M. xanthus strains. We found that activation of internal promoters in the operon was able to promote the gene transcription, but the activation efficiency was distinct from the activation of separate promoters. The transcription of genes in the operon was influenced by not only the starting promoter but also internal promoters of the operon; internal promoters affected the transcription of the following and neighboring upstream/downstream genes. Multiple interferences between internal promoters thus changed the transcriptional profile of operon genes and the production of epothilones. Better activation efficiency for the gene transcription and the epothilone production was obtained in the low epothilone-producing strains. Our results highlight that interactions between promoters in the operon are critical for the gene transcription and the metabolite production efficiency.The biosynthetic genes for secondary metabolites are often clustered into giant operons with no transcription terminator before the end. The long transcripts are frangible and the transcription efficiency declines along with the process. Internal promoters might occur in operons to coordinate the transcription of individual genes, but their effects on the transcription of operon genes and the yield of metabolites have been less investigated. Epothilones are a kind of antitumor polyketides synthesized by seven multifunctional enzymes encoded by a 56-kb operon. In this study, we identified multiple internal promoters in the epothilone operon. We performed CRISPR-dCas9–mediated transcription activation of internal promoters, combined activation of different promoters, and activation in different epothilone-producing  M. xanthus strains were grown in CYE medium  for routine growth and CMO medium (CYE medium plus 7 mL/L methyl oleate) for the production of epothilones. When appropriate, kanamycin (40 μg/mL), tetracycline (10 μg/mL), and apramycin (25 μg/mL) were added. The growth temperatures were 37°C for E. coli and 30°C for M. xanthus.Total RNA was extracted from cell cultures using BIOZOL kits  and then transcribed reversely into cDNA with PrimeScript™ reagent kit with DNAase . Primers were designed at the junction between two genes . Total Rhttps://www.fruitfly.org/seq_tools/promoter.html) to predict internal promoters in the epothilone gene cluster derived from Sorangium cellulosum strain So0157-2. The threshold was set as 0.8. At the same time, another online promoter prediction software “BPROM” (http://www.softberry.com/berry.phtml) was also employed in the prediction. The −35 and −10 binding regions were predicted in comparison to the σ70 consensus -35 (TTGACA) and -10 (TATAAT) promoter regions of E. coli. The threshold was set greater than 0.8.We used the promoter prediction software “Neural Network Promoter Prediction” . The activity of the reporter gene CAT was detected by using the CAT ELISA kit. The CAT ELISA kit was purchased from Roche and operated according to the instructions provided.The plasmids pkk-232-P E. coli . The promxdcas9 [D10A (GAC→GGC) and H840A (CAC→GCC)], linked to the EcoRI/HindⅢ site of the pSWU30 plasmid. We fused the Omega (ω) protein gene from M. xanthus DK1622 to the 3′-end of mxdcas9 with a 21-nt linker (AAGCTTTCTGGATCAAGTTCT). In front of mxdcas9 is the promoter PcuoA from M. xanthus DK1622, which is inducible by the addition of cupric ions (200 μM).In our previous work , we consThe plasmid pZJY41-sgRNA was used to express sgRNA . To consE. coli competent HB101 cells and obtained the pkk-CuOm strain. Then we transformed two plasmids into E. coli pkk-CuOm at the same time: pZJY41-sgRNA series plasmid carrying sgRNA with different spacer sequences and pKK232-Pepo series plasmid carrying an internal promoter and reporter gene CAT. These three plasmids all replicate autonomously in E. coli. Similarly, we constructed CRISPRa-dCas9 in M. xanthus by introducing plasmids pSWcuomxdCas9-ω and pZJY41-sgRNA. Notably, pSWcuomxdCas9-ω in M. xanthus was inserted at the attB site of the genome, while the plasmid pZJY41-sgRNA replicated autonomously.We transformed plasmid pSWcuomxdCas9-ω into 2O at a flow rate of 1.0 mL/min.ZE9 and mutants were grown overnight in 50 ml of CYE medium supplemented with Apra (25 μg/mL). The cultures were inoculated at a ratio of 2:100 into 50 mL of CMO medium containing 2% of XAD-16 resin. The resin was harvested with a strainer after 6 days and extracted with 3 mL of methanol by shaking at room temperature overnight. The supernatant was centrifuged for 10 min at 12,000 rpm and filtered with a 0.22-μm filter to remove the impurities. Twenty microliter of the sample was injected into a high-performance liquid chromatography  system, analyzed on a Shim-pack MRC-ODS RP C18 column , and monitored at 250 nm, with a mobile phase of 60% of methanol (HPLC grade) and 40% of HThe major epothilone products are epothilones A and B. The structural difference between the two compounds is that there is an extra methyl on 12C in epothilone B . EpothilgapA gene  was chosen as the reference gene for normalization. The transcriptional level of the epothilone gene cluster was analyzed by RT-qPCR on the LightCycler®480 system (Switzerland) with SYBR® Premix Ex Taq™ GC dye . All the primers used in RT-qPCR are listed in We collected samples continuously from the fermentation culture after 48 h of incubation. Then, total RNA of the samples was extracted using BIOZOL kits  and then transcribed reversely into cDNA with the PrimeScript™ reagent kit with DNAase . The S. cellulosum So0157-2 consists of seven ORFs transcribed in the same direction. These ORFs are shortly overlapped or separated with a short distance (15–147 bp), and no terminator structure was found in the intergenic regions  and “BPROM” (http://www.softberry.com/berry.phtml) programs, both of which have been successfully employed for promoter prediction (epoD (shown as red arrows in http://www.softberry.com/berry.phtml) revealed similar results but excavated more potential promoters, including a promoter upstream of epoD , except for epoD (The CRISPR/dCas9 activation system (CRISPRa) is derived from the RNA-mediated CRISPR Cas system by fusing the nuclease-deficient Cas9 (dCas9) with a transcription activator, which combines with the sgRNA to guide the fused dCas9 protein to the target promoter to recruit more RNA polymerases and promote transcription . CRISPRalymerase  under thlymerase , while tlymerase . The prifor epoD . Accordifor epoD .E. coli strains to activate separate promoters of the epothilone operon. According to the CAT activity assay, the transcription activities of all these promoters were significantly improved  not only promoted the transcription of epoE or epoF genes but also affected the transcription of anterior genes of the operon, and even inhibited the first epoA gene. The above results suggested that in situ activation of internal promoters might change the operon transcriptional profile, thus leading to changes in epothilone yields.Furthermore, RT-qPCR analysis of all the operon genes in different activation mutants revealed that activating a specific internal promoter promoted the transcription of not only the target gene but also the nearby genes . For exaepoA (the promoter of epoA) significantly increased the transcription of the operon genes and improved the yield of epothilones by 1.5-fold  in these epothilone-producing strains. As expected, the epothilone yields were all increased in these strains, and the highest 15-fold increase of epothilone yield was obtained in ZE5 with the DEF promoter activation  gene cluster in enteroaggregative E. coli cells; the transcription of T6SS was regulated by the main promoter, and the internal promoter allowed the optimum production of T6SS under the condition where enteroaggregative E. coli encounters competing species , an internal promoter was found in the HSRV operon, which was constitutively activated by the internal promoter and thus resulted in the accumulation of nonstructural proteins inside the host cell at the early stage of HSRV replication . This kith phase . An inte species . In homo species . Taken tE. coli, these internal promoters were constructed in different mutants and thus independently regulate the expression of the reporter gene CAT in a separated form. In M. xanthus, these internal promoters exist in the operon and may be subject to more complex regulation. The potential mechanisms for the different transcriptional activities of promoters are complex. It has been reported that upstream promoters can block the activities of downstream promoters  arrangements of promoters might lead to interactions between RNA polymerases (RNAPs), which causes transcriptional interferences and is often with important consequences for gene expression . In suchli cells . Moreoveli cells . TherefoThe inconsistency in the transcriptional levels of operon genes often limits the yield of secondary metabolites. The inconsistent expression levels of genes in operons observed in different bacterial species are challenging to the concept of operons  and also


