Inhibition of transcription caused by DNA damage-impaired RNA polymerase II (Pol II) elongation conceals a local increase in de novo transcription, slowly progressing from Transcription Start Sites (TSSs) to gene ends. Although associated with accelerated repair of Pol II-encountered lesions and limited mutagenesis, it is still unclear how this mechanism is maintained during genotoxic stress-recovery. Here we uncover a widespread gain in chromatin accessibility and preservation of the active H3K27ac mark after UV-irradiation. The concomitant increase in Pol II escape from promoter-proximal pause (PPP) sites of most active genes, PROMPTs and enhancer RNAs favors unrestrained initiation, as evidenced by the synthesis of nascent RNAs including start RNAs. Accordingly, drug-inhibition of PPP-release replenishes levels of pre-initiating Pol II at TSSs after UV. Our data show that such continuous engagement of Pol II molecules ensures maximal transcription-driven repair throughout expressed genes and regulatory loci. Importantly, revealing this unanticipated regulatory layer of UV-response provides physiological relevant traction to the emerging concept that Pol II initiation rate is determined by pause-release dynamics. A transcription-driven cellular response is activated upon UV stress. Here the authors reveal mechanistic insights into the regulatory process affecting transcription and chromatin dynamics, showing how maintaining Pol II firing safeguards the integrity of cells’ transcriptome Similarly, Pol II is regulated for transcribing regulatory non-coding regions: enhancer RNAs (eRNAs) are expressed bidirectionally from eTSSs5, while PROMoter uPstream transcripts  are produced in the opposite direction to mRNA when two stable transcripts are not initiated in very close proximity and in opposite directions 6. However, contrary to mRNAs, eRNAs and PROMPTs are short and unstable due to high early termination rates and increased susceptibility to degradation by the RNA exosome7, thus making their detection technically challenging.Pol II initiation at TSSs and release into productive elongation from PPP are ubiquitous and crucial steps regulating transcription of protein-coding genes and long non-coding RNAs8. After elongation of ~30–60 nucleotides of initiation-associated RNAs (start-RNAs)9, Pol II is paused at PPP sites by negative elongation factors DSIF and NELF10. Signal-regulated phosphorylation of these factors and of Pol II CTD serine 2 (S2P) by P-TEFb is required for productive elongation13. It recently emerged that, if this step does not occur rapidly, start-RNAs are terminated14, implying that Pol II turnover at PPP sites is high at steady state, and that replenishment of Pol II engaged in early transcription (initiation to PPP) is achieved through the continuous re-entry of pre-initiating Pol II into PICs15.Initiation of transcription in all the above regions depends on the efficient assembly of the pre-initiation complex (PIC) upstream of TSS and serine 5 phosphorylation (S5P) of Pol II C-terminal domain (CTD)16. A plethora of DNA damage response (DDR) mechanisms have evolved to guarantee the detection and removal of different types of DNA lesions, limiting the probability of mutagenesis by adjusting to the cell’s status and need for efficient recovery from DNA damage18. Nucleotide excision repair (NER) plays a vital role in sensing and removing a large panel of helix-distorting DNA adducts such as cyclobutane pyrimidine dimers (CPDs) induced by ultraviolet (UV) light, and benzo[a]pyrene guanine adducts induced by cigarette smoke17. Transcription-coupled NER (TC-NER) is promptly triggered by elongating Pol II molecules encountering DNA adducts and speeds up excision and repair in expressed loci21. In comparison, the second NER subpathway, Global Genome-NER (GG-NER), operates through the entire genome, but recognizes more stochastically helix distortions23. Importantly, given all the classes of transcripts defined above, it is estimated that the coverage of transcribed regions24 potentially scanned by TC-NER expands to more than 50% of the genome, thus qualifying transcription as a major driving force in safeguarding genomic stability.The integrity of the genetic information encoded in DNA sequence is persistently challenged by a variety of genotoxic perturbations27 due to a proportion of Pol II molecules stalling at encountered DNA damages28. Moreover, depletion of the pre-initiating hypo-phosphorylated Pol II(hypo) isoform from chromatin shortly after UV irradiation30 has led to the assumption that new transcription initiation events are transiently and globally repressed33. On the other hand, recent reports34 have revealed a functionally essential stress-dependent increase in 5′ nascent RNA (nRNA) activity globally that depends on the UV-induced increase in active P-TEFb levels36, and on the rapid dissociation of the NELF complex37. The ensuing fast and global release of de novo Pol II elongation waves from PPP sites into gene bodies boosts lesion-sensing activity and accelerates removal of DNA adducts by TC-NER in virtually all active mRNA genes25. Together, these findings substantiate the possibility that initiation of transcription might not be as severely affected by UV as previously believed.Although TC-NER depends on lesion-sensing potential by elongating Pol II molecules, transcription elongation has been shown to be transiently inhibited after UV irradiation38, it is tempting to assume that ensuring continuity in transcription initiation may bring advantages in the repair process. We thus hypothesized that the apparent loss of pre-initiating RNAPII may not be due to the absence of RNAPII recruitment at TSSs, but rather due to a decrease in the dwell time of Pol II-hypo isoform at TSS, as justified by the concomitant increase in Pol II-ser5P and Pol II-ser2P downstream of TSS25. In this way, cells would be able to uninterruptedly feed the global release of scanning enzymes into transcribable sequences, and guarantee the detection of more lesions along genes’ template strand (TS). Herein, we decipher chromatin dynamics genome-wide upon UV damage, and find a significant gain in accessibility (ATAC-seq) at the TSSs of virtually all active regulatory regions controlling mRNAs, PROMPTs, and eRNA expression. This phenomenon is underlined by the maintenance of active histone marks (H3K27ac), the lack of deposition of transcriptional silencing modifications (H3K27me3) at transcribed loci, and is correlated with the influx of Pol II into productive elongation. The paradoxical decrease in pre-initiating Pol II-hypo at these TSSs upon UV is elucidated by revealing that the presence of Pol II-hypo is rescued when PPP release is drug-inhibited. Accordingly, preserved production of start-RNAs after UV stress lies under the increased production of nRNA, and is prevented only after inhibition of transcription initiation. The identified genome-wide dependence of initiation rate on promoter-proximal pause-release dynamics explains the seamless recruitment/initiation of Pol II upon UV, in turn enabling efficient repair of the totality of the sequences encoding active regulatory regions and mRNAs.Taking also into consideration recent evidence that supports the model of disengagement of a given Pol II molecule from DNA template after damage recognition34, we first determined the genome-wide changes in chromatin accessibility. The omni-ATAC-seq protocol39 was implemented in our system involving UV-C irradiation (15 J/m2) of human skin fibroblasts synchronized in G1 . We reproducibly measured chromatin accessibility before (NO UV) and 2 h after (+UV) irradiation , and how this could be linked to the widespread PPP release of elongating Pol II and the local increase in nRNA production downstream of TSS2 FC > 1) and P value (P < 0.001), and found that 6410 loci showed particularly increased chromatin accessibility upon UV (DAR-gain)  of a number of biological pathways previously associated with DDR processes, including cellular response to stress, DNA repair, transcription regulation by TP53, and cell cycle checkpoints  by applying stringent thresholds both in terms of FC (Login) Fig.  were lin41. However, especially in the case of UV-C-induced DNA damage, little is known about the post-translational modifications (PTMs) of histones around transcriptional regulatory regions. To better interpret the increase in chromatin accessibility and clarify its possible impact on genome-wide transcription dynamics, we studied the differential presence of two histone PTMs representative for the transcription status of associated chromatin: the silencing mark H3K27me3 and the activation mark H3K27ac43.A number of studies have demonstrated that the turnover, modification, and/or degradation of histones around damage sites represent essential steps in conserved pathways that help cells deal with genotoxic stress25) from the steady state (NO UV) condition, to determine subsets of active (presence of H3K27ac and Pol II-ser2P peaks over TSS), repressed (presence of H3K27me3 peaks over TSS), and inactive loci    conditions, and focused our analysis on TSSs of mRNAs and on a robust set of eTSSs, which are known to be functional and potentially transcribed in the investigated cell type according to the FANTOM5 database (see “Methods”). We used the ChIP-seq data generated in this study (H3K27ac and H3K27me3), as well as previously published ChIP-seq data (Pol II-ser2P2)  of H3K27ac levels -seq from ref. 4, see “Methods”). First, we customized a genome annotation, which unambiguously pinpoints to the TSSs of mRNAs, PROMPTs, and eRNAs that do not overlap with regions possibly being transcribed through from neighboring/overlapping genes, promoters, or enhancers (see “Methods”). We then established three categories , as well as for 70.1% of PROMPTs  and 68.6% of eRNAs . We conclude that the PPP release of Pol II upon genotoxic stress is synchronously triggered at all active transcription units, and coincides with increased chromatin breathing. These data extend the previously characterized transcription-driven genome surveillance mechanism25 to essentially all active gene-regulatory regions, and give mechanistic insights into the synergy between the increase in chromatin accessibility and the transcriptional response observed upon UV.Using this setup, we discovered that the UV-dependent increase in chromatin accessibility Fig. , ATAC wa2 FC (+UV/NO UV)) < 0, see “Methods”) in line with earlier published data45, a local increase in nRNA synthesis downstream of TSS of all active genes is detected during the UV-recovery phase34. This observation combined with the above findings on the UV-induced chromatin opening around virtually all active TSSs, PROMPTs, and eTSSs are hardly compatible with the previously suggested model of UV-induced global inhibition of transcription initiation. We thus searched for alternative reasons that could explain reduction of Pol II-hypo levels at active TSSs/eTSSs despite increased accessibility after UV.We noted that although 63.65% of the transcribed genome shows reduction in transcription activity (coverage of the transcriptome with Log2) and left them to recover for 2 h, when the levels of Pol II-hypo are known to be severely depleted30 . The restoration of pre-initiating Pol II levels was even more pronounced when we focused on the occupancy on active TSSs, PROMPTs, and