Drosophila Tc1-like elements, Bari1 and Bari3. The AT-richness and the presence of weak core-promoter motifs make these promoters, that we have defined “blurry”, able to activate transcription of a reporter gene in cellular systems as diverse as fly, human, yeast and bacteria. In order to clarify whether the blurry promoter is a specific feature of the Bari transposon family, we have extended this study to promoters isolated from three additional DNA transposon and from two additional LTR retrotransposons.We have recently described a peculiar feature of the promoters in two Sleeping Beauty and Hsmar1, belonging to the Tc1/mariner superfamily. In contrast, this feature is not shared by the promoter of the hobo transposon, which belongs to the hAT superfamily, nor by LTR retrotransposon-derived promoters, which, in general, do not activate transcription when introduced into non-related genomes.Here we show that the blurry promoter is also a feature of two vertebrate transposable elements, Tc1/mariner superfamily with possible evolutionary and biotechnological implications.Our results suggest that the blurry promoter could be a shared feature of the members of the The online version of this article (10.1186/s13100-019-0155-6) contains supplementary material, which is available to authorized users. Transposable elements (TEs) are widespread genetic elements that have played a fundamental role in genome evolution , contribTEs, like other genes, are usually transmitted from parents to offspring, and propagate in the population. In parallel to one or several bursts of genomic expansion TEs are subjected to mutational load, denoting their a neutral mode of evolution. The overall absence of selection acting on TEs establishes a kind of “genomic homeostasis”, i.e. a balance in terms of numbers of functional and non-autonomous TE copies. When mutant copies overcome functional ones, the TE is destined to extinction in the genome in which it resides, an effect that could be also translated at the species level , 6. HoriTc1 and mariner are two related DNA transposon families, part of the Tc1/mariner/pogo/IS630  superfamily. They were first identified in nematode  and two additional LTR retrotransposons (Tirant and ZAM) from D. melanogaster. Furthermore, we included in this study the promoter of hobo, a member of the hAT superfamily . The hAtake HTT –22, haveTc1/mariner superfamily tested in this study, while the promoters of hobo, Tirant and Zam are usually functional within a limited range of genomic environments strictly related to the species of origin.Our results suggest that the blurry promoter is a feature shared by the elements of the SB and Hsmar1 were used as representative elements to gain insights into promoter features of the Tc1/mariner superfamily. The promoter of the hobo element was chosen as an outgroup to Class II of transposons, whereas Tirant and ZAM were used as representative elements of the LTR retrotransposons belonging to the Ty3-gypsy superfamily. For the sake of comparison, the results obtained from the present work have been integrated with the results obtained in our recent study . We. WeTc1/mnsposase  and alsoe tested  and a 15Drosophila S2R+ cells) or stably expressed (i.e. transformation in S. cerevisiae and E. coli) at the episomal level . We used a promoter-less luciferase cassette as a negative control for background expression correction in each experiment  were sub-cloned into pFL39, a low-copy centromeric yeast vector that allowed an easy clonal analysis in S. cerevisiae. Interestingly, and similarly to what was previously found for Bari1_p, the activity of Hsmar1_p was about 4% of the strong URA3 promoter, suggesting that it is a weak, but active promoter in yeast , but still significantly different from the promoter-less vector. Contrarily, SB_p displayed a strong activity, representing roughly 17% of the CAT promoter activity  mapping and scientific literature data mining. We scanned the sequences of SB_p, Hsmar1_p, hobo_p, ZAM_p, TIR_p and Copia_p using matrices describing the TATA-box, the initiator element (InR) and the downstream promoter element (DPE), three DNA motifs commonly found in eukaryotic promoters. The results obtained using relaxed parameters  of hobo promoter reveals a high scoring TATA-box motif at position 105–116, which is in accordance with the TATA-box annotated in the GenBank record (M69216.1), and low-scoring Inr and DPE motifs at position 156–162 and 305–309 respectively. A CAAT box (position 49–52) is also annotated in the GenBank file. No transcripts arising from within the analyzed hobo sequence could be found in the database, in accordance with the reported low transcriptional activity of hobo in D. melanogaster . Si. Si35S psh cells . Given tsh cells . MoreoveThe observation that the promoters of some transposon families display transcriptional activation of downstream genes in multiple cellular hosts enables the development of expression vectors based on blurry promoters. Enabling ectopic expression in many cellular model systems using a single recombinant construct could be useful in basic research to accelerate industrial production processes. The weakness of the promoters described here could limit their use in such applications. However, this could be a desirable feature to express genes closer to their physiological levels. To better assess the strength of TE-derived promoters a comparison with weaker promoters will be needed. Further investigations in the field of TEs and mutagenesis of preliminary characterized TE- related promoters could hopefully lead to a wider application of TE-derived regulatory sequences for the development of new biotechnological tools with a broader range of transcriptional activation in multiple hosts.Bari elements isolated from Drosophila, the SB (from fish) and Hsmar1 (human) transposons contain transcriptionally promiscuous “blurry” promoters. The blurry promoter could be a feature widely shared among the members of the Tc1/mariner superfamily.This study demonstrates that, similar to the Tc1/mariner superfamily could contribute to the success and spread of these mobile elements and might represent one of the keys towards the full understanding of the complex phenomenon of HTT.The results obtained in this work confirm our previous hypothesis that the promoters of TEs from the Given the high number of annotated and characterized TEs, we believe that other elements could also carry promiscuous promoters. Continuing effort put into the study of TE’s regulatory sequences, would enable the discovery of additional peculiar features that could be used in biotechnological applications and would allow a significant advancement in the field of TE biology.Saccharomyces cerevisiae, strain BMA64-1A , was cultured on Synthetic Complete medium [Escherichia coli cultures, strain DH5alpha, were grown on selective LB medium supplemented with ampicillin.S2R+ cells were passaged in Schneider’s Insect Medium supplemented with 10% FBS, 1% penicillin/streptomycin, and maintained at 25 °C. HeLa cells were maintained at 37 °C with 5% CO2 in Dulbecco’s Minimum Essential Medium supplemented with 10% FBS, 200 mM glutamine, 1% penicillin/streptomycin. e medium  supplemehobo, ZAM and Tirant were amplified from the D. melanogaster reference strain genomic DNA . The entire expression cassettes (i.e. promoter-luc + SV40 terminator) were cut out from the pGL3B vector and sub-cloned into pFL39 vector [Bari1 and Bari3 promoters, as well as the control promoters used in this study , have been described in [The transposable elements’ fragments analyzed in this study have been amplified using the primers listed in Table 9 vector  using eiribed in . SequencS. cerevisiae and E. coli transformants were assayed in the log-phase. We recorded three independent luciferase activity measurements per sample and the average value taken as sample measure. Experiments were made at least in triplicates.Transfections were performed in 6-well plates using TransIt LT1 , and 1 μg of the appropriate plasmid or co-transfected with the Renilla luciferase construct , according to the manufacturer instructions. Yeast transformation was performed using the TRAFO methods described in . BacteriRLUs obtained for the tested promoters, were corrected for the RLU value of the respective negative control, and normalized to the total protein content measured using the Bradford Assay or to the Renilla luciferase measure.The normalized luciferase activity of positive controls, were arbitrarily set to 100, whereas the negative controls were set to zero in each experiment. T-student test was carried out to assess the statistical significance of the differences observed between the transposons’ promoter tested and the respective promoter-less construct .www.bioinformatics.org/yapp/cgi-bin/yapp.cgi; last accessed June 2018). Matrix scan analysis was performed using Regulatory Sequence Analysis Tools (RSAT)  [Positional weight matrices used to map the TATA-box, Inr (Initiator Element) and DPE (Downstream Promoter Element) core-promoter motifs were retrieved at YAPP Eukaryotic Core Promoter Predictor (ly 2018) ).https://blast.ncbi.nlm.nih.gov/Blast.cgi) against appropriate databases.BLAST searches were performed at the NCBI Additional file 2:Table S2. Inr motif prediction. Pval cutoff 10exp-2; origin = START; only direct strand results are shown (DOCX 115 kb)Additional file 3:Table S3. DPE motif prediction. Pval cutoff 10exp-2; origin = START; only direct strand results are shown (DOCX 151 kb)Additional File 4:Accessions of cDNA/ESTs supporting TSS predicted in the sequences tested (TXT 643 bytes)Additional file 5:Figure S1. Luciferase-promoter assay in HeLa cells. Individual promoter-luciferase assay results in HeLa cells. Top and bottom whiskers: maximum and minimum values of each samples respectively. Top and bottom of boxes: 75th and 25th percentile of the samples respectively. Line through the boxes: median of each sample. X markers: mean of each samples. The statistical significance of promoter activity against the promoter-less cassette is shown. *P < 0.05; **P < 0.005; ***P < 0.001 (PDF 168 kb)Additional file 6:Figure S2. Luciferase-promoter assay in S2R+ cells. Individual promoter-luciferase assay results in S2R+ cells of D. melanogaster. Top and bottom whiskers: maximum and minimum values of each samples respectively. Top and bottom of boxes: 75th and 25th percentile of the samples respectively. Line through the boxes: median of each sample. X markers: mean of each samples. The statistical significance of promoter activity against the promoter-less cassette is shown. **P < 0.005; ***P < 0.001 (PDF 174 kb)Additional file 7:Figure S3. Luciferase-promoter assay in S. cerevisiae cells (BMA64-1A). Individual promoter-luciferase assay results in yeast. Top and bottom whiskers: maximum and minimum values of each samples respectively. Top and bottom of boxes: 75th and 25th percentile of the samples respectively. Line through the boxes: median of each sample. X markers: mean of each samples. The statistical significance of promoter activity against the promoter-less cassette is shown. *P < 0.05; **P < 0.005 (PDF 216 kb)Additional file 8:Figure S4. Luciferase-promoter assay in bacteria . Individual promoter-luciferase assay results in DH5alpha cells. Top and bottom whiskers: maximum and minimum values of each samples respectively. Top and bottom of boxes: 75th and 25th percentile of the samples respectively. Line through the boxes: median of each sample. X markers: mean of each samples. The statistical significance of promoter activity against the promoter-less cassette is shown. ***P < 0.001 (PDF 154 kb)


