Saccharomyces cerevisiae to identify histone mutants that exhibit transcriptional readthrough of terminators. Amino acid substitutions identified by the screen map to the nucleosome DNA entry-exit site. The strongest H3 mutants revealed widespread genomic changes, including increased sense-strand transcription upstream and downstream of genes, increased antisense transcription overlapping gene bodies, and reduced nucleosome occupancy particularly at the 3’ ends of genes. Replacement of the native sequence downstream of a gene with a sequence that increases nucleosome occupancy in vivo reduced readthrough transcription and suppressed the effect of a DNA entry-exit site substitution. Our results suggest that nucleosomes can facilitate termination by serving as a barrier to transcription and highlight the importance of the DNA entry-exit site in broadly maintaining the integrity of the transcriptome.Compared to other stages in the RNA polymerase II transcription cycle, the role of chromatin in transcription termination is poorly understood. We performed a genetic screen in  Packaging of the eukaryotic genome into chromatin presents a regulatory barrier to DNA templated processes. Nucleosomes, the fundamental repeating unit of chromatin, are comprised of approximately 147 bp of DNA surrounding an octamer of core histone proteins H2A, H2B, H3, and H4 . To faitS. cerevisiae. Widespread transcription termination defects can result from cell stress  is a conserved, five-subunit protein complex that associates with Pol II on gene bodies and is required for several important transcription-coupled histone modifications, including H2B K123 mono-ubiquitylation, H3 K4 di- and tri-methylation and H3 K36 tri-methylation . Pearson’s correlation coefficients calculated for each pair of biological replicates for each strain confirm a high degree of reproducibility . Our spiTo gain a better understanding of the nature of these readthrough transcripts, we generated de novo annotations from the wild-type and mutant RNA-seq datasets . De novoSNR gene termination  . In both3)  to label nascent RNA and performed sequencing analysis of the 4tU-containing transcripts  see Fig. Compare3) , both of3) .To identify the source of the intergenic nascent transcript density in the H3 T45A and H3 R52A mutants, we mapped the spike-in normalized, stranded 4tU-seq reads in the wild-type and mutant strains to protein-coding genes sorted by their relative orientation . The datBeyond its role in transcribing mRNAs and structural ncRNAs, such as snoRNAs, Pol II is responsible for the transcription of several classes of ncRNAs that arise from pervasive genome transcription and can play regulatory roles within the cell. Relative to the wild-type strain, our 4tU-seq data revealed increased transcription of most annotated CUTs , stable SET2 impair NNS-dependent termination   . We, the (SRATs) . Both DN (SRATs) . Despite3 defects  and a deletion of the second H3-H4 locus (hht2-hhf2∆). In this context, cells retained the ability to express the double mutant H3 protein  . This maH3 K56ac , suggestS. cerevisiae chromatin was treated with increasing amounts of MNase to assess bulk nucleosome stability as well as to identify suitable digestion conditions. Based on this assessment, H3 R52A nucleosomes appear more sensitive to MNase than wild-type nucleosomes, as evidenced by reduced levels of di-, tri-, and higher order nucleosomes at comparable treatments  phenotype (Bypass Upstream Activation Sequence (UAS) Requirement (Bur-) phenotype (suc2∆uas(−1900 /- 390) allele, a mutant gene lacking positive regulatory signals in the promoter; and (iii) the cryptic transcription initiation phenotype, which can be measured using a sensitive reporter that detects aberrant initiation within the FLO8 coding region, GAL1pr:FLO8::HIS3  phenotype, which measures the ability of histone mutants to overcome the absence of the SWI/SNF chromatin remodeling complex , which can be integrated into the yeast genome via standard two-step gene replacement  was used to substitute H3 K36 for an alanine residue in the H3 R52A SHIMA library plasmid . A plasmbackbone . The SB backbone  flanked lacement . PlasmidSaccharomyces cerevisiae strains used in this study are isogenic to FY2, a +GAL2 derivative of S288C (S. cerevisiae strains were grown in rich medium (YPD) supplemented with 400 µM tryptophan or synthetic complete (SC) media lacking uracil or the specified amino acids. YPSuc and YPGal media contained 1 μg/ml antimycin A and 2% sucrose or 2% galactose, respectively. Unless otherwise noted, transformations were performed with yeast strains lacking endogenous copies of the genes encoding histones H3 and H4 and containing a URA3-marked, wild-type H3-H4 plasmid. By selection on SC-Trp medium containing 0.1% 5-fluoroorotic acid (FOA), URA3 plasmids were shuffled   had 40 bp of homology to the region downstream of SNR48, and universal inside primers (EHO58 and EHO59) annealed to the URA3 gene. Fragments generated by PCR with EHO54/EHO58 and EHO55/EHO59 overlapped such that upon cotransformation into yeast, homologous recombination joined the two fragments and integrated the full-length cassette, which was confirmed by PCR. After plating to 5-FOA containing media, successful recombination resulted in a single copy of SB located 89 bp downstream of the annotated 3’-end of SNR48, replacing bp 609786 through 609883 of chromosome VII. The final recombinant was confirmed by DNA sequencing. Kluyveromyces lactis strains were grown in YPD medium supplemented with 400 µM tryptophan and Schizosaccharomyces pombe strains were grown in standard YES medium.of S288C  and are of S288C . S. cereshuffled  with TRPctively)  or a TRPYeast cultures were grown at 30°C and diluted as indicated in the figure legends. Cells were plated by pipetting (3 μl) or using a pinning tool  on control and selective media to assess specific phenotypes. Plates were incubated at 30°C for at least 3 days and imaged daily.600 = 0.8–1.0) by hot acid phenol extraction . Probe locations are as follows with numbering relative to the start codon of the indicated protein-coding gene, where appropriate: SNR47-YDR042C (−325 to −33 of YDR042C), SNR48-ERG25 (−746 to −191 of ERG25), SNR48 (−70 to +92 of the annotated SNR48 gene), SNR13-TRS31 (−231 to +449 of TRS31), and SCR1 (−242 to +283 of the annotated SCR1 gene).Total RNA was isolated from log phase yeast cultures  by bead beating in 20% trichloroacetic acid as described previously (3) membrane. Membranes were blocked with 5% milk in TBST  or 3% BSA in PBST (H3 K36me3), and then incubated with primary antibody against total H3 , HA  or G6PDH , which served as the loading control. For H3 K56ac blots , alkaline lysis protein extraction was performed as previously described  as described previously . All qPCR reactions were performed in biological triplicate and technical duplicate. Protein occupancy was calculated using the appropriate primer efficiency raised to the difference between input Ct and immunoprecipitated Ct values.Chromatin was isolated from 250 mL of yeast cells grown to log phase  and mixed in a 9:1 ratio enabling the use of mapped S. pombe reads as an internal spike-in control for RNA-seq analysis. Total RNA was isolated from this mixture of cells by hot acid phenol extraction as described previously (eviously . RNA wasS. cerevisae and S. pombe nascent RNA was labeled with 4-thiouracil (4tU) as previously described  for 30 min at room temperature in biotinylation buffer . Meanwhile, streptavidin beads  were washed in a high-salt wash buffer  and blocked  for 1 hr prior to use. A phenol chloroform extraction was used to quench biotinylation reactions by removing unincorporated biotin. Isopropanol precipitated RNA was resuspended in 100 µL nuclease-free water and incubated with blocked streptavidin beads for 15 min. Supernatant was removed and set aside as unlabeled RNA. Beads were washed twice with elution buffer  and samples were pooled for concentration via MinElute columns .escribed . Brieflyescribed . The res600 = 0.8 and crosslinked with formaldehyde at a final concentration of 1%. 100 mL of cells were pelleted, resuspended in FA buffer , and lysed by bead beating. Cell extracts containing chromatin were pelleted and resuspended in NP-S buffer , and then subjected to digestion by MNase . Mononucleosomal DNA was purified using agarose gel electrophoresis and Freeze N’ Squeeze Columns . As a spike-in control, MNase-treated K. lactis DNA, purified in the same manner as S. cerevisiae DNA, was added to achieve a 9:1 ratio of S. cerevisiae DNA to K. lactis DNA. This normalization method reports relative but not absolute differences between samples.Mononucleosomes were prepared essentially as described . BrieflyK. lactis expressing FLAG-tagged Rpb3 from the endogenous locus  and quantified on a Nanodrop OneC . Anti-Flag M2 affinity gel  was used to immunoprecipitate FLAG-Rpb3 from the chromatin.ChIPs were performed on plasmid shuffled derivatives of KY3232 as described above. us locus  was usedS. cerevisiae rRNAs for depletion. All RNA and DNA libraries were quantified using Qubit and TapeStation and pooled for paired-end sequencing on an Illumina NextSeq 500 .RNA-seq libraries were built by the UPMC Children’s Hospital Health Sciences Sequencing Core following rRNA depletion. MNase-seq and ChIP-seq libraries were built prior to sample submission to the sequencing core. Libraries were prepared using the NEBNext Ultra II kit  and NEBNext Ultra sequencing indexes  according to manufacturer’s instructions. 4tU-seq libraries were built using a custom SoLo RNA-seq library preparation kit  with custom primers targeting S. cerevisiae genome (Ensembl R64-1-1), S. pombe genome (Ensembl EF2), or K. lactis genome (Ensembl ASM251v1), using HISAT2 .Annotations for mRNA transcriptional units were generated using CDS annotations from SGD and published data for nucleosome positioning and CPS locations . Data fohttps://github.com/mae92/building_annotations_for_mRNAs_in_S.c/tree/master/Orientations.To identify gene pairs in convergent, divergent or tandem orientation, two duplicate files containing the annotations generated using the steps outlined above were compared using the closest command  in BEDtools. Using BEDtools closest in this manner allowed for a single closest gene to be identifed for each gene in the yeast genome without allowing overlapping genes to be called. Distances to upstream and downstream genes were reported as negative or positive values (in bp) allowing for files to be further manipulated based on distance and gene orientation. From this list ‘close’ genes were selected using a distance cutoff of 500 bp. Strand information along with information on positioning (upstream or downstream) mentioned above were used to separate out genes in convergent, divergent and tandem orientations. All scripts used in this analysis as well as input and output files can be found here: Yeast serial dilution growth assays, northern blots, western blots, and ChIPs were performed in biological triplicate with three independently passaged plasmid shuffle transformants. Western blot quantification is shown as the mean signal of three biological replicates with error bars representing the standard error of the mean (SEM). p-Values were calculated via unpaired Student’s t-test. ChIP-qPCR was performed in technical duplicate and technical duplicate results were averaged prior to averaging across biological replicates. ChIP-qPCR quantification is shown as the mean value with error bars representing the SEM. NGS experiments – RNA-seq, MNase-seq, 4tU-seq, and Pol II ChIP-seq – were performed in biological duplicate with two independently passaged plasmid-shuffled transformants. Agreement between replicates is shown as biplots with Pearson’s correlation. Boxplots of NGS data are presented such that the whiskers indicate the full range of the data and the boxes indicate the second and third quartiles. The dark line within the box indicates the sample median and the distance between the top and bottom of the box represents the interquartile range. For data shown as boxplots p-values were calculated via unpaired Wilcoxon Rank-Sum test. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:All reviewers agree that the authors have done a good job of addressing the previous comments, and that this work provides a significant and careful dissection of how transcription is regulated by histone-DNA interactions at the entry/exit site of a nucleosome.Decision letter after peer review:Saccharomyces cerevisiae" for consideration by eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Cynthia Wolberger as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "The nucleosome DNA entry-exit site is important for transcription termination in 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 revisions 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:While nucleosomes are assumed to act as road blocks for different steps in transcription, their impact on transcription termination has not been systematically tested. The reviewers agreed that this work provides significant and careful experimental evidence towards testing such a role. They found the experiments investigating K36 methylation and K56 acetylation changes to be compelling for ruling out indirect effects. They also felt that the experiments using the nucleosome superbinder DNA sequences helped nail down the role of nucleosome stability. While overall the findings were considered interesting and important for the gene regulation community, the reviewers have some questions about (i) the specificity of the effects on termination vs. cryptic transcription, and (ii) interpretation of the effects in terms of nucleosome occupancy vs. altered nucleosome dynamics. We ask that you address these, and other comments summarized below in a revised version.Essential revisions:1) How specifically is this a termination effect vs a general effect of cryptic transcription? The original screen was based on a termination test, but given the increase in the upstream mRNA, anti-sense transcription, and some other ncRNA, we wonder if these mutations simply deregulate transcription in general. If this is the case, the title is a bit misleading. This should be discussed.2) In terms of mechanism it is not clear how much of the effects are due to lower nucleosome occupancy and how much are due to altered nucleosome dynamics at entry/exit site. It would be ideal to test the effects of some of the key mutants such as H3R52A on DNA breathing and nucleosome stability in vitro and assess if there is a strong correlation between the in vitro and in vivo effects. However, if such experiments are not readily feasible, the authors should explicitly mention in the Discussion the challenge of deconvolving these two effects in vivo.SNR termination? The labeling of the SNR names is hard to read. It will be better to generate an overall correlation between the two mutants.3) In Figure 2A and B, do these two different mutants generate similar effect on the 4) Since different genes respond differently to these mutations , the authors should divide genes into different groups based on their read-through level and look at the termination signals of these genes (PAS or Nab / Nrd binding sites). If the authors think that the termination effects are related to nucleosome stability, they can also look at the nucleosome positioning pattern near the termination sites in these groups of genes.5) It was mentioned that the "transcript levels downstream of the CPS were elevated 1.6-fold and 1.9-fold in the H3 T45A and H3 R52A mutants". These numbers seem to be inconsistent with what was shown in Figure 3B.6) The difference in nucleosome occupancy in Figure 6 seems to be rather minor and very much relies on the spike-in normalization. The statistical significance of such difference should be estimated.7) The authors proposed a model that these histone mutants affect the elongation rate of the polymerase. Elongation rate change can have many consequences, e.g. change in start site or splicing efficiency. From the RNA-seq data, can the authors find more evidence of increase in the elongation rate? Essential revisions:1) How specifically is this a termination effect vs a general effect of cryptic transcription? The original screen was based on a termination test, but given the increase in the upstream mRNA, anti-sense transcription, and some other ncRNA, we wonder if these mutations simply deregulate transcription in general. If this is the case, the title is a bit misleading. This should be discussed.We agree that the termination defect is only one measure of transcriptional deregulation in the DNA entry-exit site mutants. While the initial premise of the study was to identify histone mutants defective in transcription termination, our comprehensive analysis revealed widespread effects on cryptic and other forms of noncoding transcription. These results highlight the importance of the DNA entry-exit site in controlling the integrity of the transcriptome. We tried to emphasize this better in the revised version. Changes can be found in the wording to the Abstract, Introduction, Results, and Discussion.In addition, we changed the title of the manuscript to: “The nucleosome DNA entry-exit site is important for transcription termination and prevention of pervasive transcription”2) In terms of mechanism it is not clear how much of the effects are due to lower nucleosome occupancy and how much are due to altered nucleosome dynamics at entry/exit site. It would be ideal to test the effects of some of the key mutants such as H3R52A on DNA breathing and nucleosome stability in vitro and assess if there is a strong correlation between the in vitro and in vivo effects. However, if such experiments are not readily feasible, the authors should explicitly mention in the Discussion the challenge of deconvolving these two effects in vivo.The reviewer makes an excellent point. Indeed, the in vivo experiments included in the manuscript cannot distinguish between effects on nucleosome occupancy and effects on nucleosome dynamics. While we agree that a detailed analysis of the dynamics of the histone mutants in vitro is an excellent idea, we feel it is outside the scope of the current study. However, we are fortunate that the lab of Tom Owen-Hughes (MCB 27: 4037) previously studied DNA entry-exit site mutants and showed that nucleosomes harboring the H3 R52A and T45A substitutions exhibit increased sliding rate and increased DNA unwrapping (by a DNA end-to-end FRET assay), relative to wild-type nucleosomes. We have now changed the text in the Discussion to acknowledge the potential role of nucleosome dynamics and also highlight the Owen-Hughes data on both the H3 R52A and H3 T45A substitutions.3) In Figure 2A and B, do these two different mutants generate similar effect on the SNR termination? The labeling of the SNR names is hard to read. It will be better to generate an overall correlation between the two mutants.SNR genes. We calculated the Pearson’s correlation coefficient between 3’ extension indexes for our H3 T45A and H3 R52A mutants which resulted in r = 0.795. We have included a scatter plot showing this result. Please see new Figure 2—figure supplement 1D.We are grateful for the suggestion. The H3 T45A and H3 R52A mutants agree well in terms of their effects at the 4) Since different genes respond differently to these mutations , the authors should divide genes into different groups based on their read-through level and look at the termination signals of these genes (PAS or Nab / Nrd binding sites). If the authors think that the termination effects are related to nucleosome stability, they can also look at the nucleosome positioning pattern near the termination sites in these groups of genes.SNR genes reduced nucleosome occupancy appears to correlate with transcriptional readthrough , this correlation did not extend to all genes. In the section describing the MNase-seq experiments, we included this sentence: “However, the degree of nucleosome loss in the mutant does not appear to be directly correlated with RNA levels at all genes, consistent with other factors, such as RNA degradation pathways, contributing to the accumulation of 3’-extended transcripts.” We also softened the language in the Discussion on this point.In response to this request, we assessed possible relationships between our RNA sequencing results and Nrd1/Nab3 PARCLIP data . We also looked for relationships between differential expression in our mutants and snoRNA type and organization using data presented in the Dieci 2009 Genomics review on snoRNAs. We did not observe an obvious relationship between snoRNA readthrough in our mutants and Nrd1/Nab3 binding, snoRNA type or genomic organization. The snoRNA genes were also clustered using our RNA-seq, 4tU-seq, and MNase-seq datasets. While at some 5) It was mentioned that the "transcript levels downstream of the CPS were elevated 1.6-fold and 1.9-fold in the H3 T45A and H3 R52A mutants". These numbers seem to be inconsistent with what was shown in Figure 3B.We are grateful to the reviewer for pointing this out. After re-analysis, we believe the discrepancy arose from plotting mean transcript levels and reporting median values. We have changed the text in the Results to highlight the modest, but statistically significant effects detected by RNA-seq downstream of the CPS: “For the region 150 bp downstream of the CPS, the increase in sense-strand RNA levels in the H3 T45A and H3 R52A mutants was modest but statistically significant .”6) The difference in nucleosome occupancy in Figure 6 seems to be rather minor and very much relies on the spike-in normalization. The statistical significance of such difference should be estimated.In response to this request, we have generated a violin plot of log2-transformed spike-in normalized read counts from our MNase-seq data in the region 150bp downstream of the CPS and found the difference between WT and the H3 R52A mutant to be statistically significant (p < 0.0001) by a Wilcoxon Rank Sum test. We have included a new figure panel showing this plot . We have also revised the Materials and methods to clearly state that our spike-in approach for the MNase-seq experiments allows us to detect relative and not absolute changes between samples. Although we did not include these data, we followed up on the MNase-seq experiments with H3 ChIP assays on a small number of loci. We found that the H3 ChIP signals are in agreement with the MNase-seq data in that we do not detect a dramatic loss of nucleosomes on gene bodies. Thus, we do not feel that our MNase-seq analysis normalized out a larger global effect of the H3 R52A mutant.7) The authors proposed a model that these histone mutants affect the elongation rate of the polymerase. Elongation rate change can have many consequences, e.g. change in start site or splicing efficiency. From the RNA-seq data, can the authors find more evidence of increase in the elongation rate?In response to this request, we analyzed our 4tU-seq data for potential splicing defects in the H3 R52A and H3 T45A mutants. To do this we calculated intron-exon splice junction ratios  for the WT and mutants. The results revealed a significant splicing defect in the mutants. Specifically, we calculated the median IEJR values for WT, T45A, and R52A to be 0.19, 0.31, and 0.27, respectively . While these data are supportive of transcription elongation rate defects in the mutants, they could equally be explained as a consequence of a defect in H3 K36 methylation. Tracy Johnson’s lab (Cell Rep. 27:3760) demonstrated a role for Set2, H3 K36 methylation and Eaf3 in regulating splicing in yeast. Since the DNA entry-exit site mutants also have defects in H3 K36 methylation, as shown in Figure 5 of our paper, it is difficult to ascribe the splicing defect to a specific molecular cause. Given this uncertainty, we have chosen not to include our splicing calculations in the revised manuscript but hope to explore the mechanistic basis for these effects in the future.