Homo sapiens and Mus musculus VDAC genes and their regulatory elements. RNA-seq transcriptome analysis shows that VDAC isoforms are expressed in human and mouse tissues at different levels with a predominance of VDAC1 and VDAC2 over VDAC3, with the exception of reproductive system. Numerous transcript variants for each isoform suggest specific context-dependent regulatory mechanisms. Analysis of VDAC core promoters has highlighted that, both in a human and a mouse, VDAC genes show features of TATA-less ones. The level of CG methylation of the human VDAC genes revealed that VDAC1 promoter is less methylated than other two isoforms. We found that expression of VDAC genes is mainly regulated by transcription factors involved in controlling cell growth, proliferation and differentiation, apoptosis, and bioenergetic metabolism. A non-canonical initiation site termed “the TCT/TOP motif,” the target for translation regulation by the mTOR pathway, was identified in human VDAC2 and VDAC3 and in every murine VDACs promoter. In addition, specific TFBSs have been identified in each VDAC promoter, supporting the hypothesis that there is a partial functional divergence. These data corroborate our experimental results and reinforce the idea that gene regulation could be the key to understanding the evolutionary specialization of VDAC isoforms.VDACs are pore-forming proteins, coating the mitochondrial outer membrane, and playing the role of main regulators for metabolites exchange between cytosol and mitochondria. In mammals, three isoforms have evolutionary originated, VDAC1, VDAC2, and VDAC3. Despite similarity in sequence and structure, evidence suggests different biological roles in normal and pathological conditions for each isoform. We compared  Different statements have been reached regarding the expression and distribution of the different isoforms, the structure of the proteins, the channels functionality, and their involvement in many cellular mechanisms  with either mouse or human recombinant VDAC1 . The human VDAC2 gene holds an additional exon2 in the transcript, responsible for its longer N-terminal sequence of additional 11 amino acids . In this work, we compared human information with those found about murine VDAC isoforms. Several transcript variants are annotated for each isoform, falling into three different groups: coding proteins; processed transcripts; or RNA involved in nonsense mediated decay  of the human VDAC1 gene suggested the generation of two transcript variants, the proteins encoded did not change the amino acid sequences derived from the canonical transcript  were predicted in murine VDAC, and the 5′-UTR located downstream was cloned and studied for its transcriptional activity by the CAT gene reporter assay. Interestingly, the putative VDAC1 promoter was the less active among the three isoforms and lacked any significant activity in antisense orientation, while VDAC2 predicted promoter showed the highest transcriptional activation level  and confirmed its role through the analysis of the best predictors of transcription available in the UCSC genome browser: CpG island location, the RNA polymerase II binding site, the chromatin-state model, and the enrichment levels of the H3K4me1 and H3K4me3 histone marks .We also investigated the promoter core structure and motif elements distribution involved in VDAC gene transcription. A general overview of VDAC promoter regions reveals that, coherently, with the analysis of mammalian promoter properties , which provided data on VDAC genes methylation status in different tissues, cell types, and/or developmental stages  and crossed the results with the data experimentally validated by ChIP-Seq (ENCODE Project v3). With this approach, we found the most relevant families of TFs regulating VDAC genes expression , EBOX (E-box binding factors), KLFS (Krueppel-like transcription factors), NRF1 (nuclear respiratory factor 1), EGRF (EGR/nerve growth factor-induced protein C and related factors), MYOD (Myoblast determining factors) families, controlling cell survival, apoptosis, proliferation, differentiation, development, and metabolism regulation. The overlap of conserved transcription motifs reveals that there was a common evolutionary path of VDAC isoform regulatory regions in mammals. Meanwhile, other distinctive TF families exclusively characterize mouse or human VDAC promoters; however, in line with the previous observation, all of them fall back on those regulators involved in the main biological processes of growth, differentiation, and development in which cell, tissue, and organs need to be controlled in physiological and pathological conditions –6.Interestingly, we found that all mouse VDAC promoters bear specific motifs in their sequences recognized by the family of the p53 tumor suppressor transcription factors, including p63 in addition to p53. Their role in cell death, cell-cycle arrest, senescence, and metabolic regulation in response to cellular stress is well-known to affect cancer development /Forkhead box (Fox); V$LBXF: Ladybird homeobox (lbx)] (Friedman and Kaestner, The promoter analysis of motifs elements involved in gene transcriptional activity and the identification of the regulators by the TFBS distribution, mainly delineate, in the VDAC gene, clusters of evolutionarily conserved transcription motifs. However, we also simultaneously found some distinctive traits corresponding to TF families regulating mouse or human specific VDAC genes. From the biological point of view, the overall picture emerging from the analysis of the specific TFBS, characterizing VDAC promoters, reports a less undifferentiated role of VDAC isoforms in mice. All the isoforms in mouse seem to be involved in the mechanism of cell stress response, in particular in tumorigenesis, while in humans, this role has been assigned to VDAC1.H. sapiens and M. musculus revealed that same functions, at least in terms of abundance/reduction of gene expression, are prevalently conserved among these two organisms, even though with some differences. The difference we found among human VDAC isoforms (Zinghirino et al., The activity of genes is a hallmark of the role of the proteins they encode. This aspect is even more relevant when analyzing a family of protein isoforms. In this work, we attempted to look for a nexus that could explain the evolution of three isoforms of the pore-forming protein VDAC and the need in the higher-evolved organisms to maintain such diversity. The comparison of promoter features of VDAC1-3 genes in close but different species as FG conceived the work. FZ, XP, AM, GN, VDP, and FG wrote the manuscript. FZ and XP equally performed the data collection and analysis. All authors contributed to the article and approved the submitted version.AM, VDP, and FG have an intellectual collaboration with the company, we.MitoBiotech.srl. The remaining 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Pou5f1 enhancer. Here, we demonstrate that this cluster or some member(s) plays an active role in the transcriptional regulation of not only Pou5f1, but also Sox2, Nanog, Klf4 and Esrrb. To clarify in vivo function of the cluster, we performed genome editing using mouse ES cells, in which each of the CFIRs was altered to the corresponding mirror repeat sequence. The alterations reduced the level of the Pou5f1 transcript in the genome-edited cell lines, and elevated those of Sox2, Nanog, Klf4 and Esrrb. Furthermore, transcription of non-coding RNAs (ncRNAs) within the enhancer was also upregulated in the genome-edited cell lines, in a similar manner to Sox2, Nanog, Klf4 and Esrrb. These ncRNAs are hypothesized to control the expression of these four pluripotency genes. The CFIRs present in the Pou5f1 enhancer seem to be important to maintain the integrity of ES cells.DNA can adopt various structures besides the B-form. Among them, cruciform structures are formed on inverted repeat (IR) sequences. While cruciform formable IRs (CFIRs) are sometimes found in regulatory regions of transcription, their function in transcription remains elusive, especially in eukaryotes. We found a cluster of CFIRs within the mouse  Genomes contain many unusual DNA structures, including cruciform structures, left-handed DNA helices, curved DNA structures, triplex structures, and G-quadruplex G4) structures. Among them, cruciform structures have the longest history of study. In 1955, two years after Watson and Crick revealed the right-handed double helical structure of DNA, Platt proposed the presence of cruciform structures ,3,4, tra structurEscherichia coli and Saccharomyces cerevisiae genomes, constructed comprehensive maps of IRs, and classified them depending on their structures and positions, as compared with gene positions. In E. coli, cruciform formable IRs (CFIRs) were significantly enriched in five regions, including the adjacent regions downstream of stop codon-coding sites and on and around the positions corresponding to mRNA ends. Furthermore, most of the CFIRs with a repeat unit length of ≥8 bp and a spacer size of ≤8 were found to be parts of the intrinsic terminators [S. cerevisiae, the close vicinity of the DNA positions corresponding to poly(A) sites was the most statistically significant region of IR enrichment, and these IRs strongly correlated with the poly(A) signal. In addition, the majority of the IRs caused low nucleosome occupancy [To gain insight into the biological role of cruciform structures, we recently performed genome-wide analyses of the minators . Regardiccupancy . These sPou5f1 (Oct3/4) has a CFIR cluster within its enhancer. Based on the FANTOM5 cap analysis of gene expression (CAGE) database [Pou5f1 enhancer produces enhancer RNAs (eRNAs), which are a signature of active enhancers [Pou5f1 was originally regarded as a “general” enhancer [To our knowledge, no report has shown the relevance of IR(s) or CFIR(s) in the functions of enhancers. Using the mouse genome, we recently performed a screening of CFIRs and found that mouse database ,17, the nhancers ,19,20. Tnhancers ,21,22. Jnhancers ,17, the enhancer ,24,25, benhancer . The SEsenhancer .Pou5f1 enhancer, we performed genome editing in which each of the CFIRs was replaced with the corresponding mirror repeat (MR). This alteration downregulated the Pou5f1 transcription, but upregulated the eRNA transcription and Sox2, Nanog, Klf4 and Esrrb transcription. The eRNAs are hypothesized to control the expression of these four pluripotency genes. A delicate transcriptional regulation network seems to exist among these genes, in which CFIRs apparently play an active role.To determine the function of the CFIR cluster present in the Pou5f1 contains 12 IRs with repeat unit (R) lengths greater than or equal to 6 bp, spacer (S) lengths between 0 and 6 bp, and an entire IR length equal to or longer than 15 bp   of the gene  . Each ofthe gene . Among tP method -based gePou5f1 transcription were first examined by qPCR. As shown in Pou5f1 mRNA level was reduced in both the MR/WT and MR/MR cells, and the effect was larger in MR/MR than MR/WT. The transcription levels of the gene in MR/WT and MR/MR were ~85% and ~75% of that in the wild-type cells, respectively. Since the leukemia inhibitory