eTSSs, where average read densities detected by Pol II-hypo ChIP-seq after DRB treatment (+UV/+DRB/X 4 h) matched the control NO UV levels (NO UV/+DRB/X 4 h) . First, as depicted in Fig.  h) Fig. . Therefo2, Supplementary Fig. t test P = 0.0145). This result was corroborated by ChIP-qPCR experiments (performed on the same chromatin extracts used above), as DRB prevented the UV-induced reduction in occupancy of Pol II-hypo at promoter/TSS-proximal regions of six active genes  for DRB).We also applied DRB just before and for 2 h after UV  could originate from increased Pol II initiation at active TSSs . We followed the experimental procedure depicted in Fig. 2). For each condition, we isolated small RNAs by size selection (<200 nucleotides), and we ligated an RNA–DNA linker to their 3′ ends. Reverse transcription (RT) was performed using a universal primer annealing to the linker sequence as previously described7. Subsequently, locus-specific qPCR reactions were performed in order to compare, in a quantitative way, the levels of start-RNAs at representative active loci for which we had identified Pol II-ser2P ChIP-seq or nRNA-seq signal (see “Methods”). Our results revealed that start-RNAs could be detected after UV treatment, validating the fact that initiation still occurs during the UV-recovery phase . Together, these results consolidate further the evidence of the non-stop recruitment and initiation of Pol II at TSSs after UV irradiation.To further verify initiation activity during UV recovery, we exploited the possibility to track start-RNAs, which directly inform on the amount of dynamically engaged Pol II located within the initially transcribed sequence  when the assay is performed in GG-NER-deficient cells (xeroderma pigmentosum (XP)-C cells). Given the strand specificity of the assay, we considered only the excision of CPD damages from template (non-coding) strand (TS) for mRNAs, PROMPTs, and eRNAs, which corresponded to the + (blue) or the − (red) strand of the genome  and CAGE4 accurately locate active TSSs . Nevertheless, we note that the variability between both directions was strikingly less for TC-NER and Pol II-hypo than for CAGE , Supplementary Fig. P = 0)).Next, we took advantage of XR-seq data (eXcision-Repair sequencing)ome Fig. , dependiSSs Fig. , left, FSSs Fig. . As expeSSs Fig. . This reSSs Fig. , and CAG46. We quantified strand-specific repair upstream and downstream of unidirectional promoters, and found that repair activity at unambiguously resolved divergent PROMPTs was stronger than expected from CAGE levels  Pol II molecules is not sufficient to enable recognition of damaged thymidine dimers  in the totality of the transcribed genome. In accordance, a recent study reports that inhibition of transcription elongation with DRB limits the extent of excision activity detected by CPD XR-seq38. Thus, sending Pol II molecules from TSS into gene bodies after UV is of pivotal importance to maximize the detection of TTs on active TS.We next assessed the biological relevance of continuous transcription initiation from active regulatory regions during the UV-recovery period. We have reported previously38) around TTs and around TSSs of mRNA genes, PROMPTs, and enhancers . We observe that when DRB was applied immediately after UV, repair of TTs located from TSS to upstream of the +1 -h wave front (WF) of UV-released Pol II (as obtained in the non-DRB control) are seriously affected . We found that significant levels of transcription-dependent excision activity were maintained at lesions located directly downstream of active genes TSSs at 4 and 8 h into the recovery process  is the result of the continuity in Pol II initiation. To confirm this directly, we mapped XR-seq reads around TTs located on TS 1, 4, and 8 h after UV in WT cells  at active genes50. Our results obtained using a range of mild UV doses (8–20 J/m2), and focusing on the early phase of recovery (1-4 h), support the fact that chromatin relaxation and maintenance of H3K27ac has an active role in the repair of transcription-blocking lesions, and give substance to previously observed rather low increase in acetylation of histones in similar conditions51. In contrast, drastic chromatin remodeling observed in murine cells at a later time during recovery (6 h) when much higher doses (80 J/m2) were used52 fits with findings signifying that cellular response to UV depends on the exposure dose54. In other words, when cells deal with unmanageable levels of damages, they need to implement more radical expression changes, which are required for the associated fate of programmed death56, a protective mechanism limiting the risk of malignant transformation.The finding that an increase in chromatin accessibility parallels the conservation of H3K27ac-modified nucleosomes at the flanks of already opened regions in response to UV irradiation is compatible with reports showing that there can be a significant gain in DNA accessibility without changes in nucleosome occupancy during rapid transcriptional induction58. Indeed, for bidirectional mRNAs and mRNA-PROMPTs, the binding of Pol II-hypo occurs at both edges of highly accessible regions  loci, and supports the idea that bidirectional transcription of divergent RNAs arises from two distinct hubs of transcription initiation (PICs), located within a single nucleosome-depleted region (NDR)see Fig. , bottom,34, (ii) P-TEFb and NELF are important regulators of UV response61, and (iii) although elongation gradually decelerates due to the encounter of Pol II with DNA lesions, significant initiation/early elongation activity is observed in the first thousand bases of actively transcribed regions34, a characteristic that has also been used for the identification of active TSSs genome-wide after UV27. These features are consistent with our finding that new Pol II molecules are constantly recruited to PICs post-UV . Such a model explains previously published data concerning the persistence of sufficiently important levels to be worthy of attention, of (i) PIC/basal transcription factors in nuclear extracts30 or upstream of genes’ TSSs (TFIIB)33 and (ii) nRNAs at the beginning of genes34 upon UV.By uncoupling TSSs of mRNA genes from those of PROMPTs and enhancers, we reveal that P-TEFb-dependent release of elongating Pol II from PPP sites extends to all actively transcribed regions see Fig. . Interessee Fig. . ConsideWe note that excision fragments (from XR-seq) are distributed more homogeneously at sense (mRNA) and antisense (PROMPT) strands of unidirectional TSSs, and at enhancers, than it could be predicted from the CAGE levels see Fig. . This fi38, and thus a fortiori preventing the beneficial effects of Pol II initiation. Indeed, upon DRB treatment, we find a drastic impairment of CPD excisions at the beginning of all classes of active transcripts  after inhibition of PPP releasesee Fig. , and in see Fig. . In addi26. We propose that keeping initiation active maximizes the probability to repair quickly all lesions located on the TS. Our model is compatible with the idea that even if a Pol II molecule is ubiquitylated and degraded after the recruitment of the NER complex38, the amount of Pol II still normally recycling after termination from all the short genes (which contain less or no lesions) will provide sufficient trailing polymerases for scanning lession-containing genes. In this way, this mechanism can sense efficiently one after the other the more distal lesions even in longer genes, and gives an alternative biological perspective to presently favored mode of action21. In agreement, we detect TC-NER activity on TS directly downstream of TSSs 4 and 8 h after UV. In addition, repair of distal damages in long genes is detected only when DRB is not applied after UV  sites67. As such, transcription of non-coding regulatory loci could serve to keep their transcription factor-binding sequences (TFBS) in check.Increase in TC-NER at regulatory regions has also been observed in 15, implying that continuous cycles of initiation followed by rapid premature termination are required for fast transcriptional induction14. Our results, showing that persistent initiation guarantees a prolonged transcription-coupled NER across the whole transcriptome, are functionally linked to the fact that DNA damage-triggered widespread PPP release of a given Pol II is sufficient to drive immediate initiation of the next Pol II  and triptolide  were used in a final concentration of 100 μΜ and 125 nM, respectively, and they were added directly in growth media at the indicated times. Cells were irradiated with UV-C   and left to recover for indicated times (see figure legends and below).Cells used in this study were VH10 hTERT-immortalized human skin fibroblasts, and were cultured, synchronized by low-serum starvation, and released in full medium as described previously25 with minor changes (see Supplementary Methods for details). Cells were mock-treated (NO UV) or treated with UV (+UV) with 15 J/m2 except if otherwise stated. Treated cells were left to recover for 2 h before harvesting or as indicated on the timeline , H3K27me3 , and 8WG16 (Pol II-hypo) . The primers used for ChIP-qPCR experiments were the following : SSBP1_F: GTGAGGGAGGAAGGGATAGC, SSBP1_R: AGGGCCAGACACCTACACAG, OSBPL9_F: ATTGGCGGCTCCCAAGAT, OSBPL9_R: GCATTGTAGTCCAGCACGAA, TRPM7_F: CCCAGGGAAACCTTCTCAG, TRPM7_R: TCGCACAATTATGAAAGACTCG, MYC_F: ACTCAGTCTGGGTGGAAGGTATC, MYC_R: GGAGGAATGATAGAGGCATAAGGAG, AKNA_F: CCGTTCCAATCCCTTACC, AKNA_R: TGGAACAAAGAATTCACAGG, APRT_F: GCCTTGACTCGCACTTTTGT, APRT_R: TAGGCGCCATCGATTTTAAG, ChIA_neg_F: AGTCTGAGCTTTGTGGACAGC, and ChIA_neg_R: CCCTCCCAGTATACAGTCTTGC. qPCR, library preparation, and next-generation sequencing were performed as previously described25. Antibodies used for western blot analysis are the following: anti-H3K27ac , anti-H3K27me3 , 8WG16 , anti-elongating RNA pol II , anti-Lamin B1 , anti-histone 4 , and anti-histone3 . Dilutions of antibodies were performed according to the manufacturer's guidelines. Time for analysis is indicated in the figures  was performed as describedres Fig. . Uncropp39 with minor modifications: (i) 70,000 cells were used per experimental condition and (ii) the DNase treatment of cells in culture medium, before the transposition reaction, was skipped. The UV dose applied for ATAC-seq experiments was 15 J/m2, and treated cells were left to recover for 2 h before harvesting.ATAC-seq method  was performed using Nextera DNA Library Prep Kit  and primers as described by Corces et al.2O, and 0.5 μl of truncated RNA ligase)  was added in a final volume of 20 μl. The reaction was incubated for 3 h at 37 °C. After H2O was added to a final volume of 100 μl, ethanol precipitation  was performed overnight at −80 °C. RNA was purified at 10 μl, and reverse transcription (RT) was performed using primer oLSC003: /5Phos/TCGTATGCCGTCTTCTGCTTG/iSp18/CACTCA/iSp18/AATGATACGGCGACCACCGATCCGACGATCATTGATGGTGCCTACAG according to Invitrogen Superscript II (Cat No. 18064014) instructions. qPCR was performed using gene-specific forward primers  and one common reverse primer (oNTI230: 5′-AATGATACGGCGACCACCGA-3′), which anneals to RT primer oLSC003 sequence. Quantitative PCR results were obtained from two independent biological experiments. Values of all qPCR replicates are supplied in the Source Data file.To isolate small RNAs , we used Qiagen miRNeasy Mini Kit and RNeasy MinElute Cleanup