Hajheidari et al.In eukaryotic cells, chromatin, a highly dynamic nucleoprotein complex, plays a critical role in controlling gene expression notably by regulating the interaction between transcription factors and regulatory elements. The structure of the chromatin is determined by epigenetic mechanisms, including DNA methylation, histone modifications, and chromatin remodeling. A growing body of evidence indicates that epigenetic regulations are involved in plant adaptation to environmental stresses, and in plant development, including flowering control, fruit and root development, as well as seed maturation and germination. Furthermore, epigenetic mechanisms have the potential to stabilize cell identity and maintain tissue organization. Hence, epigenetic diversity is now emerging as a new source of phenotypic variation to improve adaptation to changing environment and ensure yield and quality of crops. The 14 articles published in this Research Topic highlight recent progresses, opinions, and reviews to advance our knowledge in the role of the epigenome on controlling plant development, plant response to environmental stresses, and plant evolution. For instance, gene duplication and chromatin remodeling contribute to increase the morphological and cellular complexity of plants during their evolution according to Inácio et al.; Zhang et al.; Hajheidari et al.). For instance, Inácio et al. immunolocalized various epigenetic marks and correlated epigenomic changes with transcriptional regulation when studying cork formation and quality in cork oak, a genuinely forest-specific process. Furthermore, changes in the acetylation levels of the lysine 9 of the histone H3 (H3K9) and lysine 5 of the histone H4 (H4K5) were found associated with the heat stress-dependent inhibition of lateral root formation in maize . Interestingly, whereas a global increase in histone acetylation was observed in response to heat stress, H3K9 and H4K5 acetylation decreased significantly in the promoter region of the haem oxygenase-1 (ZmHO-1) and giberellic acid–stimulated like-1 (ZmGSL-1) genes, two inhibitors of lateral root formation .Chromatin modifications, including DNA methylation and histone modifications, are critical in regulating gene transcription, and thus may reprogram cell differentiation and development (Pasternak and Dudits). In addition to somatic embryogenesis, the epigenome also controls the juvenile-to-adult developmental transition notably by modulating the expression of regulatory genes. Indeed, in Arabidopsis plants, this transition is regulated by miR156/157 and its target-squamosa promoter binding protein-like gene . Other epigenetic changes controlling the juvenile-to-adult developmental transition include DNA methylation, and histone modification . Ultimately, these chemical changes lead to a remodeling of the chromatin. The SWI/SNF chromatin remodeling complexes play a central role in this biological process by controlling phytohormone biosynthesis, the establishment and maintenance of meristems, organ development, and floral transition . Supporting the central role of chromatin remodeling and histone modifications in controlling development of plant, Kang et al. studied the role of the chromatin-remodeling factor inositol auxotrophy 80 and the histone chaperones nap1-related protein 1 and 2 in modulating auxin fluxes and the activity of the inflorescence and root apical meristems. Another interesting study highlights the impact of the epigenome in controlling transcriptional initiation. The single-stranded DNA-binding protein whirly1 promoted the acetylation of H3K9 and repressed the trimethylation of H3K4 to enhance the recruitment of the RNA polymerase II on the wrky53 promoter .Plant cells have the capability to dedifferentiate in totipotent cells, a prerequisite to asexual embryogenesis. Recent papers support a role of histone deacetylation and DNA methylation in cellular reprogramming leading to callus formation and asexual embryogenesis through the regulation of key developmental genes such as Wuschel  and AFR2 genes, is critical for the proper regulation of Arabidopsis circadian rhythm. These two proteins directly bind to the circadaian clock associated 1 (CCA1) and pseudo-response regulator 9 (PRR9) promoters in order to locally deacetylate the histone H3 and negatively affect their expression. This is just a first level of the epigenetic regulation of the Arabidopsis circadian clock. Indeed, Hung et al. described a more complex transcriptional regulation of the circadian clock: the recruitment of the lysine-specific demethylase 1 (LSD1)-like 1/2 (LDL1/2) and histone deacetylase 6 (HDA6) proteins by circadian clock associated 1 (CCA1)/late elongated hypocotyl (LHY) is needed to repress the expression of timing of cab expression 1 (TOC1). Acting as a negative feedback regulatory loop, TOC1 also interacts with LDL1/2 and HDA6 proteins to repress the expression of CCA1/LHY. A broader picture of the role of the epigenome on the plant circadian clock is provided in the Du et al. review paper.Epigenetic alterations also control the response of plants to environmental stresses including light perception and various abiotic stresses . As described by Sun et al. revealed that the de novo deposition of trimethylated histone H3 lysine 27 residue in the promoter and coding sequence of plant genes is needed to repress their transcription in response to salt stress. Mechanistically, Ageeva-Kieferle et al. described in their review the role of nitric oxide as inhibitors of histone deacetylase through the S-nitrosation of selected cysteine residues. Nitric oxide also regulates the epigenome by controlling the expression of genes encoding DNA and histone methyltransferases and demethylases. Taken together, nitric oxide is a chemical agent controlling plant gene activity in response to environmental stresses notably by regulating the activity of various histone acetyltransferases, deacetylases, methyltransferases and demethylases, and DNA methyl transferases and demethylases.Environmental stresses also induce the formation of stress-responding agents such as nitric oxide. In soybean, This special topic clearly highlights the central role of the epigenome in the regulation of gene expression that influences many plant biological processes such as plant development and plant response to environmental stresses. A deeper analysis of the chromatin remodeling and transcription related mechanisms will be needed to better understand the epigenetic regulation of gene expression. Single cell -omic technologies such as single cell RNA-seq and ATAC-seq will enable further discoveries by capturing the transcriptome and epigenome for each cell composing a complex organ. While single cell RNA-seq was recently applied on Arabidopsis root protoplasts, there is a need to develop plant single cell ATAC-seq technology to gain a more complete picture of the plant cell epigenome.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The work was supported by Youth Innovation Promotion Association, Chinese Academy of Sciences (2017399), Guangdong Natural Science Foundation (2018A030313350), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA13020603), the National Science Centre, Poland UMO-2014/13/B/NZ2/01187, Natural Sciences and Engineering Research Council of Canada (06679), the Chinese University of Hong Kong direct grant for research (#4053383), the National Science Foundation (awards #1854326 and #1339194), the Spanish Ministry of Economy, Industry and Competitiveness-INIA-FEDER (RTA2017-00011-C03-01) and by the Nebraska Research Initiative core facility research grant.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.


The histone variant macroH2A1 localizes to two functionally distinct chromatin subtypes marked by either H3K27me3 or H2B acetylations, where it is thought to directly regulate transcription. The recent finding, that macroH2A1 regulates mitochondrial respiration by globally dampening PARP activity, requires the field to re-evaluate which functions of macroH2A1 are due to global effects on cellular metabolism and which are direct effects determined by macroH2A1 chromatin localization. Here, we demonstrate macroH2A1 incorporation into H2B-acetylated chromatin requires a feature in its histone-fold domain, distinguishing this process from incorporation into H3K27me3-containing chromatin in which multiple features of macroH2A1 are sufficient for targeting. In addition, we identify H2BK20 acetylation as a critical modification required to target macroH2A1 to H2B-acetylated chromatin. Our findings have allowed us to definitively establish that macroH2A1’s regulation of an important transcriptional program, the senescence-associated secretory phenotype (SASP), requires its accurate genomic localization. The histone variant macroH2A1 localizes to two functionally distinct chromatin subtypes marked by either H3K27me3 or H2B acetylations. Here the authors identify the features of macroH2A1 required for its recruitment to H2B-acetylated chromatin and identify H2BK20 acetylation as a critical requirement for this recruitment. Unlike their canonical counterparts, histone variants are incorporated in a replication-independent manner to perform a particular function predicated on its targeting to the appropriate genomic locations. There are two fundamental requirements for this targeting. (1) The histone variant must harbor a specific feature differentiating it from other histones. (2) A feature at the site of chromatin incorporation must serve as a beacon to recruit the histone variant to the appropriate genomic locations.Chromatin, made of repeating units of nucleosomes, can be locally, structurally and functionally differentiated by a variety of mechanisms including covalent post-translational modifications (PTMs) of histones, ATP-dependent remodeling and the incorporation of specialized histone variants, to regulate DNA-dependent reactions2. At three-times the size of canonical H2A, macroH2A-containing nucleosomes organize the same amount of DNA2. The linker and macrodomain emerge from the nucleosome core where they participate in the recruitment of a host of co-factors9. Generally, macroH2As create large, specialized chromatin environments, hundreds of kilobases long10. While the overall structure of the three macroH2A isoforms