Serine-5 and Serine-7 phosphorylation mark transcription initiation, whereas Serine-2 phosphorylation coincides with productive elongation. In vertebrates, the CTD has eight non-canonical substitutions of Serine-7 into Lysine-7, which can be acetylated (K7ac). Here, we describe mono- and di-methylation of CTD Lysine-7 residues (K7me1 and K7me2). K7me1 and K7me2 are observed during the earliest transcription stages and precede or accompany Serine-5 and Serine-7 phosphorylation. In contrast, K7ac is associated with RNAPII elongation, Serine-2 phosphorylation and mRNA expression. We identify an unexpected balance between RNAPII K7 methylation and acetylation at gene promoters, which fine-tunes gene expression levels.Dynamic post-translational modification of RNA polymerase II (RNAPII) coordinates the co-transcriptional recruitment of enzymatic complexes that regulate chromatin states and processing of nascent RNA. Extensive phosphorylation of serine residues at the largest RNAPII subunit occurs at its structurally-disordered C-terminal domain (CTD), which is composed of multiple heptapeptide repeats with consensus sequence YDOI:http://dx.doi.org/10.7554/eLife.11215.001 Genes are sections of DNA that encode the instructions to make proteins. When a gene is switched on, the section of DNA is copied to make molecules of messenger ribonucleic acid (RNA) in a process called transcription. These messenger RNAs are then used as templates for protein production. In animals, plants and other eukaryotic organisms, an enzyme called RNA polymerase II is responsible for making messenger RNA molecules during transcription. This enzyme is made up of several proteins, the largest of which contains a long tail, called the carboxy-terminal domain. This domain is crucial for transcription because it serves as a landing platform for other enzymes that help to make the RNA molecules.The carboxy-terminal domain contains multiple repeats of a string of seven amino acids (the building blocks of proteins). Normally, each repeat contains three amino acids called serines. However, in humans and other mammals, one of these serines is often substituted with another amino acid called lysine instead. This lysine (referred to as Lysine-7) was known to be modified by the addition of a chemical group called an 'acetyl' tag, but it was not clear how this tag affected transcription.Dias, Rito, Torlai Triglia et al. carried out an in-depth study into how Lysine-7 is modified in mouse cells, and what effects these modifications have on transcription. The experiments show that Lysine-7 can also be modified by the addition of a different chemical group, called a 'methyl' tag. This new modification is also found in flies, worms and human cells, which suggests that it is generally important for transcription.Next, Dias, Rito, Torlai Triglia et al. found that in mouse stem cells, methyl tags are added to Lysine-7 during the earliest steps of transcription, before the acetyl tags are added. Further experiments show that a balance between the addition of methyl tags and acetyl tags to Lysine-7 fine-tunes the activity of RNA polymerase II. These findings add to our understanding of how cells control the activity of RNA polymerase II at different genes. Future challenges are to find out which enzymes are responsible for adding and removing these chemical tags, and how the balance between the methyl and acetyl modifications is controlled.DOI:http://dx.doi.org/10.7554/eLife.11215.002 Transcription of protein-coding genes is a complex process involving a sequence of RNA processing events that occur at different stages of the transcription cycle. Co-transcriptional recruitment of chromatin modifiers and RNA processing machinery is modulated through a complex array of post-translational modifications at the C-terminal domain (CTD) of RPB1, the largest subunit of RNAPII. This unique domain constitutes a docking platform for protein complexes that cap, splice and polyadenylate newly-made RNAs . The CTD1-S2-P3-T4-S5-P6-S7. Extensive remodeling of the CTD occurs during distinct steps of the transcription cycle  cells, we show that gene expression levels depend on the balance between CTD-K7 methylation and acetylation.in vivo  constructs containing different number of K7-to-S7 mutations  and is the only K7 residue present in C. elegans. Mutant construct 3K has three K7 residues present at repeats 35, 40 and 47, all of which are conserved in D. melanogaster. Finally, we used a wild-type murine Rpb1 construct (8K), which contains all eight vertebrate-conserved K7 residues, as a control for expression and α-amanitin selection. We produced viable mouse NIH-3T3 fibroblast lines that express each of the four constructs and show stable YFP-RPB1 expression for more than one month in culture and after several passages under α-amanitin selection. Viability of cells expressing α-amanitin-resistant RPB1 was previously shown for K7-to-R7 mutations  or 0K constructs ; extractTo examine the effect of K7-to-S7 mutation on the subcellular localization of RPB1, we used confocal microscopy and YFP fluorescence to detect YFP-RPB1 fusion proteins and found that the typical RNAPII nucleoplasmic distribution is unaffected by K7-to-S7 mutations . ImmunofAcetylation of CTD-K7 residues was recently identified and found associated with inducible gene expression . Lysine in vivo methylation of non-canonical K7 residues of the CTD in mouse fibroblasts.To test whether mono- or di-methylation of the CTD could be identified in vivo, we performed western blotting on total extracts from NIH-3T3, 8K and 0K cell lines using the novel antibodies against CTD-K7me1 and CTD-K7me2 . K7me1 aRPB1 migrates in two major forms, a fast migrating hypophosphorylated (IIa) state and a slower migrating hyperphosphorylated (II0) state, as well as intermediate phosphorylation forms. Interestingly, we found that both the K7me1 and K7me2 antibodies detect the hypophosphorylated RPB1 and YFP-RPB1 bands . This baTo explore the extent of methylation of the eight mammalian K7 residues, we performed western blots using the NIH-3T3 cell lines engineered to express YFP-RPB1 fusion proteins bearing different numbers of K7 residues  forms of RPB1  suggestsTo further investigate whether K7 mono- and di-methylation occur upstream of elongation, we treated ES cells with flavopiridol, an inhibitor of RNAPII elongation . DepletiWe then tested whether K7me1 and K7me2 are localized within the nucleus using immunofluorescence in mouse NIH-3T3 cells . We findTo explore the role of K7me1 and K7me2 in the transcription cycle, we mapped their chromatin occupancy genome-wide using chromatin immunoprecipitation coupled to next generation sequencing (ChIP-seq) in mouse ES cells . The chrEed, Rpl13, and Tuba1a), and are detected at lower levels beyond transcription end sites (Myf5 gene). K7me1 and K7me2 occupancy at active genes, and absence at inactive, were confirmed by single gene quantitative polymerase chain reaction (qPCR) (Inspection of ChIP-seq profiles at single genes shows that K7me1 and K7me2 are highly enriched at promoters of active genes (nd sites . K7ac isn (qPCR) . In summGiven that K7me1, K7me2 and K7ac exhibit different occupancy patterns at active genes, we assessed the genome-wide promoter distribution and abundance of each mark. We first identified all genomic regions positive for CTD-K7 marks, using a peak finder suited for both sharp and broader occupancy patterns  and transcription end sites (TES) of the 15% most and least expressed genes . The novTo explore the temporal sequence of CTD modifications during the transcription cycle and how they predict gene expression levels, we performed extensive correlation analyses between different RNAPII modifications . We focuGiven that all CTD modifications correlate with each other to a certain extent, we performed partial correlation analyses to disentangle the association between K7 methylation and productive elongation after accounting for the effects of other intervening RPB1 modifications . SurprisIn these pairwise correlation analyses, we began to dissect unexpected dependencies between different CTD modifications and how they relate to productive elongation. However, the most likely scenario is that several CTD modifications jointly contribute to expression levels. To further explore whether CTD modifications work together to promote productive transcription, we employed stepwise regression models to determine the CTD modifications that best predict elongation marked by S2p levels, for a given number of predictors . These aTo investigate whether the balance between CTD-K7 methylation and acetylation at gene promoters could be predictive of gene expression, we calculated the ratio between K7me2 and K7ac and investigated how it relates to mature and nascent RNA levels. We find that the K7me2/K7ac ratio negatively correlates with mRNA expression  analyses . Interes–16). We observed a similar trend when we treated our ES cell line with TSA; genes with higher ratios tend to be upregulated , suggesting that the interactions of RNAPII with CTD-K7 modifiers and/or readers may be transient and not easily captured by in vitro biochemical approaches.The identification of modified CTD-K7 residues  expands the repertoire of CTD interactors to proteins containing domains that recognize these marks, such as PHD finger, bromo-, chromo-, tudor- and MBT-domains . Cross-tMouse ES cell line OS25  was grown on 0.1% gelatin-coated surfaces in GMEM-BHK21 supplemented with 10% fetal calf serum, 2 mM L-glutamine, 1% MEM non-essential amino acids, 1 mM sodium pyruvate, 50 μM β-mercaptoethanol , 1000 U/ml of human recombinant leukaemia inhibitory factor  and 0.1 mg/ml Hygromycin , as described previously . For inhNIH-3T3 cells (kind donation from Alex Sardini) were grown in Dulbecco's modiﬁed Eagle's medium (DMEM) supplemented with 10% fetal calf serum, 2 mM L-glutamine and 1 mM sodium pyruvate . NIH-3T3 derived cell lines 8K, 3K, 1K and 0K were grown in presence of α-amanitin selection .Mouse ES cell line (OS25) and mouse NIH-3T3 fibroblast line were negative for mycoplasma, and routinely tested for mycoplasma with PCR mycoplasma test kit , according to manufacturer’s instructions.YFP-Rpb1 amr contains a point mutation that gives resistance to α-amanitin  to remove a fragment with 1.3 kb (residues 6126 to 7428) containing the Rpb1 CTD. Different CTD sequences with wild-type (8K) and mutant repeats  were obtained by gene synthesis  in the pUC57 vector backbone. In the mutant constructs, the K7 residues were converted into the consensus S7 residues. Mutant 3K contains only three of the K7 residues present at repeats 35, 40 and 47, mutant 1K contains only one K7 residue present at repeat 35 and mutant 0K has all K7 residues mutated to S7. Wild-type and mutant CTDs were cloned from the pUC57 vectors to the YFP-Rpb1 amr backbone using the SanDI and HpaI restriction sites. Constructs were digested and sequenced to confirm the presence of the eight K7 codons at the wild-type construct, the K7 to S7 mutations at the different mutant contructs and the absence of additional sequence changes. For generation of cell lines 8K, 3K, 1K and 0K, NIH-3T3 cells growing in a 6-well dish were transfected with 3 µg of DNA using lipofectamine 2000 . α-Amanitin selection  was started 24 hr after transfection. Polyclonal cell lines were kept under selection and expanded for 1 month before being used in the experiments described.The RPB1 construct All handling of mice was approved by the Hokkaido University Animal Experiment Committee  and carried out according to guidelines for animal experimentation at Hokkaido University, where MAB Institute is located. Animals were housed in a designated pathogen-free facility at Hokkaido University. Mice were humanely euthanized via cervical dislocation by technically proficient individuals.Mouse monoclonal antibodies were produced essentially as described previously . BrieflyHybridoma clones CMA611 and CMA612 used in this study were specific for K7me1 and K7me2 peptides, respectively. Prior to purification, CMA611 and CMA612 antibody clones were isotyped as IgG2b-κ and IgG1-κ, respectively, using a Mouse Monoclonal Antibody Isotyping Kit .Clones were expanded in CD Hybridoma medium (Life Technologies) and culture supernatant was collected , filtered using a 0.45 μm membrane  and purified using HiTrap Protein A FF Sepharose columns . Elution of CMA611 clone was performed using elution buffer . Elution of CMA612 clone was performed using mouse IgG1 mild elution buffer (Thermo Scientific). Antibody elution fractions were concentrated and the buffer was replaced to PBS using centrifugal filtration with Amicon Ultra filter 50K (Millipore).3VO4 (2 mM) and PMSF  and protease inhibitor cocktail ]. To release chromatin-associated proteins, cell extracts were sheared by passage through a 25G needle, as in Whole cell extracts were collected by scraping cells in ice-cold 'lysis' buffer , scraped from dishes and nuclei were isolated with a Dounce homogenizer (tight pestle) followed by centrifugation. Nuclei resuspended (1x107 nuclei/ml) in 'sonication' buffer  were sonicated (Diagenode Bioruptor) for 30 min at full power for 30 cycles of 30 s "on" and 30 s "off" at 4°C. Chromatin was centrifuged and, after disposal of the insoluble fraction, DNA content was quantified using alkaline lysis. Both swelling and sonication buffers were supplemented with phosphatase inhibitors sodium flouride (5 mM), Na3VO4 (2 mM) and phenylmethylsulfonyl fluoride  and protease inhibitor cocktail .ChIP for RNAPII modifications was performed using fixed chromatin as described previously . Mouse EProtein G magnetic beads  were incubated (50 µl per IP) with 10 µg of bridging antibody anti-IgM/IgG (Jackson ImmunoResearch) for 1 hr at 4°C and then washed and resuspended in sonication buffer. For each immunoprecipitation, 600–700 µg of chromatin was incubated overnight with beads and the respective RNAPII or control antibody (for details about ChIP antibodies see (input Ct – IP Ct). Primer sequences are listed in Samples were incubated with 100 µg of Proteinase K , after EDTA concentration was adjusted to 5 mM, for 2h at 50°C. DNA was recovered by phenol-chloroform extraction followed by ethanol precipitation in the presence of 20 ng/ml glycogen. DNA concentration was determined by PicoGreen fluorimetric assay  and samples were diluted to a final concentration of 0.2 ng/µl. Immunoprecipitated and input samples (0.5 ng each) were analyzed by quantitative real-time PCR (qPCR) using SYBR No-Rox sensimix . Quantitative PCR 'cycle over threshold' (Ct) values from immunoprecipitated samples (RNAPII or control antibody) were subtracted from the input Ct values and the fold enrichment over input was calculated using the equation 2Prior to ChIP-seq library preparation, RNAPII enrichment and distribution were assessed by qPCR analyses using a previously characterized panel of genes . ChIP-seet al., 2012 and RNAPII K7ac from In addition to ChIP-seq datasets generated for CTD-K7me1 and K7me2 modifications in mouse ES cells, we also analyzed published ES cell ChIP-seq and GRO-seq datasets . PublishSequenced reads were aligned to the mouse genome  using Bowtie2 version 2.0.5 , with de–4 was added to FPKM values from Boxplots were produced using R. A pseudo-count of 10Average ChIP-seq profiles were generated by plotting the average depth of coverage in non-overlapping windows of 10 bp, across 5 kb genomic windows centered on TSS and TES as in . The reaGRO-seq data from th percentile  or least active (bottom 15%) were defined according to FPKM values from mRNA-seq datasets published in , and werTo relate CTD-K7 methylation to acetylation levels, we computed the ratio between the read counts of K7me1/2 and K7ac at promoters (2 kb around TSS), both scaled by 10 million over the total number of non-duplicated mapped reads of the respective dataset. The ratios K7me1/K7ac and K7me2/K7ac are highly correlated (Spearman’s correlation coefficient 0.88); for simplicity, only the K7me2/K7ac ratio was used for further in-depth analyses. The ratio of K7me2/K7ac for each active gene was used to define three quantiles .2 and Mallow’s Cp.To apply linear regression models and perform correlation analyses, the promoter read counts for the different CTD modifications were centered and scaled. The Spearman’s correlation coefficients were plotted as a matrix using the "corrplot" package in R and partial correlations calculated using the "pcor" function of "ggm" package. Traditional regression modeling using correlated predictor variables is difficult to interpret and hence we used a stepwise regression approach to better untangle the contributions of different CTD modifications. Typically, for a given number of predictor variables, we report the top five best models. Since CTD-S2p measured at 2 kb window after TES is the best predictor of mRNA amongst all CTD modifications and gene regions, we decided to study models that predict the levels of the S2p (instead of mRNA-seq) to avoid inclusion of another highly correlated variable. Model fitting was performed in R using the packages "lm" and "leaps". Model ranking was based on both adjusted RTo investigate whether the contribution of CTD modifications to S2p levels at 2 kb window after the TES, which were obtained through correlation and stepwise regression analyses, are due to co-linearity artifacts or over-fitting, we also employed the LASSO regression method implemented in the "lars" R package . We usedhttp://genmapp.org/go_elite). Over-representation analysis of the top and bottom 300 genes for K7me2/K7ac ratio (see above) was performed using as background the n = 1564 active, non-overlapping genes for which the ratio was computed. Default parameters were used as filters: z-score threshold more than 1.96, P-value less than 0.05, number of genes changed more than 2. Over-representation analysis was performed with "permute p-value" option, 2000 permutations.Gene ontology (GO) enrichment analysis was performed using GO-Elite version 1.2.5  is publicly available and described in Actb mRNA measured using primers for the exon1-exon2 junction  extraction using Phase Lock Gel tubes . Samples were DNAse I  treated according to manufacturer instructions. Total RNA (1 µg) was retrotranscribed using random primers (50 ng) and 200 U of reverse transcriptase  in a 20 µl reaction. The synthesized complementary DNA was treated with 2 U of RNAse H (NEB), diluted 1:10 and 2.5 µl were used per RT-PCR reaction. Total RNA was measured using primers designed for the 5´end of genes and levels were normalized for  review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent  To further study the interplay between K7 acetyl and me1/2 marks, a careful analysis of the potential switch in these marks following gene induction should be revealing.d of P300 or use of chemical HAT inhibitors) to deplete K7Acetyl marks may increase K7 me1/2 and thereby cause gene repression. This should be tested.2) Inhibition of HATs (i.e. K3) Correlating K7me1/2 with low activity genes needs to be tested by looking at nascent RNA as well as steady state RNA. Looking at GR0-seq or chromatin-seq RNA to focus on transcription rather than mRNA levels would be informative.4) Some understanding of which factors recognise these CTD K7me1/2 marks is needed. For example IP followed by mass spectrometry could be informative. It could also be tested whether SET1, which is recruited by CTD S5P to methylate histone H3K4 at promoters, also methylates CTD K7me1/2.We recommend that you extend your analysis as indicated above. You should also be careful not to oversell the importance of K7me1/2 versus the dominant S2P and S5P marks. Use of "correlate" rather than "depend" might be better unless more definitive data is obtained. Essential revisions: 1) To further study the interplay between K7 acetyl and me1/2 marks, a careful analysis of the potential switch in these marks following gene induction should be revealing.We thank the reviewers for encouraging us to further investigate the interplay between K7me and K7ac in the context of gene expression changes. Without knowledge of the enzymatic activities that modulate K7me directly, we consulted published data about inhibitors that could modulate K7me/K7ac ratio, specifically Trichostatin A (TSA), previously shown to up-regulate global levels of K7ac .We took advantage of published microarray data for mouse ES cells treated with the histone deacetylase inhibitor TSA . In light of our suggestion that the K7me2/K7ac relates with the potential for gene expression, we hypothesized that genes with the highest K7me2/K7ac ratios would be most sensitive to the TSA-induced global increase in K7ac, and therefore more likely to be up-regulated upon TSA treatment.Integrating data from Karantzali et al. (2008) with our ChIP-seq data defining K7me-bound genes, we find that TSA has different effects on genes in the three K7me2/K7ac groups, with genes with a High K7me2/K7ac ratio being more likely to be up-regulated after TSA, in keeping with our hypothesis. This result is consistent with the observed relationship between K7me2/K7ac ratio and the extent of productive transcription at active genes, independently of the amount of RNAPII at gene promoters. We have included these results into the main text and added a new plot in d of P300 or use of chemical HAT inhibitors) to deplete K7Acetyl marks may increase K7 me1/2 and thereby cause gene repression. This should be tested.2) Inhibition of HATs .To examine the effects of the reduction of K7ac, we treated mouse ES cells with C646 . We first confirmed the efficiency of P300 inhibition by analyzing H3K9ac levels, and found that it decreases, as expected . We then analyzed global levels of CTD-K7 methylation by western blotting, and found a reproducible increase in CTD-K7me2 after inhibition of P300 . This result combined with the observation that K7ac decreases  points to a dynamic balance between methylation and acetylation at CTD-K7 residues, which supports our interpretation of the genome-wide analyses of CTD marks.The results of P300 inhibition on total RPB1, K7me2 and S7p were integrated in the manuscript, in To test the effects of P300 inhibition on genes with different K7me/K7ac ratios, we performed RT-PCR on 6 genes with a range of K7me/K7ac ratios; To further explore the effect of K7ac loss (and K7me gain) on gene expression states, we also investigated how P300 inhibition impacts on S7p levels . This ex3) Correlating K7me1/2 with low activity genes needs to be tested by looking at nascent RNA as well as steady state RNA. Looking at GR0-seq or chromatin-seq RNA to focus on transcription rather than mRNA levels would be informative.We thank the reviewers for prompting us to investigate how the ratio between K7 methylation/acetylation relates with nascent RNA. We have analyzed nascent transcript data obtained from mouse ES cells, using published Global Run-On sequencing (GRO-seq) data from Jonkers et al. (2014). In agreement with the results obtained for mRNA-seq data , we find4) Some understanding of which factors recognise these CTD K7me1/2 marks is needed. For example IP followed by mass spectrometry could be informative.We agree with the reviewers that identifying readers of CTD K7me1/2 marks is a most pressing task, which we have tried in various ways. As alluded in the Discussion of the first version of the manuscript, we attempted to identify potential readers of K7 methylation, by performing mass spectrometry after immunoprecipitation of YFP-tagged wild-type (8K) RPB1 versus 0K mutant. However, this approach did not yield reproducible protein identifications in biological replicates, probably due to the fact that K7 methylation might be associated with a very small pool of RNAPII complexes found at the earliest stages of transcription. The staining of only small and discrete dots by IF  supports1-S2-P3-T4-S5-P6-K7). We hypothesized that this step should clear up mouse ES cell extracts from proteins that bind to non-canonical CTD sequence in the absence of K7 modifications. After this pre-clearing step, we performed pull-downs using beads carrying dimethylated K7 peptides (3 heptapeptide repeats Y1-S2-P3-T4-S5-P6-K7me2). Proteins obtained after the first and second pull-downs  were analyzed by LS-MS/MS.In response to the reviewers’ requests we attempted a peptide pull-down approach, where we designed a two-step peptide pull-down strategy to identify possible factors that recognise CTDK7me2, but not unmodified K7 peptides. Total protein extracts from mouse ES cells were first incubated with bead-immobilized peptides comprising unmodified non-canonical CTD sequence  or RNA processing . The second pull-down step with dimethylated K7 peptide did not reveal specific protein enrichment over mock background in two biological replicates. These results raise the possibility that CTDK7 methylation may act to prevent protein-protein interactions that occur in the earliest stages of transcription initiation (between transcription factors and RNAPII), which have to be disabled to allow CTD modifications that are compatible with productive elongation. Alternatively, it is possible that K7me1 and K7me2 act in concert with other CTD modifications, e.g. with S5p. In this light, more complex peptide pull-downs will be necessary where K7me2 presence/absence is combined with presence of S5p, S7p or other CTD modifications, to help understand what is the contribution of K7me2 to the ability of adjacent modifications to regulate co-transcriptional recruitment of machinery.We believe that more complex and unbiased screens for CTDK7 binders will be necessary to identify factors that can recognize CTDK7me marks.It could also be tested whether SET1, which is recruited by CTD S5P to methylate histone H3K4 at promoters, also methylates CTD K7me1/2.To test the potential role of SET1 complexes as CTD-K7 methylases, we performed western blots for K7me1 and total RPB1 on protein extracts from control mouse ES cells and conditional knockout ES cells for Setd1a and Setd1b . We find that Mll1 or Mll2 knockout is accompanied by a large global increase in total RPB1 levels, making it difficult to assess the effect on CTD methylation. These results argue for an intricate interplay between histone and RNAPII modifications.Mammalian cells have twelve H3K4 methyltransferases with redundant activity, and so it is possible that knockout of multiple enzymes may be needed to detect significant global changes in CTD-K7 methylation. Depletion of H3K4me3 levels may also have global effects on transcription that could influence the levels of CTD-K7 methylation independently of the direct effect of the methyltransferases. Alternative biochemical approaches such as CTD-K7 peptide pull down screens for methylases and/or isolation of CTD-K7 methylation activity from nuclear extracts might be needed for the identification of CTD-K7 methyltransferases. As these analyses are not conclusive at identifying K7me writers or readers we have not included these additional results in the manuscript.