Kit according to the manufacturer's instructions. In order to monitor the efficiency of the different enzymatic reactions, we included in our experiments a spike-in RNA oligonucleotide of known sequence . After purification, small RNAs and spike-in molecules were ligated to the IDT DNA linker 1 (/5rApp/CTGTAGGCACCATCAAT/3ddC/). Specifically, samples were denatured for 2 min at 80 °C and then placed immediately on ice. Ligation mix . ATAC-seq and ChIP-seq reads were subjected to quality control, data filtering, and alignment, and wig profile generation was performed essentially as described previously25 with minor modifications. Chip-seq data for Pol II-ser2P and Pol II-hypo , respectively. nRNA-seq data were generated in our lab previously25 (GSE83763) and obtained from ref. 27 (GSE75398). Downloaded data were processed as described in ref. 25  data analyses, in-house scripts and pipelines were developed to automate and analyze the data consistently (see below for details). Code is available upon request. Sequenced data and generated wig profiles are available on Gene Expression Omnibus (GEO)  and log2 fold change cutoffs were set to 0.01 and 1.5, respectively.For H3K27ac and H3K27me3 ChIP-seq alignment files, peak calling was performed using SICER version 1.172. Because of the variability of ATAC-seq fragment lengths, several runs of the peak-calling algorithm were performed, using different parameters per run, in an attempt to maximize the sensitivity of the detection of open-chromatin regions. In particular, --nomodel --shift 100 --extsize 200, --broad --shift 100 --extsize 200, --nomodel --shift 37 --extsize 73, --broad --shift 37 --extsize 73, --broad --nomodel --shift 37 --extsize 73 --keep-dup all, --broad --nomodel --shift 100 --extsize 200 --keep-dup all and --nomodel --shift 75 --extsize 150 --keep-dup all runs were combined, and detected peaks were filtered using fdr < 0.05 and fold change > 1. Only peaks present in five out of seven methods were kept for further analysis. Although the majority of peaks detected were common between conditions, a number of peaks were also detected only in NO UV and +UV. As these peaks were less in number than the common ones, and showed a rather low density of reads in another attempt to maximize the sensitivity of the detection of open-chromatin regions, and to unbiasedly investigate the changes occurring upon irradiation, we considered the union of the peaks in each condition to perform the rest of the analysis (Supplementary Table https://www.bioconductor.org/packages//2.10/bioc/html/DiffBind.html) was used, with the merged ATAC-seq peak set as a reference. Differential accessibility regions were detected and filtered by applying fold change (Log2 FC ≥ 1) and P-value  thresholds.For ATAC-seq alignment files, peak calling was performed using MACS225 around genomic regions of interest, as indicated in the figures. Heatmaps were generated directly using the software, from matrices of binned read densities (bin size is indicated in the figures) for all considered individual (n) items (metagenes). Read- density matrices were also imported in R and python custom scripts for (i) plotting average-density profiles (smoothing achieved by a moving window of the bin size as indicated) and (ii) for determination of read densities per genomic category.ATAC-seq, ChIP-seq, nRNA-seq, CAGE-seq, and XR-seq data were subjected to read-density analysis after read depth normalization of all samples per experiment. Heatmaps and average-density profiling were computed as described previouslyhttp://genome-euro.ucsc.edu/cgi-bin/hgTables). For each transcript, a biotype was assigned using BioMart (www.biomart.org), and all the small non-coding RNAs were excluded. For all the gene models containing multiple alternative transcripts, TSS neighborhoods of a 100-bp window were clustered together, and only the longest transcript was kept, resulting in 30,473 transcripts. Transcripts were then separated into three groups, based on their transcriptional activity. TSS coordinates were extended to 2 kb on each direction, and were tested for overlap with the Pol II-ser2P-UV, H3K27ac-UV, and H3K27me3-UV peak sets. Regions overlapping with at least one Pol II-ser2P-UV and H3K27ac -UV peak were characterized as active, those overlapping with a H3K27me3-UV peak, but not with a Pol II-ser2P-UV or with a H3K27ac-UV peak, were characterized as repressed, and those that did not overlap with any of the above peak sets were characterized as inactive. Any region overlapping with both H3K27ac-UV and H3K27me3-UV peaks was excluded from the rest of the analysis. This resulted in 15,819 active, 2943 repressed, and 7608 inactive transcripts , were characterized as bidirectional, while the rest of the annotations were characterized as unidirectional. Bidirectional pairs were further categorized into two groups of annotations: convergent bidirectional transcript pairs with TSSdistance ≤ 100 bp, and divergent bidirectional transcript pairs with TSSdistance > 100 bp. To optimize the categorization of convergent and divergent transcript pairs, TSS coordinates were redefined by scanning at a radius of 250 bp, to detect the nucleotide occupied by the maximum-sense CAGE signal. Any bidirectional pair with a non-significant CAGE peak in the aforementioned region was excluded from the analysis. This finally resulted in 12,859 unidirectional transcripts and 2822 active bidirectional TSS pairs, 1806 of which were characterized as divergent and 1016 as convergent (Supplementary Table To annotate transcription start sites (TSSs), all known protein-coding and non-coding RNA hg19 RefSeq transcripts release 86 was downloaded from UCSC table browser  and downstream antisense (daRNA) transcripts (referred as an ensemble to PROMPTs in this paper for convenience). Only the active unidirectional mRNA-TSSs were used. For all the genes annotated with more than one mRNA transcript, only the leftmost TSS (for + strand genes), and rightmost TSS (for − strand genes) were considered for the rest of the analysis. The antisense CAGE peak with the highest summit in the region that ranged from −2 kb upstream to +1 kb downstream of each unidirectional TSS was considered to be the main PROMPT TSS for further analyses (inter-TSS distance = mRNA TSS - CAGE PROMPT). The above procedure was also repeated for the inactive transcript set to estimate the highest CAGE summit background distribution. The putative active PROMPT CAGE summits, which were higher than the average of the summit background distribution, were considered as active. This resulted in 5366 pairs of active unidirectional—PROMPT-TSSs, which were categorized to 1444 divergent and 3922 convergent pairs, as described above , all 65,423 human enhancers from phases 1 and 2 of the FANTOM5 project from distance > 100 bp, TSSdistance = TSS coordinate forward reference − TSS coordinate reverse reference, active divergent bidirectional transcript pairs with TSSdistance > 100 bp, and active intergenic enhancers with no nearby transcripts within 10 kb and no nearby eTSSs within 2 kb. TSSs and PROMPT-TSS promoter escape indexes  were calculated as previously defined25, by taking the average coverage in rpm in the gene body  divided by the average coverage on the promoter-proximal region (Dp) ranged from 250 bp upstream to 100 bp downstream of TSS.Promoter escape analysis was performed for a subset of active unidirectional and bidirectional transcripts, PROMPTs and active enhancers. In particular, to avoid the inclusion of Pol II-ser2P reads mapped in overlapping promoters and gene bodies, only active divergent unidirectional transcript—PROMPT pairs were considered, where TSSFor enhancer escape analysis, EI was calculated as above, where density of reads at enhancer flanks (Df) is calculated for the regions ranging from −2 kb to −100 bp upstream of eTSS and from +100 bp to +2 kb downstream of eTSS, while density of reads on enhancer TSS (De) is calculated for the regions ranging from 100 bp upstream to 100 bp downstream of eTSS.20  and Chiou et al.38 . XR-seq data for wild-type (WT) cells  were obtained from Adar et al.46. Sequence read archive (SRA) data sets were downloaded from Gene Expression Omnibus using the sra toolkit prefetch (https://www.ncbi.nlm.nih.gov/sra/docs/sradownload/) command, and converted to fastq files using fastq-dump. Fastq quality control, data filtering, and short read alignment were performed as above. Meta-analysis involved that read counts were normalized to equal read depth. Heatmap read-density matrices and average read-density plots of CPD XR-seq read around potential pyrimidine dimers. For XP-C data, we focus on TpT sequences (TT) as explained previously25, and filtered the TTs overlapping with enhancer regions defined above to avoid signals generated from eTSSs. For WT cells, the list of TTs was further curated to only consider TTs in active genes (or inactive genes) located on plus strand and filtering out TTs in the region between TSS and +2 kb for CONV and DIV bidirectional and unidirectional genes with inter-TSS < 100 bp , available for XP-C mutants lacking the global genome nucleotide excision repair mechanism , were obtained from Hu et al.ftp://ftp.biosciencedbc.jp/archive/fantom5/datafiles/phase2.2/basic/human.primary_cell.hCAGE and were combined. Heatmap read-density matrices and average read-density plots were computed as described in the section “Read densities heatmaps and average plots”. Read-density matrices were calculated for both strands separately.The FANTOM5 strand-specific CAGE-seq alignment files of normal dermal fibroblast primary cells  and normal skin fibroblasts (two donors with source codes: 11553-120C5 and 11561-120D4) were downloaded from Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Reporting Summary