is conserved, an alternative splicing event renders macroH2A1.1 capable of binding the PTM poly(ADP-ribose)(PAR) produced by PAR polymerases (PARPs), which plays key roles in the ability of macroH2A1.1 to regulate gene transcription11 and DNA damage responses12. Two critical glycines (Gly224 and Gly314) are required for PAR binding11. The macrodomains of macroH2A1.2 and macroH2A2 lack these critical glycines and contain a three amino acid insertion which collectively disrupts the stabilizing interactions observed between PAR and macroH2A1.113. The relative level of macroH2A1.1 splicing is significantly perturbed across many types of cancer, resulting in reduced macroH2A1.1 expression16. Re-expression of macroH2A1.1 suppressed cancer cell proliferation18, suppressed anchorage-independent growth and cell invasiveness in breast cancer19, and suppressed metastasis of melanoma in mice18. MacroH2A1.1 is thus not only a structural component of chromatin, but also a reader of a functionally diverse PTM with roles in transcriptional regulation and DNA damage responses.MacroH2A-type histone variants, which include macroH2A1.1, macroH2A1.2, and macroH2A2, are composed of a histone-fold domain (HFD), a basic linker region and a 25 kDa carboxyl-terminal region called a macrodomain20. However, later work demonstrated that macroH2A’s roles in the cell are not so simplistic. The majority of macroH2A is found on autosomes as part of facultative heterochromatin marked by trimethylation of histone H3 on lysine 27 (H3K27me3) or as part of transcriptionally active euchromatin marked by nine histone acetylations  where it can either positively or negatively regulate transcription22. The physiological functions of macroH2A are an active area of research; it serves as a barrier to stem cell reprogramming25, as a regulator of gene expression28 during normal growth and cellular senescence30. MacroH2A1.1 plays a specialized role in transcriptional regulation; through interaction with PAR, macroH2A1.1 recruits PARP1 and CBP, leading to the acetylation of H2B K12 (H2BK12ac) and K120 (H2BK120ac)29. This mechanism plays a critical role in oncogene-induced senescence (OIS), an important tumor suppressive mechanism. In OIS, macroH2A1 serves as a component of the senescence-associated heterochromatic foci involved in transcriptional repression30. However, macroH2A1.1 is specifically required for the transcriptional activation of a myriad of cytokines, chemokines and metalloproteases that make up the senescence-associated secretory phenotype (SASP), which enables the clearance of senescent cells31.First identified as enriched on the inactive X chromosome (Xi), macroH2As were considered markers of transcriptionally repressed chromatin+ in mitochondria32 suggesting that macroH2A1 plays a global role in cellular energetics, regardless of where macroH2A1 localizes on the genome. This result requires the field to carefully re-evaluate which functions of macroH2A1 are mediated by global effects of macroH2A1 on cellular energetics and which require it to function locally through deposition at specific chromatin loci.Recently, a global role for macroH2A1.1 in the regulation of mitochondrial function and energy metabolism has been identified. Through its interaction with PAR and concomitant inhibition of PARP1 activity, macroH2A1.1 allows for greater production of NADHere, we demonstrate that the requirements for deposition of macroH2A1 into H2Bac and H3K27me3-marked chromatin are distinct. We find that the carboxyl-terminal region of the HFD of macroH2A1 is required for association with H2B-acetylated chromatin, whereas multiple features of macroH2A1 are sufficient for association with chromatin marked by H3K27me3. Furthermore, we determined that regulation of macroH2A1-dependent transcriptional programs, such as the SASP, requires its ability to accurately localize to its H2B-acetylated chromatin niche. Additionally, we identified H2BK20ac as a key PTM which serves as a beacon to target macroH2A1 into the H2B-acetylated chromatin environment. The loss of macroH2A1 from H2B-acetylated chromatin, caused either by mutating macroH2A1 itself or by ablating the H2BK20ac chromatin cue for macroH2A1 deposition, led to a defect in the upregulation of SASP transcription. Overall, our work highlights key elements regulating the deposition of macroH2A1 into H2B-acetylated chromatin and demonstrates that macroH2A1 plays critical local roles in regulating gene expression.35, we know little about the rules governing specification of macroH2A1-containing chromatin. Previous studies have demonstrated that multiple features of macroH2A1 were sufficient to target the histone variant to the predominantly H3K27me3-containing Xi, including regions in macroH2A1’s HFD and macrodomain37. Subsequently, we determined that macroH2A1 localized to H3K27me3-containing chromatin across autosomes as well10. Additionally, we determined that a similar proportion of macroH2A1 localizes to transcriptionally active chromatin  or lacking both the linker and macrodomain (macroH2A11–122-Flag) . To ensure that each ectopic macroH2A1 mutant and wild-type protein could incorporate into chromatin, we fractionated the cells into soluble and chromatin fractions and liberated the mononucleosomes from the chromatin fraction with Micrococcal nuclease (MNase) . Ectopic expression of macroH2A1.1 has been shown to induce cellular senescenceag) Fig. . In an ase) Fig. . A Flag 4. The feedback and reduction of macroH2A1.1 can be clearly observed in all the ectopic macroH2A1.2-Flag mutant lines that harbor an intact macrodomain  failed to reduce endogenous macroH2A1.1 indicating that the macrodomain plays a critical role in this compensatory feedback loop.We previously described a compensatory post-translational feedback mechanism which decreases the level of endogenous macroH2A1 when macroH2A1 is ectopically expressedain Fig. ; however4 and publicly available data38, we designed ChIP primers targeting regions with and without macroH2A1 enrichment in both H2Bac and H3K27me3-containing chromatin . Our results confirmed the previous finding showing that this chimera was accurately deposited into autosomal H3K27me3-containing chromatin  did not disrupt specification of macroH2A1-containing chromatin. However, replacement of the carboxyl-terminal half of the HFD of macroH2A1 with the homologous region of canonical H2A (macroH2A1.2ΔH2A74–122-Flag) failed to significantly correlate with wild-type macroH2A1.2, specifically in the H2Bac-marked chromatin  with the knowledge that replacement of the entire HFD or the carboxyl-terminal half of the HFD leads to aberrant macroH2A1 localization  by RT-qPCR. The results demonstrated that localization deficient macroH2A1.1-Flag mutants, macroH2A1.1ΔH2A1–122 and macroH2A1.1ΔH2A74–122, were defective in their ability to induce SASP gene expression   Fig. , leading47. Therefore, we decided to replace endogenous H2B with ectopically expressed H2B harboring lysine-to-arginine mutations at position 15 or 20 to render these sites incapable of acetylation. To replace endogenous H2B, a highly expressed protein, we generated stable cells simultaneously expressing two H2B lentiviral constructs with identical mutations  and either a carboxyl-terminal Flag or HA tag   Fig.  using a ion Fig.  and wereion Fig. .Fig. 6H21–122-Flag 68. Here, we demonstrate that H2BK20ac is an upstream PTM regulating the localization of macroH2A1 to H2B-acetylated chromatin. This result, together with the role macroH2A1 plays in promoting H2BK12ac and H2BK120ac, firmly integrates macroH2A1 as an important hub in the broader histone code.Trans-histone regulatory pathways, where modification of one histone in a nucleosome  effects a second histone modification , are central features of chromatin regulatory mechanisms. Histone variants also participate in trans-histone regulatory processes29. However, a variety of other studies failed to identify a dominant role for macroH2A1 in the regulation of gene expression70. Recently, an indirect, global role for macroH2A1.1 in the regulation of mitochondrial function and cellular energy metabolism has been identified32. By capping PAR chains, macroH2A1.1 can inhibit PARP activity reducing nuclear NAD+ flux and allowing a greater fraction of NAD+ to be available for mitochondrial respiration. This effect is global, as it does not require macroH2A1.1 to directly regulate transcription. Interestingly, recent studies of macroH2A1 knockout mice have demonstrated phenotypes consistent with altered energy metabolism71. Overall, these findings require the field to carefully re-evaluate which functions of macroH2A1 play a global role in cellular metabolism and which functions of macroH2A1 depend locally on its ability to regulate transcription. Here we demonstrate that the ability of macroH2A1 to promote H2BK12ac and H2BK120ac and regulate a key transcriptional program during senescence, the SASP, are local functions of macroH2A1 which require the carboxyl-terminal region of its HFD and H2BK20ac for its localization to target genes. These findings provide critical tools to definitively determine which of macroH2A1’s biological functions are mediated by its global effect on energy metabolism and which are a local consequence of its role in transcriptional regulation.We have operated under a model in which macroH2A1 directly regulates transcription of its target genes by cofactor recruitment and modifying local chromatin structure. There is a variety of evidence that supports this model, most notably that macroH2A1 regulated genes are typically found in or near macroH2A1-containing chromatin14 and maintained in MEM supplemented with 10% FBS. Human embryonic kidney 293T cells (HEK293T) (ATCC) were cultured in DMEM supplemented with 10% FBS. Cell lines in culture were routinely tested for mycoplasma contamination MycoAlert™ Mycoplasma Detection Kit (Lonza).IMR90 (CCL-186) (ATCC) primary human fetal lung fibroblast cells were immortalized using retroviral-mediated gene transfer of the hTERT gene (Addgene plasmid 1773) (IMR90-hTERT)V12 were gifts from William Hahn (Addgene plasmid #9051). The retroviral or lentiviral infections were performed using X-tremeGENE™ 9 DNA Transfection Reagent (Roche)4. Three days post infection, the cells were selected for infection with 1 µg/ml puromycin and/or 0.5 mg/ml neomycin.IMR90-hTERT cell lines expressing macroH2A1 constructs were generated using the pLVX-Puro expression system (Clontech). IMR90-hTERT cell lines expressing H2B constructs were generated using pLVX-IRES-Puro and pLVX-IRES-Neo vectors (Clontech). MacroH2A1 was cloned into and mutated within pLVX-Puro vector. H2B was cloned and mutated in pLVX-IRES-Puro and pLVX-IRES-Neo. Single chain H2B-H2A-type construct were fused directly, minus the stop codon in H2B and the methionine in the H2A-type histone. The H2B was expressed on the N-terminus and the H2A-type was on the C-terminus of the peptide. Lysine to arginine mutations were generated