APETALA1 (AP1), is a pioneer transcription factor. In vitro, LFY binds to the endogenous AP1 target locus DNA assembled into a nucleosome. In vivo, LFY associates with nucleosome occupied binding sites at the majority of its target loci, including AP1. Upon binding, LFY ‘unlocks’ chromatin locally by displacing the H1 linker histone and by recruiting SWI/SNF chromatin remodelers, but broad changes in chromatin accessibility occur later. Our study provides a mechanistic framework for patterning of inflorescence architecture and uncovers striking similarities between LFY and animal pioneer transcription factor.Master transcription factors reprogram cell fate in multicellular eukaryotes. Pioneer transcription factors have prominent roles in this process because of their ability to contact their cognate binding motifs in closed chromatin. Reprogramming is pervasive in plants, whose development is plastic and tuned by the environment, yet little is known about pioneer transcription factors in this kingdom. Here, we show that the master transcription factor LEAFY (LFY), which promotes floral fate through upregulation of the floral commitment factor  Pioneer transcription factors access their DNA binding motifs in closed chromatin and often act in cell fate reprogramming. Here, Jin et al. present biochemical evidence for a pioneer factor in plants and show that LFY promotes floral cell fate and locally unlocks chromatin by displacing histone H1 and recruiting SWI/SNF chromatin remodelers. The basic unit of chromatin is the nucleosome comprised of 147 base-pairs of DNA wrapped nearly two turns around the histone octamer2. Chromatin is further compacted by nucleosome/nucleosome interactions and by the linker histone H1, which associates with the dyad (midpoint) of the nucleosome and the linker DNA near the nucleosome entry and exit3. During cell fate reprogramming in eukaryotes, master transcription factors silence and activate new gene expression programs in the context of chromatin6. While it is easy to imagine how master transcription factors bind to active genes in open chromatin to trigger silencing, it is difficult to envision how these sequence-specific binding proteins can access their cognate motifs in silent chromatin to activate gene expression. This is because nucleosomes are refractory for most transcription factor binding11. However, a special class of transcription factors, termed pioneer transcription factors, can access their cognate binding motifs in the nucleosome17. These pioneer transcription factors play important roles in cell fate reprogramming. For example, the mammalian pioneer transcription factor FoxA reprograms fibroblast to hepatocytes20, while the Oct4, Klf4 and Sox2 pioneer transcription factors reprogram fibroblasts to induced pluripotent stem cells22. Defining criteria for pioneer transcription factors are (1) ability to bind cis motifs in the context of a nucleosome both in vitro and in vivo, (2) facilitating access of additional, non-pioneer, transcription factors to target loci via local chromatin opening and (3) cell fate reprograming24.Chromatin prevents expression of inappropriate or detrimental gene expression programs, allowing the formation of distinct cell types from the same genome25. Not surprisingly, many master transcription factors that reprogram cell fate have been identified in plants. For example, the bHLH transcription factors MUTE and FAMA reprogram leaf epidermal cells to guard cell fate27, while AP2 family transcription factors WOUND INDUCIBLE1 and BABY BOOM cause de-differentiation upon wounding and somatic embryo formation on seedlings, respectively29. The NF-Y complex transcription factor LEAFY COTYLEDON1 (LEC1) also promotes embryo fate in seedlings30. The helix-turn-helix (HTH) transcription factor LEAFY (LFY) is necessary and sufficient to trigger flower formation on inflorescences32 and reprograms cells in roots of growing seedlings to flower fate, when ectopically expressed together with the pluripotency factor WUSCHEL33. In root explants, inducible activation of LFY is sufficient to trigger synchronous, abundant flower formation bypassing elaboration of a shoot34. Finally, MADS box transcription factors of the SEPALLATA (SEP) family can reprogram cauline leaves into floral organs35.Plant development occurs after embryogenesis and is tuned by the environment, a likely adaptation to the sessile lifestyle36. The NF-YB homolog LEC139 has not been tested for pioneer activity in plants. In animals, NF-YB is recruited as a component of the histone fold NF-Y complex, which binds closed chromatin by displacing histones45. MADS box transcription factors and LFY associate with genomic regions in Arabidopsis that are not in an open chromatin configuration based on DNAse hypersensitivity47. While the available data hint at possible pioneer factor activity, it has not yet been established for any of these plant transcription factors whether they can indeed access nucleosome occupied binding sites in vivo or in vitro. The ability of LFY to reprogram root cells to floral fate prompted us to investigate whether LFY acts as a pioneer transcription factor. A key direct target of LFY is APETALA1 (AP1), a MADS box transcription factor that commits primordia in the inflorescence to flower fate50. LFY upregulates AP1 both directly and indirectly, via a series of coherent ‘and’ logic feed-forward loops52. The LFY binding site at the AP1 locus is critical for locus activation53 and LFY association with this site has been structurally characterized54.Pioneering activity has been proposed for several of these plant transcription factors, including LEC1AP1 locus in the context of a nucleosome. In vivo, the majority of the LFY bound sites, including that at AP1, are nucleosome occupied and isolated LFY-associated DNA fragments are co-bound by histones. LFY displaces linker histone H1 and recruits SWI/SNF chromatin remodelers at the AP1 locus, this triggers subsequent changes in chromatin accessibility. Our findings identify LFY as a pioneer transcription factor, link pioneer activity of LFY to competency for cell fate reprogramming and pave the way for understanding the molecular basis for the developmental plasticity of plants.Here we use complimentary biochemical, genomic and structural approaches to test whether LFY is a bonafide pioneer transcription factor. We find that in vitro LFY binds with high affinity and specificity to a native regulatory fragment from the AP148. Analysis of a published MNase-seq dataset55 revealed a nucleosome whose midpoint (dyad) was positioned near the functionally important53 LFY binding site at the AP1 locus . LFY bound its target site in the AP1 fragment with high affinity based on the apparent dissociation constant (KD), and with high specificity as binding was abolished when we mutated the known consensus motif57  in a coherent ‘and’ logic feed-forward loop51  digestion followed by tiled oligo qPCR. This uncovered a nucleosome centered over the LFY binding site at the AP1 locus at a similar position as in the published dataset  nuclear entry of a glucocorticoid receptor hormone binding domain fusion of LFY (35S:LFY-GR)57 identified 1177 significant  LFY binding peaks in the steroid treated tissue  MNase digestion. Nucleosome occupancy was assessed immediately prior to (one hour mock treatment) or after (one hour steroid treatment) LFY nuclear entry. All MNase-seq datasets were of high quality on the basis of characteristic phased nucleosome occupancy  and MNase digestion in root explants one hour after steroid or mock treatment Fig.  followed−50) in the MNase-seq datasets. We next compared LFY binding and nucleosome occupancy in a 2 kb region centered on the significant LFY peaks and ranked on the signal strength of the nucleosomes present under the peak summit region 62, LFY bound naked DNA  or partially (ACR7) nucleosome occupied on the basis of our ChIP-seq and MNase-seq analyses  into the 35S::LFY-GR genetic background, we treated root explants either with mock solution (no LFY chromatin association) or with dexamethasone (LFY chromatin association)34, before inducing LMI2-3HAER expression by estradiol treatment  of the LFY binding peaks overlapped with a nucleosome  or 24  h (strongly) after steroid application, while AP1 was significantly and strongly induced only 24 h after dexamethasone application . Despite their similar behavior, on the basis of gene ontology term enrichment analysis (AgriGO2 GOslim) only LFY regulated targets where LFY bound to a nucleosome occupied binding sites were linked to flower development 74 followed by quantitative PCR at the AP1, RAX1 and ACT7 loci. We did not observe increased accessibility at any of the loci tested 24 h after LFY activation . Such FFLs not only make biological processes more robust to noisy stimuli such as environmental cues, but they also represent temporal delay elements84. In one FFL, LFY activates LMI2, which upregulates AP1 together with LFY51. In another FFL, LFY directly triggers reduced accumulation of bioactive gibberellin hormone; this stabilizes the DELLA/SQUAMOSA BINDING PROTEIN9 (SPL9) transcriptional complex, which activates AP1 in parallel with LFY52.LFY is a master regulator of onset of flower formation and can reduce time to formation of the first flower in trees from decades to monthsLFY establishes competency for cell fate reprogramming to floral fate by associating with the nucleosome occupied binding site at the AP1 locus where it opens chromatin locally, concomitant with initial (low level) AP1 upregulation  transcription factors; these include not only LMI2, but also SPL9/DELLA and the florigen activator complex, which all bind near the LFY binding site at the AP1 locus to activate gene expression89. We propose that the activity of these LFY co-factors, in a manner not yet understood, contributes to further opening of the AP1 locus chromatin, enables strong AP1 upregulation and reprogramming to floral fate  and hierarchical recruitment of the LFY co-factors LMI2, SPL9/DELLA and the florigen activation complex to the AP1 locus. This ensures correct timing of the irreversible switch to flower formation, which is critical for reproductive success.Combining these and additional prior findings with those of our current study, we propose that the pioneer transcription factor ion Fig. . This loLFY Fig.  or by SWLFY Fig. . That LF80. While first ‘rules’ or characteristics of pioneering activity of transcription factors are emerging77, it is far from clear what their unique or defining set of properties is. For example, some pioneering factors act cooperatively with other transcription factors, but it is not known how widespread such interactions are90. The LFY pioneer transcription factor shares many properties with animal pioneer factors in addition to the ability to bind nucleosomes in vitro and in vivo. Like the pioneer transcription factors Oct4, Ebf1, and Rap192, LFY recruits SWI/SNF chromatin remodeling complexes to key target loci. Like FoxA16, LFY displaces linker histone H1 in vivo. Like the pioneer transcription factors Pax793, PHA-477, and Rap178, LFY rapidly associates with nucleosome-occupied binding sites at target loci, but chromatin opening is delayed relative to binding. Finally, the LFY DNA contact helix shares structural properties with strong nucleosome binders15 and LFY binds weaker motifs in nucleosomes, as described for Oct4 and