Derivatives with changed transcriptional activity were selected using a reporter plasmid pET28_eGFP. Only one cycle of mutagenesis with negative selection suppressed the activity of the main dps promoter to the background level due to a single substitution in its -10 element, while positive selection gave a sequence with improved -35 element, thus testifying feasibility of the approach. The same suppression for appY was achieved by three cycles, while eightfold transcription activation required nine iterations of mutagenesis. In both cases, the number of potential start points decreased resulting in an ordinary regulatory region with only one dominant promoter in the case of positive selection. Efficiency of H-NS binding remained virtually unchanged in all mutant constructs. Based on these findings we conclude that excessive promoters can adversely affect transcription by providing a platform for interference between several RNA polymerase molecules, which can act as a silencer at promoter-dense regions.Horizontally acquired genes are usually transcriptionally inactive, although most of them are associated with genomic loci enriched with promoter-like sequences forming “promoter islands.” We hypothesized that lateral DNA transfer induces local mutagenesis, accumulating AT base pairs and creating promoter-like sequences, whose occupancy with RNA polymerase and a specific silencer H-NS suppresses the transcription of foreign genes. Error-prone mutagenesis was implemented for the “promoter island” of a foreign gene  Horizontal gene transfer plays a pivotal role in bacterial evolution assisting in the adaptation of microbes to the environment and increasing the diversity of their populations . At leasEscherichia coli (E. coli).A typical regulatory region of bacterial genes contains one or several overlapping promoters with one or several transcription start points (TSPs) in each . HoweverE. coli this function is performed by a specific sentinel, a histone-like protein H-NS . Cells of E. coli BL21(DE3) or E. coli BL21(DE3)Δhns transformed with pGEM_H-NS-His expression vector . A fragment containing the regulatory region of the dps gene (402 bp) was obtained with primers: 5′-TCCTCTAGATGTTATGTCCCAGT-3′ and 5′-GGAAGATCTTCCTCGGAGAAACACT-3′ . A fragment with the appY-associated “promoter island” (423 bp) was amplified with primers: 5′-GATAAGATCTGCAAGTAAAAATGATACTC-3′ and 5′-CCCTTCTAGATTTGTCGCTTACAATAAA-3′. All PCR reactions were carried out using a standard protocol: 2 min melting at 95°C followed by 35 cycles: 95°C, 30 s; 55°C, 30 s; 72°C, 1 min.Plasmid pET28b-EGFP with the  protein  was used protein . At the XbaI and FastDigest BglII nucleases . Restriction was carried out in universal FastDigest buffer for 1 h at 37°C. Restriction fragments were purified with QIAquick PCR Purification Kit, while the pET28_EGFP vector processed in parallel was fractionated by gel electrophoresis in 1% agarose and purified with MinElute Gel Extraction Kit . Restriction fragments were ligated with T4 Ligase  immediately upstream of the gfp gene (12 h at 4°C) and the library of recombinant pET28b_eGFP . Amplicons of target sequences were obtained with Taq polymerase  and sequenced. The mutated inserts with the largest number of new spontaneous mutations were used for a subsequent cycle of random mutagenesis, and the whole procedure was repeated until the GFP fluorescence was not distinguishable from the previous round.After purification of DNA samples with QIAquick PCR Purification Kit , error-prone PCR was carried out with GeneMorph II Random Mutagenesis Kit . Without purification, 0.1 ng of amplicon samples were used for a second stage of PCR. Thereafter, amplicons were purified with QIAquick PCR Purification Kit and prepared for cloning into pET28_EGFP vector using FastDigest 28b_eGFP  was used28b_eGFP  using Legfp in up-regulated constructs was compared to the E. coli cells transformed with corresponding pET28b-eGFP plasmids. For visualization, cells in LB medium (1 μl) freshly transformed with each of the constructed vectors were plated on one Petri dish and grown overnight before imaging. For quantitative measurements, cells were cultured for 12 h in 4 ml of LB medium, sedimented, suspended in 1 ml of TE buffer (pH 7.5) and sonicated . Cellular debris was sedimented and fluorescence intensity of GFP was measured in supernatant using a fluorescent spectrophotometer Cary Eclipse . The total protein concentration of the supernatant measured with a NanoDrop ND-1000 spectrophotometer (United States) was used for normalization.The expression level of 2 . Samples were treated with RNase A , precipitated with threefold volume of 96% ethanol and 0.3M sodium acetate and washed with 70% ethanol. The precipitate was dissolved in 5 μl of 98% formamide with 8 mM NaOH and 4 mM EDTA, fractionated in 8% polyacrylamide gel (PAAG) with 8M urea and radioautographed.The total RNA was isolated from bacterial cells transformed with pET28b-EGFP containing different inserts as described . Ten micSDS electrophoresis was used to assess the purity of H-NS. Stacking gel contained 4% acrylamide, 0.125M Tris-HCl (pH 6.8), 0.1% SDS. Separating gel contained 12.5% acrylamide, 0.375 M Tris-HCl (pH 8.8), 0.1% SDS. Electrophoresis buffer included 25 mM Tris-HCl, 250 mM glycin and 0.1% SDS (pH 8.3). Ammonium persulfate and TEMED were added to a final concentration of 0.004 and 0.003%, respectively. Sample loading buffer contained 50 mM Tris-HCl (pH 6.8), 100 mM β-mercaptoethanol, 1% SDS, 0.01% bromphenol blue, and 10% glycerol. After sample loading the gels were run at constant current of 25 mA in stacking gel and 45 mA in separating gel. Molecular weight markers used were PageRuler Unstained Low Range Protein Ladder  and Blue Prestained Protein Standard, Broad Range . Following electrophoresis, the gels were stained in 0.25% Coumassie Brilliant Blue R-250  with 45% ethanol and 10% acetic acid  and washed in solution containing 40% ethanol and 7% acetic acid.2, 50 mM NaCl, 0.01 mM EDTA, 0.01 mM DTT and 3.3 mM imidazole (present in storage buffer of H-NS to prevent oligomerization) for 40 min at 30°C. Following the addition of glycerol to the final concentration of 10%, the samples were loaded on pre-warmed 5% PAAG. The gels were run at constant voltage of 280 V. Alternatively, H-NS protein was obtained from IPTG-induced (100 μM) E. coli BL21(DE3)Δhns cells transformed with the pGEM_H-NS-His expression vector. Cells were harvested 4 h after the induction, washed, sonicated, and cellular debris was sedimented, while the supernatant containing 0.36–0.4 μg/μl of total protein was used for complex formation. Bands were stained with AgNO3 (pure protein) according to the protocol  to form complexes with H-NS under two different experimental settings. Complexes with purified H-NS were formed in 20 μl of buffer containing 5 mM Tris-HCl (pH 8.0), 1 mM MgClprotocol  or analylication . The intE. coli gene encoding the nucleoid protein Dps  upstream from the ATG codon were previously identified as functional by high-throughput techniques . This also created a weak (p = 9.0 × 10–4) H-NS binding site in position −54 . Substitution A→T in position −268, on the contrary, improved the −10 element of P3 (TAACCA → TAACCT), but without a compensatory effect on the transcriptional activity reduced by the two aforementioned mutations. This independence of gfp expression from P3 in the plasmid pET28b_EGFP corresponds to our previous data, indicating that P3 and P2 are required for maximum transcription of gfp, but cannot ensure its expression in the absence of P1’, P1, and Pdps  and shifted its context closer to the consensus sequence . Primer extension assays confirmed the activation of Pdps , while interaction with the down-regulated construct showed only statistically insignificant tendency for increase . As promoter mutants were selected in vivo . Surprisingly, the up-regulated derivative, whose substitutions did not change any of H-NS binding sites, reproducibly formed the largest complex with a higher efficiency than the two other fragments . Assuming the slight difference between the smaller and larger complexes cannot be mediated by binding of a large protein, like RNA polymerase, we suggested that the mutation-mediated increase in binding may be due to some other transcription factors. Thus, we searched for potential binding sites of 109 regulatory proteins with RSAT matrix-scan software and found them for Dps at position −370/−363 (p = 4.7e-4) and AraC at position −94/−57 (p = 8.1e-4). Of these binding sites, the one with increased affinity for the AraC dimer (monomer MW – 33.4 kDa) may explain the observed change in the mobility of the complexes. Thus, it became clear that the functionality of the ordinary promoter region is highly sensitive to random mutagenesis and quite expectedly accumulated changes in the context of key promoter elements in response to positive or negative selection.Since in the down-regulated derivative mutation at position −47 increased the conformity to the context of H-NS binding sites, we compared the H-NS affinity to both mutated constructs with that of the initial fragment by EMSA . When pu in vivo , certainragments  and demoappY-associated “promoter island”  and the second (A+54 → G) cycles, extended the hairpin originally formed in the region of + 60/ + 71 to + 53/ + 76, and the substitution C+71 → T ensured the perfect complementarity in this hairpin . Error-prone mutagenesis created one new site at position −160 in the down-regulated mutant (p = 3.8e-3) that can contribute to the slightly increased affinity of H-NS to this construct: 110.1 ± 5.7 and 117.2 ± 0.8% in the EMSA experiments with pure protein and lysates . Multiple mutations in the activated construct created three additional sites  eliminating two binding modules at positions −102 and −45 (n = 6) in assays with pure protein and lysates  and the total number of transcription factor binding motives (96) almost the same as shown in To test the possibility that blindly selected mutations in the up-regulated derivative predominantly accumulated new motifs for activators or eliminated binding sites for inhibitors and gave the opposite effect for an inhibited construct, we grouped 109 transcription factors with known binding motifs into four functional categories . These iE. coli MG1655 genome and started a long-term evolutionary experiment that already confirmed an increased frequency of spontaneous GC to AT substitutions in the recombinant region (appY mutant with dramatically decreased number of potential binding sites for RNA polymerase.Exploiting foreign genes for rapid environmental adaptation, bacteria also have to evolve the mechanisms for their acquisition or suppression. We hypothesized that recombination increases local mutagenesis with predominant accumulation of AT base pairs. This can be achieved by deamination of cytidines or oxidation of guanines , if nuclt region . Here, wKlebsiella aerogenes (UH promoter with a strengthened −10 region was shown to block the transcription from the overlapping weaker PC site. Another way of transcription blockade is related to the inactivation of a downstream promoter under active functioning of an upstream promoter. This phenomenon can be exemplified by intragenic promoters required for the synthesis of an RNA product from within the gene of 16S rRNA and activated in the stationary phase upon repression of transcription of the major promoters of the rrn operon (The idea of the bacterial RNA polymerase to be a repressor of transcription was implicitly or directly announced before. Though transcription inhibition, as opposed to the main synthetic role, is not regarded as its general function, there are a number of studies indicating that the enzyme complex formation with DNA can suppress transcription from neighboring promoters . For inserogenes , the binn operon . A totaln operon  by Genomn operon .dps regulatory region demonstrated some mutation-driven alterations in complex formation (appY construct. If the population control over horizontally transferred genes primarily operates at the level of randomly created promoters, then a new function of RNA polymerase as a sentinel of foreign genes should be considered.Starting these experiments with promoter regions regulated by H-NS, we expected to observe changes in the number of its potential binding sites, types of complexes formed or efficiency of interaction. However, only the activated construct with the ormation . Thus, wAll datasets generated for this study are included in the article/gfp-encoding reporter plasmid. KS: initial plasmid construct. OO: bioinformatic analysis and manuscript preparation for publishing with a contribution of all other authors.AB and OG: isolation of plasmids for sequencing. AB: PCR mutagenesis and GFP fluorescence measurements for cell extracts. OA and KS: primer extension analysis. OA: electrophoretic mobility shift assay. NS and H-NS: isolation/purification. IM: 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.