using standard Dpn1 mediated site-directed mutagenesis. The commercial pBABE and pBABE-H-RasV12 expressing senescent IMR90-hTERT cells  as previously described29. Briefly, conditioned media was collected from H-RasV12-induced senescent cells or normally growing cells as a control. Starting 14 days post-infection, the cultured medium was collected from the dish every 3 days, centrifuged at 3000 rpm for 10 min, then the supernatant was filtered through a 0.2 µm pore filter . The resulting filtered medium was then mixed with an equal volume of MEM with 20% FBS and used as media for IMR90-hTERT cells expressing H2B constructs. Cells were harvested 3 days after treatment with conditioned media for RNA expression analysis.Paracrine senescence was induced using conditioned media from H-Ras4 or liberated by micrococcal nuclease digestion (MNase). Briefly, cells from a 10-cm dish were lysed in 0.1% Triton X-100 lysis buffer ) and incubated with agitation at 4 °C for 30 min. Then, the lysate was centrifuged at 14,000 rpm for 10 min at 4 °C. The supernatant was collected as the soluble detergent lysate. For acid extraction, the insoluble pellets containing the histones were resuspended in 0.5 M HCl at 4 °C for 2 h under constant agitation. The sample was spun at 14,000 rpm for 10 min at 4 °C. The resulting supernatant containing the histones was neutralized with 2 M Tris base.For immunoblots, histones were either acid extracted2, 4 mM MgCl2) and digested with MNase  for 12 min at 37 ºC. Digestion was stopped using MNase stop buffer . Ten microliters of the reaction was used to isolate DNA in order to confirm digestion to mononucleosomes. Briefly, the sample for DNA was cleared of protein with 0.4 mg/ml glycogen and proteinase K  in Txn stop buffer  at 37 °C for 30 min. The DNA was extracted with phenol/chloroform/isoamyl alcohol (25:24:1) and ethanol precipitated. The resultant mononucleosome DNA was confirmed by running on 1.5% agarose gel.For MNase digestion, the insoluble pellet was gently resuspended in MNase digestion buffer  and digested micrococcal nuclease  and digested at 37 °C for 10 min with gentle vortexing every 2 min. Digestion was stopped by adding MNase stop buffer (0.3 pellet volumes) and rocked at 4 °C 1.5 h to release mononucleosomes from euchromatin. The reaction was then spun at 13,000 rpm for 10 min. The supernatant contained the released mononucleosomes and the pellet had the nuclear mononucleosomes which were acid extracted as described above. Fifteen microliters of the supernatant of the MNase reaction was kept as input, and another 15 μl was used to confirm digestion to mononucleosomes (as described above). Then remaining supernatant was combined with an equal volume of HEGTw/300 buffer , 40 μl protein G beads and either no antibody (negative control) or antibody against Flag. The reactions were rocked at 4 °C for 2 h. The beads were spun at 4000 rpm for 3 min and washed three times in HEGTw/300 buffer. The resulting beads were resuspended in 1X Laemli buffer, boiled, and centrifuged. The resulting supernatant containing eluted mononucleosome complexes were run on 10% or 20% SDS-polyacrylamide gels and immunoblotted for indicated proteins.Cells were grown to 90% confluence in 15-cm dishes, cross-linked with 1% formaldehyde in PBS at room temperature for 10 min and then quenched in 125 mM glycine for 5 min. The cells were washed with cold PBS once and were collected by centrifugation and then sonicated in lysis buffer  to generate chromatin fragments of ~500 bp in length. The material was clarified by 10 min centrifugation at 14,000 rpm and 4 °C, and 20 μl supernatant was used as input for quantitation. The remaining supernatant was diluted ten-fold in dilution buffer  and 1 mM DTT) and precleared with 20 μl protein G–agarose beads at 4 °C for 2 h. The supernatant was used in immunoprecipitations at 4 °C overnight with antibodies against macroH2A1 (8 μl), Flag (5 μl) or histone H3 (4 μl), as indicated (Supplementary Table 4.MacroH2A1 ChIP from GEO database (GSE54847) and sequencing reads for 26 additional histone marks in IMR90 cells were downloaded from the GEO website (GSE16256) and used to determine macroH2A1 enrichment. Enrichment analysis was performed as previously describedmRNA levels were analyzed by reverse transcription followed by quantitative PCR (RT-qPCR). RNA was isolated with TriPure (Roche) according to the manufacturer’s protocol. The RNA was reverse transcribed with Moloney murine leukemia virus (MMLV) reverse transcriptase (Invitrogen) and a dT18 primer. cDNA, SYBR Green PCR master mix, and forward and reverse primers were used in 45 cycles of amplification  following 10-min incubation at 95 °C, with a LightCycler 480 (Roche). The efficiency-corrected threshold cycle (ΔCT) method was used to determine the relative levels of RNA For transcription analysis, the expression was normalized to ACTB. Melting-curve analysis was performed to ensure specificity. Primer sequences are listed in Supplementary Table Supplementary Information


Streptomyces species, that were discovered over 60 years ago. However, genome sequencing has revealed that most antibiotic-producing microorganisms encode many more natural products than previously thought. Biosynthesis of these natural products is tightly regulated by global and cluster situated regulators (CSRs), most of which respond to unknown environmental stimuli, and this likely explains why many biosynthetic gene clusters (BGCs) are not expressed under laboratory conditions. One approach towards novel natural product discovery is to awaken these cryptic BGCs by re-wiring the regulatory control mechanism(s). Most CSRs bind intergenic regions of DNA in their own BGC to control compound biosynthesis, but some CSRs can control the biosynthesis of multiple natural products by binding to several different BGCs. These cross-cluster regulators present an opportunity for natural product discovery, as the expression of multiple BGCs can be affected through the manipulation of a single regulator. This review describes examples of these different mechanisms, including specific examples of cross-cluster regulation, and assesses the impact that this knowledge may have on the discovery of novel natural products.Discovering new antibiotics is vital to combat the growing threat of antimicrobial resistance. Most currently used antibiotics originate from the natural products of actinomycete bacteria, particularly  Streptomyces bacteria . Bo. Boc redalbus S4 . Candicitalensis ,47. It wgulators . Deletiond fscMI . The genby FscRI . The S. t’s nest . Whilst ynthesis .Streptomyces clavuligerus produces both cephamycin C, a ß-lactam antibiotic, and clavulanic acid, a ß-lactamase inhibitor [S. coelicolor. Disruption of CcaR abolished both cephamycin C and clavulanic acid production in S. clavuligerus, while overexpression of ccaR led to a twofold increase in the production of cephamycin and a threefold increase in clavulanic acid after 48 hours [ceaS2, which encodes the carboxyethylarginine synthase CeaS2, responsible for committing arginine to clavulanic acid biosynthesis. The transcript levels of this mRNA were 1000–10,000× lower in the ccaR disruption mutant. A similar effect was seen for the cephamycin C pathway with the proteome of the mutant lacking multiple important biosynthetic proteins, including PcbC and CefF, the isopenicillin N synthase and deacetoxycephalosporin C hydroxylase, respectively. These changes, and others reported by Santamarta et al., account for the lack of production of both final products in the ∆ccaR mutant and reveal the coordinate regulation of cephamycin C and clavulanic acid in S. clavuligerus by CcaR [nhibitor ,52. The nhibitor . Their enhibitor . CcaR is48 hours . Using p by CcaR . Streptococcus pneumoniae [S. clavuligerus competitors.On its own, clavulanic acid has little in vitro antimicrobial activity, but it has proven effective in combination with the semi-synthetic antibiotic amoxicillin for the treatment of acute otitis media, an infection of the middle ear. High-dose formulations are also used to treat penicillin-resistant eumoniae ,57. The eumoniae . It is rStreptomyces melanosporus for its in vitro antimycobacterial activity [Streptomyces hygroscopicus, which also displays antitumor activity, through binding to and inhibiting the chaperone activity of eukaryotic Hsp90 [Streptomyces autolyticus, the geldanamycin BGC encodes the LuxR-family regulators, GdmRI and GdmRII, and the TetR-family regulator, GdmRIII. In an S. autolyticus ∆gdmRIII mutant, the production of geldanamycin was significantly reduced. Three other compounds, elaiophylin and its congeners , were markedly increased. Quantitative RT-PCR revealed that compared to the wild-type, the ∆gdmRIII mutant had reduced levels, some undetectable, of multiple geldanamycin biosynthetic gene transcripts, including gdmRI, gdmRII and the tailoring enzyme genes . The disruption of gdmRIII had the opposite effect on the elaiophylin transcriptome with a marked increase in major biosynthetic genes, including elaE, F, G and elaO, but a slight decrease in the elaI exporter gene transcript was also observed. EMSAs using purified GdmRIII confirmed in vitro binding to the promoter regions of gdmM, gdmN and elaF. DNase I footprinting revealed the conserved non-palindromic binding sequence (5′-ATNGAGGAC-3′). TetR family regulators often bind to palindromic regions, but the binding of non-palindromic regions has also been reported [The macrodiolide antibiotic elaiophylin  was origactivity  and has activity . It is oic Hsp90 ,61. In Sreported .Whilst these two antibiotics are co-produced, GdmRIII has an inverse effect on geldanamycin and elaiophylin, positively regulating the former and repressing the latter, although not completely. No studies into possible reasons for this co-production have been reported, although it has been postulated that the inverse regulation is important to control the flux of precursor metabolites. Both malonyl-CoA and methylmalonyl-CoA are utilised as substrates by both the geldanamycin and elaiophylin PKSs; thus, it is possible that GdmRIII works under nutrient-limited conditions to selectively synthesise the preferred compound .Streptomyces venezuelae has been shown to synthesise two antibiotics in vivo, chloramphenicol and the jadomycins  and pyoluteorin by gulation . Both thd plants . The chlnd wheat ,67,68. D disease ,70,71. Pseudomonas strains have been shown to produce one or other of these antibiotics, P. protogens Pf-5 is among a small subset of strains, which is known to produce both DAPG and pyoluteorin. The biosynthetic genes are encoded in distinct clusters separated by 3.7 MB, and their production is tightly coordinated; deletion of the pyoluteorin biosynthetic genes results in overproduction of DAPG in addition to the loss of pyoluteorin [P. protogens bacterial cultures leads to the repression of DAPG production. The addition of the DAPG biosynthetic intermediate phloroglucinol (PG) at nanomolar concentrations activates pyoluteorin production; however, micromolar concentrations leads to repression [2 and PG-Cl, which directly bind to and activate the LysR-family regulator PltR, inducing transcription of the pyoluteorin BGC [Although many luteorin ,73. Intrpression ,75. The orin BGC . 