Sox214. Unique properties of the LFY pioneer transcription factor include its ability to bind both fragile and stable nucleosomes (this study) and to contact a consensus motif half site as a monomer in vitro and in vivo57. Combined with the fact that the LFY binding site is palindromic57, this enables LFY to bind its cognate motif in a nucleosome even if the DNA is rotated 180° to face the histone octamers 95. Moreover, LFY bound sites cluster around the transcription start site57, which enhances nucleosome positioning97. These combined characteristics make the pioneer transcription factor LFY extremely well suited to license key developmental transitions.Animal pioneer transcription factors play important roles in developmental reprogramming in vivo, generation of iPS cells and trans-differentiation, enabled by their unique ability to bind cis motifs even when buried deep in the nucleosome (near the dyad)98, suggesting that pioneer transcription factors may be more prevalent in this kingdom. Our study sets the stage for identification and validation of further plant pioneer transcription factors, characterization of which may not only help understand the basis for plant developmental plasticity but should also contribute to elucidation of the rules for pioneer activity by transcription factors in multicellular eukaryotes and enable their future engineering for enhanced cell fate reprogramming.Plants evolved multicellularity independently from animals and can change their final body plan in response to environmental cuesArabidopsis thaliana wild-type accession Landsberg erecta48. LMI2ER in binary vector pMDC799 and SWI3B-3xHA in binary vector pGWB1100 were transformed into 35 S::LFY-GR plants. For experiments in inflorescence, plants were grown in soil at 22 °C short-day photoperiod . For all other experiments, plants were grown on 1/2 MS plates  at 22 °C in long-day photoperiod . Arabidopsis seeds were stratified at 4 °C for 3 days.35S::LFY-GR is in the 34 for 5 days. Dexamethasone  or mock (0.1% ethanol) treatments lasted the entire duration (5 day) or commenced 24 h, 6 h, or 1 h before the end of the 5th day. Tissue was harvested at ZT 6.To obtain root explants, roots from 3-week-old seedlings were harvested and placed on callus inducing medium (CIM) LMI2 fused with 3 times Hemagglutinin (HA) was amplified from a published construct51 and cloned into pENTRD-TOPO . pENTR LMI2-3HA was moved to pMDC799 by LR reaction . For pSWI3B::SWI3B-HA, a genomic fragment covering 311 bp upstream of the transcription start site and genomic SW3B coding region (2124 bp) excluding the stop codon were fused to 3xHA plus stop codon and the 163 bp 3′UTR sequence  and the resulting pSWI3B::SWI3B-3xHA-3′UTR construct was cloned into the binary vector pGWB1 by LR reaction . Constructs were introduced into Agrobacterium strain GV3101 for plant transformation103.105 using Gibson assembly  with primers listed in Supplementary Table E.coli RosettaTM2 (DE3) for protein expression.For protein expression, the coding sequences of LFY and LMI2 were moved into pE-SUMOproER35S:LFY-GR plants were harvested from 4 trays of plants. After trimming, shoot apices were submerged in 5 µM dexamethasone or mock solution for 18 h followed by addition of 5 µM beta-estradiol and a further incubation for 6 h. For LFY ChIP-qPCR and ChIP-seq, 35 S::LFY-GR root explants were treated with 5 µM dexamethasone for 1 hour prior to the end of day 5 on CIM. For each sample, 1.2 g of root explant tissue was harvested. For H1 and SWI3B-3xHA ChIP-qPCR, root explants were treated with 5 µM dexamethasone or mock solution for 24 h. For each sample, 0.6 and 1.2 grams of tissue was harvest for H1 and SWI3B ChIP-seq, respectively. At least two biological replicates were generated for each experimental condition.For LMI2 ChIP-qPCR, root explants on CIM were treated with 5 μM dexamethasone or mock solution for 18 h followed by addition of beta-estradiol  and a further incubation for 6 h. 100 seedling plates yield about 1 g of tissue. For each sample, 2 grams of tissue was harvested at the end of day 5 on CIM. For LMI2 ChIP-qPCR in inflorescence, for each sample, 2 grams of shoot apices from 25-day-old, short-day grown LMI2106 and the following antibodies: rabbit polyclonal anti-LFY antibodies57, mouse anti-HA antibody , Mouse IgG2bκ Anti-HA antibody , rabbit anti-histone H1 antibody , rabbit anti-histone H3 antibody , and rabbit anti mouse IgG . ChIP-qPCR was performed using Platinum Taq DNA Polymerase  and EvaGreen dye . Inputs from each sample were used to generate the standard curve to compute sample enrichments106. Primer sequences for ChIP-qPCR are listed in Supplementary Table ChIP was conducted using our protocol106. The eluted chromatin was next immunoprecipitated for histone H3 or IgG following an established protocol63 using commercial antibodies (above). The anti-H3 or IgG chromatin bound to protein G beads was eluted with 50 mM Tris–HCl pH 8.0, 5 mM EDTA, 20 mM DTT, and 1% SDS at 37 °C for 30 min.For sequential-ChIP, four ChIP reactions from 0.5 g root explant tissue each were combined into one biological replicate. Three such biological replicates were analyzed after 24 h steroid application Fig. . A totalFor LFY ChIP-seq, each biological replicate was generated by pooling four individual ChIP reactions (each consisting of 0.6 g of root explant tissue) prior to DNA purification on MinElute PCR columns . Three biological replicates were sequenced for each condition. Dual index libraries were prepared for the six ChIP samples (three mock and three treatment) and three input samples using the SMARTer ThruPLEX DNA-Seq Kit . After quantifying libraries using NEBNext Library Quant Kit for Illumina , libraries were pooled based on desired read depth. Single-end sequencing was conducted using NextSeq 550/500 High Output Kit v2  on the NextSeq500 platform (Illumina).107, FastQC (v0.11.5) was performed on trimmed reads to confirm high-quality sequences108. Sequencing reads were then mapped to release 10 of the Arabidopsis Genome (TAIR10)109 by Bowtie2 (—phred33 -q -x) v2.3.1110. Next, uniquely mapped reads )111 were processed following ENCODE guidelines. Significant ChIP peaks and summits  were identified in the pooled dexamethasone-treated samples using the pooled mock-treated samples as controls in MACS2 (v2.1.2)113 . This yielded 1177 significant LFY peaks. For quality control, Spearman correlation coefficients of the reads in the LFY peak regions of each biological replicate were compared using deepTools (v3.1.2)115. De novo motif analysis was conducted using HOMER v4.1116 for MACS2 q-value ≤ 10−10 peak summits (±150 base pairs) compared to genome matched background (unbound regions from similar genomic locations as the peak summits), for an example of this approach see Refs. 117.After trimming adapter sequences and low-quality reads by Trimmomatic (v0.32)46 was retrieved from GEO dataset GSE64245). Two replicates were analyzed. Inflorescence ETT ChIP-seq data65 was obtained from EBI-ENA database accession number PRJEB19862). Three biological replicates and one negative control file were analyzed. Trimming, FastQC, filtering  and mapping were done as described for LFY ChIP in root explants (above). For inflorescence ChIP seq, peaks were called using MACS2 (v2.1.2)113 using the same parameters as for the ChIP-seq analysis in root explants. Since no control files were available, we used more stringent criteria for peak calling . This identified 1952 significant LFY binding peaks. For ETTIN ChIP seq, peaks were called using  as described for LFY ChIP in root explants. A total of 670 significant peaks were identified.LFY ChIP-seq data (15day-old 35S:LFY)69 were retrieved from DDBJ Sequence Read Archive (DRA) under the accession number DRA008014 for histone H3, H3K4me3 and H3K27me3 in the Columbia ecotype. Trimming, FastQC, filtering for low quality reads and mapping were as described for LFY ChIP-seq (above). For all histone modification ChIP-seq, peaks were called using MACS2113 callpeak command -f BAM —call-summits —keep-dup auto —nomodel —extsize 138 -g 101274395, using the histone H3 file as the control file and the respective histone modification file as the treatment file. For H3K4me3 and H3K27me3 peaks, q-value ≤ 10−100 was used for significance calling and a total of 6032 and 1359 peaks were identified, respectively.Histone modification ChIP-seq data from root explants cultivated for 14 days on CIM118. Control samples consisted of 0.2 g tissue per replicate processed without fixation. For Supplementary Fig. 106 and FAIRE-qPCR was conducted as previously described119. Primer sequences for FAIRE-qPCR were designed to query published DNase I hypersensitivity sites near each candidate locus73 and are listed in Supplementary Table For FAIRE-qPCR, 35S:LFY-GR root explants were treated with 5 μM dexamethasone or mock solution for five-days or for the last 24 h on CIM. For Fig. 122 with the following modifications. After washing isolated nuclei twice with HBB buffer , isolated chromatin was digested with 0.5 unit/µl or 0.05 unit/µl  of Micrococcal Nuclease  for 3 min at 37 °C to obtain high or low digestion of chromatin, respectively. Subsequent steps were performed as in121. Mono-nucleosomes were excised from 1.5% agarose gels and purified using the QIAquick gel extraction kit . Purified DNA was diluted 50 times for qPCR analyses.For MNase-qPCR and MNase-seq, 35S::LFY-GR root explants were treated with 5 μM dexamethasone or mock solution for the last hour of the 5-day CIM incubation. 0.6 g of tissue was harvested per replicate into liquid nitrogen. Nuclei and chromatin were isolated following standard protocolsAP1 regulatory region and are listed in Supplementary Table 122. Tiling primers were designed based on our analysis of an Arabidopsis vegetative stage MNase-seq dataset55 retrieved from DDJB Sequence Read Archive (DRA) accession number SRP045236 samples SRX669229).Tiling primers (each primer pair covering 80 bp with 40 bp overlap between neighboring primer sets) were used for MNase-qPCR spanning the 110 with —no-unal -S —chunkmbs 200 —best -m 1 parameters. Using the mapped reads, DANPOS (v3.0.0)123 was employed (-q 50 smooth_width 10 -H 1 -m 1 —mifrsz 40 -u .1) to identify nucleosome occupied regions using q <10−50 as cutoff on the replicates, except for low MNase digestion, dexamethasone treated, samples where the replicates were pooled prior analysis. A published MNase-seq data from inflorescences64  was analyzed as described above except DANPOS q <10−300 was used to control for higher background. A total of 317,312 (Low MNase Mock treatment), 237,480 (Low MNase Dex treatment), 482, 659 (High MNase Mock treatment), 376,790 (High MNase Dex treatment) and 438712 (Inflorescence)64 significant nucleosomes were called.For MNase-seq (low MNase digestion in mock or steroid treated LFY-GR and high digestion MNase digestion in mock or steroid treated LFY-GR) two independent replicate libraries were constructed as described above for ChIPseq, quantified using NEBNext Library Quant Kit for Illumina , pooled and paired-end sequenced using NextSeq 550/500 High Output Kit v2  on the NextSeq500 platform (Illumina). Quality control and filtering were identical to the ChIP-seq analysis. Mapping was performed using the paired-end mode of bowtie (v1.2.2)16 were generated for regions spanning ±1000 bp of the transcription start sites (TSS) of protein-coding genes. To