Changes in available nutrients are inevitable events for most living organisms. Upon nutritional stress, several signaling pathways cooperate to change the transcription program through chromatin regulation to rewire cellular metabolism. In budding yeast, histone H3 threonine 11 phosphorylation (H3pT11) acts as a marker of low glucose stress and regulates the transcription of nutritional stress-responsive genes. Understanding how this histone modification ‘senses’ external glucose changes remains elusive. Here, we show that Tda1, the yeast ortholog of human Nuak1, is a direct kinase for H3pT11 upon low glucose stress. Yeast AMP-activated protein kinase (AMPK) directly phosphorylates Tda1 to govern Tda1 activity, while CK2 regulates Tda1 nuclear localization. Collectively, AMPK and CK2 signaling converge on histone kinase Tda1 to link external low glucose stress to chromatin regulation. AMPK orant does . The β sant does  and a spant does .Snf1 interacts with and phosphorylates several proteins involved in nutritional stress responses . Its subINO1 gene promoter to regulate its transcription  residues, which is required for in vivo Tda1 activity. CK2 regulates H3pT11 by controlling Tda1 nuclear localization. Hence, Tda1 acts as a signaling platform that can combine Snf1 and CK2 signaling, thereby connecting external nutrient availability to transcription regulation in the nucleus.snf1Δ mutant showed significantly reduced global H3pT11 levels in saturated cultures  and γ subunit (Snf4) deletion mutants showed a comparable level of H3pT11 to WT  showed a slightly increased level of H3pT11. As Gal83 dictates Snf1 nuclear localization upon low glucose stress  as a guide, we tested H3pT11 levels of deletion mutants of the kinases, which are known to interact with the Snf1 complex. Unexpectedly, the yeast Nuak1 homolog tda1Δ mutant showed a significantly reduced H3pT11 levels  predicts that Tda1 may contain a kinase domain at the N-terminus. In this regard, we made several Tda1 fragments (Tda1 N1–N5) to define the kinase domain  mass spectrometry analysis. Surprisingly, MudPIT analysis revealed that Snf1 phosphorylates multiple serine and threonine residues of the Tda1C fragment . We catesnf1Δ background  mutant  mutant . At non-pression . These rcka1Δ background showed similar activity against H3 T11 to Tda1 purified from WT  predicts that Tda1 contains a calcium/calmodulin-dependent kinase domain at its N-terminus. We found that its functional kinase domain resides between aa 1 and 380 , Nuak1 has been proposed as a human ortholog of yeast Tda1  and then broken up by bead beating. The crude cell extracts were incubated with 125U benzonase and 500 µg heparin for 15 min at RT to remove nucleic acid contamination, then the extracts were further clarified by ultracentrifugation. The clarified extracts were incubated with anti-FLAG M2 affinity resin (Sigma) for 4 hr at 4°C with gentle rotation. Proteins bound to the resin were washed three times with buffer A, then washed once with buffer B . Bound proteins were eluted in buffer B containing 0.5 mg/mL 3xFLAG peptides.Tda1 and Snf1 proteins with 3xFLAG tag were purified as previously described  with minXenopus core histones were purified as previously described  was transformed into Tuner DE3 pLysS competent cells (Novagen), and then histone protein expressions were induced in 2X YT media by 0.5 mM IPTG for 24 hr at 37°C. Cell pellets were sonicated in high salt extraction buffer , and then clarified extracts were incubated with TALON metal affinity resin (Clontech) for 3 hr at 4°C. Resin-bound proteins were eluted by 250 mM imidazole, and then eluted proteins were dialyzed in high salt extraction buffer for overnight at 4°C.Yeast Cka1 and Tda1 genes were amplified by PCR from yeast genomic DNA and then cloned into pGEX4T-1  vector. GST-Snf1 catalytic domain (Snf1-CAT) expression vector was purchased from Addgene (#52683). Those DNA constructs were transformed in Rosetta2 (Novagen) competent cells, and protein expressions were induced by 0.5 mM IPTG for 18 hr at 16°C. Bacterial cell pellets were resuspended in TAP extraction buffer, then sonicated to disrupt cell walls. Crude extracts were incubated with glutathione sepharose 4B  resin for 3 hr at 4°C. Resin-bound proteins were eluted in 50 mM Tris pH 8.0 buffer containing 20 mM glutathione. escribed  with minYeast cellular fractionation was carried out as previously described  with min Yeast RNAs were prepared as previously described . Five toAll in vitro kinase assays were done in NEBuffer for protein kinase (NEB) at 30°C. As a phosphate donor, 5 mM ATP or GTP was used. Recombinant Snf1 catalytic domain (Snf1-CAT) was phosphorylated by 1.4 pmol of recombinant human CaMKK2 (Abnova) for 2 hr at 30°C for its activation . When Td18 reverse phase , strong cation exchange particles , and reverse phase (E. coli proteins (downloaded from NCBI on 2013-11-06), 177 usual contaminants such as human keratins, IgGs, and proteolytic enzymes, 4476 ‘shuffled’ sequences, and four yeast proteins sequences . Peptide/spectrum matches were sorted and selected using DTASelect (Trichloroacetic acid (TCA) precipitated proteins were urea-denatured, reduced, alkylated, and digested with endoproteinase Lys-C (Promega) followed by modified trypsin (Promega) as previously described . Peptidese phase . Loaded se phase . Tandem se phase  against TASelect  with theTASelect . To estiTASelect . In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:This paper does an excellent job demonstrating that AMPK and CK2 signaling converge on histone kinase Tda1 to link external low glucose stress to chromatin regulation. The insight provided into how Tda1 is a novel mediator of H3T11P is exciting. This work is of wide interest to the chromatin, transcription, nutrient signaling community.Decision letter after peer review:eLife. Your article has been reviewed by three peer reviewers, including Xiaobing Shi as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Jessica Tyler as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Brian D Strahl (Reviewer #3).Thank you for submitting your article "Yeast Nuak1 phosphorylates histone H3 threonine 11 in low glucose stress by the cooperation of AMPK and CK2 signaling" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.eLife but that some conclusions require a modest amount of additional new data, as they do with your paper, we are asking that the manuscript be revised to either limit claims to those supported by data in hand, or to explicitly state that the relevant conclusions require additional supporting data.We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, when editors judge that a submitted work as a whole belongs in eLife, either of which would be linked to the original paper.Our expectation is that the authors will eventually carry out the additional experiments and report on how they affect the relevant conclusions either in a preprint on bioRxiv or medRxiv, or if appropriate, as a Research Advance in Summary:This is an excellent study from the Workman group that delineates the mechanism by which H3T11 becomes phosphorylated during nutrient  deprivation in yeast to regulate gene transcription. Previous studies from the Workman lab and others have revealed that H3T11 is phosphorylated from yeast to humans, and that this modification is important for transcriptional regulation. Specifically in yeast, the Workman group recently demonstrated that pyruvate kinase and CKII are required for H3T11P; however, whether additional enzymes were also involved and the interplay between kinases in mediating this mark was not well understood. As CKII was determined to not be the main kinase for H3T11P under glucose starvation, the Workman group sought to identify the relevant kinase(s). The authors found that Snf1 of the AMPK signaling pathway is required for proper H3T11P, but that not all of the regulatory subunits of AMPK (β and γ) are needed, thereby suggesting a novel connection of Snf1 with the terminal kinase. A search for this kinase found, surprisingly, that Tda1 (yeast NUAK1 homolog) is the bona fide kinase that mediates this modification. They show that Tda1 is activated by the nutrient regulated kinase Snf1, they identify the sites phosphorylated by Snf1, and they show that the nuclear translocation of Tda1 is dependent on Cka1 signaling. In all, this work describes an elegant molecular pathway wherein multiple singling pathways and kinases converge to regulate a key kinase that mediates an important chromatin modification required for glucose-deprived gene regulation.eLife with minor modifications.Overall, this study is thorough, and the conclusions are strong. This paper does an excellent job demonstrating which kinases are responsible for phosphorylating which sites. The insight provided into how Tda1 is a novel mediator of H3T11P is exciting, as well as the interesting connections made to its nuclear localization. Taken together, this work would be of wide interest to the chromatin, transcription, nutrient signaling community. Therefore, I support its publication in Essential revisions:1) A major weakness in this story is the lack of any indication that phosphorylation of histone H3T11 is biologically significant. Do cells lacking this signaling pathway or tda1 deletion have any phenotypes or defect in gene expression? Some experimental data to demonstrate that phosphorylation of H3T11 is important for some aspects of nutrient stress response would increase the significance of this paper tremendously.2) It is interesting that Snf1-mediated phosphorylation of S483/T484 is critical for Tda1 kinase activity. However, the in vivo function of this phosphorylation event is not very clear. It will help to complete the circuit in the paper if it can be experimentally tested if mutation of S483/T484 leads to defective gene activation of glucose sensitive target genes. It would be assumed but not formally shown. Also, it would also be interesting to see if a phospho-memetic (S/T to E) mutation of Tda1 might bypass the need for Snf1/CKII, etc.3) Similar experiments to test the role of CK2-mediated Tda1 phosphorylation would also strength the paper greatly.4) The conclusion that Snf1 is a kinase that can phosphorylate H3S10 is arguable. The authors may want to provide more biochemical data using recombinant proteins and orthogonal approaches, or tune down their conclusion. Essential revisions:1) A major weakness in this story is the lack of any indication that phosphorylation of histone H3T11 is biologically significant. Do cells lacking this signaling pathway or tda1 deletion have any phenotypes or defect in gene expression? Some experimental data to demonstrate that phosphorylation of H3T11 is important for some aspects of nutrient stress response would increase the significance of this paper tremendously.Life (PMID: 29938647), we reported genome-wide ChIP-seq results of H3pT11 in low glucose YPgly media (GSE111218). We also reported RNA-Seq results of several mutants whose H3pT11 was impaired (i.e. H3T11A mutant) in YPgly media (GSE111217). In that manuscript, we reported a solid relationship between the transcription activation of low glucose stress responsive genes and H3pT11 signal increase upon media shift from YPD to YPgly media. In addition, we found the significant transcriptional defects of several low glucose stress responsive genes in H3T11A mutant. In this regard, we believe that we successfully proved a biological significance of H3pT11 in low glucose condition in our previous work. Thanks to the reviewer’s comment we realize that we need to better review the biological significance demonstrated in our previous study in the Introduction of this manuscript. We have added a summary of the previous manuscript to the revised manuscript (Introduction).We thank the reviewer for this comment. In our previous work, also published in eIn addition, as we already know the target genes of H3pT11, we selected several genes whose transcription are impaired in H3T11A mutant in low glucose stress. We tested their gene expression levels in tda1Δ mutants to see if tda1Δ showed a similar phenotype compared to H3T11A mutant. We added a new Figure 6—figure supplement 2, describing this result.2) It is interesting that Snf1-mediated phosphorylation of S483/T484 is critical for Tda1 kinase activity. However, the in vivo function of this phosphorylation event is not very clear. It will help to complete the circuit in the paper if it can be experimentally tested if mutation of S483/T484 leads to defective gene activation of glucose sensitive target genes. It would be assumed but not formally shown. Also, it would also be interesting to see if a phospho-memetic (S/T to E) mutation of Tda1 might bypass the need for Snf1/CKII, etc.In newly made Figure 6—figure supplement 2, we compared the expression levels of four H3pT11 target genes  in WT, tda1Δ, and Tda1 S483A/T484A (AA) mutant. The data clearly showed that AA mutant failed to restore H3pT11 target gene expression levels compared to WT. Indeed, AA mutant showed a similar level of transcription to tda1Δ mutant. The AA mutant did not show transcription defects at non-target genes (PMA1 and MET13). These results suggest the critical role of Tda1 S483/T484 phosphorylation in the regulation of Tda1 activity.Based on the reviewer’s suggestion, we made Tda1 S483D/T484E (DE) mutant and tested whether the potential phosphorylation mimic mutant could bypass the requirement of Snf1 or CK2. Interestingly, we found that DE mutant, as well as AA mutant could not restore H3pT11. The result is shown in Although we showed that Tda1 phosphorylation at S483/T484 is critical for the regulation of Tda1 activity , we do not know the exact mechanism how the modification controls Tda1 activity. This needs further investigation in the near future. If we can understand the detailed mechanism of Tda1 activity control by phosphorylation, then we may understand why the DE mutant failed to mimic Tda1 phosphorylation.3) Similar experiments to test the role of CK2-mediated Tda1 phosphorylation would also strength the paper greatly.We appreciate the reviewer’s attention on this point. We showed that the Tda1 C terminal was phosphorylated by Cka1 at serine 578  using in vitro kinase assays. We tested Tda1 S578A mutant for H3pT11 and found that the Tda1 S578A mutant did not show any defect in H3pT11 , suggesting that Cka1 may not regulate Tda1 by direct phosphorylation at serine 578. Thus far, we were not able to find any evidence that Cka1 could phosphorylate Tda1 at sites other than S578, or control Tda1 nuclear localization by direct phosphorylation. The mechanism how CK2 regulates Tda1 nuclear localization needs more investigation in the near future.4) The conclusion that Snf1 is a kinase that can phosphorylate H3S10 is arguable. The authors may want to provide more biochemical data using recombinant proteins and orthogonal approaches, or tune down their conclusion.We agree with reviewers comment that Snf1 as a H3S10 kinase is arguable. As H3pS10 is dispensable for H3pT11 onset upon low glucose stress , we believe that Snf1 as a histone H3 S10 kinase is not a critical part of this study. In this regard, we revised manuscript as below.From “Snf1 phosphorylates H3 at serine 10 at the INO1 gene promoter…”to “Snf1 is required for H3 serine 10 phosphorylation (H3pS10) at the INO1 gene promoter…”From “Snf1 can phosphorylate histone H3 at serine 10 in yeast.”to “Snf1 is required for H3pS10 at the INO1 gene promoter in yeast.”In addition, we changed Figure 4D. in the previous Figure 4D, we showed that yeast flag purified Snf1 could phosphorylate both H3S10 and H3T11 in vitro. To avoid confusion, we repeated the experiment without flag purified Snf1. Instead, we added GST-Cka1 as a new control.