2 and PG-Cl, act as both intra- and inter-cellular signals orchestrating the activation or repression of DAPG and pyoluteorin production. DAPG is toxic to the bacterivorous amoeba Acanthomoeba castellanii, whilst pyoluteorin has no effect [DAPG and pyoluteorin have been shown to function as antimicrobials and as cell-cell signalling molecules, whereas the intermediates, PG-Clo effect . Antibioo effect ,77. Firmicutes, been found throughout the bacterial phyla [Escherichia coli, Crp and its effector molecule cAMP affect the expression of hundreds of genes mediating general carbon catabolite repression (CCR). Crp has over 70 different DNA binding sites in E. coli, and while it does not appear to have the same CCR effect in Streptomyces spp., it still plays an important role in global regulation as S. coelicolor ∆crp mutants display distinct morphological and developmental defects [S. coelicolor has been the favoured model organism for five decades. ACT, RED, CDA and yCPK are regulated by the CSRs ActII-4, RedD/RedZ, CdaR and CpkO, respectively, which are, in turn, controlled individually or together by global regulators, such as MtrA, which will be discussed later [crp disruption mutant and the crp complement strain revealed further coordinate regulation with Crp inducing transcription of the NRPS gene cluster SCO6429-38 and repressing the albaflavenone BGC. Overexpression of crp in the wild Streptomyces spp. strain WAC4988 resulted in the production of unidentified secondary metabolites with antibacterial activity, supporting the idea that Crp globally regulates secondary metabolism in streptomycetes [The examples above focus on coordinate regulation between two BGCs, and there are also global regulators, which have a more pleiotropic effect. The Cyclic AMP receptor protein (Crp) has, barring al phyla . In Esch defects ,80. Crp  defects . In addied later . ChIP-seS. coelicolor, as Crp controls many of the conspicuous antibiotics, including ACT, RED, CDA and yCPK [Pleiotropic regulators, such as Crp, which seemingly modulate the production of numerous antibiotics, likely act as the canvas on which CSRs and other cellular regulatory elements act to add detail and fine-tuning to the secondary metabolome. The presence of certain carbon sources can require sweeping changes to both primary and secondary metabolism. Crp regulates the cellular response to such signals, which is particularly visible in the case of and yCPK . S. coelicolor, S. venezuelae, Saccharopolyspora erythraea and Mycobacterium species, including M. tuberculosis, from which its name originates (M. tuberculosis regulator A) and in which it is essential [M. tuberculosis, MtrA has been shown to regulate expression of dnaA and dnaN, which encode for the DNA replicator activator and DNA clamp of DNA polymerase III, whilst also sequestering the chromosome origin oriC region. This sequestration of oriC is thought to prevent unwanted DNA replication during the post replication period and keep the cell cycle advancing towards division [Streptomyces spp., deletion of mtrA disrupts regular development, with mutants failing to produce aerial mycelium in a media-dependent manner [The response regulator MtrA is highly conserved among Actinobacteria and forms one part of the MtrAB two-component system. It has been characterised as a master regulator in ssential ,82,83. Tdivision . In Stret manner . S. coelicolor and S. venezuelae where it is a pleiotropic regulator of secondary metabolism. In S. coelicolor, ChIP-seq of MtrA reveals binding upstream of actII-orf4 and redZ, CSRs for the pigmented antibiotics ACT and RED. Whilst RedZ and ActII-orf4 are activators of their respective pathways, MtrA also binds between the divergent actII-orf1 and actII-orf2 genes, which encode a repressor and putative ACT transporter, respectively. This suggests that MtrA can directly modulate the antibiotic expression both positively and negatively. Deletion of the sensor kinase gene mtrB and, in effect, the loss of the regulatory function of MtrA result in the overproduction of both ACT and RED [S. venezuelae, a similar effect is observed with an ∆mtrB mutant producing >30× more chloramphenicol than the wild-type. ChIP-seq analysis showed MtrA binding between the divergent cmlN and cmlF genes, which encode for the chloramphenicol transporters required for production, and upstream of clmR, the CSR in this gene cluster [jadR1 and jadR2, which, as discussed earlier, are involved in the regulation of jadomycin biosynthesis and antagonistic regulation of chloramphenicol. Thus, it is possible that MtrA could activate or repress jadomycin production in a similar manner to its regulation of ACT in S. coelicolor by selectively activating the transcription of either the repressor jadR2 or the activator jadR1 depending on the requirements of the cell at that time [Regarding antibiotic production, MtrA has been best studied in  and RED . In S. v cluster ,62. MtrAhat time . S. venezuelae ∆mtrB mutant decoupled chloramphenicol production from this developmental schedule, resulting in constitutive production of the antibiotic. As MtrA has been shown to regulate cell cycle progression and development, it is likely that the MtrAB two-component system plays an important role in coordinating not only the production of multiple antibiotics but also the timing of antibiotic production with the correct developmental stages [In streptomycetes, the production of secondary metabolites is intimately linked to the developmental transition from vegetative hyphae to aerial hyphae and the onset of sporulation ,86. Intel stages ,43. Aspergillus nidulans, has been shown to encode 53 secondary metabolite BGCs, but the majority remain silent under normal laboratory conditions. One such cryptic cluster is the NRPS BGC, coding for the inpA and inpB genes, that is predicted to be responsible for the production of a tripeptide antibiotic. Overexpression of the putative activator at the inp locus, scpR, not only induces overexpression of the inp genes but also the upregulation of genes from the afo BGC and subsequent over-production of the novel polyketide metabolite asperfuranone. The biosynthesis of these two products is not linked, as deletion of the core NRPS genes from the inp cluster does not affect asperfuranon biosynthesis; therefore, it is predicted that scpR is able to control the expression of biosynthetic genes from both pathways due to the presence of a common motif within the intergenic regions of both gene clusters [Studying the regulation of secondary metabolite biosynthesis, especially cross-cluster regulation, has great potential value as we can use the knowledge gained to improve the production of secondary metabolites within strains of interest or even to discover new molecules from previously cryptic BGCs. Overexpressing pathway activators or inactivating pathway repressors is a well-established method of inducing the expression of cryptic BGCs and discovering new natural products ,7,87. Hoclusters . Aspergillus can combine polyketide biosynthesis with terpenoid precursors encoded by distinct BGCs to produce complex monoterpenoids that have a variety of biological activities [Studying regulation and coordinate regulation can also be used to inform on the biosynthesis of a molecule. For example, the need to cross-regulate multiple BGCs may indicate a biosynthetic link between two pathways, where one BGC synthesises the precursor molecule and enzymes from a distinct BGC elsewhere in the genome convert the molecule into the final product. For example, members of the fungal genus tivities . The regThe limitation of using CSRs that coordinately regulate multiple BGCs as a tool for natural product discovery is that the genetic manipulation of the regulators usually needs to occur in the native host. Often, when non-model organisms are isolated that encode cryptic BGCs, the genes are captured and expressed in an organism that is easier to manipulate in the laboratory. However, on heterologous expression of a secondary metabolite BGC, any effects of cross-regulation by a CSR are lost as the other BGC(s) is/are likely not present. In the past, it has been challenging to genetically manipulate non-model actinomycetes and other antibiotic-producing microorganisms but recent advances, such as the development of widely applicable CRISPR/Cas9 protocols, has increased the efficiency with which these genetic mutations can be made ,91. UnfoStreptomyces platensis NRRL 18993, Streptomyces sp. YIM56141and Streptomyces amphibiosporus ATCC 53964, respectively. The genes, mgsA and chxA are SARP-like regulators encoded within the iso-migrasratin and cycloheximide BGCs, respectively. No CSRs have been identified within the lactimidomycin BGC, but overexpression of either mgsA or chxA in S. amphibiosporus ATCC 53964 results in significantly increased titres of lactimidomycin [Streptomyces natalensis. However, pimM has also been shown to increase production levels of candicidin, amphotericin and filipin when transformed into Streptomyces albus, Streptomyces nodosus and Streptomyces avermitilis, respectively, indicating that some regulators can be fully exchangeable between related BGCs [S. coelicolor has also been shown to regulate the expression of a previously cryptic type II PKS BGC from Streptomyces antibioticus when the cluster is cloned into the former [In some instances, it is possible to exploit CSRs from within certain families of secondary metabolites in organisms other than the native host to increase the production of related natural products. For example, the glutarimide-containing polyketides iso-migrastatin, cyclohexamide and lactimidomycin are secondary metabolites with important biological activities, including cytotoxicity and antimicrobial activity, that are encoded by idomycin . Anothere former . These eCoordinate regulation has made it increasingly clear that BGCs do not always form fully self-contained secondary metabolite production clusters as was once thought. The production of these compounds can be costly to the host, and they are often produced as effectors in response to specific signals. As such their production is tightly controlled by a network of multi-level regulators, from global regulators, such as Crp, to CSRs like FscRI, and can comprise the coordinate regulation of multiple BGCs within a single organism. It might be considered that a CSR that regulates more than one BGC is, in fact, a global regulator. However, we take the term global regulator to mean a transcription factor that regulates different types of genes and not just antibiotic biosynthesis genes within BGCs. Given the examples discussed above, the coordinate regulation of BGCs is likely to be a much more common occurrence than was previously considered, and its value should not be underestimated.With the impending threat of an antimicrobial resistance crisis and the urgent need for new antimicrobials, significant effort has been made into the discovery of new antibiotic-producing BGCs. It may be possible to rewire entire BGCs to introduce constitutive promoters and express these metabolites; however, this may still prove difficult without understanding the regulatory elements found in other BGCs. Juxtaposed to this is the potential for vastly increasing our antimicrobial repertoire through the exploitation of coordinate regulation. Decoupling antagonistic regulatory elements  would allow the expression of increased numbers of secondary metabolites, whilst heterologous expression of global regulators might lead to the expression of previously cryptic BGCs, such as those already been shown using Crp ,42. This