Bacillus subtilis. In this study the genome-wide gene regulation of a CcpA knockout and three specific CcpA mutants were studied by transcriptome analysis, to further elucidate the function of specific binding sites in CcpA. The following three amino acids were mutated to characterize their function: M17(R) which is involved in DNA binding, T62(H) which is important for the allosteric switch in CcpA upon HPr-Ser46-P binding, and R304(W) which is important for binding of the coeffectors HPr-Ser46-P and fructose-1,6-bisphosphate. The results confirm that CcpA was also involved in gene regulation in the absence of glucose. CcpA-M17R showed a small relief of Carbon Catabolite Control; the CcpA-M17R mutant regulates fewer genes than the CcpA-wt and the palindromicity of the cre site is less important for CcpA-M17R. CcpA-T62H was a stronger repressor than CcpA-wt and also acted as a strong repressor in the absence of glucose. CcpA-R304W was shown here to be less dependent on HPr-Ser46-P for its carbon catabolite control activities. The results presented here provide detailed information on alterations in gene regulation for each CcpA-mutant.Carbon catabolite control is required for efficient use of available carbon sources to ensure rapid growth of bacteria. CcpA is a global regulator of carbon metabolism in Gram-positive bacteria like  Bacillus subtilis uses a global transcriptional regulator, carbon catabolite protein A (CcpA), to employ CCC. Together with the seryl phosphorylated form of the histidine-containing protein, HPr, are the main coeffector for transcriptional regulation of various operons, it is involved in regulation of carbon utilization, overflow metabolism, amino acid anabolism and nitrogen assimilation    or, in much fewer cases, as an activator, i.e., when the cre site is upstream of the promoter, . However, there are also exceptions to this rule: the cre site of the levanase operon is upstream of the promoter but it is repressed by CcpA  to use carbon sources in a preferential manner .CcpA belongs to the LacI family . Threonine 62 is the last residue of the allosteric switch domain, mediating the signal of HPr-Ser46-P binding to the DNA binding domain 2SO4, 0.5 mM Mg2SO4, 10 μM MnSO4, 22 mg/l Ferric Ammonium Citrate, 250 μM L-Tryptophan, and 0.4% (w/v) glucose. C-medium supplemented with glutamate contains 0.03% (w/v) L-Glutamate and when it was also supplemented with branched chain amino acids it contained 0.25% (w/v) L-Isoleucine, 0.25% (w/v) L-Leucine, 2.5% (w/v) L-Valine, and 2.5% (w/v) L-Methionine. Uridine 5′-monophosphate (Sigma) was added to C-medium in a final concentration of 20 mg/l.PCR and DNA purification were done as previously described  and the second flanking region was downstream of ccpA (primers ytxD_FW and ytxD_REV). PCR with Pfux7 as polymerase was also used to amplify pUC18 and the spectinomycin gene from pDOW01  was done using the uracil-excision DNA engineering method  were used to check whether the plasmid was inserted via double recombination and whether the ccpA gene was removed.Colony PCR and PCR on isolated chromosomal DNA  glucose and grown at 200 rpm and 37°C until the OD600 was 0.3. Now 100 ml of cell culture was harvested by centrifugation  and used for RNA isolation. Cell pellets were resuspended in 400 μl TE (DEPC) buffer, and transferred to screw-cap tubes with 0.5 g glass beads , 50 μl 10% SDS and 500 μl phenol/chloroform:IAA (a 1:1 pre-made mixture of phenol acid and chloroform:IAA (24:1); from this mixture the organic phase was used). The screw-capped tubes were placed in a bead beater for two times 1 min to lyse the cells. Total RNA was isolated with the High Pure RNA Isolation Kit (Roche). The RNA concentration was measured with the NanoDrop  and the quality was checked on a 1% agarose gel supplemented with 1% bleach.Lysogeny Broth (LB), supplemented with 2 μg/ml erythromycin when necessary, was inoculated with B. subtilis ccpA-wt cells and cDNA from B. subtilis ccpA-mutant cells were mixed in a 1:1 ratio and hybridized overnight on home-made DNA microarray aminosilane glass slides  and 20 μg of total RNA were used for cDNA synthesis as described before . The images of the scans were analyzed with ArrayPro4.5 .Further analysis was done with PrePrep  under accession number GSE69575.The microarray data is available at the NCBI GEO database  at an OD600 of 0.3. The pellets were resuspended in 200 μl 50 mM Tris-Cl pH 7.4, a spatula tip of glassbeads  was added and subsequently cells were lysed by mini-bead beating . After centrifugation  the supernatant was transferred to a clean tube. Total protein was measured with the DC Protein Assay (BioRad) to ensure that equal amounts of protein were used. 30 μl of 2x SDS loading buffer was added to 20 mg of total protein in a volume of 30 μl and heated for 5 min at 90°C before loading on a 12% SDS-PAGE gel. After electrophoresis, the proteins were transferred to a PVDF membrane . The PVDF membrane was incubated in PBST + 5% (w/v) skim milk at 4°C overnight. Next morning, the PVDF membrane was washed three times10 min with PBST and subsequently incubated at room temperature for 2 h in PBST + 5% skim milk + 1:10,000 dilution of anti-CcpA antibody  and always compared to the transcriptome of B. subtilis pWH-ccpA-wildtype. All strains were grown in LB, either in the presence or absence of 1% glucose. Our goal was to study whether all genes of the regulon will be up- or down-regulated equally or in a differential way. The results will be presented for each mutant separately in the sections below.The effects of the CcpA knockout and of the point mutations in the CcpA protein  were studied on the transcriptome level at the early exponential growth phase . When the ccpA knockout strain was grown in LB then the expression of 89 genes was changed . The findings from the ccpA knockout strain served as a reference to interpret the results from the CcpA-mutants. Our main questions were: are the CcpA-mutants more or less active in repression or activation than the wildtype? Are all genes affected to similar extents or in a differential way?The Table 5) indicating that the function of the CcpA-M17R mutant was similar to the CcpA-wt, but with lowered affinity for most binding sites. When the CcpA-M17R mutant strain was grown in LB + 1% glucose the gene regulation for 129 genes (115 up and 14 down) differed from CcpA-wt  had a changed gene regulation compared to CcpA-wt  when the strain was grown in LB + 1% glucose. When the CcpA-R304W strain was grown in the absence of glucose the number of affected genes was almost as high; there were 206 genes affected (155 up and 51 down) of which 151 genes were unique for CcpA-R304W Figures  showing For a more detailed look on the differences between the CcpA mutants all genes with a different regulation were functionally analyzed by categorizing those genes into Clusters of Orthologous Groups (COGs) Figure . When thccpA knockout strain shows a growth defect in glucose minimal medium , a G (2x), or an A (1x) on position 12, but those six had a T on position 11 (5x) or position 13 (1x). This finding could mean that the arginine on position 17 in CcpA also contacted a thymine in the cre site and that the longer sidechain of arginine could reach further into the surrounding  and thereby changes the concentration of intracellular inducers  was mapped . Thus, it is impossible to find the whole CcpA regulon in the ΔCcpA experiment. The other genes found in this study that do not belong to SubtiWiki's CcpA regulon could be unidentified members of the CcpA regulon, or a member of the class 2 genes .The CcpA protein consists of a N-terminal DNA binding domain and a core domain for coeffector and HPr binding, linked together with a hinge helix region. The threonine on position 62 is located in the core protein, just above the hinge-helix, and is important for the conformational change in the CcpA protein upon HPr binding  and glucose-6-phosphate (G6P) is more difficult. Earlier work shows that the affinity of HPr-Ser46-P to CcpA increased more than twofold by the presence of 40 mM FBP or G6P (Seidel et al., It is known that a ptsH-S46A strain lost part of its repressing power (Deutscher et al., The regulation of genes in some COGs really differed from CcpA-wt, i.e., they were not regulated by the CcpA-wt but only by the CcpA-mutant. Genes in COGs for Energy production [C], carbohydrate metabolism [G], translation [J], posttranslational modification [O], and inorganic ion transport [P] are more strongly repressed in the CcpA-T62H and CcpA-R304W mutants than in the CcpA-wt strain in LB + 1% glucose (Figure In general, we should take into account that the CcpA mutants studied here could have effects on gene regulation which cannot be seen in LB medium with a CcpA mutant because there might be unknown triggers for CcpA which are absent in LB ± glucose. Furthermore, LB medium without added glucose still contains low amounts of glucose and other repressing sugars which might still control the most sensitive target genes. The mutations might uncouple regulation from these triggers. Future experiments in chemically defined medium or in a HPr knockout in the CcpA mutant strains could be done to reveal triggers involved in CcpA dependent gene regulation which were not present when the strains were grown in LB.cre sites in presence or absence of HPr-Ser46-P.In conclusion, the point mutations in CcpA in this study were made to get more insight into the gene regulation mechanisms of CcpA. The main finding here was that the M17R mutation resulted in a small relief of CCR. CcpA-T62H was a stronger repressor than CcpA-wt and can do CCR also in the absence of glucose. CcpA-R304W had a strong regulatory affect in presence and absence of glucose and we suggest that CcpA-R304W is less dependent on HPr-Ser46-P, FBP, and G6P. However, SPR measurements should show the binding affinity of the CcpA mutants to the RD and OK were financially supported by a specific SYSMO2 grant from ALW-NWO. GS was financially supported by a specific SYSMO2 grant from the BMBF.