Abcg2 gene, is reported to influence the pharmacokinetics of substrate drugs during clinical therapy. The aim of this study was to clarify the mechanisms that regulate the transcription of the chicken Abcg2 gene through cloning and characterization of its promoter region. Results showed that the Abcg2 gene is transcribed by a TATA-less promoter with several putative Sp1 sites upstream from two putative CpG islands. A luciferase reporter assay conducted both in chicken leghorn male hepatoma (LMH) cells and chicken primary hepatocytes mapped a basal promoter to nucleotides −110 to +30, which is responsible for the constitutive expression of Abcg2. The 5′-region upstream of the basal promoter was characterized by both positive and negative regulatory domains. Further, using the cell-based reporter gene assay combined with RT-PCR and drug accumulation analysis, we found that four xenobiotics, daidzein, clotrimazole, ivermectin, and lipopolysaccharide (LPS), influence the expression and function of BCRP through significant regulation of the Abcg2 gene promoter. Interaction sites with the Abcg2 gene promoter of these four selected regulators were clarified by progressive deletions and mutation assays. This study shed some light on the regulatory mechanisms involved in chicken Abcg2 gene expression and the results may have far-reaching significance regarding the usage and development of veterinary drugs.Breast cancer resistance protein (BCRP), an ATP-binding cassette (ABC) half transporter encoded by the  ABCG2, encoded by the Abcg2 gene) is a known member of the ATP-binding cassette (ABC) transporter family that utilizes the hydrolysis of ATP to energize the efflux of a broad range of substrates, including commonly used antimicrobial agents licensed in veterinary medicine [It is currently recognized that the impact of drug transporters on clinically relevant drug disposition and drug–drug interactions (DDIs) is as significant as that of drug-metabolizing enzymes ,2. Breasmedicine ,4. The Fmedicine ,6. Unpremedicine ,8,9. TheAbcg2 gene promoter lacks a TATA box, but possesses several putative Sp1 sites downstream from a putative CpG island. Furthermore, they also found positive and negative regulatory domains upstream from the core promoter [Abcg2 gene. Benoki et al. found a novel constitutive androstane receptor (CAR), a responsive element in the distal promoter of human Abcg2 that enhances the expression of the reporter gene via CAR binding [Abcg2 gene [Abcg2 promoter through the estrogen response element in T47D:A18 cells [Abcg2 promoter via PR elements in breast cancer cells [Abcg2 is also modulated by a remarkable diversity of xenobiotics, including many widely used drugs such as imatinib, toremifene, buprenorphine, norbuprenorphine, and R-methadone [Abcg2 gene as a result of a number of factors, including DNA elements, transcription factors, and compounds. Species-specific variations are also present in regard to regulation [Abcg2 expression is regulated. Therefore, based on the pivotal role of BCRP in the dispositions of some veterinary-labeled drugs, the aim of this study was to investigate the core promotor and cis-acting elements to address the mechanisms involved in the regulation of the chicken Abcg2 gene. The current work aimed to further enrich the current knowledge regarding the factors affecting transporter gene expression, including genetic and pharmacologic regulation of promoter activity and hence the transcription of transporter genes.Although post-transcriptional and translational regulation are involved in BCRP regulation ,11,12, ipromoter . Later, promoter  identifi binding . Recent cg2 gene ,20,21. F18 cells , whereaser cells . Other dethadone ,26,27,28gulation . TranspoThe pGL3-basic vector and Renilla luciferase assay vector pRL-CMV were generously provided by Professor Qian. Rifampicin, clotrimazole, lipopolysaccharide (LPS), and mitoxantrone were purchased from Sigma . Berberine and daidzein were obtained from Aladdin . Enrofloxacin, florfenicol, tilmicosin, sulfadiazine, ciprofloxacin, doxycycline, cefuroxime sodium, and ivermectin were kind gifts from China Institute of Veterinary Drug Control . All other chemicals were of analytical grade and were obtained from local suppliers, unless otherwise mentioned.2. The chicken leghorn male hepatoma cell line (LMH) was obtained from American type culture collection . The cells were grown in roswell park memorial institute (RPMI) medium supplemented with 10% fetal bovine serum, 1% glutamine, and 1% penicillin and maintained at 37 ℃ in 5% CO2.Chicken primary embryonic hepatocytes were isolated from the livers of 9-day old embryonated eggs according to the method described by previous studies ,31. In bAbcg2 gene was amplified via reverse transcriptase polymerase chain reaction (RT-PCR) using adult chicken liver-derived genomic DNA as a template. Based on the chicken genomic sequence 423767, we designed primers to amplify the promoter region of chicken Abcg2, i.e., 5′-GAGTCCAGAGAGGGCCATGAA-3′ (sense primer) and 5′-AAAAGGCCCAGCAGAA-3′ (antisense primer), generating a product of 2411 bp from −1071 to +1339 (relative to the transcription start site). The amplicons were verified by sequencing. The putative transcription factor binding sites of the chicken Abcg2 gene promoter were then predicted using AliBaba2.1 (accessed on March 2019) [The promoter region of the chicken ch 2019) . The CpGAbcg2 gene were constructed in this study. The deletion fragments of the chicken Abcg2 promoter  were separately cloned into a pGL3-basic reporter plasmid via homologous recombination; the generated plasmids were named pGL3-D1, pGL3-D2, pGL3-D3, pGL3-D4, and pGL3-D5. Mutated vectors of the putative chicken xenobiotic receptor (CXR) (−649/−638), nuclear factor-kappaB (NF-κB) (−395/386), glucocorticoid receptor (GR) (-191/-182), and specificity protein 1 (Sp1) (−39/−30) binding sites were generated from pGL3-D1, pGL3-D2, pGL3-D3, and pGL3-D4 using PCR-based, site-directed mutagenesis and named CXR-mut, NF-κB-mut, GR-mut, and Sp1-mut, respectively. The putative estrogen receptor (ER) (−228/−219) binding site mutated vector was also generated from pGL3-D2 and named ER-mut. All of the constructed plasmids were verified by DNA sequencing. The forward and reverse primers set for the plasmid construction were listed in A series of plasmids containing fragments with various promoter region sizes of the chicken ® 2000  was then used to mediate thte transfections using 0.5 µg of the reporter constructs and 2 ng of Renilla luciferase assay vector pRL-CMV to correct the transfection efficiency. A promoter-null plasmid, pGL3-Basic, was also included in the transfection assay as a negative control. After 12 hours, the transfection medium was replaced with the fresh medium either alone or containing individual concentrations of xenobiotics. Cells were incubated for another 6 h and the luciferase activity was measured using a Dual Luciferase Reporter Assay Kit . Xenobiotics, including natural compounds (daidzein and berberine), commonly used drugs  and LPS, were used in the transfection assay. The concentrations of the above xenobiotics were selected according to the cytotoxicity assay results, where the concentrations were based on cell viabilities of no less than 90% compared with the control group [The LMH cells and chicken hepatocytes were seeded in 24-well culture plates and cultured overnight until grown to 70%–80% confluence in their respective mediums, as mentioned in ol group . The finAbcg2 was quantified using SYBR Green Realtime PCR Master Mix  with a real-time PCR detection system . Chicken β-actin was chosen to be the internal control. The relative levels of Abcg2 were analyzed using the 2−ΔΔCt method [The LMH cells and chicken hepatocytes were seeded in 12-well plates and incubated overnight until grown to 70%–80% confluence. The cells were then treated with individual xenobiotics for 6 h. The final concentration of each xenobiotic was 30 µM, except ivermectin (10 µM) and LPS (50 µg/mL). After treatment, the total RNA was isolated using TRIzol Reagent  according to the manufacturer’s instructions and treated with DNase to remove any DNA contamination. RNA was quantified using a photometer  at 260/280 nm. The RNA integrity was confirmed via 1% agarose gel electrophoresis and ethidium bromide staining. Accordingly, cDNA was synthesized using a reverse transcription kit  according to the manufacturer’s protocol. Chicken t method .The LMH cells were seeded in 24-well plates and incubated overnight until grown to 70%–80% confluence and were then treated with individual concentrations of xenobiotics for 6 hours. The final concentrations of the xenobiotics were the same as above. After treatment, the LMH cells were washed with phosphate buffer saline (PBS) and incubated with 5 µM mitoxantrone (a substrate of BCRP) for 60 min. Later, the cells were trypsinized and washed with PBS three times. The fluorescence signals of the samples were recorded using a fluorescence-activated cell sorter (FACS)Calibur with CellQuest Prosofware under 488 nm excitation for mitoxantrone.t-test. p < 0.05 was considered to be significant and p < 0.01 was considered to be extremely significant.All experiments were performed in triplicate and repeated at least in 3 independent experiments. The data were presented as the mean ± standard deviation (SD) of 3 replications. The mean values between the groups were compared using the two-tailed Student’s Abcg2 gene from the chicken genomic DNA , NF-κB (−395/−386), ER (−228/−219), GR −191/−182), and AP-1 (−24/−15 and −22/−13) were recognized in this region , generated five sequentially truncated luciferase reporter constructs, and transfected them into the LMH cells and chicken primary hepatocytes. The transcription activities of the different Abcg2 gene promoter constructs relative to the activity of the pGL3-Basic vector were detected both in the LMH cells and the chicken hepatocytes. In the LMH cells (p < 0.01). Transcriptional activity was lower and only 6.7-fold higher than that of the pGL3-Basic vector when the pGL3-D2 (−509/+30) construct was used, indicating the possibility of positive regulatory element(s) between −1,071 bp and −509 bp. Removing the next 285 bases resulted in an increase in luciferase activity of the pGL3-D3 (−224/+30) construct, which was greater than that seen in construct D2 (a change from 6.7-fold to 8.9-fold), thereby further suggesting the presence of negative regulatory element(s) between bases −509 and −224. The removal of the upstream region (−224/−111) from pGL3-D3 led to a slight decrease in the luciferase activity of construct D4 (−110/+30) (7.3-fold), implicating a weak increase in activity in this region. When the sequence was deleted as far down as −27 bp (pGL3-D5), the luciferase activity fell almost to the same level as pGL3-Basic. Similar trends were also observed when the constructs were transfected into the chicken hepatocytes , the mutation in the region of putative CXR binding site caused a significant decrease (p < 0.05) in the relative luciferase activity levels to 80% and 48% in the LMH cells and chicken primary hepatocytes, respectively , indicating that a negative regulatory element exists in the chicken Abcg2 promoter.We employed site-directed mutagenesis, which disturbed the recruitment of the transcription factors, to further determine the contributions of different transcription factor binding sites to the activity of the chicken shown in . The mutAbcg2 promoter from the commonly used drugs licensed in veterinary medicine, including clotrimazole, enrofloxacin, ciprofloxacin, florfenicol, tilmicosin, sulfadiazine, cefuroxime sodium, doxycycline, and ivermectin, as well as the natural compounds berberine and daidzein. LPS was also testes. As shown in Abcg2 gene promotor, causing 3.2- and 4.9-fold inductions in the LMH cells  the luciferase activity levels of the Abcg2 promoter, with the reporter gene constructs being 0.56- and 0.6- fold in the LMH cells and chicken hepatocytes, respectively . However, daidzein, clotrimazole, enrofloxacin, sulfadiazine, doxycycline, cefuroxime sodium, and ivermectin caused significant increases in Abcg2 