Rosmarinus officinalis, Withania somnifera, and Sophora japonica have a long history of human use to promote health. In this study we examined the cellular effects of a combination of extracts from these plant sources based on specified levels of their carnosol/carnosic acid, withaferin A, and luteolin levels, respectively. Individually, these bioactive compounds have previously been shown to activate the nuclear factor erythroid 2-related factor 2 (Nrf2) transcription factor, which binds to the antioxidant response element (ARE) and regulates the expression of a wide variety of cytoprotective genes. We found that combinations of these three plant extracts act synergistically to activate the Nrf2 pathway, and we identified an optimized combination of the three agents which we named PB125 for use as a dietary supplement. Using microarray, quantitative reverse transcription-PCR, and RNA-seq technologies, we examined the gene expression induced by PB125 in HepG2 (hepatocellular carcinoma) cells, including canonical Nrf2-regulated genes, noncanonical Nrf2-regulated genes, and genes which appear to be regulated by non-Nrf2 mechanisms. Ingenuity Pathway Analysis identified Nrf2 as the primary pathway for gene expression changes by PB125. Pretreatment with PB125 protected cultured HepG2 cells against an oxidative stress challenge caused by cumene hydroperoxide exposure, by both cell viability and cell injury measurements. In summary, PB125 is a phytochemical dietary supplement comprised of extracts of three ingredients, Rosmarinus officinalis, Withania somnifera, and Sophora japonica, with specified levels of carnosol/carnosic acid, withaferin A, and luteolin, respectively. Each ingredient contributes to the activation of the Nrf2 pathway in unique ways, which leads to upregulation of cytoprotective genes and protection of cells against oxidative stress and supports the use of PB125 as a dietary supplement to promote healthy aging.Bioactive phytochemicals in  Oxidative stress and the diminishment of the body’s antioxidant defenses are usually associated with aging and with a variety of disorders and diseases ,2,3,4,5.HMOX1 we also measured heme oxygenase-1 protein levels (HMOX1) after PB125 treatment. We also demonstrate protection of HepG2 by PB125 against an oxidative stress challenge caused by cumene hydroperoxide exposure.Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that regulates the gene expression of a wide variety of cytoprotective phase II detoxification enzymes and antioxidant enzymes through its binding to the antioxidant-responsive element (ARE) in the promoter region of these genes. Relevant to oxidative stress, the ARE is a promoter element found to regulate expression of many antioxidant enzymes ,21,22,23The functionality of Nrf2 cytoprotective signaling appears to diminish with age ,25,26,27® to activate the Nrf2 pathway to promote healthy aging. PB125 is based on a combination of compounds found in Rosmarinus officinalis, Withania somnifera, and Sophora japonica  pyrene, a fairly common environmental pollutant found in charred meat and cigarette smoke, into potent human carcinogens [CYP1A1 is a phase I cytochrome cinogens . Other scinogens ,110. Thecinogens . A commocinogens . These pVGF encodes a 68 kDa polypeptide, comprising 615 amino acids, that is cleaved to ten or more polypeptides which have been associated with a number of neuroendocrine roles [VGF was reduced in parallel with development of ALS symptoms, leading the authors to hypothesize that restoring VGF expression in spinal cord motor neurons could therapeutically benefit clinical ALS [Snf2h gene which impairs cerebellar development, producing mice with poor motor function, progressive ataxia, and death between postnatal days 25 and 45 [Snf2h-cKO mice. VGF production was a major cause of this effect. Snf2h-cKO mice treated with full-length VGF-encoding adenoviruses removed the requirement of exercise for survival. These results suggest that increased expression of VGF might represent a therapeutic strategy for cerebellar ataxia and other pathologies of the central nervous system. [VGF gene. Stephens et al. found that VGF regulates secretory granule formation in pancreatic islet beta cells. VGF loss-of-function studies in both isolated islets and conditional knockout mice caused a profound decrease in stimulus-coupled insulin secretion. Moreover, VGF was required for exit of granule cargo from the trans-Golgi network and for proinsulin processing. It also functioned to replenish insulin granule stores following nutrient stimulation. The studies suggest that loss of VGF could be involved in the development of islet beta cell dysfunction in type 2 diabetes [ne roles . While tical ALS . VGF serical ALS . Exercis5 and 45 . They sh system. . Also ofdiabetes . Our genWe have constructed a potent Nrf2 activating composition that combines several novel features in addition to the synergistic interactions we have previously described . A key a


Dosage compensation between XX female and XY male cells is achieved by a process known as X chromosome inactivation (XCI) in mammals. XCI is initiated early during development in female cells and is subsequently stably maintained in most somatic cells. Despite its stability, the robust transcriptional silencing of XCI is reversible, in the embryo and also in a number of reprogramming settings. Although XCI has been intensively studied, the dynamics, factors, and mechanisms of X chromosome reactivation (XCR) remain largely unknown. In this review, we discuss how new sequencing technologies and reprogramming approaches have enabled recent advances that revealed the timing of transcriptional activation during XCR. We also discuss the factors and chromatin features that might be important to understand the dynamics and mechanisms of the erasure of transcriptional gene silencing on the inactive X chromosome (Xi). MECP2 (X chromosome inactivation (XCI) is a process that evolved in mammals to balance gene expression between XX female and XY male cells . Early iMECP2 . While mMECP2 . Today, Mecp2 allele in female cells can be beneficial  . During neficial .in vitro procedures have been used  analyses  are good candidate inducers of epigenetic memory erasure, but their precise role during XCR remains unclear. For XCR to take place, the expression of the long non-coding RNA (lncRNA) e erased . The enre erased . The dynanalyses . HoweverRecent advances in sequencing have enabled researchers to define the precise timing of X-linked gene reactivation for many genes in multiple experimental systems. While several mechanistic insights have been obtained, the precise factors and mechanisms that underlie the coordinated reactivation of silenced genes on the Xi during XCR remain to be defined. Here, we identify and discuss new studies focusing on the dynamics of XCR, as well as the factors and possible mechanisms underlying reversal of gene silencing.in vivo , which mediates ubiquitination of histone H2A, in turn leading to the recruitment of other PRC complexes and of PRC2  on the Xi in somatic cells restores TAD formation but does not compromise XCI , XCR may operate via at least three phases: initiation, progression, and completion of XCR. Transcriptional activation of early genes and the start of Xist repression may represent the initiation stage of XCR  of female mouse embryonic fibroblasts (MEFs) undergoing reprogramming to iPSCs has been reported . A groupe of XCR .trans-acting factors. Further work is needed to elucidate the starting chromatin states of the imprinted and random Xi.The reversal of random XCI during iPSC reprogramming takes much longer than the erasure of gene silencing during the reversal of imprinted XCI in the ICM. During iPSC reprogramming, XCR takes 1 week, while it is completed in hours in the ICM . In addiXist at E7.0 during the migration to genital ridges with a few genes re-acquiring bi-allelic expression  experiments during mouse spermatogenesis have identified a strong enrichment for H3K9me3, a chromatin modification associated with gene repression, in promoters of spermatid-specific genes expressed in spermatocytes . These oXist deletion on the Xi of differentiated cells strengthens TADs but gene silencing is maintained suggesting that TAD formation is not sufficient for XCR  have been implicated in the developmental timing of XCI  and the expression of Tsix during PSC differentiation  is considered a robust hallmark of naïve pluripotency in mice and humans . Severalpression . Indeed,went XCR . In addintenance . Moreove and XCR .Xist transcription in mESCs  which is known to trigger the activity of Xist by binding to its promoter  PSCs to a naïve-like PSC state . In thishylation . RNA-FIShylation , in agrehylation . Thus, dpression . Howeverpression , such thpression . TherefoXACT reactivation , the ubiquitin-ligase Rlim, whose expression correlates with XCI  will need to be developed, similar to the one described in TFs such as OCT4, NANOG, SOX2, PRDM14, and ESRRB might be directly involved in XCR and have also been implicated in ilencing . Their bwith XCI , and thewith XCI , might pAltogether, the dynamics of XCR is likely the outcome of the combinatorial effect of lncRNAs, TFs, genome topology, and chromatin modifications. Understanding the reversal of gene silencing is important for at least three reasons. First, reactivation of silenced genes represents a potential therapy for diseases, such as Rett Syndrome. This strategy might extend to targeted reactivation of silenced tumor suppressor genes in cancer. Second, it is likely that revealing mechanisms of XCR will uncover gene regulation principles that are generally applicable, as it has already repeatedly been done in the past . Third, IT, AJ, and VP wrote the manuscript with input from all the authors. JC and LV prepared the figures with the help of IT, AJ, and VP. All authors listed have approved the manuscript for publication.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. The reviewer CR declared a past co-authorship with one of the authors VP to the handling Editor.