build the heatmap, read counts were normalized by sequencing depth and counted in 20 bp bins for each gene. Gene regions were sorted by the sum of their normalized read values and averaged in groups of 5. Horizontal Bartlett smoothing124 was applied using an 11-bin window.To validate MNase digestions, read enrichment heatmaps115. To test the reproducibility of the three MNase-seq datasets, Pearson correlation coefficients of the reads from pooled bigwig files in the LFY peak regions were used. Matrices and graphs were generated using deepTools115.Heatmaps comparing transcription factor and nucleosome occupancy were centered on the ChIP-seq summits and ranked by the DANPOS summit occupancy value of the nucleosome that most overlapped with the ChIP summit (±75 bp). ChIP or MNase signals ± 1 kb of the summit were visualized using deepTools v3.1.2intersect function125 and separated into two region files: LFY binding site nucleosome occupied or LFY binding site nucleosome free. Binding peaks whose summit overlapped with a significant nucleosome based on the DANPOS thresholds described above in at least three of the four MNase datasets for root explants were considered nucleosome occupied.To classify ChIP peaks into nucleosome bound vs. naked DNA bound sites, significant ChIP summits were overlapped with significant nucleosome regions using BEDTools v2.26.0 E.coli RosettaTM2 (DE3)  were induced during exponential growth with 1 mM IPTG followed by incubation at 18 °C overnight in LB supplemented with 0.2% glucose. Cell pellets were lysed using 500 mM NaCl, 20 mM Tris-HCl pH 8, 5 mM imidazole, 5% glycerol supplemented with cOmpleteTM EDTA-free protease inhibitor cocktail , 2 mM MgCl2 and 1ul/ml benzonase nuclease . After sonication on ice, cell debris was removed by centrifugation . Proteins were affinity purified from the supernatant using Ni-NTA agarose  and passed through a HiTrap Q HP anion exchange chromatography column . Protein concentrations were determined by SDS-PAGE, using a BSA standard curve run on the same gel.pE-SUMOpro-LFY and pE-SUMOpro-LMI2 in 126. A 152 bp fragment region from the AP1 regulatory region (TAIR10 Chr1:25986457 – 25986608) with the LFY binding site in the center and containing the LMI2 binding site served as wild type nucleosomal template. The LFY binding site mutated fragment was generated by changing GGAAGGACCAGTGGTCCGTA to GGCAGGAAAAGTAATCCGCA, while the LMI2 binding site mutated fragment replaced CCGTCAAT with GGAGACCG.Recombinant human full-length histones H2A, H2B, H3, and H4, which share 80–95% amino acid identity with their Arabidopsis counterparts, were expressed and purifiedAP1 regulatory region DNA fragments containing BamHI sites were cloned into pUC19. At least 1 mg of the resulting plasmids was isolated using Plasmid Mega Kit , digested with BamHI, followed by fragment purification by gel electrophoresis and dialysis15. Subsequently, we Cy5 labeled the three DNA probes 117. We conducted nucleosome assembly with Cy5-labeled DNA fragments by mixing purified core histone dimers and DNA at 1:1 molar ratio in 2 M NaCl supplemented with 0.1 mg/mL BSA15. Cy-5 labeled DNA was then assembled around core histones by stepwise dialysis with decreasing concentration of salt and urea as previously described15. Initially, assembled nucleosomes were run on a native gel to see whether multiple nucleosome bands formed—an additional 2 hr 42 °C heat shift was performed if multiple nucleosome conformations were present as previously described15. Glycerol gradients of the dialyzed assembled nucleosomes were employed to separate free DNA from nucleosomes and the fractions collected were then dialyzed in 10 mM Tris–HCl pH = 8.0 for 1 h in 4 °C to remove glycerol as previously described15. Finalized dialyzed nucleosomes were concentrated using Amicon Ultra-0.5 ml (ultracel 10 K)  at 16,500 × g for 10 min in 4 °C.After synthesis (GeneScript), 15. Briefly, Cy-5 labeled DNA fragments and nucleosomes were diluted to 10 nM concentration in EMSA buffer (10 mM Tris-HCl (pH 7.5), 1 mM MgCl2, 10 μM ZnCl2, 1 mM DTT, 10 mM KCl, 0.5 mg/ml BSA). Serial dilutions of transcription factors were conducted in EMSA buffer to achieve the desired concentrations (ranging from 1 nM to 500 nM depending on the reaction). To test for affinity, 10 μl of diluted proteins of various concentrations were added to 10 μl of Cy-5 labeled DNA or nucleosomes. Reactions were then incubated at room temperature for 30 min in the dark followed by analysis in 5% nondenaturing polyacrylamide gels at 100 V for 75 min.DNA and nucleosomal binding reactions were performed using established proceduresDs were calculated from two separate EMSA binding curves per sample, each representing one independent experiment. Image analysis was conducted using image J127. The experimental data was analyzed using the ‘non-linear regression’ function with ‘One site – Total’ in GraphPad Prism (v8.0) software128. Bmax less than 1 and R2 values between 0.8 and 0.99 were met to ensure actual fit of data128. KDs were either computed from the reduction in DNA or nucleosome bound fractions  or based on the first appearance of a DNA or nucleosome bound complex (designated specific KD) as in Ref. 15.Apparent K129. RNA secondary structure was removed by a 5 min 65 °C incubation followed by immediate cool down. mRNA was selected with OligodT25 dynabeads . Reverse transcription was performed using the SSIII RT kit  followed by end repair of cDNA using an enzyme mixture of T4 PNK and T4 DNA polymerase (Enzymatics Y9140-LC-L). After generating a 3′ A-overhang by Klenow HC , adapters were ligated with T4 DNA ligase (Enzymatics T4 DNA ligase (Rapid) #L603-HC-L, 600 U/μl). One-sided selection with SPRIselect beads  was conducted before library amplifications with P5 and P7 index primers). Library quantification was performed with the NEBNext Library Quant Kit for Illumina . Single-end sequencing was conducted using NextSeq 550/500 High Output Kit v2  on the NextSeq500 platform (Illumina).For RNA-seq, two biological replicates were generated for each treatment (mock or steroid) and timepoint . Root explants were treated with either 5 µM dexamethasone or mock solution for 24 h, 6 h, or 1 h before the end of the 5-day incubation on CIM plates. RNA from each sample (ca. 0.2 g) was purified using the RNeasy mini kit  after TRIzol  extraction130 mapping algorithm to the Araport11131 annotation of the Arabidopsis genome 130. Specific read-coverage was assessed with HT-Seq 132. For quality control, Spearman correlation coefficients of the reads in all protein coding and miRNA genes (Araport 11) of all biological replicates were compared using deepTools115.Quality control and filtering were identical to the ChIP-seq analysis. Reads were mapped using the STAR (v2.7.3)133 parameters with normal shrinkage and adjusted134p-value cutoff of less than 0.01133. This yielded 54, 189, 2042 differentially expressed genes 1, 6, and 24 h after dexamethasone relative to mock treatment, respectively.Pairwise differential expression analyses were performed by comparing pooled normalized read counts from dexamethasone- to mock-treated samples using default DESeq2 (v3.9)131. Two rounds of annotation were performed. First, all peaks that were 3 kb upstream, or within genic regions were annotated to that gene. Second, orphan peaks were annotated to the nearest LFY dependent gene within 10 kb of the peak. LFY dependent genes are defined as genes that displayed rapid changes in gene expression after LFY-GR activation (this study and Ref. 57). Plant GOSlim analyses were performed in AgriGO v2.0135.Significant LFY ChIP peaks were annotated to release 11 of Arabidopsis genome annotation (Araport11)5NL0), LFY (PDB: 2VY1), FoxA2 (PDB: 5X07) and ARF1 (PDB: 4LDX) were visualized and aligned using PyMOL v2.3 128.Structures of linker histone H1  of at least two independent biological replicates. The hypergeometric test137 was used to test whether two datasets significantly overlapped.For all qPCR data, the Kolmogorov–Smirnov (K–S)Further information on research design is available in the Supplementary InformationPeer Review FileDescriptions of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Reporting Summary


The epigenetic inheritance relies on stability of histone marks, but various diseases, including aging-related disorders, are usually associated with alterations of histone marks. Whether and how the proteasome is responsible for maintaining the histone marks during transcription and aging remain unclear. The core histones can be degraded by the atypical proteasome, which contains the proteasome activator PA200, in an acetylation-dependent manner during somatic DNA damage response and spermiogenesis.Methods: By utilizing a substitute of methionine to label proteins metabolically, we analyzed histone degradation genome-wide by sequencing the DNA fragments following pulse-chase assays. The genome-wide RNA-sequencing analysis was performed to analyze transcription and chromatin-immunoprecipitation (ChIP)-sequencing was used for analyses of histone marks. The experimental models included gene-manipulated cells (including both mouse and yeast), mouse liver, and mice.Results: Degradation of H4 or the transcription-coupled histone variant H3.3 could be suppressed by deletion of PA200 or its yeast ortholog Blm10. The histone deacetylase inhibitor accelerated the degradation rates of H3, while the mutations of the putative acetyl-lysine-binding region of PA200 abolished histone degradation in the G1-arrested cells. Deletion of PA200 dramatically altered deposition of the active transcriptional hallmarks (H3K4me3 and H3K56ac) and transcription, especially during cellular aging. Furthermore, deletion of PA200 or Blm10 accelerated cellular aging. Notably, the PA200-deficient mice displayed a range of aging-related deteriorations, including immune malfunction, anxiety-like behavior and shorter lifespan.Conclusion: PA200 promotes the transcription-coupled degradation of the core histones, and plays an important role in maintaining the stability of histone marks during transcription and aging. The current model for epigenetic inheritance relies on stability of histone marks. The core histones, including H2A, H2B, H3 and H4, form an octamer to pack DNA into the nucleosome, the basic unit of chromatin Proteasomes catalyze degradation of most cellular proteins, and consist of a 20S catalytic particle and one or two activators, such as the 19S regulatory particle, PA28α/β, PA28γ and PA200/PSME4 In this study, we show that the core histones are degraded in the G1-arrested cells, and present evidence that PA200 promotes the transcription-coupled degradation of the core histones, and maintains the stability of histone marks during transcription and aging.Detailed methods are available in +/+, PA200-/- MEF cells, and PA28γ-/- MEF cells were isolated from the embryos of the wild-type, PA200-deficient mice and PA28γ-deficient mice, respectively. The permanent wild-type and PA200-deficient MEF cells were obtained from the mice as described MEF cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 μg/ml streptomycin, 1% non-essential amino acids and 200 μM of β-mercaptoethanol. Primary PA200All yeast strains were grown at 30°C in yeast peptone dextrose (YPD). The yeast strain BY4741 (MATa his3△1 leu2△0 met15△0 ura3△0) was from Dr. Wei Li. The strain YHS539 (MATa BY4741 yak1-GFP (kan), blm10::Nat hat9) was obtained from Dr. Daniel Finley. The yeast strain Blm10 O/E (MATa BY 4741 blm10::NAT GPD-HA-Blm10) was obtained as described The pulse-chase assay was employed to determine histone degradation by labeling proteins with Aha After pulse-chase analysis as described as above, the DNA fragments associated with the newly-synthesized H3/H4 proteins were then purified with DNA purification columns  and processed for ChIP-seq as described later.-/- samples.We used MAnorm to statistically compare the signal density of ChIP-seq data at chase times 0 and 4 h. To identify the degraded region of the G1-arrested cells, we selected the unique peaks which were present in ChIP-seq at 0 h, but were absent at 4 h. We selected the common peaks whose read density at 0 h was significantly larger than that at 4 h. Then, we defined the PA200-depdentdent histone degradation regions, which were not overlapped between the wild-type and the PA200Old yeast cells were isolated by using EZ Link Sulfo-NHL-LC-LC-Biotin (Thermo Scientific) to label cell surface proteins (Invitrogen) 2, 10 mM β-glycerophosphate, 5 mM tetrasodium pyrophosphate, 1 mM NaF, 1 mM Na3VO4, and a mixture of protease inhibitors. Protein samples were separated by SDS-PAGE and transferred onto PVDF membranes. Antibodies or antisera against specific proteins were used as primary antibodies to detect the corresponding proteins. Peroxidase-conjugated anti-mouse IgG (1:5000) or anti-rabbit IgG (1:5000) was used as the secondary antibody. The protein bands were visualized by ECL detection system (Millipore) or ODYSSEY (LI-COR).Cells were lysed in the buffer containing 20 mM Tris-HCl, pH 8.0, 100 mM KCl, 0.2% Nonidet P-40, 10% glycerol, 1 mM ZnClCells were plated on coverslips, cultured in a six-well plate and then subjected to appropriate treatments. Cells were fixed with 4% formaldehyde, blocked with 5% normal goat serum for 60 min at 37 °C, and then incubated with anti-PA200 or anti-H4K16ac antibody overnight at 4 °C, followed by incubation with Alexa fluor 594-conjugated anti-rabbit IgG or Alexa fluor 488-conjugated anti-mouse IgG for 60 min at room temperature. The nucleus was stained with DAPI. Finally, the coverslips were examined with a confocal laser scanning microscope.https://www.ncbi.nlm.nih.gov/bioproject) with accession number PRJNA451247. The data that support the findings of this study are available from the authors upon request. Correspondence and requests for materials should be addressed to X.B.Q. (xqiu@bnu.edu.cn) or K.L. (linkui@bnu.edu.cn).All sequencing data, including RNA-seq, ChIP-seq, genome-wide analysis of histone degradation, and WGBS, have been deposited at the BioProject database , azidohomoalanine (Aha). In this assay, Aha was co-translationally incorporated into proteins and subsequently ligated with biotin. Thus, following chase in the regular medium with Met, old histones with Aha could be purified with streptavidin for analysis of degradation A. In thee.g., H3.1), is only integrated into chromatin during transcription Because amanitin was added for 24 h before pulse-chase analysis, Rbp1 was not detectable even at 0 h of the chase. Probably because H3.3, but not other species of H3 . These results further support our previous notion that acetylation is involved in the degradation of the core histones by the PA200-proteasome We previously showed that the PA200-proteasomes promote the acetylation-dependent degradation of the core histones during DNA repair and spermiogenesis By utilizing Aha to label proteins metabolically, we performed genome-wide analysis of histone degradation (GAHD) by sequencing the DNA fragments purified together with histones following 2 h-pulse labeling with Aha and chase in the regular medium for 0 or 4 h. In the wild-type MEF cells at 0-h chase, the levels of the Aha-labeled core histones (primarily H3 and H4) peaked at transcriptional start sites (TSS) genome-wide  in the PA200-deficinet MEF cells at 0 h of chase , down-regulated DEGs , and not significantly changed genes (Cers2 and Gpc4) on H3K4me3 or H3K56ac in the G1-arrested were further validated by quantitative PCR  nor the down-regulated DEGs  showed these effects in the PA200-deficient MEF cells that were not arrested (Hoxc13 and Hoxa3) were further validated by quantitative PCR analysis -sequencing analyses of histone marks, which revealed a slight down-regulation of H3K4me3 on the gene bodies and 21 kb upstream of TSS and 21 kb downstream of TTS  regions in the G1-arrested PA200s Figure A. The uptive PCR B-C. In carrested B-C. KEGGarrested A-D. H3K4arrested E-F. It iarrested A-H. Repranalysis I-J. Furt-/- MEFs  Cdkn2b (INK4bp15) Hmgb1Sod1Cdkn2a (INK4ap16) Bmp4, and decreased on gene bodies of some aging-suppressing genes, such as Hmgb1 and Sirt2-/- MEF cells . Enrichment of H3K4me3 or H3K56ac at promoters was generally in a positive correlation with the changes in the RNA levels of specific genes  in primary MEF cells cultured for 0 (young) or 30 (old) days were further validated by quantitative PCR (We next found that deletion of PA200 reduced the proliferative potential B, and intive PCR E. Meanwhtive PCR B. Deletitive PCR F-H. The tive PCR C. In ordtive PCR D, which tive PCR , support-/- mice had more memory and fewer naive CD4 T cells, however, these differences were not significant in the younger cohort (Figure To further explore whether PA200 participates in regulation of aging, we analyzed the wild-type and PA200-deficient mice for distinct parameters of aging. The aging-related increase in memory T cells and reduction in naive T cells are important predictors of senescence t Figure A. Renal t Figure B. We als-/- mice than that in the wild-type mice. Unlike the wild-type mice, which showed an 8% decrease in gastrocnemius muscle fiber diameter between 3 and 12 months old, the PA200-deficient mice had a 15% decrease with age (Figure Gastrocnemius muscle fiber diameter declined with age more seriously in PA200e Figure C, suggese Figure D-E, indie Figure F. Finalle Figure G. Taken e.g., by degrading histone chaperones that assist assembly of the nucleosome, the transcription-coupled degradation of histones has not yet been validated. We have previously shown that the PA200-containing proteasomes can promote the acetylation-dependent degradation of the core histones during somatic DNA repair and spermatogenesis in vitroThe pattern of the histone post-translational modifications has been proposed as “histone code” The 19S complex of the 26S proteasome was shown earlier to function independently of the 20S catalytic particle, playing a direct and non-proteolytic role in RNA polymerase II-mediated transcription Partial histone loss across the genome has been shown to be associated with aging in both yeast and human cells Although the cellular aging can prevent tumor development early in life, aging is also marked by an increase in tumorigenesis It has been a mystery how histone marks remain stable during transcription. Histones are frequently evicted from the nucleosome and then re-assembled in a transcription-coupled manner, a process referred to as nucleosome turnover The proteasome activator PA200 promotes the transcription-coupled degradation of the core histones, and plays an important role in maintaining the stability of histone marks during transcription and aging.Supplementary figures and tables.Click here for additional data file.


Heterochromatin Protein 1 (HP1) is a highly conserved protein that has been used as a classic marker for heterochromatin. HP1 binds to di- and tri-methylated histone H3K9 and regulates heterochromatin formation, functions and structure. Besides the well-established phosphorylation of histone H3 Ser10 that has been shown to modulate HP1 binding to chromatin, several studies have recently highlighted the importance of HP1 post-translational modifications and additional epigenetic features for the modulation of HP1-chromatin binding ability and heterochromatin formation. In this review, we summarize the recent literature of HP1 post-translational modifications that have contributed to understand how heterochromatin is formed, regulated and maintained. Heterochromatin is tightly packed DNA which is usually transcriptionally inactive. It is established in early development through controlled epigenetic processes  that neeHeterochromatin is characterized by the presence of specific epigenetic marks that are important for its establishment, maintenance and regulation. One of the best-known heterochromatin markers is Heterochromatin Protein 1 (HP1): it recognizes and binds to di- and tri-methylated lysine 9 in histone H3 (H3K9me2/3) to form a repressive chromatin environment ,6. How HCBX5); HP1β, encoded by CBX1; HP1γ, encoded by CBX3. Recently, a shorter HP1γ splice variant was also reported [HP1 is a conserved family of proteins that was discovered as a suppressor of variegation through studies on position-effect variegation (PEV) in Drosophila . In mammreported .CBX5, CBX1 and CBX3 null mutant mice revealed different phenotypic outcomes for the three isoforms: CBX5−/− mice presented a normal phenotype, CBX1−/− led to perinatal lethality, and CBX3−/− to infertility [HP1α, HP1β, and HP1γ are approximately 65% identical, but they present distinct localization patterns and fulfil different functions within the cells: while HP1α is located predominantly at constitutive heterochromatin, HP1β and HP1γ are uniformly distributed in the cell nucleus and are found in both hetero- and euchromatic regions ,11,12. Certility ,14.The HP1 family belongs to a superfamily of proteins that contain conserved chromatin organization modifier (chromo) domains. The chromodomain is a 30–60 amino acids long sequence  situatedThe HP1 chromo- and chromoshadow domains are highly conserved: the chromodomain from mouse HP1β can functionally replace the one of S. pombe HP1  and exprHP1 binds to H3K9me3 through its chromodomain; how this interaction occurs has recently been reviewed . HoweverH3S10ph also takes place in interphase, where it has been associated with transcription activation ,30,31. IHistone acetylation also influences the HP1-chromatin binding ability. In fission yeasts, H3K4 acetylation at pericentric heterochromatin by Mst1 enhances the switch between Clr4 (Su(var)39 homologue) and Swi6; its mutation to a non-acetylable residue decreases Swi6 pericentric occupancy and reduces heterochromatin formation . In SaccDespite the importance of histone modifications in regulating heterochromatin formation, post-translational modifications of HP1 itself have been studied less, but there is evidence showing that they can greatly modulate its chromatin binding ability.HP1 isoforms can be modified at several residues: 35 modification sites have been detected for HP1α, 34 for HP1β, and 37 for HP1γ . These mPhosphorylation is the most abundant and studied HP1 modification. One of the first indications that HP1 phosphorylation was important for heterochromatin assembly was reported in the mid-1990s, when Eissenberg et al. (1994) showed that HP1 becomes hyperphosphorylated in Drosophila embryos at about the same time when heterochromatin is first visible . Since tN-terminal tail of HP1α (S11–S14)—lacking in HP1β and HP1γ—seems to be critical for HP1α binding to H3K9me3 [N-terminal tail, allowing the negatively charged neighboring acidic amino acids (15EDEEE19) to interact with the positively charged sequence surrounding H3K9me3 (8RKmeSTGGKAPRK18) [Recent studies reported that HP1 can form liquid droplets upon H3K9me3 binding, which leads to the accumulation of several repressive factors that can physically sequester and compact the nearby chromatin ,47,48. T H3K9me3 ,50,51. TKAPRK18) . Casein KAPRK18) ,54. CKIIKAPRK18) .CKII also phosphorylates other HP1 sites. HP1β S89 and S175 have been reported as targets of CKII, but these phosphorylations do not seem to affect its chromatin binding ability . HoweverOther kinases have been identified to target HP1 sites. The serine/threonine-protein kinase Pim-1  has beenHP1 proteins can also be acetylated in the hinge, chromo- and chromoshadow domains , althougHP1 can also be methylated at several residues ,44; monoThe specific HP1 methyltransferases are still unknown. However, as LeRoy et al. (2009) pointed out, all three HP1 isoforms can directly interact with the methyltransferase Su(var)39H1 through its chromoshadow domain , so it mLysine formylation has been reported in histones and other nuclear proteins and linked to chromatin function and regulation . LeRoy eAnother known modification of HP1 is peptidyl citrullination, where an arginine residue is converted to citrulline, a non-encoded amino acid, losing a positive charge and reducing the hydrogen bonding ability. Wiese and colleagues  found thSeveral studies have shown the implication of SUMO proteins in transcription repression regulation and maintenance of silenced heterochromatin . HP1α caIn SUV39H1-depleted cells, SUMOylated HP1α was targeted to the chromatin but it could not be maintained , suggestUbiquitination is yet another important post-translational modification of HP1 and several groups have reported degradation of HP1 by several ubiquitin ligases.During DNA damage, RAD6 has been linked to the ubiquitination of HP1 at K154, which promotes its degradation, forming an open chromatin state that will enable DNA repair .This degradation system could be very important for the remodelling of chromatin that occurs during DNA damage. In fact, this targeted disruption could be coordinated with the eviction of histones at sites of DNA breaks, also proteasome-mediated, in order to facilitate the recruitment of repair factors .The ubiquitin ligase RNF123 has also been shown to ubiquitinate and contribute to the degradation of HP1α and HP1β, but not HP1γ, upon lamin A/C mutations . HECW2, When cells enter mitosis, chromatin is subjected to several changes that allow the formation of mitotic chromosomes for proper segregation of the genetic material. In addition to H3S10ph as a regulator of HP1-chromatin binding through mitosis, PTMs of HP1 itself also play a role in proper mitotic progression.Early in this century, Mink et al. (2000) already reported that HP1α and HP1γ are hyperphosphorylated during mitosis . Aurora Although the majority of HP1α is removed from the chromatin during mitosis, a small fraction is retained at the centromeres  where itHow HP1α achieves this function at the centromeres has been studied by several groups, yet some aspects remain unclear. Although the role of H3K9me3 for HP1α-chromatin binding has been well studied, some evidence shows that the histone variant H2A.Z is also important for HP1α function at the centromeres ,76,77. HHP1α was also identified as one of the first known binding partners of the chromosomal passenger complex (CPC) , a cruciHP1α–INCENP appears to be important for some Aurora B functions: while HP1α–INCENP disruption does not alter the levels of Aurora B-mediated H3S10 and CENP-AS7 phosphorylation , the phoShugoshin (Sgo1) is a protein that prevents premature cohesin dissociation . Sgo1 alAlthough the role of HP1α in pericentric heterochromatin and chromosome segregation has been addressed, the implications of HP1β and HP1γ still remain controversial. Some studies report that all three HP1 isoforms co-immunoprecipitate with INCENP , while oHP1α PTMs are also important for centromeric targeting; specifically, phosphorylation of the hinge region seems to regulate HP1α centromeric localization during mitosis. In early prophase, Aurora B phosphorylates HP1α at S92 ; this moThe role of epigenetics in human diseases has gained interest in recent years as our knowledge on epigenetic-regulated processes has increased. How chromatin is organized and structured has profound implications on both gene expression and genome stability and chromosome segregation, which can result in abnormal cell functions and lead to the origin of different diseases. Thus, studying the mechanisms involved in the regulation of chromatin architecture is crucial, not only to understand many aspects of cell biology, but also to address treatment of some diseases.Protein regulation by post-translational modifications has been known for many years and it is now accepted that just studying the protein itself is not sufficient to fully understand its role. In this review, we summarized the recent literature that encompasses HP1 post-translational modifications and how they affect its chromatin binding, phase separation behavior and heterochromatin formation. Although several studies have identified the specific enzymes involved and the implications these modifications might have,


Liver cancer is one of the most common cancer types worldwide and the fourth leading cause of cancer-related death. Liver carcinoma is distinguished by a high heterogeneity in pathogenesis, histopathology and biological behavior. Dysregulated signaling pathways and various gene mutations are frequent in hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (iCCA), which represent the two most common types of liver tumors. Both tumor types are characterized by telomere shortening and reactivation of telomerase during carcinogenesis. Continuous cell proliferation, e.g., by oncogenic mutations, can cause extensive telomere shortening in the absence of sufficient telomerase activity, leading to dysfunctional telomeres and genome instability by breakage–fusion–bridge cycles, which induce senescence or apoptosis as a tumor suppressor mechanism. Telomerase reactivation is required to stabilize telomere functionality and for tumor cell survival, representing a genetic risk factor for the development of liver cirrhosis and liver carcinoma. Therefore, telomeres and telomerase could be useful targets in hepatocarcinogenesis. Here, we review similarities and differences between HCC and iCCA in telomere biology. Liver cancer is predicted to be the sixth most common tumor disease worldwide and the fourth leading cause of cancer-related death . Liver cThe major risk factors in hepatocarcinogenesis are chronic viral infections with hepatitis B virus (HBV) and hepatitis C virus (HCV), heavy alcohol consumption, obesity, type 2 diabetes, smoking and long-term exposure to aflatoxin B . Most liTelomere shortening and reactivation of telomerase, two common hallmarks of carcinogenesis, are described in a broad range of human cancers, including liver cancer ,20,21. TINK4a and p21WAF1/Cip1 and are positive for senescence-associated β-galactosidase staining. A disruption of the p53-signaling pathway overcomes the senescence checkpoint and leads to further cell division of hepatocytes with already-shortened telomeres until the telomeres become critically short. At this point, the cells enter the crisis checkpoint, which is characterized by massive cell death ,106TERT  hotspot ,112,114 r family ,84, leadr family . Recentlsequence .TERT promoter mutation frequency in HCC differs geographically. By comparing different studies, Pezzuto et al. [TERT promoter mutations seem to be most common in Europe (56.6%). In Africa, TERT promoter mutations were identified in 53.3% and in Asia in 42.5% of HCCs. The overall TERT promoter mutation rate in America is around 40% [Interestingly, the o et al.  reportedound 40% .TERT promoter region in HCC, TERT promoter mutations are less frequently analyzed in cholangiocarcinoma [TERT promoter mutations in 31–47% of the HCCs. Nakamura et al. analyzed the largest dataset of 145 iCCAs (named as ICCs in the study). A TERT promoter mutation was detected only in one iCCA sample. Furthermore, the same study also analyzed a set of 86 extrahepatic cholangiocarcinomas, with none of them exhibiting a TERT promoter mutation [In contrast to frequent mutations in the arcinoma ,125,126.TERT promoter mutation was detected in iCCA samples in the majority of studies [TERT promoter mutations were observed in iCCA [While no  studies ,96,116,  in iCCA ,124,126 TERT promoter mutations in 5.12% (4/78) of intrahepatic cholangiocarcinoma and in 53.3% (8/15) of combined hepatocellular cholangiocarcinoma [The study by Fujimoto et al. was focused on different subtypes of liver cancer displaying biliary phenotype (LCB). They described arcinoma .TERT promoter mutations in HCC/iCCA may reflect the presence of the HCC part consisting of the TERT promoter mutation or indicate that both tumor types may arise from the same cell type of origin  . Taken tany HCCs .TERT promoter activation and eventually to telomerase reactivation. Frequent amplifications of the TERT gene have been shown in human cancer cell lines and in human tumors [TERT gene was found in around 22% of hepatocellular carcinoma and shows a higher incidence in poorly differentiated hepatocellular carcinoma [TERT mRNA level did not correlate with the number of TERT gene copies, as was also reported in colorectal cancer [TERT) were observed with a reduced overall survival, independently of other clinicopathological parameters in patients with hepatocellular carcinoma [TERT amplifications do not always lead to increased TERT mRNA levels, which are by themselves not assignable to increased TERT translation levels.Structural and numerical aberrations in the organization of the genome can lead to n tumors ,128. Thearcinoma . Neverthl cancer ,130. Focarcinoma . HoweverTERT gene have been shown in several cancers like neuroblastoma, renal cell carcinoma, sarcoma and prostate carcinoma, and also in liver carcinoma [TERT promoter region [TERT promoter region might be a reason for a low rate of TERT promoter mutations in HBV-related HCC, which is known to induce telomerase transcription [Structural rearrangements in the arcinoma ,132,133.r region ,135,136.cription ,119. ThiTERT promoter region and thereby lead to overexpression of the TERT gene [TERT overexpression and telomerase reactivation [AAV2 (adeno-associated virus type 2) is the second virus reported to integrate into HCC cell lines and hepatocellular carcinoma of human patients . The insERT gene . The AAVtivation .TERT gene regulation in hepatocytes and HCC. The RB/E2F gene regulatory circuit regulates TERT promoter activity during liver regeneration and cancer [TERT regulation is supported by a recent report showing that the Human Krüppel related 3 (HKR3) is capable of repressing TERT gene expression in HCC cell lines, resulting in the subsequent activation of CDKN2A (encoding the p16 tumor suppressor factor) and cell cycle inhibition as a tumor suppressor mechanism [TERT gene expression in HCC cell lines, indicating a role of telomerase activation in promotion of HCC [TERT promoter has been shown to correlate with increased patient mortality [Studies addressing telomerase activation in liver cells mainly focused on d cancer . The rolechanism . In anotn of HCC . Concurrortality .TERT gene expression via nucleosome remodeling in HCC cell lines [TERT gene activation to c-MYC, which