mRNA (p < 0.05). In addition, LPS significantly downregulated Abcg2 mRNA in both cell types (p < 0.01). In summary, the variation trends of Abcg2 mRNA were in line with the variation trends of the Abcg2 promoter after all xenobiotic treatments, except for berberine and tilmicosin. Thus, the chicken Abcg2 promoter was activated or inactivated by a remarkably diverse group of synthetic compounds that correspondingly upregulated or downregulated Abcg2 gene expression.Next, we sought to determine whether the xenobiotics, which affected the activity of the atocytes A,B. Aftep < 0.05) the accumulation of mitoxantrone in LMH cells. Among them, berberine and LPS significantly increased (p < 0.01) mitoxantrone accumulation in LMH cells, showing relative fluorescence intensities of 157% and 121%, respectively, and revealing that they could block the mitoxantrone efflux from the cells. However, daidzein, clotrimazole, and ivermectin significantly decreased (p < 0.01) mitoxantrone accumulation, with relative fluorescence intensities of 71%, 75%, and 69%, respectively.A mitoxantrone (a substrate of BCRP) accumulation assay was further carried out in the LMH cells to detect whether the efflux ability of BCRP was changed by the above xenobiotics. The results in Abcg2 mRNA through the chicken Abcg2 promoter and influence the function of BCRP. Therefore, we further identified the interaction sites with the Abcg2 gene promoter of these four significant regulators using progressive deletions and the mutation assay.The above results indicated that daidzein, clotrimazole, ivermectin, and LPS may regulate the expression of p < 0.01). However, the induced function of daidzein disappeared when the pGL3-D2 construct was transfected. Therefore, we assumed that the daidzein may regulate the chicken Abcg2 gene promoter via the putative CXR binding site. To confirm this hypothesis, we introduced mutations into the CXR binding site, then transfected it into LMH cells and chicken hepatocytes and determined the luciferase activity levels. As shown in Abcg2 gene promoter induced by daidzein.Abcg2 gene promoter only when the pGL3-D1 construct (p < 0.01) was transfected compared with pGL3-D2 construct (p > 0.05) in both the LMH cells and chicken hepatocytes  inhibitory effect on the Abcg2 gene promoter when the pGL3-D1 and pGL3-D2 constructs were transfected into both cell types  TCA played an essential role in regulating the chicken CYP2H1 gene [Abcg2 gene, which enhanced chicken Abcg2 gene expression. Similar to the putative CXR element, an ER element was also identified as an enhancer of the chicken Abcg2 gene. This result was consistent with the previous data showing that ER elements enhance the expression of human BCRP mRNA [Abcg2 gene expression. The same results were also acquired in our previous study on the porcine Abcb1 gene promoter [Abcg2 gene due to weakened chicken Abcg2 gene expression, similar to a study reported by Ogretmen and Safa [Cis-acting elements, including enhancers and silencers regulate, gene transcription efficiency through complex interplays with specific transacting factors. Detecting multiple cis-acting elements is critical to understanding transcriptional regulatory mechanisms and gene expression patterns. The results of this study demonstrated that four distinct cis-acting elements, including putative CXR, ER, SP1, and NF-κB, were responsible for chicken 2H1 gene . We founCRP mRNA . The Sp1CRP mRNA . An analpromoter . The NF-and Safa , who fouAbcg2 is also important for predicting drug–drug interactions in veterinary medicine and may be a strategy regarding the development of new drugs. Utilizing a cell-based assay to detect xenobiotic effects on the chicken Abcg2 promoter could be a practical approach in the screening of BCRP inducers or inhibitors, as evidenced by similar studies regarding the screening of Klotho and PXR gene regulators [Abcg2 gene promotor, except for florfenicol, ciprofloxacin, and LPS. Our previous studies showed that many of the compounds, such as berberine, enrofloxacin, and tilmicosin, are substrates of chicken BCRP [Abcg2 promoter, mRNA expression levels, and the transport function of BCRP, thereby suggesting that the net absorption of BCRP substrate drugs increases when bacterial infection occurs in chicken; although this might be favorable in many cases, it bears the risk of undesirable side-effects of the drug with a small margin of safety [Understanding whether a drug is an inducer or an inhibitor of chicken gulators ,47. The ken BCRP . These ff safety ,49.Abcg2 promotor and identified its transcriptional factor binding sites contributing to the complex transcriptional regulation of Abcg2 by compounds. Nevertheless, limitations exist in this work, including that the involvement of transcription factors regulating the chicken Abcg2 gene and mediation of xenobiotic regulation were not investigated. These issues will be addressed in our further studies.In conclusion, we comprehensively described the characterization of the chicken


Saccharomyces cerevisiae. For this, we used new constructs designed to induce expression of mRNA transcripts in trans with respect to the SSA system. We show that unscheduled and recombinogenic DNA:RNA hybrids that trigger the SSA event are formed in cis during transcription and in a Rad51-independent manner. We found no evidence that such hybrids form in trans and in a Rad51-dependent manner.DNA:RNA hybrids constitute a well-known source of recombinogenic DNA damage. The current literature is in agreement with DNA:RNA hybrids being produced co-transcriptionally by the invasion of the nascent RNA molecule produced in cis with its DNA template. However, it has also been suggested that recombinogenic DNA:RNA hybrids could be facilitated by the invasion of RNA molecules produced in trans in a Rad51-mediated reaction. Here, we tested the possibility that such DNA:RNA hybrids constitute a source of recombinogenic DNA damage taking advantage of Rad51-independent single-strand annealing (SSA) assays in the yeast  R loops are structures formed by a DNA:RNA hybrid and the complementary displaced single stranded DNA (ssDNA). They were observed naturally as programmed events in specific genomic sites such as the S regions of Immunoglobulin genes in mammals or mitochondrial DNA , where tThe increasing number of reports showing R loop accumulation in different organisms from bacteria to human cells, and the relevance of their functional consequences, whether on genome integrity, chromatin structure and gene expression suggest that most DNA:RNA hybrids are compatible with a co-transcriptional formation . This isSaccharomyces cerevisiae , and another one providing the in trans LacZ transcripts (p:LacZtet)  . The bac mutants .LacZ recombination system is a leu2 direct-repeat construct carrying the LacZ gene in between and under the GAL1 inducible promoter so that this construct is transcribed as a single RNA unit driven from the GAL1 promoter . As a control of no expression in trans, we used transformants with an empty plasmid to avoid any possible effect from leaky transcription from the tet promoter in the presence of doxycycline.The GL-promoter . Single-t manner . To provLacZ recombination system and the p:LacZtet construct were used to assay SSA events in the four different possible conditions: i) no transcription, with GL-LacZ construct turned transcriptionally off (2% glucose) and an empty plasmid; ii) transcription in trans, with GL-LacZ construct turned transcriptionally off (2% glucose) and the p:LacZtet construct; iii) transcription in cis, with GL-LacZ construct turned transcriptionally on  and an empty plasmid; and iv) transcription in cis and in trans, with GL-LacZ construct turned transcriptionally on  and the p:LacZtet construct , in which the LacZ sequence was inverted so that the LacZ transcript produced in trans would not be able to anneal with the transcribed DNA strand of the GL-LacZi system (hpr1∆ cells when the LacZ sequence was transcribed in agreement with previous reports and with the fact that it has been shown that it is the length (and the GC content) but not the orientation of the lacZ sequence what impairs transcription and triggers hyper-recombination , RNA driven from an ectopic locus (transcription in trans) led to a partial suppression of the hyper-recombination. The reason for such suppression might involve the potential ability of the remotely produced RNAs to interfere with transcription occurring at the GL-ongation , one posi system . We detebination . SurprisLacZ gene is inefficient and leads to unstable RNA products, particularly in tho mutants (LacZ (p:LacZ400tet)  . Strikinrnh1∆ rnh201∆ cells elevated the recombination frequency when transcription was stimulated in cis, as expected. Importantly, the recombination frequencies were not altered by producing transcripts in trans, arguing again against the recombinogenic potential of putative DNA:RNA hybrids formed with RNAs produced in trans.The THO complex is thought to prevent R-loops mainly by promoting a proper mRNA-protein assembly , whereasLacZ sequence  or when the RNA coating functions preventing DNA:RNA hybrid formation are impaired (tho mutants), arguing against the idea that harmful DNA:RNA hybrids could spontaneously form in trans and constitute a menace for genome integrity. Co-transcriptional R-loops are responsible for the hyper-recombination of hpr1∆ as reported previously  for which the action of Rad52 is sufficient . Our coneviously . Putativeviously  reduced inverted  and prevonstruct , which wkgrounds , suggestkgrounds  or when promoter  and 4.S. cerevisiae, in which an induced-DSB triggered recombination between two other homologous fragments at different chromosomes  and its isogenic hpr1∆::HIS3 mutant U678-1C (MATa) and U678-4C (MATα), mft1∆::KANMX mutant (WMK.1A) (rnh1Δ::KANMX rnh201Δ::KANMX (RNH-R), rad51∆::KANMX (WSR51.4A) (hpr1∆::HIS3MX rad51∆::KANMX (HPR51.15A) from this study. rnh1Δ::KAN rnh201Δ::KAN (HRN2.8A) and the wild-type HRN2.8A were from hpr1∆HIS3 mutant were made in this study by insertion of the GL-LacZ::NATMX at the LEU2 locus in Chromosome III of a W303-1A strain corrected for RAD5 (Strains used were the wild-type W303-1A ((WMK.1A) , rnh1Δ::SR51.4A) , and hprfor RAD5 .LacZ  to repress transcription from the plasmids . BrieflyGCGCCGTGGCCTGAT), LacZT1-Rv (GTGCAGCGCGATCGTAATC), Intergenic-Fw (TGTTCCTTTAAGAGGTGATGGTGAT) and Intergenic-Rv (GTGCGCAGTACTTGTGAAAACC). The exact values obtained are shown in Mid-log cultures were grown with either glucose or galactose and with or without 5 μg/ml doxycycline (kept in the dark). Total RNA was obtained using Qiagen’s RNeasy kit and used for cDNA synthesis with the QuantiTect Reverse Transcription kit with random primers (Qiagen) according to instruction. Real-time quantitative PCR was performed using iTaq universal SYBR Green (Biorad) with a 7500 Real-Time PCR machine (Applied Biosystems). Primers sequences used for this analysis were LacZT1-Fw  overnight 37°C as RNase H control. RNA-DNA hybrids were immunoprecipitated using S9.6 monoclonal antibody (hybridoma cell line HB-8730) coupled to Dynabeads Protein A (Invitrogen) for 2 hr at 4°C and washed 3 times with 1x binding buffer. DNA was eluted in 100 μL elution buffer  treated 45 min with 7 μL proteinase K 20 mg/ml at 55°C and purified with Macherey-Nagel DNA purification kit. Primers sequences used for this analysis were LacZT1-Fw (GCGCCGTGGCCTGAT) and LacZT1-Rv (GTGCAGCGCGATCGTAATC). The average and SEM of at least three independent transformants was plotted but the numerical data can be seen in DNA:RNA hybrids were measured in cultures with either glucose or galactose and either with or without 5 μg/ml doxycycline (kept in the dark). Cultures were collected, washed with chilled water, resuspended in 1.4 mL spheroplasting buffer  and incubated at 30°C for 30 min. The spheroplasts were pelleted (5 min at 7000 rpm) rinsed with water and homogeneously resuspended in 1.65 mL of buffer G2 . Samples were treated with 40 μl 10 mg/ml RNase A for 30 min at 37°C and 75 μl of 20 mg/ml proteinase K (Roche) for 1 hr at 50°C. DRIP was performed mainly as described  with fewSpontaneous Rad52-YFP