Many active eukaryotic gene promoters exhibit divergent noncoding transcription, but the mechanisms restricting expression of these transcripts are not well understood. Here, we demonstrate how a sequence-specific transcription factor represses divergent noncoding transcription at highly expressed genes in yeast. We find that depletion of the transcription factor Rap1 induces noncoding transcription in a large fraction of Rap1-regulated gene promoters. Specifically, Rap1 prevents transcription initiation at cryptic promoters near its binding sites, which is uncoupled from transcription regulation in the protein-coding direction. We further provide evidence that Rap1 acts independently of previously described chromatin-based mechanisms to repress cryptic or divergent transcription. Finally, we show that divergent transcription in the absence of Rap1 is elicited by the RSC chromatin remodeler. We propose that a sequence-specific transcription factor limits access of basal transcription machinery to regulatory elements and adjacent sequences that act as divergent cryptic promoters, thereby providing directionality toward productive transcription.  •Expression of divergent noncoding RNAs is repressed by Rap1•Rap1 prevents initiation of divergent noncoding transcription near its binding sites•Rap1 provides directionality toward productive transcription Wu et al. characterize how divergent noncoding RNAs are repressed at highly expressed gene promoters in yeast. The authors identified that the sequence-specific transcription factor Rap1 restricts transcription initiation in the divergent direction locally near its binding site, thereby controlling promoter directionality. Precise control of gene expression is critical for all cellular functions. How and when genomes produce coding messenger RNAs and prevent the expression of unwanted RNAs has been a long-standing question of interest. In this context, an apparent paradox exists: genomic locations of coding gene transcription also produce aberrant noncoding transcripts. The transcriptionally active coding gene promoters, which often express noncoding transcripts in the antisense direction , are a mDivergent noncoding transcription is present across eukaryotic species. A large fraction of all noncoding transcripts emanate from divergent or bidirectional gene promoters . TypicalIn budding yeast, 138 genes encode for the protein subunits of the ribosome. These highly expressed ribosomal protein (RP) genes account for approximately half of all RNA polymerase II transcription . TranscrHere, we describe how divergent noncoding transcription is repressed at highly active RP gene promoters. We find that depletion of Rap1, but not other transcription factors important for RP expression, causes transcription in the divergent direction. Rap1 represses noncoding transcription typically within 50 bp of the Rap1 motif, which is uncoupled from transcription regulation in the protein-coding direction. We further show that Rap1-mediated repression of divergent transcription is distinct from known chromatin-based mechanisms. Thus, a sequence-specific transcription factor controls promoter directionality by repressing transcription in the divergent direction. Our work adds a new layer of regulation to various mechanisms that limit expression of aberrant transcripts and defines how promoter directionality is controlled.In budding yeast, a large fraction of bidirectional promoters express noncoding transcripts, also known as cryptic unstable transcripts (CUTs) or stable unannotated transcripts (SUTs), in the divergent direction . TranscrRPL43B and RPL40B genes to study, since both promoters are directly adjacent to a divergent noncoding transcript: IRT2 and SUT242, respectively  showed strong induction of IRT2 . IRT2 and iMLP1 expression increased simultaneously as Rap1 protein levels decreased . IRT2 and iMLP1 expression levels increased in RPL43B-bsΔ and RPL40B-bsΔ, respectively, to a level comparable to Rap1-depleted cells (RAP1-AID + IAA)  B. InitiaIRT2 is part of a regulatory circuit that facilitates expression of IME1, the master regulator of entry into meiosis  to 16 in the RPL43B-bsΔ mutant  did not show IRT2 or iMLP1 expression . We performed RNA-seq on both polyadenylated (poly(A)) and total RNA A. As exppression A and S2Dsequence A. ConseqOur data from example loci indicate that Rap1 mediates repression of noncoding transcription close to the Rap1 binding sites. To systematically determine how Rap1 depletion affects noncoding transcription, we binned RNA-seq data in windows of 50, 100, 200, and 500 bp up- and downstream of 564 annotated Rap1 sites B Lieb e. For smaNext, we analyzed the RNA-seq data to identify features of cryptic transcript repression by Rap1. First, we determined whether there is a bias for the orientation of Rap1-repressed transcripts. We selected 141 Rap1 binding sites at well-annotated gene promoters regulated by Rap1 for further analysis (mostly RP genes) . ExpressRPL43B promoter (bsΔS), IRT2 was expressed and the size of the transcript approximated the spacer sequence plus IRT2 (S + IRT2) indicating that initiation of IRT2 occurs downstream of the Rap1 binding sites. Strikingly, we observed a similar pattern when we integrated the spacer directly downstream (SD), but not upstream (SU), of the Rap1 binding sites relative to RPL43B. The spacer sequence had no effect on Rap1 binding to the RPL43B promoter  (SUT129 promoter (R1p) lowered yellow fluorescent protein (YFP) levels, while PPT1 (mCherry) activity increased (SUT129 promoter (R1p) activity increased to match control plasmid (pPS) levels upon Rap1 depletion (RAP1-AID + IAA). The repression of SUT129 by Rap1 was not dependent on transcription regulation in the coding direction because in RAP1-AID (IAA or NT) cells the PPT1 signal matched WT  D and S3Aon (pPS) . A Rap1 ncreased E. SUT129tched WT B. Finalle effect E. Thus, RPL43B promoter, a cluster of multiple TSSs was detected in a region of 35 bp up- and downstream of the Rap1 motifs in Rap1-depleted cells, and to a lesser extent, in WT cells  in WT and Rap1-depleted cells A–S4C. AtNext, we computed the changes in TSS signals between WT and Rap1-depleted cells. The TSS-seq data matched the RNA-seq data well over a wide range of expression D. ASc1 aRAP1-AID cells (IRT2 and iMLP1 expression upon Rap1 depletion (RAP1-AID + IAA), whereas the empty vector (EV) control displayed divergent transcription , the silencing domain (Rap1Δ764–827), or the AD plus an adjacent sequence (Rap1Δ631–696) failed to repress IRT2 and iMLP1 (RPL43B (IRT2) and RPL40B (iMLP1) promoters , and the toxicity domain (Tox) . We assend iMLP1 C. Exceptromoters D and S5Dpression . We founcription . In concGiven that the Rap1Δ631–696 mutant displayed divergent transcription but maintained its ability to bind the Rap1 motif, we hypothesized this mutant may associate differently with corepressors or activators of divergent transcription. To identify candidate regulators, we affinity-purified Rap1 from micrococcal nuclease (MNase) solubilized chromatin and used proteomics mass spectrometry to identify associated proteins A van We. We compRSC (remodels the structure of chromatin) is an ATP-dependent chromatin remodeling complex, and it generates NDRs at promoters to facilitate gene activation . The ATPSTH1-AID + IAA) had no effect on IRT2 and iMLP1 expression  increased IRT2 expression. When we compared iMLP1 expression patterns from our data to a published dataset, we found that five mutants overlapped, which we decided to study further  that displayed no detectable divergent transcription in the five mutants, while there was a clear signal in Rap1-depleted cells  contributes to exclusion or activity of other factors. The Rap1 C terminus may modulate the affinity and binding mode of the DNA binding domain . ConsistWe find that Rap1 controls the activity or action of RSC, because in Rap1-depleted cells RSC elicits divergent transcription. Our data indicate that Rap1 restricts RSC to stimulate productive transcription in the protein-coding direction. Recently, it was shown that RSC helps maintain an NDR in the absence of Rap1 . A RSC-dDecades of work have shown that eukaryotes have adopted redundant strategies to limit expression of aberrant noncoding RNAs. Mis-regulation of divergent noncoding transcripts could have negative effects on local or global gene expression, especially in gene-dense genomes such as that in budding yeast. Repression of cryptic transcription nearby regulatory elements, as we have shown for Rap1, could be a conserved property of sequence-specific transcription factors and other DNA binding proteins.folkert.vanwerven@crick.ac.uk).Further information and requests for resources and reagents should be directed to the Lead Contact, Folkert van Werven . Cells were collected immediately after resuspending in SPO media.Cells were grown in YPD media , uracil (24 mg/L) and adenine (12 mg/L). Cells were cultured with shaking in conical flasks at 30°C. For single molecule RNA FISH experiments described in For auxin induced depletion experiments, 3-indole-acetic acid (IAA) (Sigma-Aldrich) was used to induce AID-tagged protein depletion. 1 M IAA stocks were prepared in dimethyl sulfoxide (DMSO) and added directly to cultures to a final concentration of 500 μM.RAP1-AID, FHL1-AID, IFH1-AID, SFP1-AID, SPT6-AID, SPT16-AID, and STH1-AID), which contains three copies of the V5 epitope and the IAA7 degron , and STH1-AID-FLAG allele contains six copies of the FLAG epitope and IAA17 residues 71-114 (AID-FLAG) (Oryza sativa TIR1 (osTIR1) ubiquitin E3 ligase from the GPD1 promoter (gift from Leon Chan). osTIR1 plasmids were linearized by digestion with PmeI and integrated at either the HIS3 or LEU2 locus.A one-step tagging procedure was used for generating C-terminal auxin-inducible degron (AID) alleles (7 degron . The RAPID-FLAG) . The AIDRAP1-AID or RAP1-AID-MYC strain backgrounds. The truncation or domain deletion mutants described in RAP1 plasmids (gift from Amanda Johnson and Tony Weil) by NotI and XhoI digestion into plasmid 372 to generate plasmids 471-474, and by SacI and KpnI digestion to generate plasmids 477-483 (All Rap1 mutants and truncation constructs were expressed from single copy integration plasmids (gift from Wendell Lim) in  477-483 . Three cRAP1 expression plasmids containing C-terminal point and patch mutations were re-cloned from plasmids described previously (gift from Cynthia Wolberger) by Gibson-style cloning (HIS3 locus.Single copy integration  cloning . In shorpPPT1-pSUT129 fluorescent reporter plasmid was described previously (gift from Sebastian Marquardt)  or XmnI  restriction sites. The PPT1-SUT129 locus was replaced by digesting the plasmid with EcoRI and integrating the reporter construct by transformation as described above. All plasmids are listed in The mCherry-YFP rquardt) . The Rapi (pH 7), 0.05 M MgCl2, 1.2 M sorbitol), and resuspended