factor (LIF) is required to maintain mouse ES cells in an undifferentiated state [Pou5f1 were gradually reduced in all of the cell lines as time elapsed, the extents were higher in MR/WT and MR/MR cells, especially in the latter , MR/WT and MR/MR cells, effects of the genome editing on ed state ,31, we ae latter b. RegardPou5f1 and the two eRNA species.Active enhancers are transcribed to generate ncRNAs, which are referred to as eRNAs ,19,20,32Pou5f1 reportedly acts as a super-enhancer [Pou5f1, Sox2, Nanog, Klf4, and Esrrb [Sox2, Nanog, Klf4, and Esrrb. Interestingly, the qPCR analysis showed that the transcription levels of these genes all exhibited the same changes in the following sense: the mRNA levels increased in both genome-edited cell lines, and those in MR/WT were higher than those in MR/MR , PE, and DE (Pou5f1 expression [Pou5f1 expression.POU5F1 (OCT3/4) is a key TF for maintaining pluripotency in ES cells and iPS cells ,35,36,37, and DE a [23,24,pression ,44,45,46Pou5f1 TSSs [Pou5f1. Although the negative effects of MRs 2–7 were not large, the presence of the effect was obvious and ~20% transcriptional reduction occurred in MR/MR cells  binding to the 2B sequence, which may have impaired some mechanism in Pou5f1 expression (POU5F1 binding-implicated positive feedback mechanism may exist). This scenario seems plausible if the protein binding induces DNA bending that entails unwinding. In this hypothetical case, a torsional stress imposed on the 2B region by the binding of POU5F1 (and SOX2) can be alleviated by the cruciforms formed on CFIR6 and/or CFIR7, which can rewind structurally unfavorable local DNA unwinding (melting), but MRs 6 and 7 cannot do this. Thus, this editing could have destabilized the binding of these TFs or even inhibited their binding. Many reports have described protein-induced DNA bending and unwinding [E. coli promoter is wrapped around RNA polymerase (DNA is “intensely bent” on the enzyme), it also induces DNA unwinding [The 2B sequence is located between CFIRs 6 and 7 and does not overlap with them a. Distannwinding ,50,51,52nwinding . These DPou5f1 expression in mouse cells [Pou5f1 expression, at least in mice. Although CFIR7 partially shares a sequence with 2A, the alteration of CFIR7 to MR7 did not impair the 2A sequence itself . If this is the case, then most or all of these CFIRs may be used as “absorbers” of the increased negative superhelical density in the region, which seems beneficial in Pou5f1 transcription, at least. Multiple or consecutive cruciforms may be further stabilized by inter-loop base-pairing, such as that found between the D-loop and TΨC-loop in tRNA [We have mainly focused on CFIRs 6 and 7 to explain the phenomena caused by the editing. Finally, we must also discuss the possible functions of all six CFIRs. An important hint may be that cruciform formation can absorb the increased negative superhelicity that leads to DNA strand separation ,63,64. T in tRNA ,66.2. For the 2H2OP method [The mouse ES cell line E14Tg2a  was mainP method -based geEcoRV site of pUC57, was prepared by Genewiz, Inc. . To generate pUC57/KI-puro, a DNA fragment encoding the puromycin-resistance gene in pSF-MinCMV-Puro  was obtained by PCR, using the primers puro-fw-NcoI and puro-rv-XhoI (NcoI and XhoI sites of pUC57/KI-neo. The CRISPRdirect web tool (https://crispr.dbcls.jp/) [BbsI site of px330-U6-Chimeric-BB-CBh-hSpCas9 , to generate px330-gRNA1 to px330-gRNA4. The region containing gRNA expression cassettes in each construct was amplified by PCR, using the primers U6-gRNA-Fw-AflIII and U6-gRNA-Rv-HindIII or U6-gRNA-Fw-HindIII and U6-gRNA-Rv-AflIII   was usedv-AflIII , and insThe genomic DNA was purified using a conventional method. The sequence of the edited region was amplified by PCR with the primers KIcheck-fw and KIcheck-rv . The resTotal RNA was isolated from the cells with a RNeasy Mini Kit  and QIAshredder , and treated with RQ1 RNase-free DNase , according to the manufacturers’ protocols. The resulting sample was subjected to reverse transcription with ReverTra Ace . Briefly, 1 µg of purified total RNA was used in the reaction in the presence of 5 pmol of oligo(dT)20 , and 25 pmol of random primer .The qPCR was performed using the primer sets shown in


Epigenome editing methods enable the precise manipulation of epigenetic modifications, such as histone posttranscriptional modifications (PTMs), for uncovering their biological functions. While histone PTMs have been correlated with certain gene expression status, the causalities remain elusive. Histone H3 Lysine 27 acetylation (H3K27ac) and histone H3 Lysine 4 trimethylation (H3K4me3) are both associated with active genes, and located at active promoters and enhancers or around transcriptional start sites (TSSs). Although crosstalk between histone lysine acetylation and H3K4me3 has been reported, relationships between specific epigenetic marks during transcriptional activation remain largely unclear. Here, using clustered regularly interspaced short palindromic repeats (CRISPR)/dCas-based epigenome editing methods, we discovered that the ectopic introduction of H3K27ac in the promoter region lead to H3K4me3 enrichment around TSS and transcriptional activation, while H3K4me3 installation at the promoter cannot induce H3K27ac increase and failed to activate gene expression. Blocking the reading of H3K27ac by BRD proteins using inhibitor JQ1 abolished H3K27ac-induced H3K4me3 installation and downstream gene activation. Furthermore, we uncovered that BRD2, not BRD4, mediated H3K4me3 installation and gene activation upon H3K27ac writing. Our studies revealed the relationships between H3K27ac and H3K4me3 in gene activation process and demonstrated the application of CRISPR/dCas-based epigenome editing methods in elucidating the crosstalk between epigenetic mechanisms. Genome-wide profiling and gene expression analysis enabled by the next generation sequencing (NGS) technologies have established the correlation between the distribution of certain histone PTMs with particular transcriptional status6. For instance, Histone H3 Lysine 27 acetylation (H3K27ac) preferentially resides at promoters and enhancers and is strongly correlated with active transcription10. Histone H3 Lysine 4 trimethylation (H3K4me3) is typically located around the transcriptional start site (TSS) of active genes, whereas active enhancers are not enriched with H3K4me3 but with histone H3 Lysine 4 monomethylation (H3K4me1)12. However, correlations between histone PTMs and gene activities mapped by these approaches cannot provide mechanistic information in these epigenetic regulatory processes and neither the causal relationships between histone PTMs and transcriptional activities14. Furthermore, combinations of histone PTMs have been proposed to constitute “histone codes” that cooperatively regulate gene expression19. The co-localization of H3K27ac and H3K4me3 at active promoters of active genes revealed by NGS analyses21 suggests potential interplay between these two histone PTMs in transcriptional regulation19. However, the distinct roles of these two active histone marks in transcriptional activation and the underlying molecular mechanisms of their interplay are still not fully understood18.The covalent histone post-translational modification (PTM) is one of the major epigenetic mechanisms where a variety of chemical functional groups can be dynamically added to or removed from both tails and globular domains of histones22. WDR5, however, is also a subunit in H3/H4 acetyltransferase complexes23, making it difficult to elucidate the causal relations of these two PTMs using this method18. Other studies carried out in Drosophila and human cells employing genetic manipulation approaches have revealed that H3K27ac and H3K4me3 are interdependent on each other20. However, how the roles and relationships between these two active marks during the process of transcriptional activation remain to be uncovered. Though pharmacological inhibition26 and genetic manipulation have facilitated the understanding of histone PTMs in epigenetic regulation, these methods cause global alteration of epigenome, resulting in complex secondary effects which may pose obstacles to elucidate the underlying epigenetic mechanisms.Several studies have revealed the crosstalk between H3K4me3 and histone acetylation. H3K4me3 was shown to facilitate H4K16ac enrichment in certain genomic contexts from loss-of-function studies of WDR5, a subunit of COMPASS H3K4me3 methyltransferase complex32. Using this approach, p300 acetyltransferase have been targeted locus specifically to install H3K27ac at promoters and enhancers leading to transcriptional activation of targeted genes33. Furthermore, through a chemical inducible CRISPR/dCas9-based epigenome editing method, which allowed precise temporal mapping of epigenetic and gene expression events, the casual relationship between H3K27ac and transcription has been revealed34. It is known that crosstalk between different epigenetic marks and mechanisms collectively regulate gene activities36. Applying CRISPR/dCas9 and zinc finger protein-based methods, artificially installed H3K4me3 at permissive genome loci has been shown to enable sustained gene reactivation through the crosstalk between H3K4me3 and histone H3 Lysine 79 monomethylation (H3K79me)35.Clustered regularly interspaced short palindromic repeats (CRIPSR)/dCas9-based epigenome editing technologies allow programmable locus-specific manipulation of epigenetic environment without global disruption, which is advantageous in investigating epigenetic regulation in gene activities33 and a newly engineered protein dCas9-SET(CD), containing the core domain of SET1 (an H3K4me3-specific histone methyltrasferase39)  to target the promoters of IL1RN34  and may cause transcription repression40. We decided to use the minimal SET domain  without additional motifs to remove any functions that may interfere with H3K4me3 editing and complicate the resulting gene activity37. Meanwhile, a dCas9-fusion with catalytically inactivated SET(CD) mutant (S208I)39 (termed as dCas9-SET*) was also cloned to confirm the H3K4me3 