foci from mid-log growing cells carrying plasmid pWJ1344 were visualized and counted by fluorescence microscopy in a Leica DC 350F microscope, as previously described . More th2 at pH 7) and then digested by adding 10 mM DTT and 150 mg/ml of Zymolyase 20T to the same buffer. The digestion was performed for 10 min (37°C) and stopped by mixing the samples with the solution 2 . Later, spheroplasts were centrifuged carefully 8 min at 800 rpm, lysed with 1% vol/vol Lipsol and fixed on slides using Fixative solution . The spreading was carried out using a glass rod and the slides were dried from 2 hr to overnight in the extraction hood.The procedure performed is similar to For the immuno-staining, the slides were first washed in PBS 1X in coplin jars and then blocked in blocking buffer  over 10 min in humid chambers. Afterwards, slides were incubated with the primary monoclonal antibody S9.6 (1 mg/ml) in a humid chamber 1 hr at 23°C. After washing the slides with PBS 1X for 10 min, the slides were incubated 1 hr at 23°C in the dark with the secondary antibody Cy3 conjugated goat anti-mouse  diluted 1:1000 in blocking buffer. Finally, the slides were mounted with 50 μl of Vectashield  with 1X DAPI and sealed with nail polish. For each experiment, more than 100 nuclei were visualized and counted to obtain the fraction of nuclei with DNA:RNA hybrids. The average and SEM of at least three independent transformants was plotted but the numerical data can be seen in  In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:Saccharomyces cerevisiae the authors test whether R-loops can form in trans, meaning that a transcript from locus A could lead to R-loop formation in locus B. Moreover, they test whether R-loop formation is dependent on Rad51, the eukaryotic RecA family recombinase. Using their genetic system and the S9.6 antibody to detect R-loops in wild type and strains with mutations known to affect R-loop, conclusive data are shown that R-loops only form in cis and that R-loops in this genetic system are independent of Rad51. Overall, this work significantly enriches the discussion in the R-loop field and provides an alternative view point of an earlier publication that suggested R-loop formation in trans being catalyzed by Rad51.Using a clever genetic system in the budding yeast Decision letter after peer review:eLife. Your article has been reviewed by three peer reviewers, including Wolf-Dietrich Heyer as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Jessica Tyler as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Catherine H Freudenreich (Reviewer #2).Thank you for submitting your article "Harmful DNA:RNA hybrids are formed in cis and in a Rad51-independent manner" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.eLife'. Please let us know if you would like to pursue this option. As the editors have judged that your manuscript is of interest, but as described below that additional experiments are required before it is published, we would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). First, because many researchers have temporarily lost access to the labs, we will give authors as much time as they need to submit revised manuscripts. We are also offering, if you choose, to post the manuscript to bioRxiv  along with this decision letter and a formal designation that the manuscript is 'in revision at Summary:Saccharomyces cerevisiae the authors test whether R-loop can form in trans, meaning that a transcript from locus A could lead to R-loop formation in locus B. Moreover, they test whether R-loop formation is dependent on Rad51, the eukaryotic RecA family recombinase. Using their genetic system and cytological analysis of Rad52 foci and the S9.6 antibody to detect R-loops in wild type and strains with mutations known to affect R-loop, conclusive data are shown that R-loops only form in cis and that R-loops in this genetic system are independent of Rad51. Overall, this work significantly enriches the discussion in the R-loop field and provides an alternative view point of an earlier publication that suggested R-loop formation in trans being catalyzed by Rad51.Using a clever genetic system in the budding yeast Essential revisions:1) The pGal promoter induces very high transcription in the presence of galactose (often 500X or more induction). The level is likely very different (much less) for the tet promoter, which is generally only induced 2-3X upon addition of doxycycline. This could significantly affect the results – e.g. the cis vs. trans effects could really be a matter of different transcription levels. Transcription levels from each promoter really need to be determined- this is a very important control. The exact induction conditions used, including concentrations and induction times, need to be spelled out in the Materials and methods and should be consistent with those used during the RT-PCR experiment to test transcript levels. In the absence of being able to do the experiment on the constructs used , at least it could be cited if this lab has used the same promoters and induction conditions in the past, and a caveat inserted if transcription levels are different. It would also be good to switch the promoters and make sure the result holds, as there could be issues of differences in timing of transcription as well.LacZ reporter is essentially as high as the regular lacZ construct. This result implies that hybrid formation is insensitive to the orientation of the reporter when in many reported cases, R-loop formation is strongly orientation-dependent. Of course, another hypothesis is that  transcription itself triggers recombination, not R-loops. Without data on R-loop formation, one cannot disentangle transcription from co-transcriptional R-loop formation. The authors must use DRIP-based assay to quantify R-loop levels in the various sequence contexts and under the various genetic backgrounds to establish that their assay is reflective of R-loop levels. Using bisulfite-based readouts to measure R-loop distributions and lengths across the LacZ region would be even better. Without this data, the claim that this new genetic assay can "infer the formation of recombinogenic DNA:RNA hybrids" is unsubstantiated.2) In Figure 2 the authors relate recombination frequencies in their assays to RNA:DNA hybrid formation without measuring hybrids directly. This is a major weakness that significantly limits data interpretation. For instance, I am very surprised that the "cis" recombination frequency of the inverted 3) Source data:The source data file should be labeled better. Missing are:-what the numbers in the table are (rates of Leu+ x 10^-4?)-which data goes with which figure panel-average and SEM numbers should be shown in the data tableThe exact p values not reported and could be added to source data file.N values can be discerned from the source data file but it would be nice for them to be stated in the figure legends. Essential revisions:1) The pGal promoter induces very high transcription in the presence of galactose (often 500X or more induction). The level is likely very different (much less) for the tet promoter, which is generally only induced 2-3X upon addition of doxycycline. This could significantly affect the results – e.g. the cis vs. trans effects could really be a matter of different transcription levels. Transcription levels from each promoter really need to be determined- this is a very important control. The exact induction conditions used, including concentrations and induction times, need to be spelled out in the Materials and methods and should be consistent with those used during the RT-PCR experiment to test transcript levels. In the absence of being able to do the experiment on the constructs used , at least it could be cited if this lab has used the same promoters and induction conditions in the past, and a caveat inserted if transcription levels are different. It would also be good to switch the promoters and make sure the result holds, as there could be issues of differences in timing of transcription as well.Thank you for this comment. We have addressed this issue by switching the promoters  and, indeed, the result holds. In addition, we have checked the transcription levels by RT-PCR  and indicated the exact time and conditions of induction of transcription in the Materials and methods section for further support.As a consequence, it now reads:LacZ recombination system enhanced recombination as previously published , again no significant stimulation of recombination was detected when RNAs were produced in trans in either wild-type, hpr1∆ or rnh1∆ rnh201∆ cells even when the RNA was generated from the strong GAL1 promoter.’‘Given that the levels of transcription from the GAL and tet promoters, used for the constructs are very different , we decided to perform recombination tests with similar constructs in which the promoters were switched. […] Figure 4 shows that, whereas transcription at the TL-2) In Figure 2 the authors relate recombination frequencies in their assays to RNA:DNA hybrid formation without measuring hybrids directly. This is a major weakness that significantly limits data interpretation. For instance, I am very surprised that the "cis" recombination frequency of the inverted LacZ reporter is essentially as high as the regular lacZ construct. This result implies that hybrid formation is insensitive to the orientation of the reporter when in many reported cases, R-loop formation is strongly orientation-dependent. Of course, another hypothesis is that  transcription itself triggers recombination, not R-loops. Without data on R-loop formation, one cannot disentangle transcription from co-transcriptional R-loop formation. The authors must use DRIP-based assay to quantify R-loop levels in the various sequence contexts and under the various genetic backgrounds to establish that their assay is reflective of R-loop levels. Using bisulfite-based readouts to measure R-loop distributions and lengths across the LacZ region would be even better. Without this data, the claim that this new genetic assay can "infer the formation of recombinogenic DNA:RNA hybrids" is unsubstantiated.hpr1∆ and rnh1∆ rnh201∆ mutants as expected.We have determined the RNA:DNA hybrids at the various sequence contexts by DRIP as suggested. Results are shown in new Figure 3. We have observed that DNA:RNA hybrids accumulate in both, hpr1∆ when the transcribed sequence is inverted has been previously reported. In those previous studies we showed that the length (and the GC content) and not the orientation of the sequence impairs transcription and triggers hyper-recombination . This is now remarked on as follows:On the other hand, the hyper-recombination of hpr1∆ cells when the LacZ sequence was transcribed in agreement with previous reports and with the fact that it has been shown that it is the length (and the GC content) but not the orientation of the lacZ sequence what impairs transcription and triggers hyper-recombination .’‘We detected a strong hyper-recombination in hpr1∆ since, which was reported previously , as now stated in the Discussion. We aim to probe that RNAs do not have any recombinogenic potential when produced from an ectopic locus, in trans, in contrast to RNAs produced in cis. We hope we have now clarified the message.Thus, R-loop formation was also expected in the inverted sequence as indeed it is shown in the new results of Figure 3. Perhaps we did not explain ourselves properly. In this study we do not pursue to show that co-transcriptional R-loops are responsible for the hyper-recombination of 3) Source data:The source data file should be labeled better. Missing are:-what the numbers in the table are (rates of Leu+ x 10^-4?)-which data goes with which figure panel-average and SEM numbers should be shown in the data tableThe exact p values not reported and could be added to source data file.N values can be discerned from the source data file but it would be nice for them to be stated in the figure legends.Performed as requested. The source data files now include average and SEM values as well as all the both the exact p values and the asterisks for the statistics depicted in all the graphs. Thank you.