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We performed a detailed ChIP-seq analysis in cells of lymphoid and endothelial origin and compared H3K4me3, H3K27me3, polII, and LANA occupancy. On viral episomes LANA binding was detected at numerous lytic and latent promoters, which were transactivated by LANA using reporter assays. LANA binding was highly enriched at H3K4me3 peaks and this co-occupancy was also detected on many host gene promoters. Bioinformatic analysis of enriched LANA binding sites in combination with biochemical binding studies revealed three distinct binding patterns. A small subset of LANA binding sites showed sequence homology to the characterized LBS1/2 sequence in the viral terminal repeat. A large number of sites contained a novel LANA binding motif (TCCAT)3 which was confirmed by gel shift analysis. Third, some viral and cellular promoters did not contain LANA binding sites and are likely enriched through protein/protein interaction. LANA was associated with H3K4me3 marks and in PEL cells 86% of all LANA bound promoters were transcriptionally active, leading to the hypothesis that LANA interacts with the machinery that methylates H3K4. Co-immunoprecipitation demonstrated LANA association with endogenous hSET1 complexes in both lymphoid and endothelial cells suggesting that LANA may contribute to the epigenetic profile of KSHV episomes.Kaposi's sarcoma-associated herpesvirus (KSHV) is a γ-herpesvirus associated with KS and two lymphoproliferative diseases. Recent studies characterized epigenetic modification of KSHV episomes during latency and determined that latency-associated genes are associated with H3K4me3 while most lytic genes are associated with the silencing mark H3K27me3. Since the latency-associated nuclear antigen (LANA) (i) is expressed very early after  KSHV is a DNA tumor virus which is associated with Kaposi's sarcoma and some lymphoproliferative diseases. During latent infection, the viral genome persists as circular extrachromosomal DNA in the nucleus and expresses a very limited number of viral proteins, including LANA, a multi-functional protein. KSHV viral episomes, like host genomic DNA, are subject to chromatin formation and histone modifications which contribute to tightly controlled gene expression during latency. We determined where LANA binds on the KSHV and human genomes, and mapped activating and repressing histone marks and RNA polymerase II binding. We found that LANA bound near transcription start sites, and binding correlated with the transcription active mark H3K4me3, but not silencing mark H3K27me3. Binding sites for transcription factors including znf143, CTCF, and Stat1 are enriched at regions where LANA is bound. We identified some LANA binding sites near human gene promoters that resembled KSHV sequences known to bind LANA. We also found a novel motif that occurs frequently in the human genome and that binds LANA directly despite being different from known LANA-binding sequences. Furthermore, we demonstrate that LANA associates with the H3K4 methyltransferase hSET1 which creates activating histone marks. Eukaryotic DNA is packaged into chromatin which plays a central role in the regulation of all DNA processes including replication, transcription, and repair. Chromatin contains nucleosomes with DNA wrapped around the core histones H2A, H2B, H3, and H4. Nucleosomes carry epigenetic information in the form of post-translational histone modifications. N-terminal histone modifications including acetylation, methylation, phosphorylation, and sumoylation are important in partitioning chromatin into transcriptionally active or repressive domains  that exist in protein complexes. A single enzyme can be responsible for the three states of methylation in a progressive manner, or different enzymes may be required for different methylation states. Mammalian cells contain 10 different H3K4 KMTs, which include the hSET complex, mixed lineage leukemia 1 to 5 (MLL1-5) complexes, Set7/9, Smyd1, Smyd3, and Prdm9, which are largely not redundant Kaposi's sarcoma-associated herpesvirus  is a γ is a named. In addition to Kaposi's sarcoma (KS) which targets endothelial cells, KSHV is associated with two lymphoproliferative disorders: primary effusion lymphoma (PEL) and a subset of multicentric Castleman's disease (MCD). Although the majority of cells in KS tumors are latently infected, both latent and lytic phases of KSHV infection contribute to pathogenesis and tumorigenesis LANA is a multifunctional protein with important roles during viral latency. LANA is the only viral protein required for episome maintenance by supporting KSHV latent DNA replication and tethering the viral episome to cellular chromosomes. Tethering is mediated by LANA via interactions with the viral terminal repeats and the core histones H2A/H2B We hypothesized that LANA plays a role in the establishment and maintenance of the KSHV epigenome. To address this question and to identify viral and cellular genes potentially regulated by LANA, we performed genome-wide ChIP-seq analyses for LANA, Pol II, and histone modifications. Our data confirm that during latency both active H3K4me3 and repressive H3K27me3 marks are associated with the viral episomes Histone marks on KSHV genomes have previously been mapped by PCR-based ChIP assays and ChIP-on-chip assays using tiling array hybridization Based on studies demonstrating that the patterns for acetylated H3K9 and H3K14 were almost identical with H3K4me3 on the KSHV epigenome ChIP-seq tags mapped to KSHV were used for peak analysis by CisGenome We focused in more detail on H3K4me3, H3K27me3, and Pol II occupancy at the +/−2kb region surrounding known transcription start sites (TSS) of selected viral genes . PromoteFor validation, quantitative ChIP-PCR assays confirmed ChIP-seq results for the promoter regions of LANA, vIRF1, vIL6, RTA, and K7, and the coding region of the late lytic gene ORF19, which was enriched with H3K27me3 . In summin vivoTo date, epigenetic modifications have only been mapped in cells of lymphoid and epithelial cells but not in endothelial cells, which give rise to KS. To address this gap in the literature, we performed ChIP-seq assays in long-term infected TIVE-LTC cells, which contain BCBL-1 derived episomes H3K4me3 occupancy showed significant differences in TIVE cells compared to BCBL-1 cells within the latency-associated region. A prominent H3K4me3 peak within the ORF73 (LANA) promoter  observed in BCBL-1, was demonstrably reduced in TIVE-LTC cells. Instead, a strong H3K4me3 peak appeared at nt 126,280 within the LANA coding region, which was not present in BCBL-1 cells  panel B.In addition to its role in latent DNA replication and episomal maintenance, LANA is a key regulator of host and viral gene expression. LANA binds to DNA directly in a site-specific manner, or indirectly through protein-protein interactions with multiple chromatin associated proteins including core Histone H2A and H2B, CREB2, mSin3, RING3, MeCP2, SSRP1, and P53  in vivo. Furthermore, this LANA peak completely overlaps with three CTCF binding sites (nt 127 514–127 693) in vitroLANA ChIP-seq revealed at least 17 distinct LANA peaks in both BCBL-1 and TIVE-LTCs and observed occupancy patterns are almost identical between both cell types . Howeverin vitro replication assays in vitro EMSA assays. Unexpectedly, numerous LANA peaks are located within promoters of IE, E, and late genes, including ORF16, ORF33, ORF39, ORF48, ORF58, ORF64, and vIRF-1 and -3 (LTI), which is RTA-responsive and induced during lytic replication LANA negatively regulates RTA expression and it was demonstrated that the RBP-Jk sites within the RTA promoter are critical for LANA-dependent regulation 1 and -3 . To deteAfter viral mapping, unmapped reads from LANA ChIP-seq were mapped to the human genome hg19, and 2180 and 2951 unique peaks were identified in BCBL-1 and TIVE-LTC, respectively. In agreement with immunofluorescence data on LANA binding to mitotic chromosomes A list of all gene loci enriched by LANA ChIP-Seq in BCBL-1 cells and TIVE-LTC cells is provided in the supplement  and S3. Gene Ontology (GO) analysis was performed by using DAVID  and S5. Recently, Lu et al. performed LANA ChIP-seq in BCBL-1 cells and reported 256 enriched genes To test LANA regulation of IQGAP3 in cells, we determined IQGAP3 transcript levels in BCBL-1 and LANA-inducible BJAB cells. IQGAP3 mRNA levels are about 2-fold higher in BCBL-1 cells and induction of LANA in BJAB-Tet on-LANA cells moderately induced IQGAP3 transcription . TogetheTCCAT)3 formed from overlapping the motifs in TCCAT)3 motif formed a complex with LANA-C that was visible on longer exposures comparable to those used to detect binding of LANA-C to LBS2. The complex was supershifted in the presence of V5 mAb, confirming specificity. This demonstrates that the 15 nt (TCCAT)3 sequence  and in TIVE-LTC cells 205 out of 2951 (6.9%) peaks contained sequence similarity to LBS1/2 . Hence, sequence  present Next, we asked whether LANA-bound promoters were enriched for specific host transcription factors. Genome-wide occupancy data for many transcription factors are available through the Encyclopedia of DNA Element (ENCODE) Consortium. As the closest available cell line to BCBL-1, we mined ChIP-seq data from GM12878 cells, a B cell lymphoma cell line, for which 43 genome-wide transcription factor binding profiles are available 2 = 0.9)  . This suin vitro translated LANA, but did not detect direct interaction with these proteins (data not shown). In summary, these data show that LANA interacts with endogenous hSET1 complexes either directly or through protein-protein interaction. Recruitment of hSET1 complexes to specific chromatin loci has been reported to be mediated by a number of transcription factors or co-activators, including E2F, NF-E2, and USF1 de novo infection by interacting with HCF-1, which subsequently binds to and recruits hSET1 The strong correlation of LANA and H3K4me3 peaks  raised tde novo infection or during long-term latency.Previous reports by several independent groups demonstrated the importance of H3K27me3 deposition for both the establishment and maintenance of KSHV latency As with two previous studies, we found that in BCBL-1 latency-associated genes are enriched with H3K4me3 and PolII but depleted of H3K27me3  that is highly prevalent in the human genome and was highly enriched in both cell types (TCCAT)3 motif was lower than LBS1 but comparable to LBS2, the low affinity site within the TR TCCAT)3 consensus sequence demonstrates that LANA can directly bind to host cellular DNA via two distinct sequence motifs. This interpretation is also supported by cluster analysis using seqMiner algorithms, which revealed a number of distinct binding patterns across all LANA-enriched promoters 3, which occurs with high frequency in the human genome.Mercier et al. Motivated by LANA's preferential association with H3K4me3 mark-containing promoters, we asked whether LANA interacts with KMTs and demonstrated that LANA efficiently immunoprecipitates with hSET1, the main H3K4 methylase in mammalian cells We propose a working model for the establishment of the viral epigenome which integrates recent findings affecting i) the epigenetic variation of KSHV episomes de novo infection an early burst of promiscuous transcription which includes both LANA and the RTA gene occurs which leads to co-transcriptional H3K4me3 deposition at many promoters After We envisage competition between PRC2 silencing telomerase-immortalized human umbilical vein endothelial cells (TIVE-LTC) 2 atmosphere. BCBL-1 and BJAB cells were cultured in RPMI 1640 medium supplemented with 10% FCS and antibiotics at 37°C under 5% CO2 atmosphere.293 cells, the human embryonic kidney cells, and KSHV long term-infected 5′-GATCGGTACCACAACCCAGTCTCTAAACCAG-3′ and 5′-GACTACGCGTGTTCCTTAGGCTGCCCC), ORF16 (5′- CCGCTCGAGCGGTTGTCAACCAACCAGTCAATCACC and 5′- CCCAAGCTTGGGGCAAAACGTCCTCGTCCATT), ORF39 (5′- GGGGTACCCCGAATGATGTTTGTCTTCGCC and 5′- CCGCTCGAGCGGCGCATGTTTCTCGGTCTTTT), ORF48 (5′- GGGGTACCCCTCTACCATGGAAGCCGGCAA and 5′- CCGCTCGAGCGGGGATACACACCTCCATGTTC), and vIRF1 (5′- GGGGTACCCCATGGAAGCCGGCAACAGTCCT and 5′- CCGCTCGAGCGGACACCTCCATGTTCAGTCAC). Corresponding promoter regions were PCR amplified, and cloned into plasmid pGL3-basic at appropriate sites.Primer pairs were designed to amplify the promoter region (+/−2 kb relative to the transcription start site) for IQGAP3 , rat IgG , rabbit against H3K4me3 , H3K27me3 , rabbit anti Pol II , rat LANA monoclonal antibody , rabbit anti hSET1 , rabbit anti RbBP5 , rabbit anti ASH2L , and rabbit anti MLL1 .7 cells were lysed in 1 ml ice-cold Farnham lysis buffer  with protease inhibitors. The nuclei were spun down and resuspended in 1 ml RIPA buffer  with protease inhibitors. Chromatin was sheared to about 250 bp fragments with 5 sets of 30-second pulses using a Sonic Dismembrator (Fisher Scientific) set to 50% of maximum power. Chromatin from 6×107 cells was incubated with 10 µg primary antibody  and 100 µl magnetic beads  at 4°C overnight on a rotator. Beads were washed 5 times with LiCl wash buffer and once with TE. The immune complexes were eluted with 200 µl elution buffer twice at 65°C for 1 hour. The combined eluates were de-crosslinked at 65°C overnight. DNA was extracted once with phenol/chloroform and precipitated with ethanol. 20 µg glycogen was added as DNA carrier. DNA pellets were washed once with 70% ethanol and resuspended in 40 µl H2O.ChIP experiments were performed as described before with minor modifications 5′-pGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG and 5′- ACACTCTTTCCCTACACGACGCTCTTCCGATCT) were ligated to both ends of the DNA fragments. DNA within the range of 150 bp to 300 bp was gel-purified and PCR-amplified for 18 cycles with primer 5′ -AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT and 5′- CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCT. The resulting libraries were gel-purified, quantified with QuantIT dsDNA Assay Kit (Invitrogen) and sequenced with Genome Analyzer IIx (Illumina) in Department of Molecular Genetics and Microbiology (MGM) at University of Florida (UF).100 ng ChIP-enriched DNA or control IgG ChIP DNA was blunt-ended with T4 DNA polymerase and Klenow DNA polymerase (NEB) and phosphorylated with T4 PNK (NEB). Addition of an “A” base to the 3′ ends of the blunt phosphorylated DNA fragments was performed in the presence of Klenow exo- (NEB). Subsequently, adapters . The captured DNA was eluted and purified. The DNA was re-amplified for 22 PCR cycles with primers (5′-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT and 5′-CAAGCAGAAGACGGCATACGAGCTCTTCCGATCT), and purified using Agencourt AMPure XP beads. The libraries were quantified with QuantIT dsDNA Assay Kit (Invitrogen) and sequenced as above. HTS sequencing generated 20.8 million, 17.1 million, and 20.5 million tags for H3K4me3, H3K27me3, and Pol II ChIP-seq, respectively. After enrichment 42.76%, 65.4%, and 87.58% of H3K4me3, H3K27me3, and Pol II ChIP-seq tags were mapped to the viral genome compared to 0.014%, 0.028%, and 0.007% without enrichment; hence enrichment efficiency was about 2,000- to 4,000-fold.The biotin-labeled RNA baits specific for KSHV genome was customized with eArray XD (Agilent) with help from Agilent. The RNA baits are 120 nt long with 4× tiling frequency. TIVE-LTC ChIP-seq libraries were constructed as above. After adapter ligation, DNA fragments between 150 bp and 300 bp were gel-selected and amplified with 10 cycles in first PCR. Samples were purified with Agencourt AMPure XP beads (Beckman Coulter). The KSHV-specific DNA sequences were enriched with SureSelect Enrichment System (Agilent) according to the manufacturer's instruction. The purified 1http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE52421).After completion of Illumina sequencing, the raw output of BCL files were converted to the FASTQ format using the Illumina BCL Converter. Bowtie Peak finding was performed with CisGenome v1 In order to examine the reads that might align to the junction of two TRs and that might be unaligned to the reference genome with only one TR at the end, we attached the sequence of a half or an entire TR to the viral genome and ran the alignment pipeline against these new reference .Visualization in IGV indicated that the chromatin mark H3K4me3 usually existed near LANA binding signals. We applied CisGenome on H3K4me3 ChIP-seq data but did not obtain reasonable peaks. The finding was consistent with the consensus that most peak calling programs were not designed for histone data and therefore performed poorly in identifying these diffuse signals To study the potential regulatory function of LANA in gene expression, we mapped the reads aligned to the human genome to the promoter regions. We downloaded transcript information of Ensembl build 62 from UCSC Table Browser and defined promoters as the +/−2 Kb region of the transcription start site (TSS) of annotated transcripts. The genome positions of peaks were compared to the promoter positions using BEDTools intersectBed and any overlap was reported. The distances between the center of the peaks and their adjacent TSS's were plotted for each lane. Putative targets genes of LANA regulation were compared between BCBL1 and TIVE-LTC, as well as with previous publications.Similarly, the reads aligned to the KSHV genome were also mapped to KSHV genes, the genome positions of which were downloaded from the NCBI Nucleotide database (NC_009333.1) and parsed using a custom Perl script.GCCCCATGCCCGGGCGG , GCCGCAGGCCCCGGCGG , ATTGTCCCGGGCGCCGCG, and CCGGGTCTCCAGGGCGCGCCGCGTG. For this purpose, we used an EMBOSS tool FUZZNUC The peaks were scanned for LANA consensus LANA binding sites (LBSs): To study the tissue-specific co-occupancy of LANA and other TFs, we downloaded the peak files of all ENCODE TFBS ChIP-seq experiments in GM12878 . A total of 43 TFs were available in GM12878 The peak files were mapped to the hg19 promoter regions. A peak overlapping at least one base with the promoter was considered to be a regulator of the promoter, the same as we mapped our LANA peaks. The transcripts with both LANA and ENCODE TFBS peaks in their promoters were identified. The distance between the center of the LANA peak and the ENCODE peak within the same promoter was calculated and plotted (e.g. CTCF and STAT1 in GM12878 vs. BCBL1). In the cases that multiple peaks exist in a promoter, the smallest distance was retained.http://rsat.ulb.ac.be/rsat/) We used the “peak motifs” tools from Regulatory Sequence Analysis Tools  according to the manufacturer's instruction using the following program: Step 1: 95°C 20 sec; Step 2: 95°C 3 s, 60°C 30 sec, repeat 39 times; Step 3: melting curve from 60°C to 95°C. 32P]-ATP, and incubated with purified LANA-C protein as described previously DNA duplexes were created by annealing the oligonucleotide pairs listed in Supplementary 5 BJAB, BCBL-1, TIVE, or TIVE-LTC cells were harvested and washed once with PBS. Cells were lyzed with 100 ul RIPA buffer. 20 ul of the cells lysate was separated in 8% SDS-PAGE gels and transferred to PVDF membrane. Membranes were blocked for 2 hours in T-TBS buffer containing 5% fat-free milk. Primary antibodies against specific proteins were diluted according to manufacturers' instruction and hybridized with membrane at 4°C overnight. After washing, 1∶5000 diluted corresponding secondary antibodies conjugated with peroxidase were incubated with the membrane for 1 hour at room temperature. After final washing, the blots were developed with ECL substrates (Millipore) and exposed to films.Western blot analysis was performed as previously described 7 cells were harvested and resuspended in 1 ml hypotonic buffer . Nuclei were spun down and resuspended in hypertonic buffer . Cells were lyzed at 4°C for 15 minutes with rotation. Dilution buffer which had the same ingredients as hypertonic buffer except for NaCl was added to make the final NaCl concentration 150 mM. Cell lysate was centrifuged for 10 minutes and pre-cleared with 50 µl protein A/G beads. A small portion of the lysate was saved as input. Cell lysate was incubated with 10 µg antibody at 4°C overnight with rotation. 100 µl protein A/G beads were added and incubated for 2 hours. The beads were collected by pulse centrifugation and washed 5 times with 1 ml ice-cold PBS with 0.1% Tween-20. Proteins were eluted with 1× Laemmli buffer and subjected to Western blot analysis.IP experiments were performed as described before with minor modifications Figure S1Bland-Altman Plot to investigate the agreement between two replicates of the ChIP-seq experiments. RPKM was calculated for each sample after Bowtie alignment. The Y axis is the difference between two biological replicates and the X axis is the average between biological replicates. Green lines are the average of all differences +/−1.96× (standard deviation of the differences), indicating 95% confidence interval. The red line is drawn at zero.(EPS)Click here for additional data file.Figure S2Distribution of H3K4me3, H3K27me3, Pol II, and LANA within the latency-associated region. Detailed wiggle plots generated with UCSC genome browser illustrating nearly identical occupancy between latently infected BCBL-1 cells (A) and TIVE-LTC cells (B). Note distinct LANA, H3K4me3, and polII peaks within LANA ORF in TIVE-LTC cells (B).(EPS)Click here for additional data file.Figure S3LANA binding within the terminal repeat and ORF50 regions. Wiggle plots visualizing LANA occupancy on the viral TR and ORF50/RTA region. (A) Identical peak recovery from BCBL-1 and TIVE-LTC cells for binding to the LBS1/2 binding site within TR. Note very high recovery of LBS1/2 from BCBL-1 cells (y-axis). (B) Identical peak recovery within exonic sequences of RTA gene from both cell types. No LANA occupancy upstream of the RTA TSS was observed.(EPS)Click here for additional data file.Figure S4Examples for cell type-specific and common LANA peaks within host promoters. Wiggle plots generated with UCSC genome browser on selected LANA CHIP-seq-enriched promoters demonstrating cell type-specific (A–C) and (E–H) and common (D) occupancy between BCBL-1 and TIVE-LTC cells.(EPS)Click here for additional data file.Figure S5Flow diagram of LANA peak identification and annotation. Details about algorithms and programs used at each step are described in the Material and Methods Data analysis section.(PPTX)Click here for additional data file.Table S1Comparison of KSHV nts sequences in Accession number NC_009333.1 and U75698.(DOCX)Click here for additional data file.Table S2Potential cellular targets regulated by LANA in BCBL-1 cells.(XLSX)Click here for additional data file.Table S3Potential cellular targets regulated by LANA in TIVE-LTC cells.(XLSX)Click here for additional data file.Table S4GO analysis of LANA targets in BCBL-1 cells.(XLSX)Click here for additional data file.Table S5GO analysis of LANA targets in TIVE-LTC cells.(XLSX)Click here for additional data file.Table S6Oligonucleotides used in EMSA assays.(XLSX)Click here for additional data file.


To elucidate the principles governing these architectural functions, we use two insulators, Homie and Nhomie, that flank the Drosophila even skipped locus. We show that homologous insulator interactions in trans, between Homie on one homolog and Homie on the other, or between Nhomie on one homolog and Nhomie on the other, mediate transvection. Critically, these homologous insulator:insulator interactions are orientation-dependent. Consistent with a role in the alignment and pairing of homologs, self-pairing in trans is head-to-head. Head-to-head self-interactions in cis have been reported for other fly insulators, suggesting that this is a general principle of self-pairing. Homie and Nhomie not only pair with themselves, but with each other. Heterologous Homie-Nhomie interactions occur in cis, and we show that they serve to delimit a looped chromosomal domain that contains the even skipped transcription unit and its associated enhancers. The topology of this loop is defined by the heterologous pairing properties of Homie and Nhomie. Instead of being head-to-head, which would generate a circular loop, Homie-Nhomie pairing is head-to-tail. Head-to-tail pairing in cis generates a stem-loop, a configuration much like that observed in classical lampbrush chromosomes. These pairing principles provide a mechanistic underpinning for the observed topologies within and between chromosomes.The chromosomes of multicellular animals are organized into a series of topologically independent looped domains. This domain organization is critical for the proper utilization and propagation of the genetic information encoded by the chromosome. A special set of architectural elements, called boundaries or insulators, are responsible both for subdividing the chromatin into discrete domains and for determining the topological organization of these domains. Central to the architectural functions of insulators are homologous and heterologous insulator:insulator pairing interactions. The former (pairing between copies of the same insulator) dictates the process of homolog alignment and pairing in  Drosophila, the former defines the topology of individual looped domains in cis, while the latter dictates the process of homolog alignment and pairing in trans. Here we use two insulators from the even skipped locus to elucidate the principles governing these two architectural functions. These principles align with several longstanding observations, and resolve a number of conundrums regarding chromosome topology and function.The chromosomes of multicellular animals are organized into a series of topologically independent looped domains. This domain organization is critical for the proper utilization and propagation of the genetic information encoded by the chromosome. A special set of architectural elements, called boundaries or insulators, are responsible for both subdividing the chromatin fiber into discrete domains, and determining the topological organization of these domains. Central to the architectural functions of insulators are heterologous and homologous insulator:insulator pairing interactions. In  The highly regular and reproducible physical organization of chromosomes in multicellular eukaryotes was recognized a century ago in cytological studies on the lampbrush chromosomes that are found in oocytes arrested at the diplotene phase of meiosis I –3. At thSubsequent studies have shown that the key features of chromosome architecture evident in lampbrush and polytene chromosomes are also found in diploid somatic cells –13. One That TAD boundaries correspond to insulators is consistent with the known properties of these elements. Insulators subdivide the chromosome into functionally autonomous regulatory domains. When interposed between enhancers or silencers and target promoters, insulators block regulatory interactions. They also have an architectural function in that they can bring distant chromosomal sequences together, and in the proper configuration can promote rather than restrict regulatory interactions ,21. Morecis. One of these is the basis for partner choice. Is choice based simply on proximity, or is there an intrinsic partner preference? A second concerns the topology of the loop formed by interacting partners in cis. Do the partners interact to form a stem-loop-like structure, or does the interaction generate a circular loop (“circle-loop”)? The answer to this question will depend upon whether there is an orientation dependence to the interactions between two heterologous insulators. In flies, homologs are typically paired in somatic cells, not just in cells that are polyploid [trans. If this is case, there are similar questions about the rules that govern trans interactions. Is there a partner preference in the interactions that mediate homolog pairing? Is there an orientation dependence, and if so, what is the topological outcome of the looped domains generated by insulator interactions in paired chromosomes in cis and in trans?The notion that insulators are responsible for subdividing eukaryotic chromosomes into a series of looped domains raises questions about the rules governing loop formation in olyploid . This meeven skipped (eve) locus to address the questions posed above about the architecture of eukaryotic chromosomes. The eve domain spans 16 kb and is bordered upstream by the Nhomie  insulator and downstream by Homie (Homing insulator at eve) [eve encodes a homeodomain transcription factor that is required initially for segmentation, and subsequently in the development of the CNS, muscles, and anal plate [eve gene activate transcription in a pair-rule pattern. Later in development, around the time that germband retraction commences, mesodermal (Me) and neuronal (CNS) enhancers turn on eve expression in a subset of cells in each of these tissues. These late enhancers continue to function once germband retraction is complete, while another enhancer (APR) induces transcription in the presumptive anal plate. Located just upstream of eve is CG12134, while the TER94 gene is downstream. Unlike eve, both of these genes are ubiquitously expressed throughout much of embryogenesis.In the studies reported here, we have used insulators from the  at eve) ,26. eve al plate ,28. It hal plate ,29–31, aal plate . In earleve locus. Second, when the homed transgene carries a reporter, it is expressed in an eve-like pattern, the completeness of which diminishes with distance. Early stripe and later CNS expression are limited to 200 kb from eve, mesodermal expression has an intermediate distance dependence, while anal plate ring (APR) expression is seen at distances of several Mb. We showed previously that reporter expression at a site within the hebe gene 142 kb upstream of eve requires Homie [eve (see below).The Homie insulator has two striking properties . First ies Homie . Since oes Homie ,33, we ues Homie  to map ceve-promoter-lacZ (lacZ) reporter and Homie. One of them is inserted into the chromosome so that Homie is oriented in the same direction , lacZ is regulated by both the hebe CNS enhancer (arrow in bottom panel) and the eve enhancers . When the transgene is inserted in the opposite orientation so that the lacZ reporter is closer to the eve locus (rd column: transgene #2), Homie blocks the hebe enhancer, and only the eve pattern is seen . Finally, as expected, the reporter in the DNA control transgene , but is regulated by the hebe enhancer (arrow in bottom panel). In this case, the reporter is separated from the hebe enhancer by DNA, not Homie. These results show that Homie induces a long-range interaction between a reporter transgene located many kilobases away and endogenous eve enhancers, and that this interaction is not sensitive to the orientation of the transgene in the chromosome.  Furthermore, the long-range looping interactions between the transgene and the eve locus do not change the local enhancer blocking activity of the Homie insulator.The transgenes have an eve elements, we used 11 primers spanning the locus . In transgene #4 . The lacZ reporter is not, however, activated by the hebe enhancer, as it is insulated by Homie. A quite different result is observed for the GFP reporter. First, unlike lacZ, it is not subject to regulation by the eve enhancers. Second, it is subject to regulation by the hebe enhancer (eve locus is reversed (GFP is regulated by the eve enhancers (green arrows), while lacZ is not. And, since the orientation of the transgene in the chromosome remains the same, the hebe enhancer still activates GFP  of expression . In the reversed . Here, Geve locus are orientation-specific. With respect to the endogenous Homie insulator (see below for Nhomie), the pairing interactions could be head-to-head or head-to-tail  or a stem-loop . However, in both cases, the eve enhancers are brought into close proximity to the lacZ reporter. Note that as in lacZ reporter and the enhancers in the eve locus on opposite sides of the paired insulators, which would not be conducive for productive regulatory interactions (not illustrated).Why are the regulatory interactions in eve locus that interacts physically with Homie at -142 kb  enhancers [lacZ reporter. The transgenes were inserted into a site far away from endogenous eve , while there is virtually no Me-driven expression (red arrows). As predicted, the presence of a forward-oriented Homie in the enhancer transgene substantially augments transvection (rd panel). Not only is APR expression much stronger (green arrow), but Me-driven expression is also clearly observed (red arrow).To test these predictions, we generated two transgenes, one containing the nhancers , and thenhancers ), orientnhancers , we expenhancers ,51 when To confirm that stable pairing is head-to-head, we reversed Homie in the enhancer transgene . In this configuration, head-to-head pairing would introduce an S-shaped double loop. As illustrated in GFP and lacZ that have either DNA or Homie inserted between the reporters  substantially enhances APR lacZ transcription, and turns on lacZ in the mesoderm. By contrast, there is only a slight increase in APR GFP expression, while mesoderm expression is detectable, but only weakly. The differences in transvection for the two reporters are consistent with the topology generated by head-to-head, not head-to-tail pairing  so that the two enhancers are 3’ of Nhomie  determine their orientation dependence, we combined a Nhomie-lacZ reporter with two different Homie-enhancer transgenes. In the one in which the enhancers are 5’ of Homie .To confirm these results, we combined the dual eve insulators can interact specifically with themselves and with each other over large chromosomal distances, we took advantage of attP 25C1, located 2 Mb distal to 23C4. A Homie:lacZ transgene was inserted at 25C1. It was combined with an enhancer transgene at 23C4 containing either DNA or Homie (su(Hw) insulator. On the other hand, when both the reporter and the enhancer have a Homie insulator, the APR enhancer is able to activate lacZ expression  at 23C4 and 25C1, then crossed into the same animals. eve transcription unit and its associated enhancers and Polycomb silencers, linked together at the base by the Nhomie and Homie insulators. 3C experiments with Homie as the anchor confirm that Nhomie and Homie contact each other in the eve locus  and Mcp insulators each can mediate regulatory interactions between transgenes inserted at distant sites [Fab-7 insulator can also mediate long-range regulatory effects [A role in homolog pairing was first suggested by the discovery that the nt sites ,70. The  effects . Further effects ,36. Thes effects ,48,72,73trans-regulatory interactions between enhancers on one homolog and a reporter on the other  dCTCF) [While there is compelling evidence that insulator self-interactions are responsible for homolog pairing, many issues remained unresolved. Perhaps the most important is the nature of the code used for self-recognition and orientation. The best hint comes from bypass experiments using multimerized binding sites for Su(Hw), dCTCF, or Zw5. Homologous multimer combinations give bypass, while heterologous combinations do not. However, bypass is observed for composite multimers when they are inserted in opposite orientations  dCTCF ↔ dCTCF Su(Hw)), but not the same orientation  dCTCF ) dCTCF) . These fcis could subdivide chromosomes into looped domains. Subsequent work has confirmed this conclusion [cis insulator interactions generate loops, they provided no information about the topology of these loops, or how they are arranged.The first direct evidence that insulators generate loops came from 3C experiments on the mouse β-globin and the fly 87A7 heat shock loci ,74. Thesnclusion . HoweverCis interactions could, a priori, be either head-to-head like self-association in trans, or head-to-tail. The consequences are quite different. Head-to-head interactions generate a circle-loop, while head-to-tail interactions generate a stem-loop  would require coiling of successive homologous circle-loops on each homolog in the same direction, one inside the other , sister chromatids are aligned. Maintaining this alignment may facilitate epigenetic mechanisms that template chromatin structures from one cellular generation to the next, such as the copying of histone modifications onto both daughter chromosomes. The simpler topology of stem-loops could facilitate this sister chromatid pairing, as well as their separation during mitosis. Second, recent studies on the relationship between loop domains and CTCF insulators showed that in more than 90% of the cases, the CTCF binding sites on opposite ends of a loop are in opposite orientation [cis could generate a series of circular loops that extend out from the same side of the main axis. This configuration would be favorable for crosstalk between regulatory elements and genes in adjacent loops. By contrast, head-to-tail pairing, where adjacent stem-loops extend out in opposite directions , either the lacZ or GFP coding region, and the eve 3'-UTR, +1300 to +1525 bp. Enhancers are: eve APR,+3.0 to +4.1 kb; eve Me, +5.7 to +6.6 kb, each cloned in plasmid attB∆2 [eve [See d attB∆2  for trand attB∆2  using ΦCd attB∆2 . Target B∆2 [eve ; 23C4 Click here for additional data file.S2 Figeve enhancers and a transgenic promoter-reporter located at -142 kb relative to the endogenous eve transcription start site. Embryonic stages are indicated on the left. The sequences tested .The assay of (TIF)Click here for additional data file.


This study explored the role of G9a- and enhancer of zeste homolog 2 (EZH2)-mediated methylation of histone H3 lysine 9 (H3K9me3) and histone H3 lysine 27 (H3K27me3) in COX-2 silencing in IPF. Chromatin immunoprecipitation (ChIP) and re-ChIP assays demonstrated marked increases in H3K9me3, H3K27me3, and DNA methylation, together with their respective modifying enzymes G9a, EZH2, and DNA methyltransferases (Dnmts) and respective binding proteins heterochromatin protein 1 (HP1), polycomb protein complex 1 (PRC1) and methyl CpG binding protein 2 (MeCP2), at the COX-2 promoter in lung fibroblasts from patients with IPF (F-IPFs) compared with fibroblasts from nonfibrotic lungs. HP1, EZH2, and MeCP2 in turn were associated with additional repressive chromatin modifiers in F-IPFs. G9a and EZH2 inhibitors and small interfering RNAs and the Dnmt1 inhibitor markedly reduced H3K9me3 (49−79%), H3K27me3 (44−81%), and DNA methylation (61−97%) at the COX-2 promoter. These reductions were correlated with increased histone H3 and H4 acetylation, resulting in COX-2 mRNA and protein reexpression in F-IPFs. Our results support a central role for G9a- and EZH2-mediated histone hypermethylation and a model of bidirectional, mutually reinforcing, and interdependent crosstalk between histone hypermethylation and DNA methylation in COX-2 epigenetic silencing in IPF.—Coward, W. R., Feghali-Bostwick, C. A., Jenkins, G., Knox, A. J., Pang, L. A central role for G9a and EZH2 in the epigenetic silencing of cyclooxygenase-2 in idiopathic pulmonary fibrosis.Selective silencing of the cyclooxygenase-2 (COX-2) gene with the loss of the antifibrotic mediator prostaglandin E The most well-characterized histone methylations are the di- and trimethylation of histone H3 lysine 9 H3K9me2/3) and histone H3 lysine 27 trimethylation (H3K27me3), which repress gene expression, and histone H3 lysine 4 trimethylation (H3K4me3), which is associated with gene activation . H3K9me2/3 and hiromoters , 7.de novo Dnmts, whereas Dnmt1 is the primary Dnmt for the maintenance; however, the functions of these Dnmts overlap extensively  is a major prostanoid in lung structural cells, including fibroblasts, and is derived mainly from the inducible cyclooxygenase-2  is a deadly respiratory disease of unknown etiology with a median survival of 3–4 yr and no effective therapy . IPF is position . Animal fibrosis , 17; in fibrosis , 19. Morge fluid  and COX-ge fluid  are also2 production. These findings demonstrate a novel and central role for G9a- and EZH2-mediated histone methylation in COX-2 epigenetic silencing in IPF and provide a functional connection between histone methylation and DNA methylation. The intrinsic reversibility and mutual dependence of these epigenetic changes may prove to be beneficial in the reactivation of the antifibrotic COX-2 gene in IPF.We have demonstrated previously that COX-2 gene transcription was defective in F-IPFs compared with that in fibroblasts from nonfibrotic lung (F-NLs) due to deficient histone acetylation as a result of decreased recruitment of HATs and increased recruitment of the HDAC-containing transcriptional corepressor complexes to the COX-2 promoter . Howeverin vivo. The cells were growth arrested in serum-free medium for 24 h before stimulation with recombinant human interleukin-1β  in serum-free medium. At the indicated time points, the cells were harvested for subsequent analysis, and their responses were compared.F-IPFs and F-NLs from the explanted lungs of patients with IPF who underwent lung transplantation at the University of Pittsburgh Medical Center  and from normal lung tissues obtained from organ donors under a protocol approved by the University of Pittsburgh Institutional Review Board were cultured as described previously . The 6 FCT−ΔΔ method, where ΔΔTC is the difference between the threshold cycle (TC) for the bound fraction and the TC for the input fraction. The association of acetylated and methylated histones H3 and H4 with the COX-2 promoter DNA was further normalized to the association of total histones H3 and H4 with the COX-2 DNA.ChIP assays were performed using reagents and protocols from a ChIP-IT Express Kit  as described previously . AntibodRe-ChIP assays were conducted by using a Re-ChIP-IT Express Kit (Active Motif) as described previously . After thttp://www.zymoresearch.com). The only difference is that each contains either 100% unmodified cytosines or 5-methylcytosines. Since the sequence and extent of cytosine modification are known, these DNA standards were also used to spike the chromatin to demonstrate the specificity of the MeDIP assay. The immunoprecipitated antibody-DNA complexes were then collected, and DNA was extracted, purified, and subjected to real-time PCR amplification of the minimum COX-2 promoter region (positions −299 to+6) with key transcription factor binding sites -6,7-dimethoxy-N-[1-(phenylmethyl)-4-piperidinyl]-4-quinazolinamine trihydrochloride hydrate]; ref. To assess the role of G9a HMT, Dnmt1, and HDACs in COX-2 repression in F-IPFs, inhibitors for these enzymes were used. The G9a HMT-specific inhibitor BIX-01294 [2-, EZH2 , or ALLStars negative control siRNA  was diluted in 1.6 ml of culture medium, 48 μl of HiPerFect Transfection Reagent was added to the diluted siRNA, and the preparation was mixed and incubated at room temperature for 10 min. The transfection mixture was added to cells in a 100-mm culture dish in a total volume of 4 ml, and the cells were incubated for 3 h, after which the culture medium was made up to 7 ml with a final siRNA concentration of 5 nM, and cells were incubated for a further 48 h. Cells were then serum starved for 24 h before being treated without or with IL-1β (1 ng/ml) for 2, 4, and 24 h for analyses of epigenetic modifications, G9a, EZH2, and COX-2 mRNA and protein expression, respectively.All transient transfections were conducted by using HiPerFect Transfection Reagent according to the recommended protocol of the manufacturer . A total of 2 × 102 concentration in the culture medium was measured by a commercially available enzyme-linked immunosorbent assay kit .The PGEsem from experiments with 3−6 separate cell lines performed in duplicate. A nonparametric ANOVA was used to evaluate the statistical significance of the mean values between F-NLs and F-IPFs; this was followed by a Wilcoxon rank sum test for comparisons between F-NLs and F-IPFs at specific time points. A value of P < 0.05 was considered statistically significant.The data are presented as means ± P<0.01 at 1 h after stimulation compared with unstimulated) but not in F-IPFs, and the level of H3K4me3 at the COX-2 promoter was significantly lower in F-IPFs than in F-NLs . In contrast, under both unstimulated and cytokine-stimulated conditions, H3K9me3 and H3K27me3 at the COX-2 promoter were elevated in F-IPFs compared with those in F-NLs . In addition, the association of the 2 major HMTs G9a and EZH2, responsible for H3K9me3 and H3K27me3, respectively , but not the HMT SUV39H1, also responsible for H3K9me3 (E), with the COX-2 promoter was also consistently increased in F-IPFs compared with that in F-NLs with or without cytokine treatment. The data thus suggest that histone H3K9 and H3K27 are hypermethylated at the COX-2 promoter in F-IPFs as a result of increased recruitment of G9a and EZH2, respectively.We have previously shown that both histones H3 and H4 are insufficiently acetylated at the COX-2 promoter in F-IPFs compared with those in F-NLs . Becausein F-NLs A. In conin F-NLs A. In conin F-NLs A. In conA, BFig. 2). Because HP1 may act as an adapter in mediating histone deacetylation and DNA methylation through its recruitment of the respective epigenetic enzymes, we went on to explore the association of HP1 with G9a, Dnmts, and transcriptional repressors NCoR, CoREST, and mSin3a by a Re-ChIP assay. As shown in C, D, enrichment of the COX-2 promoter DNA was observed in both primary and secondary IPs in F-IPFs, demonstrating a physical association of HP1 with G9a, Dnmt1, Dnmt3a, NCoR, CoREST, and mSin3a at the COX-2 promoter. EZH2 is the catalytic subunit of PRC2, which also includes EED  and that EED was associated with NCoR, CoREST, and mSin3a at the COX-2 promoter in F-IPFs . These observations suggest that G9a-mediated H3K9me3 and EZH2-mediated H3K27me3 are associated with additional chromatin modifiers to the COX-2 promoter through HP1 and EZH2/EED, respectively, which may result in DNA methylation and histone deacetylation and reinforcement of COX-2 epigenetic silencing in F-IPFs.Because methylated histone H3K9 and H3K27 serve as a binding platform for HP1 and PRC1, respectively, we anticipated that the respective association of HP1 and PRC1 with H3K9me3 and H3K27me3 at the COX-2 promoter could be increased in F-IPFs. Using a Re-ChIP assay, we indeed detected HP1 and PRC1 in the IPs of H3K9me3 and H3K27me3, respectively, at the COX-2 promoter under unstimulated conditions A, B. Becudes EED , 7. We fAFig. 3). The 2 regions upstream  of the minimum COX-2 promoter were also more methylated in F-IPFs than in F-NLs but to a much lesser extent than that for the minimum COX-2 promoter, whereas no meaningful methylation was observed in the 2 regions downstream  of the minimum COX-2 promoter (A). Because DNA methylation is catalyzed by Dnmts, we next assessed whether the recruitment of Dnmts to the COX-2 promoter was increased in F-IPFs. By using a ChIP assay, we found that the association of Dnmt1 and Dnmt3a with the COX-2 promoter was significantly increased in F-IPFs compared with that in F-NLs under unstimulated conditions and marked, but not statistically significant, increases were maintained after cytokine stimulation . However, no difference was observed between F-IPFs and F-NLs in the global protein expression of Dnmt1 and Dnmt3a as analyzed by Western blotting (data not shown). These findings suggest that the COX-2 promoter DNA is methylated in F-IPFs as a result of increased recruitment of Dnmts to the COX-2 promoter rather than increased expression of Dnmt proteins. Because methylated DNA serves as a binding platform for MeCP2, we anticipated that the MeCP2 association with the COX-2 promoter could be increased in F-IPFs. ChIP analysis indeed revealed that there was a trend for increased association of MeCP2 with the COX-2 promoter in F-IPFs compared with that in F-NLs with or without cytokine treatment, although the differences were not statistically significant . Because MeCP2 can mediate gene silencing through recruitment of chromatin modifiers, we went on to explore the association of MeCP2 with HMTs, Dnmts, and transcriptional repressors by a Re-ChIP assay. As shown in Fig. 3E, F, enrichment of the COX-2 promoter DNA was observed in both primary and secondary IPs in F-IPFs, demonstrating physical association of MeCP2 with G9a, EZH2, Dnmt1, Dnmt3a, NCoR, CoREST, and mSin3a at the COX-2 promoter and suggesting that DNA methylation, like histone methylation, could also recruit additional epigenetic enzymes to the COX-2 promoter through MeCP2 and lead to reinforced COX-2 epigenetic silencing in F-IPFs.Because the COX-2 promoter is known to contain CpG islands, and COX-2 repression is commonly associated with DNA methylation, we anticipated that the COX-2 promoter DNA could be methylated, thereby contributing to the COX-2 gene repression in F-IPFs. By applying the MeDIP assay with an antibody against 5-methylcytosine and amplifying the immunoprecipitated COX-2 promoter DNA by real-time PCR using specific primers for different regions of the COX-2 promoter, we revealed that the minimum COX-2 promoter region (−299/+6) was significantly more methylated in F-IPFs than in F-NLs under unstimulated conditions A. The 2 AFig. 4); this result was accompanied by reduced HP1 association with the COX-2 promoter . Similarly, treatment of F-IPFs with DZNep, either alone or with IL-1β, decreased H3K27me3 at the COX-2 promoter by 44 and 49%, respectively, compared with that in untreated cells, whereas treatment with RG108 reduced H3K27me3 by 65 and 46%, respectively . Treatment of F-IPFs with the inhibitors also resulted in an increase in histone H3 and H4 acetylation without cytokine stimulation compared with that in untreated cells, and a further increase was generally observed after IL-1β stimulation . The results show that repressive histone methylation at the COX-2 promoter in F-IPFs can be removed by inhibition of not only the activity of respective HMTs but also the activity of Dnmt1 and that histone acetylation at the COX-2 promoter in F-IPFs can be increased by inhibition of the activity of HMTs and Dnmt1. The data also strongly suggest that G9a and EZH2 play a central role in mediating repressive histone modifications at the COX-2 promoter in F-IPFs and that Dnmt activity is required for this process.We have previously shown that HDAC inhibitors increase histone acetylation at the COX-2 promoter site and restore COX-2 expression in F-IPFs . To deteAFig. 5); this result was also accompanied by reduced HP1 association with the COX-2 promoter (B). Similarly, like EZH2 inhibition by DZNep, treatment of F-IPFs with EZH2 siRNA, either alone or with IL-1β, resulted in a marked decrease in H3K27me3  at the COX-2 promoter compared with that for untreated cells . Treatment of F-IPFs with G9a siRNA and EZH2 siRNA also led to a slight increase in histone H3 and H4 acetylation without cytokine stimulation compared with that in untreated cells, and a marked increase was observed after IL-1β stimulation ; this was accompanied by significant increases in the recruitment of HAT cAMP response element binding protein binding protein (CBP), p300, and p300/CBP-associated factor (PCAF) to the COX-2 promoter . In addition, G9a siRNA and EZH2 siRNA restored the response of F-IPFs to IL-1β to increase the active histone modification H3K4me3 at the COX-2 promoter . The data confirm the effect of G9a and EZH2 inhibitors in Because pharmacological inhibitors of epigenetic enzymes can have off-target effects, to further validate the role of G9a and EZH2 in mediating COX-2 repression in F-IPFs, we applied siRNA to knock down G9a and EZH2 expression in these cells. Like G9a inhibition by BIX-01294, treatment of F-IPFs with G9a siRNA but not the control siRNA, either alone or with IL-1β, markedly reduced H3K9me3 at the COX-2 promoter by 79 and 60%, respectively, compared with that in untreated cells A; this rA, C), we went on to assess whether G9a and EZH2 could be required for COX-2 promoter DNA methylation. As shown in AFig. 6, treatment of the cells with either BIX-01294, DZNep, or RG108 reduced DNA methylation by 62, 83, and 64%, respectively, at the minimum COX-2 promoter region (−299/+6), with significant inhibition by DZNep. The inhibitors also similarly reduced the binding of MeCP2 to the COX-2 promoter, although the reduction was not statistically significant . Furthermore, both G9a and EZH2 siRNAs but not the control siRNA also markedly reduced DNA methylation at the minimum COX-2 promoter by 86 and 97%, respectively, compared with that for untreated F-IPFs, to a level lower than that observed with F-NLs , thereby confirming the effect of the G9a and EZH2 inhibitors. The combined results of Because Dnmt activity could be required for G9a- and EZH2-mediated histone H3 hypermethylation at the COX-2 promoter in F-IPFs A, C, we AFig. 7). Western blotting analysis revealed no COX-2 protein expression when the cells were treated with the inhibitors or with IL-1β alone; however, when the cells were treated with the inhibitors and IL-1β together, marked increases in COX-2 protein expression were observed (B). To find out whether the restoration of COX-2 protein expression in F-IPFs was accompanied by PGE2 production, PGE2 in the medium was also analyzed. As shown in Fig. 7C, no increase in PGE2 production was observed when the cells were treated with the inhibitors alone, and IL-1β alone stimulated a small increase in PGE2 production; however, when the cells were treated with IL-1β and BIX-01294, DZNep, or RG108, further increases by 1.78-, 2.77-, and 1.54-fold, respectively, were observed compared with those for IL-1β alone. The results show that cytokine-induced COX-2 expression in F-IPFs can be restored by inhibition of the activity of either G9a, EZH2, or Dnmt1, leading to the reproduction of PGE2.To investigate whether G9a and EZH2 inhibition could lead to COX-2 gene derepression, F-IPFs were treated with BIX-01294, DZNep, or RG108, either alone or with the potent COX-2 inducer IL-1β. BIX-01294 and DZNep alone, but not RG108, caused a slight but significant increase in COX-2 mRNA expression, as analyzed by real-time RT-PCR, whereas marked increases in COX-2 mRNA were observed when the cells were treated with BIX-01294, DZNep, or RG108 and IL-1β together, and marked increases in COX-2 mRNA  were observed compared with that for IL-1β alone A. WesterA, BFig. 8, G9a and EZH2 siRNAs, but not the control siRNA, reduced their respective mRNAs by 96 and 76% compared with those in the control cells. Knocking down G9a and EZH2 with the siRNAs alone did not increase COX-2 mRNA; however, when the cells were stimulated with IL-1β, marked increases in COX-2 mRNA  were observed compared with those for IL-1β alone . Western blotting also revealed that G9a and EZH2 siRNAs almost abolished the expression of their respective proteins, whereas the control siRNA and IL-1β had no effect . No COX-2 protein was detected in cells treated with G9a and EZH2 siRNAs or IL-1β alone; however, when the cells were stimulated with IL-1β, marked increases in COX-2 protein expression were observed . The results therefore strongly suggest that G9a and EZH2 play a central role in mediating the epigenetic silencing of COX-2 in IPF.To explore the effectiveness of G9a and EZH2 siRNA in F-IPFs, G9a and EZH2 mRNA expression was analyzed after siRNA transfection. As shown in 2 production. The data strongly suggest that G9a- and EZH2-mediated histone methylation plays a central role and acts interdependently with DNA methylation in the epigenetic silencing of COX-2 in IPF.We have previously demonstrated that histone deacetylation as a result of decreased recruitment of HATs and increased recruitment of HDACs to the COX-2 promoter is involved in COX-2 silencing in F-IPFs . AlthougIt is well established that G9a- and EZH2-mediated histone H3K9 and H3K27 hypermethylation contributes to the epigenetic silencing of tumor suppressor genes. G9a and EZH2 are up-regulated in a number of different cancers , 30 and G9a-mediated H3K9 hypermethylation (both H3K9me2 and H3K9me3) serves as a docking site for the chromatin modifier protein HP1, which in turn recruits additional chromatin modifiers to reinforce gene silencing , 5. EZH22 production despite similar effects on methylation status at the COX-2 promoter may also suggest off-target effects of the inhibitors on genes that may have control of COX-2 expression and function. It is therefore necessary to apply siRNA knockdown to validate the findings with epigenetic inhibitors. We revealed in our current study that successful siRNA knockdown of G9a and EZH2 recapitulated the effects of G9a and EZH2 inhibitors. This finding is consistent with recent findings that G9a knockdown reduces H3K9 methylation and the recruitment of HP1, Dnmt1, and HDAC1 to the promoter of the cell adhesion molecule Ep-CAM and restores its expression in lung cancer cells . These observations provide a hint that repressive epigenetic modifications at the COX-2 promoter could be introduced, at least to some extent, by TGFβ1 treatment and that endogenously produced PGE2 after COX-2 reexpression could play a key role in preventing and reversing lung fibroblast activation in IPF. However, further studies are needed to understand the mechanisms and order of epigenetic events leading to the epigenetic silencing of COX-2 and other antifibrotic genes in IPF.It has been increasingly recognized that F-IPFs represent a persistently activated phenotype of lung fibroblasts and play a key role in the development and progression of IPF. Evidence is emerging to support the concept that these cells are epigenetically reprogramed so that a group of antifibrotic genes, including COX-2, Thy-1, and IP-10, are epigenetically silenced in a way similar to the epigenetic silencing of tumor suppressor genes in cancers. Here we provide further evidence that deregulated histone and DNA methylation contributes to the epigenetic silencing of COX-2 in F-IPFs. However, one clear limitation of the current study is that it explored the epigenetic events at the end of the disease process. Although the results are novel and valuable in understanding the epigenetic mechanisms involved in the epigenetic silencing of antifibrotic genes in IPF, little is known about the causes and order of events by which repressive patterns of histone and DNA methylation are established and maintained during the disease development and progression. It is of great importance to identify the critical initiating event that triggers antifibrotic gene silencing to understand the mechanisms underlying persistent fibroblast activation and pulmonary fibrogenesis. It is also plausible to propose that environmental and endogenous factors represent key signals that trigger distinct epigenetic changes and influence the order of epigenetic events, leading to antifibrotic gene silencing during pulmonary fibrogenesis. Preliminary data from our ongoing studies show that chronic treatment of F-NLs with the profibrotic cytokine transforming growth factor-β1 (TGFβ1) markedly reduces IL-1β-induced COX-2 expression, which is accompanied by increased DNA methylation and decreased histone H3 and H4 acetylation at the COX-2 promoter, and that PGEFig. 9). Our data also suggest that the machinery for the expression of COX-2 and probably other antifibrotic genes in IPF remains intact and functional and can mediate reexpression if the repressive epigenetic modifications are removed. Thus, the epigenetic enzymes regulating these epigenetic modifications and their crosstalk in these cells may represent a key therapeutic target to reactivate silenced antifibrotic genes for treating this progressive and fatal disease.Regardless of the hierarchical order of events, our observations support a central role for G9a- and EZH2-mediated histone hypermethylation and a model of bidirectional, mutually reinforcing, and interdependent crosstalk between histone hypermethylation and DNA methylation in the epigenetic silencing of COX-2 and potentially other antifibrotic genes in IPF (