was the first transcription factor shown to regulate TERT expression by direct promoter binding [c-MYC amplification correlates with liver cancer progression, mainly HCC, and to a lesser extent, iCCA [TERT gene expression through SP1 or YAP1, respectively [On the other hand, the bromodomain PHD finger transcription factor (BPTF) seems to regulate ll lines . BPTF is binding ,145,146.nt, iCCA ,148. In ectively . The exaTERT promoter methylation and histone acetylation may be involved in the regulation of TERT transcription and hence telomerase activity. The mechanism of MAD1-mediated repression involves modification of the chromatin structure by histone deacetylation [TERT gene expression and telomerase activity [HDAC1 causes the repression of the TERT gene and telomerase activity [TERT regulation during carcinogenesis remains pending.There are a number of studies indicating that tylation . In factactivity , whereasactivity . To whatTERT gene expression takes place indirectly, namely via the promoter methylation of genes for transcription factors which are responsible for TERT gene expression. A recent report showed the cooperation between TERT and the transcription factor SP2 to stimulate DNMT3B transcription, while TERT depletion inhibited DNMT3B expression [TERT and DNMT3B expression predicted shorter survival in HCC patients based on the TCGA database [The overexpression of DNMTs is thought to be responsible for suppressing the expression of tumor suppressor proteins by methylating the promoters of their genes, which leads to the early switching off of these factors. DNMT3B in particular is strongly overexpressed in breast tumors and correlates with the degree of tumorigenesis . It can pression . Higher database .TERT promoter was studied in some detail. Initial reports regarding the methylation state and the activity of the TERT promoter were contradictorily unequivocal: while Devereux et al. [TERT expression, Guilleret et al. [TERT promoter. Moreover, studies on TERT promoter methylation in HCC revealed contradictory results. Zhang et al. [TERT promoter in HCC patients of the Han Chinese population showed a nearly 56-fold increase of TERT expression from the hypermethylated promoter. In contrast to that study, an analysis of 106 patient tissues (64 with HCC and 42 without liver disorders) and hepatocarcinoma cell lines revealed that the TERT promoter was methylated in normal liver but was hypomethylated in most of the hepatocellular carcinomas [On the other hand, the methylation status of the x et al.  and Dessx et al.  did not t et al.  found a g et al.  observedrcinomas .TERT promoter using more advanced technologies, arguing that the methylation analysis of a small number of CpG sites may not represent the methylation landscape of the whole TERT promoter [TERT hypermethylated oncological region (THOR) defining a 433 bp genomic region within the TERT promoter as a cancer-associated epigenetic mechanism of TERT upregulation. This region encompasses 52 CpG sites and is located upstream of the TERT promoter mutations (TPMs) [TERT promoter mutations or the alternative lengthening of telomeres (ALT). In line with these results, Esopi et al. [TERT promoter sequences in cancer cells is associated with TERT repression, while the remaining unmethylated allele marked with an open chromatin is largely responsible for the TERT expression in cancer cells [TERT promoter mutations, the expressed allele is mutated, while the WT allele is silenced [TERT promoter activity can be regulated by epigenetic mechanisms in a tissue- and cancer-type-specific manner.Recent studies point out the importance of a comprehensive mapping of the methylation landscape within the promoter . Using as (TPMs) . Five Cps (TPMs) . Tumors i et al.  describei et al. . Intereser cells . In cancsilenced ,161. In The fact that telomerase is repressed in somatic tissues but active in cancer cells qualifies it as an ideal target for cancer therapy. On the other hand, impairment of telomere length and/or structure could be a target for tumor therapy as telomere length in cancer cells is usually shorter than in the corresponding normal tissue cells. Moreover, due to their high turnover, functional telomeres have to be rebuilt more often in tumor cells than in normal cells. Thus, deterioration of the intact telomere structure by compounds may represent an attractive target to cure cancer.TERT promoter-driven oncolytic adenovirus (Telomelysin (OBP-301)) or TERT promoter-driven pro-apoptotic protein  to take advantage of high telomerase activity in cancer cells for therapy options. For further reading, we refer to a recent excellent article, which discusses this topic in greater detail [Several telomerase inhibitors have been used in preclinical studies and clinical trials, involving telomerase inhibitor molecules, such as the antisense oligonucleotide Imetelstat GRN163L) or the small-molecule inhibitor BIBR1532, as well as the G-quadruplex stabilizers . There are also interesting alternative strategies such as telomerase vaccination  to induce anti-tumor responses or tumor cell lysis through r detail . FurtherL or the TERT promoter mutations were more frequently observed in HCCs compared to iCCAs. The low frequency of TERT promoter mutations reported in iCCA might be due to a not-detected HCCs part (mixed differentiation) of the analyzed tumors. This links to the phenomenon that some tumor entities lacking specific mutations in the TERT promoter region and where other mechanisms leading to telomerase activation exist. As described above, the TERT promoter region contains binding sites for many transcription factors, i.e., c-MYC, E2Fs, and others, which contribute to the tissue-specific regulation of TERT gene expression [TERT expression is regulated differentially in HCC and iCCA. The identification of the regulatory mechanisms contributing to telomerase reactivation in HCC and iCCA could shed light on the differential tumor initiation and progression pathways and may provide alternative and specific therapy options.The biology of telomere maintenance plays a major role in the process of cirrhosis formation as well as in initiation and progression in liver carcinoma. A shortening of telomeres occurs due to progressive chronic liver diseases and highly correlates with an increased tumor risk. Telomere shortening occurs in HCC as well as in iCCA. In addition, telomerase reactivation is detectable at a high frequency in both tumor entities. The mechanisms of telomerase activation seem, at least in part, to be different between the two liver cancer entities. pression . It woulTERT promoter mutations occur early during liver carcinogenesis highlights the importance of telomerase activity for tumor cell survival. Two possible scenarios are conceivable how telomerase contributes to tumorigenesis in liver cancer. On the one side, telomerase reactivation before entering the crisis checkpoint may stabilize critically short telomeres, providing growth advantage for cells with oncogenic mutations (The observation that utations . In melaOn the other side, early reactivation of telomerase may be related to its non-canonical functions. There is strong experimental evidence indicating that oncogenic mutations or mutations which result in genome instability induce cellular senescence by inducing telomere replication stress ,165. ThiHowever, telomerase activity can alleviate telomere replication stress by a yet-unidentified mechanism and promote tumorigenesis ,166,167.Whether telomere maintenance or even elongation of telomeres during chronic liver diseases or an inactivation of the telomerase could be beneficial for the treatment of liver carcinoma remains to be elucidated in future studies.


Drosophila. These subunits were stably tethered to a transgene reporter carrying the hsp70 core promoter. The tethered subunits mediate transcription of the reporter in a pattern that is generated by enhancers close to the insertion site in multiple loci throughout the genome. Both tethered SAYP and BAP170 recruit the whole PBAP complex to the reporter promoter. However, we found that BAP170-dependent transcription is more resistant to the depletion of other PBAP subunits, suggesting that BAP170 may play a more critical role in establishing enhancer-dependent transcription.The chromatin remodeler SWI/SNF is an important participant in gene activation, functioning predominantly by opening the chromatin structure on promoters and enhancers. Here, we describe its novel mode of action in which SWI/SNF factors mediate the targeted action of an enhancer. We studied the functions of two signature subunits of PBAP subfamily, BAP170 and SAYP, in  The evolutionary conserved SWI/SNF class of chromatin remodelers plays essential roles in multiple processes of cell biology, from the regulation of transcription to chromosome segregation, DNA replication, and DNA repair ,2,3. In Drosophila, as in multiple species, the SWI/SNF remodeler, the Brahma complex, exists in two different forms: BAP (BAF in human and SWI/SNF in yeast) and PBAP (PBAF in human and RSC in yeast) , JFL-attB [lexAop-hsp70-LacZ-DadEnh], JFL-attB [dppEnh-lexAop-hsp70-LacZ], and JFL-attB [lexAop-hsp70-LacZ-dppEnh] were prepared by cloning PCR fragments of the Dad and dpp enhancers in the JFL-attB vector. The JFL-attB vector has been prepared in our lab by modifying pJFRC18-8XLexAop2-mCD8::GFP as follow: (i) the NdeI/XbaI fragment corresponding to the mini-white cassette was recovered from pBPnlsLexA::GADflUw, filled, and cloned into the EcoRV site of pBluescript to obtain the white-PBS; (ii) the white cassette from pJFRC18-8XLexAop2-mCD8::GFP was removed by EcoRV/HindIII digestion and mini-white was reintroduced in the opposite orientation as a HindIII/BamHI(filled) fragment from white-PBS to obtain the JFK plasmid; (iii) the GFP cassette was then removed from JFK by BglII/XbaI digestion, and the LacZ gene was introduced as a BamHI/XbaI fragment of pCasper-AUG-beta-gal to obtain the final JFL-attB plasmid. Primers for cloning the Dad and dpp enhancers in JFL are described in the Constructs for enhancer proximity tests JFL-attB =nos-phiC31\int.NLS}X; P{y[+t7.7]=CaryP}attP2. The fly stocks carrying the TM6B, Tb balancer were used in all experiments; the genotypes are indicated in the figure legends.P element-based transgenic lines were generated by injections into escribed , using trbo Δ2–3 . The chren-GAL4, UAS-GFP; Ms1096-GAL4; UAS-GFP; UAS-RNAi-SAYP [Vdrc105946]; UAS-RNAi-BAP170 [Vdrc34582]; UAS-RNAi-Brm [Vdrc37721]; UAS-RNAi-Mor [Vdrc6969]; UAS-RNAi-PB [Vdrc108618]. The RNAi lines were chosen on the basis of their ability to induce clear defects with tubulin-GAL4 and en-GAL4 drivers, as well with other GAL4 lines in the literature , LexA , FLAG , myc, GFP (Sigma), PolII CTD, histone H3 , H3ac , SAYP , BAP170 Immunofluorescence analysis on polytene chromosomes was made according to the protocol .Whole L3 larvae were taken for analysis as described in . BrieflyLexAop-LacZ plasmid was used for calibration, the data were normalized to the RpII locus [hsp70 genes as well, but this hybridization was neglected because the genes are at great distance  away from Dad. Primers for qPCR are given in the A 3C library was obtained essentially as described in  with minII locus . The ancIn these experiments, error bars indicate SDs of quadruplicate PCR technical replicates from at least three independent biological repeats.