writing was indeed resulted from the n-SET domain methyltransferase activity37. The expression of these dCas9 fusion proteins in cells were confirmed by western blotting  systems, even though we could successfully install H3K4me3, we did not observe the transcriptional activation of IL1RN, GRM2 genes, based on the mRNA level changes of these genes determined using RT-qPCR ) for H3K4me3 writing. Previously reported dCas9-based H3K4me3 editors used the SET domain from PRDM9ing Fig. . To testIL1RN-specific sgRNA. We observed that the dCas9- SET(CD) with NT-sgRNA did not lead to changes of the H3K4me3  using dCas9-SET(CD) and MYOD1 sgRNAs34  overexpression, we transfected cells with dCas9-SET(CD) along with either non-targeting (NT) sgRNA or me3 Fig. a or H3K2me3 Fig. b level aRNA Fig. c. These s34 Fig. a. We obss34 Fig. b, the les34 Fig. c and MYORNA Fig. d were noOverall, the observed results suggested that deposition of H3K27ac can induce the writing of H3K4me3, but not the other way around, and H3K27ac is sufficient to activate the expression of the tested genes but not H3K4me3. Collectively, it suggested that H3K27ac acts upstream to H3K4me3 and plays a more significant role in transcriptional activation.49. Previous studies showed that the recognition of H3K27ac at enhancers by BET proteins promoted the binding of super elongation complex to chromatin and lead to transcriptional activation10. JQ1 is a potent and selective inhibitor of BET bromodomains. It blocks the binding of readers to acetylated histones and suppress subsequent gene expression51. To confirm dCas9-p300-installed H3K27ac was indeed responsible for the observed gene activation through BRD binding, we tested if blocking BRD-H3K27ac recognition abolishes downstream H3K4me3 writing and gene activation. HEK293T cells transfected with dCas9-p300 and sgRNAs targeting IL1RN or GRM2 locus for 24 h were treated with 2 52 (or DMSO as a control) for another 24 h. Cells were then harvested and subjected to subsequent qPCR assays to determine the changes in IL1RN and GRM2 gene activities, and ChIP-qPCR assays to quantify the level changes of H3K27ac and H3K4me3. We observed that the JQ1 treatment abolished the dCas9-p300/H3K27ac-induced expression of IL1RN and GRM2 when compared to the DMSO treatment conditions  family proteins, including BRD2 and BRD4, are readers that recognize histone acetylation and recruit corresponding cellular machineries to initiate downstream molecular events, including transcription53. It has been reported that BRD4 not only recognizes histone acetylation, but also enhances p300 histone acetyltransferase (HAT) activity through protein–protein interactions outside p300 catalytic HAT domain52. It was shown that the inhibition of BRD4 by JQ1 lead to the reduction of p300 activity and the global downregulation of H3K27ac52. However, we did not observe level changes in dCas9-p300-induced H3K27ac upon JQ1 treatment at the targeted IL1RN or GRM2 loci, which may due to the fact that only the p300 HAT domain was used in the dCas9-p300 fusion protein. Alternatively, it has also been shown that BRD2 does not share the same property as BRD4 in stimulating p300 HAT activity52. It is possible that BRD2, instead of BRD4, functions as the reader for the artificially installed H3K27ac induced by the dCas9-p300 at the tested genomic loci.BRD2 and BRD4 are two of the histone acetylation readers in mammalian BET family proteins and have been reported to have distinct functionsIL1RN and GRM2 loci. HEK293T cells were transfected with plasmids encoding dCas9-p300 or dCas9-PYL with sgRNAs for IL1RN or GRM2 locus and then harvested 48 h after transfection. The samples were subjected to ChIP-qPCR assays against BRD4 and BRD2 to evaluate their level changes at the designated loci  and performed ChIP-qPCR assays against BRD2 and BRD4 to examine the disruption on their recruitment to the RD2 Fig. a,c, but RD2 Fig. b,d, were55. However, we cannot rule out the possibility that H3K4me3 is a by-product of gene expression in our studies. Given the dynamic nature of epigenetic regulation in gene expression56, uncovering the relationships between different epigenetic marks and mechanisms requires new tools capable of resolving the temporal order and kinetics of molecular events occurred in these processes57. Inducible epigenome editing methods allowing precise temporal control have shown great potential in dissecting complex epigenetic mechanisms59. We anticipate that applying these temporally controlled technologies with genome targeting methods such as CRISPR methods can offer unparalleled insights in epigenetic functions and interplays of histone PTMs58.In this study, we investigated the potential crosstalk between two active histone PTMs, H3K27ac and H3K4me3, and their roles in transcriptional regulation using CRISPR/dCas9-based epigenome editing approaches. We observed that H3K27ac writing at promoters through the dCas9-p300 editing system induced H3K4me3 installation around TSS and resulted in active transcription, whereas H3K4me3 artificially installed by dCas9-SET(CD) was unable to activate targeted genes or induce H3K27ac enrichment. BRD2, but not BRD4, was shown to be recruited after H3K27ac writing. The inhibition of BRD2 by JQ1 under the dCas9-p300-induced H3K27ac writing condition coincided with the abolished H3K4me3 installation and the impaired expression of dCas9-p300 targeted genes. Our results suggested that BRD2 translated the acetylation of H3K27 into transcriptional activation and potentially mediates the crosstalk between H3K27ac and H3K4me3 in the CRSIPR/dCas9-based epigenome editing systemsHEK293T cells were maintained in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum  in humid atmosphere containing 5% carbon dioxide. Cells were cultured at 37 °C. Cells were platted into 10 cm dishes or 24-well plates one day prior to transfection to allow the confluency of 70%-80% for subsequent transit transfection. For transfection of the cells in a 10 cm dish, 8 60 (amino acid: 1414–1707) was obtained by polymerase chain reactions (PCRs) with CloneAmp HiFi PCR Premix . The point mutation (S208I) of SET catalytic domain of SET1A was introduced through overlapping PCR approach.The DNA fragment of the minimal SET catalytic domain of methyltransferase SET1ASET(CD) F: 5’-GGCGCGCCGCCTACGAGCCACGCAGTGAGSET(CD) R: 5’- GTTTAGGGAGCCCCGGCAGCTSET*(S208I) F: 5’-TACGTGCAGGAGGGCATTGGCATTAGCTACCTGTTCCGGGTGGACSET*(S208I) R: 5’-GTCCACCCGGAACAGGTAGCTAATGCCAATGCCCTCCTGCACGTAChIP assays were performed with the guidance of SimpleChIP Plus Sonication ChIP protocol from Cell Signaling Technology (CST). To crosslink proteins to genomic DNA, the final concentration of 1% formaldehyde was gently added to the cell culture dish and incubated for 10 min at room temperature. Then 1 ml 1 M glycine was added into each 10 cm dish containing 10 ml cell culture medium and incubated for 5 min to quench the crosslink. The cell culture medium was gently removed and the fixed cells were washed twice with 10 ml cold PBS with 1X protease inhibitor cocktail . Cells were scraped followed by centrifugation at 1000 g for 5 min at 4 °C to collect cell pellets. Chromatins were obtained with cell lysis buffer  and nuclear lysis buffer  according to the protocol. Chromatin was sonicated at 4 °C by Bioruptor Pico (Diagenode) with the program of 10 cycles of 30-s on and 30-s off. An IP reaction was set up with 410 Total RNA was isolated from harvested cells by Trizol reagent  along with chloroform, isopropanol and 75% ethanol treatment according to the manufacture’s standard protocol. To extract RNA from cells seeded in 10-cm dish, cell culture medium was firstly removed and 2 ml Trizol reagent was added directly into the cell culture dish followed by pipetting the lysate several times. Cells with Trizol were collected into centrifuge tubes and were incubated at 4 °C for 3–5 min to allow complete dissociation of proteins and nucleic acids. 0.2 mL chloroform per 1 ml Trizol reagent was added to each tube. With the cap of microcentrifuge tube secured, the tube was shaken vigorously for 2 min and incubated at 4 °C for 10–15 min. Samples were centrifuged for 15 min at 4 °C and the aqueous phase containing RNA was transferred into a new tube. Next, 0.5 mL isopropanol per 1 mL Trizol reagent was added into the new tube with aqueous phase to precipitate RNA. The tube was incubated at 4 °C for 10–30 min followed by centrifugation at 13,500 rpm at 4 °C. Supernatant was discarded carefully and the gel-like crude RNA precipitate was saved and washed with 1 mL 75% ethanol per 1 ml Trizol reagent used. The RNA in ethanol was pelleted with centrifugation and air dried. The dried RNA was dissolved in 50–100 Reverse transcription was performed with 50–200 ng total RNA as template using iScript super mix . A 20 The 0.5 GAPDH was used as housekeeping gene internal control and was in parallel detected with a SYBR green mix  and in-house designed primers. In ChIP-qPCR analysis, 2% input was applied for further normalization and was in parallel assayed by a SYBR green mix  as suggested by the CST SimpleChIP Plus Sonication protocol.Further analysis of the Ct values was calculated as describe in our previous publication with minor modifications. For gene expression analysis, The thermal profiles of qPCR reactions are listed below:Step 1 Polymerase activation and DNA denaturation for 30 s (cDNA) at 95 °C or for 3 min (genomic DNA/ChIP DNA).Step 2 Denaturation at 95 °C or 98 °C for 15 s.Step 3 Annealing/ extension and plate read at 60 °C for 1 min.Go to step 2 and amplification for 40 cycles.Step 4 Dissociation at 95 °C for 15 s, then 60 °C for 1 min and 95 °C for 15 s.t-test. In this manuscript, a p value less than 0.05 was marked as *, less than 0.01 as ** and less than 0.001 as ***. Detailed p values were listed in the supplementary information.All of the above statistical analyses were performed by GRAPHPAD Prims 6 with default settings. In brief, statistical significance of differences between experimental and control groups in ChIP-qPCR assays and gene expression qPCR assays was evaluated by multiple Supplementary Information.