in phosphate-sorbitol buffer before imaging. Imaging was performed using a 100x oil objective, NA 1.4, and SOLA SE light engine (Lumencor) on a Nikon Eclipse Ti-E imaging system (Nikon). We used 500 ms exposure time using GFP and mCherry filters to quantify YFP and mCherry levels, respectively. An ORCA-FLASH 4.0 camera (Hamamatsu) and NIS-Elements AR software (Nikon) were used to collect images.Cells were grown in YPD to the exponential phase and fixed with formaldehyde (3.7% w/v) for 15 min. Fixed cells were washed with phosphate-sorbitol buffer  . ROIs we600 0.4 in sterile water. Serial dilutions (5-fold) were spotted onto YPD agar plates in the presence of IAA or DMSO. Cells were incubated at 30°C for 2 days before imaging.Cells were grown to saturation in YPD media, then diluted to ODYeast cells were collected from cultures by centrifugation, washed with sterile water, and snap-frozen in liquid nitrogen. RNA was extracted from yeast cell pellets using Acid Phenol:Chloroform:Isoamyl alcohol  and precipitated in ethanol with 0.3 M sodium acetate. RNA was resuspended in DEPC-treated sterile water.i) buffer (pH 6.8)) at 70°C for 10 min. Denatured samples were mixed with loading buffer  and separated on an agarose gel  by electrophoresis for 2 hr at 80 Volts. Total RNA was transferred onto positively charged nylon membranes (GE Amersham Hybond N+) by capillary transfer and rRNA bands were visualized by methylene blue staining .Northern blots were performed as previously described . RNA sam32P] . The oligonucleotide sequences used to generate target-specific DNA templates for IRT2, iMLP1, and SNR190 northern blot probes are found in The membranes were blocked for at least 3 hr at 42°C in hybridization buffer , 2 g/L BSA, 2 g/L polyvinyl-pyrolidone, 2 g/L Ficoll 400, 1.7 g/L pyrophosphate, 50 mM Tris pH 7.5) or ULTRAhyb Ultrasensitive Hybridization Buffer (Thermo Fisher Scientific) before hybridization. The radioactively labeled probes were synthesized using a Prime-it II Random Primer Labeling Kit (Agilent), 25 ng of target-specific DNA template, and dATP [α-IRT2, iMLP1, and SNR190 levels were estimated from northern blots using ImageJ , anti-hexokinase rabbit , anti-Myc tag mouse monoclonal , anti-HA tag mouse IgG , anti-FLAG tag monoclonal mouse IgG1 , anti-mouse IgG antibody HRP-linked , anti-rabbit IgG antibody HRP-linked .The following antibodies were used for western blotting also see . Anti-V5RPL43B and RPL40B promoters were determined by qPCR using PowerUp SYBR Green Master Mix (Thermo Fisher Scientific) and a QuantStudio 3 instrument (Applied Biosystems). As a negative control, we used a primer pair directed to the ACT1 ORF 3′ end. The oligonucleotide sequences used for ChIP experiments are in ChIP experiments were performed as previously described . In shorin situ hybridization (FISH) was performed as previously described (IME1 (AF594) and the internal control ACT1 (Cy5) . Cells were imaged using a 100x oil objective, NA 1.4, and SOLA SE light engine (Lumencor) on a Nikon Eclipse Ti-E imaging system (Nikon). DIC, DAPI, AF594 (IME1), and Cy5 (ACT1) images were collected every 0.3 micron (20 stacks) using an ORCA-FLASH 4.0 camera (Hamamatsu) and NIS-Elements AR software (Nikon). ImageJ was used to generate maximum intensity Z projections of the images (http://rajlab.seas.upenn.edu/StarSearch/launch.html) was used to quantify transcripts in single cells. Comparable thresholds were used to count RNA foci in single cells. Only cells positive for the internal control ACT1 were quantified for analysis (n = 139 cells).Single molecule RNA fluorescence escribed . In shore images . SubsequTotal RNA from yeast was incubated with rDNase (Machery-Nagel) and column purified (Machery-Nagel) prior to sequencing library preparation. 1 μg of intact yeast total RNA was depleted of ribosomal RNA (rRNA) using a commercial kit ) for total RNA sequencing and 500 ng of RNA was used for polyadenylated (polyA) RNA sequencing. Libraries were prepared using the TruSeq Stranded Total RNA kit or TruSeq stranded mRNA kit (Illumina) according to the manufacturer’s instructions (10 or 13 PCR cycles). Each library was sequenced on the HiSeq 2500 or 4000 platform (Illumina) and generated ∼45 million 101 bp strand-specific paired-end reads per sample, on average.+ RNA together with in vitro spike-ins was first subjected to zinc-mediated fragmentation (Ambion) at 70°C. The reaction was subsequently cleaned up using RNeasy MinElute columns (QIAGEN) to isolate RNA fragments with mode length of ∼200 nucleotides. These fragments were incubated with shrimp alkaline phosphatase  at 37°C to remove the 5′ phosphate groups of non-capped fragments, followed by acid phenol/chloroform extraction and ethanol precipitation as described above. With the exception of a “no decapping” control sample from wild-type  cells, dephosphorylated fragments were next treated with Cap-Clip Acid Pyrophosphatase (CellScript) to remove the 5′-terminal caps from fragments representing the bona fide 5′ ends of transcripts. After another round of acid phenol/chloroform extraction and ethanol precipitation, all samples were treated with T4 RNA ligase 1 (NEB) to introduce a custom adaptor sequence to the 5′ uncapped ends of fragments. Excess adapters were removed via a column clean up step. First strand cDNA synthesis was performed using Superscript IV Reverse Transcriptase (Thermo Fisher Scientific), and second strand synthesis was performed using a KAPA HiFi HotStart ReadyMixPCR Kit (KAPA Biosystems) after RNase H (NEB) and RNase cocktail digestion (Ambion). Double-stranded cDNA was quantified by Qubit fluorometric quantitation (Thermo Fisher Scientific) and used as inputs for library preparation using a KAPA Hyper Prep Kit (KAPA Biosystems) and KAPA Single-Indexed adapters for Illumina platforms (KAPA Biosystems). Libraries were quantified by Qubit and sequenced on the HiSeq 4000 platform (Illumina), and typically generated ∼39 million 76bp strand-specific single-end reads per sample. ∼16 million single-end reads were generated from the “No decapping” control library.To obtain libraries representing the 5′ ends of polyadenylated and capped transcripts (TSS-seq), approximately 7-9 μg of poly(A)S. cerevisiae genes downloaded from the Ensembl genome browser . The parameters used were “–star-output-genome-bam–forward-prob 0,” and all other parameters were kept as default. Differential expression analysis was performed with the DESeq2 package (version 1.12.3) (Adaptor trimming was performed with cutadapt (version 1.9.1) with parameters “–minimum-length=25–quality-cutoff=20 -a AGATCGGAAGAGC -A AGATCGGAAGAGC” . The RSE 1.12.3)  within t2(Fold change) values from DESeq2 analysis were plotted using the geom_violin  and geom_boxplot functions (outlier data points not shown in boxplot but included in violin plot). Volcano and scatterplots were generated using the geom_point function in ggplot2 or Graphpad Prism (version 7.02). Screenshots of RNA-seq and TSS-seq data were taken using the Integrative Genomics Viewer .A list of experimentally determined Rap1 sites was obtained from a high-resolution ChIP-exo dataset . For difS. cerevisiae genome . Uniquely mapped alignments corresponding to the sense and antisense strands were obtained using SAMtools view (version 1.3.1) by using the flags “-q 1 -F 20” and “-q 1 -f 16,” respectively (st transcribed 5′ nucleotide within ± 75 bp of annotated TSSs (2(fold change) value > 1 (fold change > 2), comparing RAP1-AID +IAA over wild-type cells, was considered an increase.Adaptor trimming was performed with cutadapt (version 1.9.1)  with parectively . BedGrapectively  with theectively . Coveragectively . To calcted TSSs , on the GSE56994) (GSE65594) (GSE73337) (Publicly available datasets for Sth1 ChIP-seq (GEO: SE56994) , Sth1 MNSE65594) , and MNaSE73337)  describeSE73337)  using deSE73337)  command 2, 20 mM HEPES (pH 7.8), 0.1% v/v Triton X-100, 5 mM β-mercaptoethanol, 1X cOmplete protease inhibitor (Roche)). The pellet was then collected by centrifugation , washed once in NIB buffer, centrifuged again, and resuspended in 4.5 mL NIB buffer with 2 mM CaCl2. Samples were treated with 3000 U of micrococal nuclease  for 4 min at 30°C and reactions were stopped by addition of EDTA to 10 mM, then transferred onto ice. The concentration of NaCl was adjusted to 150 mM and samples were clarified by centrifugation at 16000 x g for 10 min at 4°C. The supernatant was taken as the chromatin extract.Chromatin extracts were prepared as previously described . In shorAnti-V5 tag immunoprecipitation was performed on approximately 15 mg of chromatin extract from cells expressing full-length Rap1-V5 , Rap1-V5, Rap1-V5, or containing an empty vector control . 100 μL of anti-V5 agarose affinity gel antibody (Sigma-Aldrich) was incubated with chromatin extracts for 4 hr at 4°C. Agarose beads were washed 5 times with 1 mL NIB wash buffer (NIB buffer with 350 mM NaCl) and proteins were eluted in SDS-PAGE sample buffer by heating at 95°C for 5 min.2 transformed, and the dataset was filtered for proteins having at least three values in at least one group (each group consisting of triplicate injections). The remaining missing values were imputed using default Perseus settings by drawing from a simulated noise distribution with a down shift of 1.8 and a width of 0.3 compared with the log2 LFQ intensity distribution. Two-sample t tests were performed with a permutation-based FDR set at 0.05. Proteins that were enriched >2-fold with p < 0.05 (comparing Full-Length versus Untagged Control samples) were subjected to SGD Gene Ontology Slim Mapper Process Analysis (https://www.yeastgenome.org/cgi-bin/GO/goSlimMapper.pl). Volcano plots were generated using Graphpad Prism (version 7.02). The processed mass spectrometry data are available in Eluted proteins were subjected to SDS-PAGE, migrated approximately 1 cm into the gel , and stained with InstantBlue Protein Stain (Expedeon). Proteins were in-gel digested using trypsin, and peptides were analyzed with an Orbitrap-Fusion Lumos mass spectrometer coupled to an Ultimate3000 HPLC equipped with an EASY-Spray nanosource (Thermo Fisher Scientific). Label-free quantification (LFQ) was performed using MaxQuant software (v1.6.01) . PerseusFor the GO-Slim terms in A table of oligonucleotides used in this study is available in Details of statistical tests used, sample number, and number of independent experiments are included in the relevant figure legends. p values were calculated using the Students’ t test, with p < 0.05 considered significant. Error bars are described in individual figure legends as ± standard error of the mean (±SEM) or 95% confidence intervals. Standard box-and-whisker plots were generated showing the median value , lower and upper quartiles (lower and upper hinges), and lowest and highest values .GSE110004.The accession number for the RNA sequencing and TSS sequencing data reported in this paper is GEO: