1 ml of lentivirus-containing 293T medium and centrifuged again at 450 g for 1.5 h at room temperature. The cells were then resuspended in 5 ml of RPMI medium and transferred to flasks. After 48 h, further 6ml RPMI were added, containing puromycin so that final puromycin concentration was 1 μg/ml.Primary B cells were infected with EBV to produce LCLs as described previously . For len2. Newly infected cells were kept at 5% CO2 for 2 weeks. All cells were cultured in RPMI-1640 medium (Invitrogen), supplemented with 10% foetal bovine serum, penicillin and streptomycin. Puromycin was added at 1 μg/ml when selection was required. The activating ligand 4-hydroxytamoxifen (HT) was added to 400 nM and doxycycline (DOX) to 500 ng/ml, where indicated. These supplements were both added to cultures every time fresh medium was added to the cells (three times per week).LCLs were grown at 37°C in 10% COCo-immunoprecipitations and western blots were performed as previously described . Supplem6 fixed cells was made in 1 ml swelling buffer  and incubated at 4°C with rotation for 20 min. They were then centrifuged at 375g for 5 min at 4°C and the supernatant discarded. The remaining nuclei were re-suspended in 1ml of sonication buffer  and incubated on ice for 30 min. Lysate was then sonicated for 1 h using a Covaris M220 Focused-ultrasonicator with a milliTUBE holder . Sonicated lysate was centrifuged at 12 000g for 10 min at 4°C and supernatant was diluted with 3 ml of sonication buffer. From the input sample 5% was kept at 4°C as a control and the rest was incubated overnight with 16 μg of α-FLAG antibody and 120 μl of ChIP-grade protein G magnetic beads  at 4°C in a 15 ml Falcon tube on rollers. The following day the beads were washed with the wash buffers described previously  and DNA between 100–500 bp was excised and purified using the Qiagen MinElute gel purification kit, according to manufacturer's instructions. At least 1 ng of DNA for each sample was then sent to the Harvard Biopolymers facility for library construction (ChIP-Seq Wafergen) and sequencing .6 for 3A-TAP, 69 × 106 for TAP-3B, 25.7 × 106 for 3C-TAP and 34 × 106 for the non-tagged LCL. Binding regions were identified using the MACS . Genes were described as within contact domains if at least the transcription start site (TSS) was contained within a domain. Peaks were described as being within a contact domain if at least one base pair was shared. Pearson's chi-squared test was performed using a 2 × 2 contingency table (Sequence reads were mapped to the human genome (vhg19) downloaded from UCSC  using BWthe MACS  peak-calcy table  in each ChIP-seq was performed to study the localization of EBNA3A, EBNA3B and EBNA3C across the host genome. The LCLs used were created by infecting primary B cells from a single donor with recombinant EBV expressing epitope-tagged EBNA3A, EBNA3B or EBNA3C. EBNA3A and EBNA3C were tagged at the C-terminus ,42 with For the ChIP, a commercial antibody directed against the FLAG epitope was used. As a negative control, the same ChIP procedure was performed on an LCL of the same genetic background (i.e. same donor) infected with EBV that does not express tagged proteins. Pooled ChIP DNA from eight  or 12 (3A-TAP and non-tagged) precipitation experiments was then tested by QPCR to verify that there was significant enrichment at known targets of EBNA3A, EBNA3B and EBNA3C that could be detected in the material from tagged cell lines relative to the non-tagged LCL Figure . The ChIhttps://www.encodeproject.org/ .To relate the peaks identified by our ChIP-seq with EBNA3-regulated genes revealed by microarrays from LCLs ,15,17 daThe percentage of contact domains that contain EBNA3 peaks and also the relevant regulated genes was calculated Figure . This waTo further assess the significance in the associations detected, random ‘peaks’ were generated for each EBNA3. These were a list of genomic locations—the same in number as the real peaks, with the same lengths as the real peaks, but with their locations determined randomly. The percentage of contact domains with EBNA3-regulated genes that also contain real peaks is considerably higher than for the same domains also containing randomly permuted ‘peaks’, in every case, apart from domains containing EBNA3B up-regulated genes . TherefoThere are contact domains that contain more than one EBNA3-regulated gene. For each EBNA3, all such domains were identified, as was the direction of regulation for each EBNA3-regulated gene within them Table . RemarkaExtensive ChIP-seq data are available for the localization of cell transcription factors in LCL GM12878. Data from 90 different ChIP-seq experiments for transcription factor localization, uniformly analysed and available from the ENCODE project 29,  were useExpression of RUNX3 is induced during the infection of B cells, because the gene encoding this transcription factor is a direct target of the EBV transactivator EBNA2 –57. WithAs mentioned before, RUNX proteins bind to DNA as a heterodimer with CBFβ and together they form core-binding factor (CBF). Consequently, an anti-CBFβ antibody was used to immunoprecipitate CBFβ from LCL protein extracts and, as expected, RUNX3 co-immunoprecipitated with CBFβ (not shown). EBNA3A co-immunoprecipitated very weakly with CBFβ, but EBNA3C and EBNA3B did so significantly—the latter most robustly Figure . EBNA2, Since the EBNA3s all appear in complexes with CBF, lentiviruses that express shRNAs targeting RUNX3 and CBFβ were constructed to assess the role of these factors in EBNA3 recruitment to chromatin. Two independent lentiviruses expressing different shRNAs were produced for each mRNA and these depleted the target proteins to a similar extent in three independent experiments, however here only one of each is shown Figure . All theAnti-FLAG ChIPs were performed on these cells, followed by Q-PCR to assess the levels of EBNA3-TAP bound at EBNA3 peaks associated with selected EBNA3-regulated genes Figure –H. EnricA lentivirus expressing shRNA for IRF4 was also produced, in order to assess the role of this factor in a similar experiment. IRF4 was only knocked down in the 3C-TAP cell line , becauseAs a control for RUNX3, CBFβ and IRF4 depletion experiments, EBNA2 ChIPs were performed in a similar manner, on the same batch of chromatin, after their infection with each shRNA-expressing lentivirus to show that the trend of reduced EBNA3 enrichment after knock-down was specific. EBNA2 enrichment at known EBNA2 peaks was not diminished after knockdown of any of these transcription factors and in most cases more EBNA2 was observed , most liFor EBNA3B, a site where no RUNX3 binds was used as a control to show that diminished binding was only found at CBF sites, after depletion of either RUNX3 or CBFβ . We wereINK4Ap16-null and expresses EBNA3C fused to a modified estrogen receptor (3CHT) that renders 3CHT active only in the presence of 4-hydroxytamoxifen (HT)  . In the g EBNA3C . The cong EBNA3C ,64, and Cultures of 3CHT or 3AERT2 LCL never exposed to HT were split and to one half of each HT was added for 5 days. Cells with and without HT were harvested for protein and RNA extraction or fixed for ChIP. The time course for 3CHT was repeated three times and 3AERT2 twice. Stabilization of 3CHT and 3AERT2 was verified by Western blot Figure . The abiWhen HT was added to the LCL 3CHT cells never exposed to HT, occupancy of both CBFβ and RUNX3 was increased at loci where EBNA3 was found, but not at loci with no EBNA3 binding Figure . ActivatEBNA3C was shown to co-immunoprecipitate with CBFβ from whole cell extracts, CBFβ was found to be required for efficient EBNA3C localization to chromatin and EBNA3C was shown to stabilize CBF on chromatin. We therefore wanted to determine whether CBF was also important for EBNA3C to act as a regulator of host genes. For this the pLKO-Tet-On lentiviral system for doxycycline (DOX) inducible knock-down of CBFβ was used  Figure . Two indIn this study, we performed ChIP-seq to assess genome-wide localization of EBNA3A, EBNA3B and EBNA3C. Similar studies have revealed many aspects of the binding-site characteristics and of EBNA3 function ,27–29. HComparing the EBNA3 MACS peaks called, we see that in general there is significant co-localization between EBNA3C and EBNA3A  and between EBNA3C and EBNA3B , but it appears that any co-localization between EBNA3A and EBNA3B occurs in the presence of EBNA3C, with only three regions of EBNA3A-EBNA3B exclusive co-localization Figure . The epiAnother striking observation concerning EBNA3 peaks was revealed by examining the same loci, but in uninfected primary B cells Figure . For allChromatin looping seems to be a very important mediator of EBNA3 function, as has been shown previously for individual genes ,27. It hcis context of the gene proximal regions, which might only be relevant in determining whether looping occurs, but not for its functional outcome.Contact domains with more than one regulated gene revealed another important aspect of EBNA3 regulation, namely that regulated genes in contact with the same peaks are regulated in the same direction, either all being activated or all being repressed. This implies that factors determining the direction of regulation reside at the peak region and are not dependent on the ChIP-seq data also highlighted the CBF component RUNX3 as the most commonly co-localising factor with the EBNA3s and an obvious candidate for further study. This seemed particularly germane since infection of B cells with EBV changes the composition of CBF, from RUNX1-CBFβ heterodimers to RUNX3-CBFβ heterodimers, by inducing RUNX3 and suppressing RUNX1 . EBNA2 –57 and RCo-immunoprecipitation of EBNA3A, EBNA3B and EBNA3C with RUNX3 partner CBFβ supported this hypothesis. There is a stronger physical association with EBNA3B than with EBNA3C (or EBNA3A—which co-precipitates very weakly under the same conditions) Figure . Althougcis elements making regions of the genome more susceptible to EBNA3 binding. These are likely to be where chromatin is open or opens as a consequence of B cell activation, thus being available for binding of multimeric complexes containing EBNA3s and then being stabilized as a result of combinatorial binding.However, it also seems that the relationship between EBNA3C and CBF during recruitment to chromatin is reciprocal—EBNA3C also facilitates robust stabilization or increased binding affinity of RUNX3 and CBFβ onto chromatin Figure . This isIrrespective of the precise sequence of events or variety of factors involved, CBF is clearly very important. The high degree of co-localization, the co-immunoprecipitation and the requirement for CBF in efficient targeting of EBNA3B and EBNA3C to chromatin are consistent with this. Moreover, it could be significant that CBF has also been shown to directly recruit polycomb repressive complex 1 (PRC1) subunit BMI1 , a factoThe ChIP-seq data reported in this paper have been deposited in the GEO database under accession number GSE88729.Supplementary DataClick here for additional data file.


Saccharomyces cerevisiae Med6p (scMed6p), we generated a series of scMed6p mutants harboring a small internal deletion. Genetic analysis of these mutants revealed that three regions (amino acids 33–42 (Δ2), 125–134 (Δ5), and 157–166 (Δ6)) of scMed6p are required for cell viability and are located at highly conserved regions of Med6 homologs. Notably, the Med6p-Δ2 mutant was barely detectable in whole-cell extracts and purified Mediator, suggesting a loss of Mediator association and concurrent rapid degradation. Consistent with this, the recombinant forms of Med6p having these mutations partially (Δ2) restore or fail (Δ5 and Δ6) to restore in vitro transcriptional defects caused by temperature-sensitive med6 mutation. In an artificial recruitment assay, Mediator containing a LexA-fused wild-type Med6p or Med6p-Δ5 was recruited to the lexA operator region with TBP and activated reporter gene expression. However, the recruitment of Mediator containing LexA-Med6p-Δ6 to lexA operator region resulted in neither TBP recruitment nor reporter gene expression. This result demonstrates a pivotal role of Med6p in the postrecruitment function of Mediator, which is essential for transcriptional activation by Mediator.Med6 protein (Med6p) is a hallmark component of evolutionarily conserved Mediator complexes, and the genuine role of Med6p in Mediator functions remains elusive. For the functional analysis of  Promoter-specific mRNA synthesis requires a minimal set of proteins comprising Pol II (RNA polymerase II) and associated GTFs , which is defined as in vitro “basal transcription” . RegulatS. cerevisiae -UTP and CTP. The purified reaction products were analyzed on the 7% denaturing PAGE and autoradiography.Reconstituted in vitro transcription was performed as described previously . Either β-galactosidase activity of cultured cells was measured in triplicate by the permeabilized-cell method [4 for 2 h to induce the expression of Med6 proteins. Preparation of yeast whole-cell extracts (WCEs), western analysis, and immunoprecipitation of Mediator were performed according to the protocols described previously [4. Cells were harvested, fixed in 1% formaldehyde solution, and sonicated for the fragmentation of chromatin according to the protocol described by Kuo and Allis (1999). WCEs containing chromatin fragments were immunoprecipitated with the use of appropriate antibodies and subjected to a decrosslinking reaction. Purified DNAs were subjected to PCR (25 cycles) with the use of oligomers specific to promoter regions of LacZ reporter or GAL1-10 genes, respectively.For the analysis of reporter gene expression in yeast, yeast strains were grown to the mid-log phase in selective synthetic complete media and l method . To inveeviously . For chrMED6 genes harboring own promoter and terminator regions , Δ5 (residues 125–134), or Δ6 (residues 157–166) were unable to support the cell growth.For the unbiased and systematic analysis of yMed6p, a series of Med6p mutants harboring a small internal deletion was made. We designed 10 amino acid deletions throughout the entire Med6p for approximately every 30 amino acid interval and introduced these into  Figures . The mutreatment . Despitereatment  and 3. NMED6 function(s), closely matched the highly conserved regions of Med6 homologs  spanning this region had no obvious effect on cell survival , resulting in the removal of the first region specific for scMed6p. Interestingly, this mutant could fully complement the  Figures  and 3. I Figures  and 3. TTaken together, the functional domains of yMed6p essential for cell viability were mapped to the highly conserved regions of Med6p, whereas the scMed6p-specific region and the divergent region of Med6ps were not required for essential function(s) of scMed6p.med6 deletion mutants showed any growth defects or transcriptional defects in vivo, such as ts lethality or limited carbon source utilization, as observed in med6 ts mutants [Although the deletion analysis indicated that substantial parts of scMed6p were dispensable for cell viability, a significant level of sequence conservation is apparent at these regions, including Δ1 and Δ7 regions. Thus, we investigated whether the viable  mutants . All of MED6 is essential for galactose-induced GAL1 gene activation [β-galactosidase activity of the GAL1UAS-TATA-LacZ reporter was measured for the viable mutants under induction conditions. None of the viable med6 deletion mutants were defective in transcriptional activation of the GAL1 promoter  by Gal4-VP16 over basal transcription from GCN4:G-template, respectively . However, Med6p-Δ5 and -Δ6 mutants were not able to complement this defect (lanes 9 and 10), in good agreement with their in vivo phenotypes. Notably, Med6p-Δ2, which did not support cell viability, could partially restore the transcriptional defect of med6-ts2 holoenzyme to half that of the wild-type . These results indicate that the Med6p regions which are essential for in vivo function (cell viability) are also required for activation function of Med6p in vitro.Next, we investigated whether scMed6p regions essential for viability are also required for Med6p-dependent transcriptional activation in vitro. For this, we purified recombinant Med6p having mutation . When trmed6-ts mutants is deficient in the med6-ts protein but the free form of med6-ts protein was not detected during column purifications, possibly due to its rapid degradation [In our previous reports, the Pol II holoenzyme prepared from radation , 20. ThilexAop-LacZ reporter gene. As expected from the expression data, LexA-Med6p-Δ2 was completely inactive for reporter gene activation . As shown in lexAop region with an efficiency similar to that of wild-type (irrespective of growth conditions). However, the association of TBP with the promoter region was observed only in the strains expressing LexA-fused wild-type Med6p or Med6p-Δ6, in agreement with reporter gene expression , and TBP (for transcription initiation) with a target promoter region (8xpression .GAL1-10 promoter region under either repression (glucose) or induction conditions  to monitor the effect of med6 mutations on the recruitment of Mediator to a natural promoter. LexA-fused Med6p-Δ5 and -Δ6 were recruited to the GAL1-10 promoter as well as wild-type Med6p upon galactose induction, indicating these mutations did not impair Mediator recruitment in the endogenous promoter (α-TBP) was also seen in all cases, as predicted by the action of native form of Mediator containing endogenous wild-type Med6p for the activation of GAL1-10 promoter  identified by complementation assays were mapped to the highly conserved regions of Med6ps, whereas yeast-specific (spacer region) and mostly divergent regions (one third of C-terminus) of scMed6p were not required for its essential function(s) Figures  and 2. TOne interesting point is that some residual activity to support in vitro transcription was retained in Med6p-Δ2, showing a discrepancy with its in vivo defect. Although we cannot explain clearly this phenomenon, we surmise that this partial activity might have resulted from an in vitro dosage effect. If Med6p-Δ2 has a normal activation function but has a defect in Mediator association, Med6p-Δ2 could provide in vitro activation function to some extent via a dosage effect when an excess amount of Med6p-Δ2 is added (which is not applicable in vivo).med6-ts proteins detected in WCEs seem to be associated with Mediator in vivo. The observed deficiency and the degradation pattern of Med6p-Δ2 in WCEs suggest that the Δ2 region of scMed6p might be involved in the association with other Mediator subunit(s)  . Based onterface . Since ttructure . In thisMediator . The othPrevious biochemical analyses and artificial recruitment experiments using mutant Pol II holoenzymes suggested that facilitated recruitment of a holoenzyme to a promoter is necessary but not sufficient for transcriptional activation and that an unknown biochemical activity is required for transcriptional activation (postrecruitment step) . Consistβ3 sheet of scMed6p . Future studies on the detailed functions of these essential domains of Med6p should be helpful for the understanding of action mechanisms of Mediator with regard to its structural and functional relationship.Collectively, all these results consistently support a notion that Med6p has a pivotal role in the postrecruitment function of Mediator during transcriptional activation, which is conceptually accepted as the conformational changes, isomerization, or modulation of Pol II activity. In addition, our results also suggest there might be functional differentiation between the Δ5 and Δ6 regions  might be involved in Mediator association and/or protein stability, and the second  and third  regions have a role in postrecruitment activity of Mediator complex, such as signal-relay or conformational changes. This study proved the previous notion that Med6p is involved in the postrecruitment function of Mediator complex, which is required for gene activation by Mediator.