Toxoplasma gondii is a protozoan parasite that differentiates from acute tachyzoite stages to latent bradyzoite forms in response to environmental cues that modify the epigenome. We studied the distribution of the histone variants CenH3, H3.3, H2A.X, H2A.Z and H2B.Z, by genome-wide chromatin immunoprecipitation to understand the role of variant histones in developmental transitions of T. gondii parasites.T. gondii epigenome including genes that are poised for expression.H3.3 and H2A.X were detected in telomere and telomere associated sequences, whereas H3.3, H2A.X and CenH3 were enriched in centromeres. Histones H2A.Z and H2B.Z colocalize with the transcriptional activation mark H3K4me3 in promoter regions surrounding the nucleosome-free region upstream of the transcription start site. The H2B.Z/H2A.Z histone pair also localizes to the gene bodies of genes that are silent but poised for activation, including bradyzoite stage-specific genes. The majority of H2A.X and H2A.Z/H2B.Z loci do not overlap, consistent with variant histones demarcating specific functional regions of chromatin. The extent of enrichment of H2A.Z/H2B.Z (and H3.3 and H2A.X) within the entire gene (5’UTR and gene body) reflects the timing of gene expression during the cell cycle, suggesting that dynamic turnover of H2B.Z/H2A.Z occurs during the tachyzoite cell cycle. Thus, the distribution of the variant histone H2A.Z/H2B.Z dimer defines active and developmentally silenced regions of the Histone variants mark functional regions of parasite genomes with the dynamic placement of the H2A.Z/H2B.Z dimer implicated as an evolutionarily conserved regulator of parasite and eukaryotic differentiation.The online version contains supplementary material available at 10.1186/s12864-022-08338-6. Eukaryotic cells have specialized macromolecular complexes that organize and pack DNA inside the nucleus. These multiprotein complexes include histones  and non-histone proteins. DNA is wrapped around an octamer formed by two copies of each histone to form the nucleosome. Nucleosome assembly is initiated with the association of tetrameric H3-H4 with DNA, followed by two dimers of H2A-H2B. A fifth histone, H1 or linker histone, helps to pack 20 bp of additional internucleosomal DNA [Histones are small, basic proteins that represent about 50% of the total volume of chromatin , 3. The Toxoplasma gondii chromatin structure has been implicated as an important regulator of gene expression. T. gondii is an opportunistic parasite that infects between 10 and 90% of the population depending on the country [T. gondii infection is asymptomatic in healthy people, untreated clinical toxoplasmosis is lethal in immunocompromised individuals such as transplant or HIV patients. In addition, toxoplasmosis is an important cause of congenital defects or fetal demise when women become infected during pregnancy. In humans and other intermediate hosts, T. gondii tachyzoite stages are associated with acute disease, while bradyzoites within tissue cysts persist in a chronic phase. The ability of the parasite to persist as a latent bradyzoite form is critical for disease pathogenesis and the developmental transition from tachyzoite to bradyzoite is likely driven by epigenetic processes involving histone PTM and histone localization [ country . Althouglization .T. gondii expresses four canonical histones, represented by single copy genes, except for H2B that is encoded by two separate genes on different chromosomes [omosomes , 10. H2Bomosomes , 12 . H4omosomes . H2A hasomosomes  . To datT. gondii  [T. gondii [T. gondii centromeres are arranged in a single apical cluster, represented by a dot that is attached to the centrocone during all cell cycle by immunolocalization [H3.3 differs from H3 by just four amino acids in s 88–91) . Althougs 88–91) . CenH3 i. gondii . T. gondlization .T. gondii H2A.X has the conserved SQ(E/D) φ motif important for DNA repair (φ represents a hydrophobic amino acid and S is a serine that is phosphorylated in response to DNA damage). Serine 132 of H2A.X is phosphorylated in tachyzoite forms of T. gondii and plays a role in DNA repair [A repair , 17. TgHA repair .T. gondii tachyzoites. H2B.Z interacts with H2A.Z but not H2A.X [T. gondii tachyzoites revealed that both H2B.Z and H2A.Z are hyperacetylated in their N-terminal domains, a classic hallmark of active chromatin [H2B.Z (previously known as H2BV), is a variant unique to Apicomplexa , 18, 19 ot H2A.X . Mass sphromatin , and bothromatin . By conthromatin .T. gondii genome, using Chromatin Immunoprecipitation. Our findings implicate histone variant exchange in regulation of stage-specific transcription and expand our understanding of histone variants in eukaryotes.In this study, we surveyed the genome-wide location of histone variants across the T. gondii histone variants or endogenous epitope-tagged proteins, and imaging by deconvolution fluorescence microscopy  followed by next generation sequencing [ChIP followed by tiling microarray hybridization (ChIP-chip) or high throughput sequencing (ChIP-seq) are widely used for studying genome-wide protein-DNA interactions. In previous work, we applied ChIP-chip to protozoa . We stanquencing . The digquencing , 22.T. gondii, both ChIP-seq and ChiP-chip were performed using antibodies described above and epitope-tagged lines in the Type I RH strain background. A summary of experiments and platforms used is shown in additional file To study the genome-wide localization of histone variants in T. gondii, H2A.X and H2A.Z are found in different nucleosomes [The correlation between histone variant locations was examined by correlating the read densities of each histone variant ChIP-seq dataset Fig. B. H2A.Z leosomes , 14. TheIn tachyzoites, a majority of genes were marked by H3K4me3 (~ 75%), H2A.Z (~ 80%) and H2B.Z (~ 90%) , CenH3 is absent and H2A.X and H3.3 are enriched  was used to calculate the statistical likelihood of enrichment or overrepresentation of genes in a particular gene set compared to a randomized gene set of the same size, yielding a p-value of enrichment which we term ‘enrichment score’. Overrepresented gene sets were defined as those with a p-value of < 0.05 in the hypergeometric test [To investigate whether this trend occurs genome-wide, we extracted lists of genes marked by histone variant peaks and performed gene set enrichment analysis using previously defined gene sets , 33. Thiric test , 34, 35.As a comparison, the same analysis was performed on H3K4me3, H2A.X and H3.3 peaks. As expected, since H3K4me3 is exclusively found at promoters in every organism to date, tachyzoites genes are overrepresented among genes with 0–40% H3K4me3 coverage, and genes with 40–60% H3K4me3 coverage are enriched in tachyzoite and not bradyzoites genes. Genes marked with 80–100% H2A.X and H3.3 are statistically enriched for tachyzoite genes, but not bradyzoite genes Fig. D. TogethT. gondii, we used the enrichment analysis method described above to compare genes with different amounts of histone variant coverage to cell cycle gene sets defined previously [T. gondii cell cycle, there are two waves of transcription corresponding to G1 and S/M phases [Canonical histones play a critical role in cell cycle control. Their synthesis is coupled to DNA replication during S-phase and is essential for chromatin assembly. In contrast, histone variants are synthesized throughout the cell cycle and whether they affect cell cycle progression is unclear . To inveeviously  . DuringM phases . G1-reguT. gondii G1 is divided into G1a followed by G1b, substages of G1 that are characterized by different sets of cell cycle expressed genes. Differentiated bradyzoites are predominantly in G1 [Genes with low amounts of H2A.Z and H2B.Z coverage (0–20% and 20–40%) are enriched in G1 genes, while genes expressed later in the cell cycle have higher amounts of coverage Fig. . Interesly in G1  and singly in G1 .Fig. 6H2Turning to S/M-regulated genes, genes with both low and high levels of H2A.Z and H2B.Z coverage are overrepresented in genes upregulated in mid-S/M (3–5 h) phase. This is the time period during which parasites begin to express bradyzoite markers that differentiate to bradyzoites , and manA similar analysis showed that genes with high coverage (80–100%) of H2A.X and H3.3 are enriched in G1 genes upregulated between 5 and 7 h of the cell cycle Fig. . Genes wT. gondii, an opportunistic parasite with worldwide distribution, has several developmental transitions that are regulated by epigenetic processes. Here, we provide a genome-wide overview of localization of T. gondii histone variants, which are linked to chromatin structure and regulation of gene expression. T. gondii has five histone variants: H3.3, H2A.X, H2A.Z, H2B.Z and CenH3. CenH3 has a conserved function at centromeric regions in eukaryotes including T. gondii [. gondii  and we c. gondii  or sequePlasmodium falciparum, H3K4me3 and H3K9ac were shown to be present on promoters of active genes in schizont stage but on both active and silent genes in ring stages [Plasmodium varies from T. gondii in having a longer 48-h asexual cell cycle (vs 6–8 h for T. gondii), therefore chromatin state and gene expression can be examined simultaneously in synchronized cells, an approach that is technically challenging given the short life-cycle of T. gondii and the large amounts of biological material required for N-ChIP.In tachyzoite stages, H3K4me3 is absent on promoters of bradyzoite genes and other silent genes. In the Apicomplexan g stages . The difT. gondii promoters, H3K4me3 colocalizes with H2A.Z and H2B.Z, whereas, when present on gene bodies, H2A.Z and H2B.Z are not accompanied by H3K4me3, suggesting an additional role for these histone variants, perhaps in gene silencing. An open chromatin state, marked by H2A.Z/H2B.Z, H4ac and H3K4me3, is required for expression of genes in asexual stages of both Plasmodium spp. and T. gondii [At . gondii , 40. H2A. gondii  and coloP. falciparum, the H2B.Z/H2A.Z dimer is enriched at TSS and 5′-UTR and is associated with well-known activation marks H3K4me3 and H3K9ac as well as H3K18ac and H3K27ac [var genes encoding variant antigens, but is present at the promoter of the single active var copy [H2A.Z is the most evolutionarily conserved histone variant, while H2B.Z is specific to Apicomplexan parasites. Both these variants differ from canonical histones within the N-terminal domain. H2A.Z has an additional ~ 20 aa region that is extensively modified by PTM [ H3K27ac . The H2Avar copy .T. gondii, these broad H2A.Z/H2B.Z peaks may play a role in state-specific gene regulation during the tachyzoite-bradyzoite transition.Bradyzoite genes are silent in tachyzoites and rarely expressed in the RH strain. H2A.Z/H2B.Z coverage of silent bradyzoite genes extends from the promoter along the gene body, but the levels detected are lower than the prominent peaks seen at promoters of active tachyzoite genes. We also observed enrichment of H2A.Z and H2B.Z on gene bodies on a subset of merozoite genes  is enriched at promoters of poised and active genes, but deacetylated H2A.Z  is spreal domain , a classl domain . Taken ttivation .T. gondii, H2A.Z and H2B.Z each have seven ubiquitination sites in both N- and C-terminal domains with unidentified functions [T. gondii. Acetylated H4K31 is enriched in promoters of active genes, whereas methylated H4K31 is found in silent regions and the body of inactive genes [T. gondii gene expression and developmental transitions.Several other PTM have been detected on H2A.Z , 48. Whiunctions . Ubiquitunctions , residueunctions  and tranunctions , respectunctions . Acetylave genes . CollectT. gondii.In mammalian cells, deacetylated H2A.Z localizes to centromeres and co-localizes with the classic centromeric marks H3K4me2, H3K9me2 or H3K9me3 , 53. In T. gondii [H2A.X is commonly associated with double strand DNA break repair, including in . gondii , but als. gondii . H2A.X l. gondii . In contT. gondii  replaces canonical H3 in centromeric chromatin [T. gondii, H3.3 and CenH3 both appear to be present in centromeric regions, as confirmed by mass spectrometry (unpublished data). In contrast to T. gondii, P. falciparum H3.3 is not present at centromeric regions [Drosophila melanogaster and humans, where centromeric chromatin was shown to contain CEN-PA containing nucleosomes interspersed with canonical H3-containing nucleosomes [Functional regions of chromatin, telomeres and centromeres, have distinct structures and composition. This study provides insight into the composition of nucleosomes present in these regions in dii Fig. . In eukahromatin . In T. g regions , suggestleosomes .Fig. 7MoT. gondii. In addition, H3.3 and H2A.X were both detected at telomeric regions in T. gondii. In mammalian cells, H2A.X is important for the correct arrangement of telomere ends during meiotic prophase [In mammalian cells, CEN-PA is recruited to centromeric regions by formation of double stranded breaks to which H2A.X is also recruited . Given tprophase , while Hprophase .T. gondii integrates signals to either initiate the differentiation program or begin DNA synthesis and cell replication.As well as histone variants having a potential role in stage-specific gene expression, we also noted a cell cycle-dependent pattern of variant histone distribution. In these experiments with asynchronous cultures, which are primarily in G1 phase, genes with less H2A.Z/H2B.Z coverage (covering only the 5′ end of the gene) are those upregulated at an earlier time point of cell cycle compared to those with more coverage. Genes where the H2A.Z/H2B.Z dimer colocalizes with H3K4me3 on promoters and also localizes on gene bodies, are genes expressed in mid-S/M phase. The timing of the tachyzoite-bradyzoite developmental transition is also linked to the cell cycle , 38, 62,T. gondii. We hypothesize that variant histones regulate stage-specific gene expression by demarcating functional regions of chromatin. H2A.Z/H2B.Z dimer is implicated as a critical player in regulation of both gene activation and gene silencing. Future work is needed to determine how the localization of histone variants evolves during T. gondii bradyzoite differentiation and the mechanisms by which H2A.Z/H2B.Z and H2A.X/H2B dimers are exchanged.Together, this work suggests that histone variants regulate cell cycle and stage-specific gene expression in 2. Confluent HFF cells were infected with tachyzoite forms of T. gondii  were grown in Dulbecco’s Modified Eagle Medium (DMEM), supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin and maintained at 37 °C with 5% COHΔhxgprt ) at a raΔhxgprt background [H2B.Z ORF in the plasmid pTUB8mycGFPtailTy-HX in frame with a c-Myc tag at the N-terminal end using selection for HXPRT into the RHΔhxgprt strain. The H3.3-HA was cloned by placing the H3.3 ORF in-frame with a C-terminal HA tag and was also transfected into the Type I RHΔhxgprt background, and stable clones created using selection for HXPRT. For IPs that required antibodies, parasites were purified from host cell debris using a 3.0 μm Nucleopore filter (Whatman) until no human cells were visualized. After two washes with cold PBS, cell pellets were frozen in liquid nitrogen.Endogenous epitope tagged variant H2A.X histone strain in the Type I RHckground  was subj6 freshly lysed tachyzoites. After 24 h, samples were fixed with 4% p-formaldehyde in PBS for 20 min at room temperature (RT). Fixed cells were permeabilized with 0.25% of Triton X-100 for 10 min at RT followed by blocking with 1% of bovine serum albumin (BSA) for 30 min. Primary antibodies were diluted in PBS and incubated for 2 h at RT. For the tagged lines, the following commercial antibodies were used: H3.3-HA tag ; H2A.X-myc and H2B.Z-myc . We also used specific polyclonal antibodies against T. gondii histone variants H2A.Z, H2B.Z and H2A.X .Following incubation, cells were washed twice with PBS and incubated with secondary antibodies (Alexa fluor 488-Molecular Probes Cat No A-11034) and 10 μg/mL of 4′,6-diamidino-2-phenylindole (DAPI) to detect nucleic acids. Slides were mounted with Vectashield (Vector Laboratories) and analyzed with a Delta Vision Elite Core Deconvolution microscope. Imaging was performed on an inverted Olympus IX71 microscope with 60x objective ; Z-stacks of 0.1 μm were collected and deconvolution was performed using SoftWoRx 3.6.0. Images were processed with ImageJ Fiji 1.46 J, Adobe Photoshop CS3 and Adobe Illustrator CS3.8) were resuspended in Suspension Solution (SS)  and lysed in the same solution with 0.2% NP-40 for 5 min on ice. Lysed parasites (nucleus-enriched) were washed once with SS and once with MNase Solution . MNase  Cat # 70196Y) digestion was performed in 100 μl of the same solution and 5 U of the enzyme for 5 min at 37 °C. The reaction was stopped by adding 200 mM EDTA and incubating for 5 min on ice. After centrifuging, supernatants were stored (sample 1). Pellet were resuspended in 1 ml Lysis Buffer  and dialyze overnight with lysis buffer .Freshly collected or frozen parasites (3-5 × 103) twice for 15 min each at room temperature with rotation. A sample of input DNA was saved before antibody incubation and the final eluted samples were treated with proteinase K for 1 h at 45 °C.After dialysis, the supernatant was combined with sample 1 and diluted 10 fold in dilution buffer . The sample was precleared with magnetic beads for 30 min at 4 °C and incubated with 20 μl polyclonal antibodies or 2 μl anti-HA, Myc or H3K4me3 (Millipore cat No 07–473) overnight at 4 °C with rotation. DNA-protein-antibody complexes were recovered by magnetic beads coupled to Protein A (Thermo Fisher Cat No 10001D) for 2 h at 4 °C and beads were washed 3 times with the following solutions before washing 6 times with TE (Triton-EDTA) buffer: Low Salt Wash Buffer: 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, 150 mM NaCl, pH 8.1; high salt wash buffer: 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, 500 mM NaCl, pH 8.1; LiCl wash buffer: 0.25 M LiCl, 1% NP40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, pH 8.1. Elution was performed with freshly made elution buffer . The concentration of the purified DNA was determined with the Quant-iT™ PicoGreen system (Invitrogen). ChIP samples were submitted to the Einstein Epigenomics Shared Facility (ESF) for sequencing. Adapters were ligated to DNA to create the Illumina library and sequenced using a Hiseq2500 sequencing machine (Illumina). This involves a first step of cluster generation in situ on the flow cell followed by massively-parallel sequencing using fluorescently-labeled reversible terminator nucleotides. Clustered templates were sequenced base-by-base during each read. Images generated by the Hiseq2500 were processed by Hiseq Sequence Control Software (HCS V2 with RTA v1.17 and CASAVA 1.8 software packages). These software components perform the functions of image analysis (Firecrest) and base-calling (Bustard).https://cutadapt.readthedocs.io/en/stable/) and aligned to the T. gondii ME49 genome  using Bowtie2 (http://bowtie-bio.sourceforge.net/bowtie2/index.shtml). For coverage plots, aligned BAM files were normalized by library size and converted to bigwig format using deeptools bamCoverage (http://deeptools.readthedocs.io/en/).Illumina adapter sequences were trimmed from reads using cutadapt (http://deeptools.readthedocs.io/en/latest/) to calculate Pearson correlations between the read densities of samples followed by visualization in a heat map generated using deeptools plotHeatmap (http://deeptools.readthedocs.io/en/latest/content/tools/plotHeatmap.html).Bigwig files were compared by cluster analysis using deeptools computeMatrix  and Homer FindPeaks (http://homer.ucsd.edu/homer/). Peak lists were compared using the findOverlapsOfPeaks function in in the Bioconductor R package ‘ChIPpeakAnno’ (http://bioconductor.org/packages/release/bioc/html/ChIPpeakAnno.html). For histone variants where the enriched domains were generally less than 5000 bp, MACS2 was applied ; for proteins with enriched domains larger than 5000 bp  and with high similarity to input, Homer was used (http://homer.ucsd.edu/homer/motif/). A minimum FDR of 0.05 was applied.Peaks were called using MACS2 (Pair files (IP and input) were combined and processed using NimbleScan software (NimbleGen) according to the software manufacturer’s instructions. A minimum FDR of 0.05 was applied to define peaks. Scaled log transformed ratio files (IP/input) were generated in NimbleScan and visualized with IGV software.http://bioconductor.org/packages/release/bioc/html/DiffBind.html). Peak lengths and numbers were calculated from peak files determined from NimbleScan (ChIP-chip), MACS2 (ChIP-seq) and Homer (ChIP-seq).Correlations between ChIP-seq and ChIP-chip were calculated at the peak level using the Bioconductor R package ‘diffbind’ (http://bioconductor.org/packages/release/bioc/html/ChIPpeakAnno.html) was used to generate a merged BED file containing all overlapping peaks with a minimum of 50 bp overlap. Peaks were annotated using ChIPpeakAnno annotatePeakInBatch function using a GFF genome annotation file downloaded from ToxoDB.org  was used to plot composite ChIP-seq profiles by averaging the read density of library size-normalized BigWig files over various genomic regions, with 500 bp upstream and downstream flanking regions.Deeptools plotProfile , compared to a FASTA file containing either the entire T. gondii genomic sequence or promoter regions as the background.DNA sequences from overlapping H2A.Z, H2B.Z and H3K4me3 peaks were extracted and searched for motifs using the commandline version of Homer  and IGV format file for visualization. The ratio file was used to determine statistically significant peaks of ChIP-chip signal enrichment using NimbleScan. ChIP-seq data was preprocessed by trimming adapter sequences from raw reads and assessing quality using FASTQC. Reads were mapped to the T. gondii genome and converted to BigWig format for visualization and downstream analysis. Statistically significant peaks were identified using either the program MACS2, using parameters for detection of narrow or broad peaks, or the program Homer, with or without an input file as a control. Correlations between BAM files were determined and graphically represented using the function computeMatrix from the package deeptools. Read density plots were generated from BigWig files. Correlations between statistically significant (FDR < 10%) ChIP-seq and ChIP-chip peaks were calculated and a clustered heat map was generated using deeptools. Peaks detected using both platforms and peaks detected in replicates were intersected to provide a set of reproducible peaks for each histone variant. Reproducible peaks were annotated to determine closest genomic features including genes. Genes where histone variants were localized to were subjected to enrichment analysis to determine whether they are enriched for genes with particular function, localization or specific timing of expression. In addition, DNA sequences corresponding to reproducible peaks were analyzed to determine whether they contained motifs that could be bound by transcription factors.Additional file 2. Correlations between ChIP-chip and ChIP-seq datasets. A. Genome browser view of ChIP-chip ratios over various genes in T. gondii. ChIP-chip outputs plot the scaled log-transformed ratio of experimental IP to input. Genes are identical to those displayed in Fig. B. Plot showing number of peaks detected using ChIP-seq and ChIP-chip platforms. The mean number of peaks detected in replicates are shown. C. Correlations between all peaks detected by ChIP-seq or ChIP-chip were calculated from peaks called with Homer , NimbleScan  or MACS . Data was analyzed and plotted using the R package Diffbind. Color bar above the heat map indicates factor (histone variant – see heat map labels), technique , and replicate . The heat map color key is shown in the upper left with darker blue indicating the strongest correlations. As shown in the heat map, the H2A.Z, H2B.Z and H3K4m3 peaks from ChIP-chip and ChIP-seq showed the strongest correlations. Peaks for each variant or histone modification are the intersection of peaks from each biological replicate (ChIP-seq and ChIP-chip). D. Plot showing lengths of peaks from ChIP-seq and ChIP-chip platforms. Data from replicates were combined into one sample.Additional file 3. Correlation between ChIP-seq datasets. Clustered heat map of correlations between all ChIP-seq alignment files for: A. 1 kb bins genome-wide; B. Restricted to promoter regions only. Samples are indicated on the Axes  is plotted. B, C, D. Plots depicting the percentage of ChIP-seq peaks with different locations of ChIP-seq peaks relative to the annotated promoter  (B), transcriptional start site (TSS) (C), gene start codon (D).Additional file 5. Average histone variant ChIP-seq profiles across different genomic features. Plots show normalized read density  for each ChIP-seq sample. A. RNA gene classes include: transcribed genes mRNA, ribosomal RNA rRNA, transfer RNA tRNA. B. Genes with different mRNA expression levels in tachyzoites. T. gondii genes were split into 5 equal groups based on their expression level in tachyzoites (determined from RNA-seq of T. gondii RH strain tachyzoites [C. G1- and S/M- regulated genes. T. gondii genes were previously classified those that are transcribed in accordance with G1 or S/M phase of cell cycle [hyzoites ). Averagll cycle . AverageAdditional file 6. Stage-specific gene regulatory patterns of histone variants. Genes with different amounts of histone variant coverage were compared to sets of genes upregulated in different parasite life cycle stages  of enrichment is plotted for genes with different levels of coverage for H2AZ and H2BZ. p-value of < 0.05 in the hypergeometric test was considered significant and is indicated by the dotted grey line.fined in , 29. –lo


Transcription restart after a genotoxic challenge is a fundamental yet poorly understood process. Here, we dissect the interplay between transcription and chromatin restoration after DNA damage by focusing on the human histone chaperone complex HIRA, which is required for transcription recovery post UV. We demonstrate that HIRA is recruited to UV-damaged chromatin via the ubiquitin-dependent segregase VCP to deposit new H3.3 histones. However, this local activity of HIRA is dispensable for transcription recovery. Instead, we reveal a genome-wide function of HIRA in transcription restart that is independent of new H3.3 and not restricted to UV-damaged loci. HIRA coordinates with ASF1B to control transcription restart by two independent pathways: by stabilising the associated subunit UBN2 and by reducing the expression of the transcription repressor ATF3. Thus, HIRA primes UV-damaged chromatin for transcription restart at least in part by relieving transcription inhibition rather than by depositing new H3.3 as an activating bookmark. How gene expression reactivation is promoted following DNA damage is not yet clear. Here, the authors identify a role for the human histone chaperone complex HIRA in transcription restart following UV damage independently of its function in new H3.3 histone deposition. Yet, modulations in transcriptional programmes are indispensable for the cellular response to environmental cues, including the response to genotoxic stress. DNA damage indeed elicits substantial rewiring of transcription, with the induction and repression of damage-responsive genes2. DNA double-strand breaks lead to the local inhibition of ongoing transcription through damage signalling pathways and to the production of small non-coding RNAs5, while other DNA lesions, including UV photoproducts, physically block the progression of elongating RNAPII8. Moreover, following the rapid and local inhibition of elongating RNAPII at sites of UV damage, UV irradiation triggers global repression of transcription initiation, which also affects undamaged genes14. The repair of transcription-blocking UV lesions by the dedicated Transcription-Coupled Repair (TCR) machinery, a subpathway of Nucleotide Excision Repair (NER)16, is necessary for the recovery of transcriptional activity17. TCR involves RNAPII backtracking, release from its template, and also ubiquitylation and proteasomal degradation in case of persistent stalling22. The ubiquitin-dependent degradation of RNAPII is a major determinant in transcription regulation following UV damage22. The release of UV–stalled RNAPII entails eviction of ubiquitylated RNAPII from chromatin by the Valosin-Containing Protein (VCP) segregase24, which targets RNAPII to proteosomal degradation in yeast and human cells26. Importantly, failure to recover from UV-induced transcription inhibition has deleterious consequences including cell death and disease. It is indeed one of the main features of Cockayne Syndrome17. However, the molecular mechanisms underlying transcription restart after UVC damage repair are still poorly understood. Several transcriptional regulators have been involved in this process35 in addition to core TCR components and factors modulating their stability36, but how they possibly cooperate in controlling transcription restart after UVC damage remains elusive. In line with the central role of chromatin dynamics in transcriptional regulation37, chromatin changes associated with the DNA damage response are of functional importance for restoring transcriptional activity38. In particular, several histone chaperones39 and modifying enzymes40 were shown to promote transcription recovery following UVC damage in mammalian cells, including the H3K79 methyltransferase DOT1L28, and the core histone chaperones Facilitate Chromatin Transcription (FACT)30 and Histone Regulator A (HIRA)41.The maintenance of gene expression programmes established during organism development is critical for preserving cell functions and identities45. Interestingly, HIRA activity is intimately linked to transcriptional regulation as this chaperone deposits H3.3 at gene bodies and regulatory elements such as promoters and enhancers in mammalian cells47. In response to UVC irradiation, HIRA is recruited to damaged chromatin regions in a ubiquitin-dependent manner by a mechanism that is not yet fully elucidated, and deposits newly synthesised H3.3 histones29. Based on the observation that HIRA was only transiently recruited to damage sites and released long before transcription restart, we proposed a bookmarking mechanism whereby this histone chaperone was priming chromatin for transcription recovery29. An attractive candidate for the activating bookmark was H3.3 because of the strong connection between the deposition of this histone variant by HIRA and transcriptional activation. Indeed, HIRA-mediated H3.3 deposition plays a critical role in transcriptional reprogramming in Xenopus48 and mouse cells49, and is key for reaching the full dynamic range of transcription in mouse developing oocytes50. Furthermore, unscheduled H3.3 deposition by HIRA governs a pro-metastatic transcriptional programme in breast cancer cells51. HIRA-mediated H3.3 deposition is also required for ribosomal gene transcription in the mouse zygote52, for myogenic gene transcription in mouse myoblasts53, for the transcription of angiogenic genes in endothelial cells54, and for viral gene expression55. However, besides these multiple links with active transcription, H3.3 deposition by HIRA is also required for the establishment of silencing at developmental genes in mouse embryonic stem cells56, raising questions about the contribution of H3.3 to HIRA function in transcription recovery after UVC damage repair. Here, by dissecting the mechanisms of HIRA-mediated transcriptional regulation following UVC damage in human cells, we rule out the contribution of new H3.3 deposition to transcription restart and uncover a non-canonical function of HIRA in this process that promotes transcription recovery on a genome-wide scale.HIRA is an evolutionarily conserved histone chaperone complex that promotes the deposition of H3.3 histones on DNA in a replication-independent manner57, Ubinuclein 1 (UBN1)58 or the less characterised UBN259, and Calcineurin-binding protein 1 (CABIN1) subunits60 . Central to the complex, HIRA serves as a scaffold, stabilising the other core subunits. Consistent with this, we observed that HIRA knock-down in HeLa and U2OS cells led to a strong down-regulation of UBN1 and CABIN1 while ASF1A levels remained unaffected. In contrast, knocking down UBN1 or CABIN1 did not have a marked impact on HIRA levels  was reduced to a comparable extent upon knock-down of HIRA, UBN1 or CABIN1  in cells knocked down for individual subunits of this histone chaperone complex , and we measured repair synthesis by 5-ethynyl-2’-deoxyuridine (EdU) incorporation at sites of local UVC irradiation35 , which was impaired in VCP-depleted cells (siVCP) and abolished in Transcription-Coupled Repair-deficient cells (siERCC6) as expected66  as a global regulator of transcription post UVC in human fibroblasts, controlling transcription recovery of thousands of genesd81 Fig. . Interesd81 Fig. . We obtad81 Fig. . Simultad81 Fig. , suggestd81 Fig. , as repo85. Interestingly, we found that ASF1B, and not the preferred interacting partner ASF1A, was upregulated after UVC damage , in particular of those that required HIRA for transcription recovery (64%), were found to be targeted by ATF3 post UVC in a previous studyPCR Fig. . NotablyPCR Fig. , further59. We confirmed the contribution of UBN2 to new H3.3 deposition at UVC damage sites in human cells  First, we uncover a non-canonical function for HIRA in transcription regulation following UVC damage, which does not involve new H3.3 deposition. (2) Furthermore, we show that HIRA primes UV-damaged chromatin for transcription restart by relieving transcription inhibition rather than by depositing an activating bookmark. HIRA indeed operates, at least in part, through reducing the levels of the transcription inhibitor ATF3. (3) Thus, we delineate two functionally distinct HIRA-dependent pathways: a local and early response to UV damage resulting in H3.3 deposition, and a global and late response leading to transcription restart on a genome-wide scale  and reflect a more global impact of HIRA on nuclear transcription (trans-effect). In line with this, we found that the CABIN1 subunit of the HIRA complex, which stimulates HIRA recruitment to UV damage sites41. These findings highlight important functional differences between the related subunits UBN1 and UBN2, which deserve further investigation. Whether the HIRA subunit functions in transcription restart in complex with CABIN1, UBN1/2 and H3.3 or as part of another protein complex is still to be determined. Recent biochemical and genomic data suggest that HIRA may operate independently of the histone chaperone complex in some instances. Indeed, gel filtration analyses of the HIRA complex in human embryonic stem cells identified a fraction containing the HIRA subunit but not CABIN1 nor H3.392. In addition, ChIP-seq analyses revealed that HIRA binds more than a thousand sites across the human genome in the absence of UBN1 and these sites are not enriched in H3.383, pointing to a function of HIRA independent of the H3.3-chaperone complex, which deserves additional investigation. Further distinguishing the HIRA subunit from CABIN1 and UBN1, mutations in the plant ortholog of HIRA lead to a significantly more pronounced developmental phenotype than mutations in UBN1 or CABIN193. Interestingly, like HIRA, other histone chaperones also control responses to DNA damage independently of their histone binding activity, as shown for ASF1A in DNA double-strand break signalling94. Histone chaperones thus appear as multifunctional proteins, which are pivotal for coordinating chromatin dynamics with critical nuclear functions such as transcription and damage signalling.Our analysis of HIRA complex subunits showed that only HIRA and UBN2, and not UBN1 and CABIN1, are needed for transcription recovery post UVC. These results are in line with a multiomic analysis of the UV response in human fibroblasts, which identified HIRA and UBN2 but not UBN1 and CABIN1 as required for transcriptional activity following UVC damage2 in Dulbecco’s modified Eagle’s medium  supplemented with 10% foetal bovine serum (EUROBIO), 100 U/ml penicillin and 100 μg/ml streptomycin (Life Technologies). U2OS cells stably expressing H3.3-SNAP95 were maintained in the same medium in the presence of 100 μg/ml G418 (Invitrogen).HeLa , XP4PA-SV , and U2OS cells  were grown at 37 °C and 5% COThe VCP inhibitor NMS-873  and the transcription inhibitor Flavopiridol  were added to the culture medium 2 h before UV irradiation of the cells and kept until the time of cell fixation.siRNA purchased from Eurofins MWG Operon  for 30 min at 37 °C followed by a 30 min wash in fresh medium and a 2 h chase. Cells were then locally irradiated with the UVC lamp and the SNAP-tagged histones neosynthesized during the chase time were pulse-labelled by incubating cells with a fluorescent SNAP reagent  for 15 min at 37 °C, followed by 45 min–1 h 45 min incubation in fresh medium. Cells were then processed for immunostaining. For cells harvested 24 h post irradiation, the quenching reagent was added again after the pulse step to avoid labelling of additional histones by free fluorescent SNAP reagent remaining in the cells.2, except for analysing RNAPII degradation  were covered with a polycarbonate filter  and irradiated with 150 J/m2 UVC for analysing HIRA and H3.3 accumulation at UV sites or with 500 J/m2 UVC in the TCR assay and when staining for ATF3 and VCP.Cells were irradiated with UVC (254 nm) using a low-pressure mercury lamp (Vilbert-Lourmat). Conditions were set using a VLX-3W dosimeter (Vilbert-Lourmat). For global irradiation, cells in PBS  were exposed to 10 J/mTotal extracts were obtained by scraping cells on plates or resuspending cell pellets in Laemmli buffer  followed by 5 min denaturation at 95 °C.For western blot analysis, extracts were run on 4–20% Mini-PROTEAN TGX gels (Bio-Rad) in running buffer  and transferred onto nitrocellulose membranes (Protran) with a Trans-Blot SD semi-dry transfer cell (Bio-Rad). Total proteins were revealed by Pierce® Reversible Stain (Thermo Scientific). Proteins of interest were probed using the appropriate primary and HRP (Horse Radish Peroxidase)-conjugated secondary antibodies (Supplementary Table Cells grown on glass coverslips (VWR) were either fixed directly with 2% paraformaldehyde and permeabilised with 0.2% Triton X-100 in PBS, or pre-extracted before fixation with 0.5% Triton X-100 in CSK buffer . Pre-extraction was required only for visualising new H3.3-SNAP deposition at damage sites. For CPD staining, DNA was denatured with 0.5 M NaOH for 5 min. Samples were blocked in 5% BSA  in PBS supplemented with 0.1% Tween before incubation with primary and secondary antibodies conjugated to Alexa–Fluor dyes (Molecular Probes) (Supplementary Table http://imagej.nih.gov/ij/) and ICY  softwares.Epifluorescence imaging was performed with a Leica DMI6000 epifluorescence microscope using a Plan-Apochromat 40x/1.4 oil or 63x/1.3 oil objective. Images were captured using a CCD camera (Photometrics) and Metamorph software. Images were analysed with ImageJ  in PBS containing 0.05% Tween and 0.5 mg/ml RNase A (USB/Affymetrix). The DNA content was analysed by flow cytometry using a BD FACScalibur flow cytometer and CellQuest Pro software (BD Biosciences). The cell cycle distribution was estimated with the Watson model on FlowJo software (TreeStar).2 and incubated with 20 μM 5-ethynyl-2’-deoxyuridine  and 1 μM 5-Fluoro-2’-deoxyuridine  for 3.5 h at 37 °C. FUdR decreases endogenous thymidine production thus favoring EdU incorporation. EdU incorporation was revealed with the Click-iT EdU Imaging kit (ThermoFisher Scientific) using Alexa Fluor 594 dye according to the manufacturer’s instructions. After EdU revelation, cells were fixed and processed for CPD detection by immunofluorescence. Quantification of repair synthesis was performed on Image J software using a custom macro: S phase cells were excluded from the analysis based on pan-nuclear EdU signal and then EdU and CPD intensities were measured at the damage sites .GGR-deficient XP4PA-SV cells were starved overnight in medium containing 0.5% serum to reduce the number of replicating cells. The following day, the cells were subject to local UVC irradiation at 500 J/m2) at different time points post siRNA transfection (48 h or 70 h) to be subject to nascent RNA labelling all at the same time (71 h post siRNA). siRNA efficiency was confirmed both at 48 h (time of the first irradiation) and 72 h post-transfection (time of fixation). Cells were incubated in DMEM supplemented with 0.5 mM ethynyl-uridine (EU) for 1 h at 37 °C, rinsed in PBS before fixation in 2% paraformaldehyde. EU incorporation was revealed with Click-iT RNA Imaging kit (ThermoFisher Scientific) using Alexa Fluor 594 dye according to the manufacturer’s instructions. Alternatively, cells were incubated with 4 mM bromouridine (BrU) in conditioned DMEM for 30 min at 37 °C, followed by detergent extraction with 0.5% Triton X-100 in CSK buffer and fixation in 2% paraformaldehyde. BrU was revealed by immunofluorescence using an anti-BrdU antibody  for 30 min at 37 °C. Cells were exposed to global UVC irradiation at different time points post siRNA transfection  to be subject to nascent RNA labelling with BrU all at the same time (72 h post siRNA). Non-irradiated cells were used as controls. The cells were lysed in TRIzol reagent (Invitrogen) and BrU-containing RNA was isolated on goat anti-mouse IgG Dynabeads  coupled with 2 μg anti-BrdU antibody67 (Supplementary Table 67. All primary sequencing data files have been deposited in NCBI’s Gene Expression Omnibus (GEO) with the accession number GSE151833.For nascent RNA sequencing from UV-irradiated cells69 because RNA sequencing data are normalised to total read counts in each sample (RPKM), which is not suited for the detection of global expression changes such as those observed after UVC irradiation. For this, we multiplied all RPKM values by correction factors corresponding to mean nascent RNA amounts per cell nucleus measured by EU staining for each siRNA condition and at each time point post UVC  on UV-repressed genes that recover transcription better in the absence of HIRA compared to all UV-repressed genes as background (only common genes from experiments #1 and #2 were included). The positions of ATF3 ChIP-seq peaks 31 were intersected with the positions of the nascent transcripts extended by 5 kb upstream to include promoter regions.To determine the dependency of UV-repressed genes on HIRA or ERCC6 for transcription recovery, we defined as independent genes those whose transcription recovery 24h30 post UVC in siHIRA/ERCC6 was equal to the transcription recovery observed in siLUC + /-10%. Stimulated genes were those with at most 90% transcription recovery in siHIRA/ERCC6 compared to siLUC, and inhibited genes were those with at least 110% better recovery in siHIRA/ERCC6 compared to siLUC. Gene ontology analyses were performed with GOrillaATF3 gene in HeLa cells were from Pchelintsev et al., 201383. Log2 fold change relative to input was obtained by bigwigCompare and plotted with Integrative Genomics Viewer (IGV 2.3). Regulatory regions were defined by enrichments in H3K4me1 and H3K27ac marks based on ENCODE data.ChIP-seq data for HIRA binding to the GAPDH housekeeping gene product, a long half-life transcript, whose levels remain fairly stable over 24 h post UVC irradiation.For qRT-PCR on total RNA, RNA was extracted from cells with TRIzol™ Reagent following the manufacturer’s instructions (Invitrogen) and precipitated in isopropanol. RNA samples were subject to DNA digestion with Turbo DNA-free (Invitrogen) before reverse transcription with Superscript III RT using random primers . Quantitative PCR reactions were carried out with the indicated exonic primer pairs  for 30 min at 37 °C before RNA extraction as above. Turbo reaction products were used for pull-down of BrU-labelled RNA using goat anti-mouse IgG Dynabeads  coupled with 1 μg anti-BrdU antibodyp values for mean comparisons between two groups were calculated with a two-sided Student’s t-test with Welch’s correction. Multiple comparisons were performed by one-way ANOVA with Bonferroni posttest. ATF3 transcript levels relative to siLUC were compared to a theoretical mean of 1 by one-sample t-test (two-sided). Analyses of transcription recovery 24 h post UVC (EU or BrU incorporation and RT-qPCR assays) were performed by two-way ANOVA with Bonferroni posttest. Correlations were evaluated using the Spearman rank test. ns: non-significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001. Confidence interval of all statistical tests: 95%.Heatmaps were established with R software. Overlaps on Venn diagrams were analysed by Fisher’s exact test using R software. Other statistical tests were performed using GraphPad Prism. Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Reporting Summary