R,6R;2S,6S)-hydroxynorketamine (HNK), which also appear to play a major role in ketamine’s rapid antidepressant effects. An HMC3 human microglial cell line was used as a model system to test a possible role for ketamine in immune response regulation that might contribute to its antidepressant effects. Our results highlight the fact that ketamine and its two active metabolites can regulate the type I interferon pathway mediated, at least partially, through signal transducer and activation of transcription 3 (STAT3) which plays a major role in the immune response. Specifically, STAT3 downstream genes that were modulated by either ketamine or its active metabolites were enriched in the “response to type I interferon” pathway. Our data also suggest that STAT3 might play a role in ketamine’s antidepressant effects, mediated, at least in part, through eukaryotic elongation factor 2 (EEF2), resulting in the augmentation of brain-derived neurotropic factor (BDNF) expression and promoting the synthesis of synaptic proteins postsynaptic density protein 95 (PSD95) and synapsin I (SYN1).Inflammation is an important biological process which contributes to risk for depression, in part as a result of the production of proinflammatory cytokines and of alterations in glutamatergic neurotransmission. Ketamine has anti-inflammatory properties which might contribute to its antidepressant effects. This study was designed to clarify mechanisms of action for ketamine and its active metabolites, (2 All drug treatment conditions were optimized as described in our previous study (The present study was designed to 1) identify molecular signatures for ketamine and its two active metabolites in human microglial cells, the predominant immune cells in the brain which play a central role in immune response regulation in the central nervous system, and 2) to determine possible molecular mechanisms underlying ketamine’s antidepressant effects. Our studies began with a determination of genome-wide expression profiles of HMC3 cells in response to treatment with either ketamine or the two known ketamine active metabolites -2-amino-2-(2-chlorophenyl)-6-hydroxycyclohexanone hydrochloride  (lot number: NCGC00378227-18), and -2-amino-2-(2-chlorophenyl)-6-hydroxycyclohexanone hydrochloride  (lot number: NCGC00373033-12) were synthesized and characterized in Dr. Craig Thomas’s laboratory at the National Center for Advancing Translational Sciences . Before treatment with ketamine or the two active metabolites, cells were cultured in EMEM media containing 5% (V/V) charcoal stripped FBS for 24 h, and were subsequently cultured in FBS free Roswell Park Memorial Institute (RPMI) media for another 24 h. Cells were then treated with ketamine or with the ketamine metabolites -HNK or -HNK for 24 h in serum and phenol red-free medium. In some experiments, cells were exposed to 0.1 nM E2  together with 400 nM ketamine, -HNK, or -HNK, at concentrations similar to plasma concentrations observed during ketamine therapy  were cultured in Eagle’s Minimum Essential Medium (EMEM)  supplemented with 10% fetal bovine serum (FBS) . Ketamine hydrochloride was purchased from Pfizer . Both ( therapy .https://david.ncifcrf.gov/) . RNA-seq experiments were conducted by the Mayo Clinic Center for Individualized Medicine Medical Genomics Facility. RNA-seq libraries were prepared with Ovation RNA-seq system v2 kit (NuGEN) according to the manufacturer’s instruction, and were sequenced using an Illumina HiSeq 2000 with eight samples in each lane using 100 bp paired end index reads. Fastq files containing paired RNASeq reads were aligned with Tophat 2.0.12  against rf.gov/) . RNA-seq7™ Real-Time PCR System . The 2-ΔΔCt method was employed for statistical data analysis.The PCR reactions contained 100 ng of total RNA, 5 µl of 2X VeriQuest SYBR green qPCR master mix , 0.1 µl of DNA polymerase, 1 µl of gene specific primer, and distilled water up to 10 µl final volume per reaction. Primer sets for real-time PCR are listed in HMC3 cells were grown on glass coverslips and treated with ketamine or the two active metabolites for 24 h. Cells were then fixed in 4% paraformaldehyde at room temperature for 15 min. Cells were washed in cold phosphate-buffered saline (PBS) and permeablized with 0.2% Triton X-100 in PBS. After blocking for 1 h with 3% bovine serum albumin (BSA), cells were incubated with anti-STAT3 or anti-SIN3A antibodies overnight at 4°C. The secondary antibody was used at 1:1,000 dilution for an hour. Antibodies for immunofluoresence are listed in 7) were resuspended in 1 ml immunoprecipitation (IP) lysis buffer containing 5 µl protease inhibitor cocktail  and were incubated on ice for 30 min. Cells were then centrifuged at 12,000 g at 4°C for 15 min. Supernatants were collected. Protein A agarose  was prepared and washed with IP lysis buffer. A pre-cleaning step was performed in order to clean the background. Cell lysates containing protein A agarose beads were rotated at 4°C for an hour. Supernatant was collected after centrifugation. At this point, input (50 µl) was collected and stored at −80°C. Anti-STAT3 (1:50) antibody  was used to perform IP. IgG  was used as negative control. Specifically, IP samples containing protein A agarose beads were rotated at 4°C overnight. Immunoprecipitates were washed three times with ice cold lysis buffer, and proteins were eluted with 50 µl 2X Laemmli loading buffer. Proteins pulled down by anti-STAT3 antibody were separated on 4–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels for mass spectrometry . In some experiments, proteins were transferred onto polyvinylidene difluoride (PVDF) membranes. After blocking, membranes were incubated with primary antibodies against STAT3 or SIN3A or EEF2 at 4°C overnight. The washed membranes were then incubated with secondary antibody  for an hour at room temperature. The membranes were visualized using super signal ECL substrate . In some experiments, cytoplasmic and nuclear extracts were isolated using nuclear extract kit following the manufacture’s protocol .HMC3 cells . DNA-STAT3 complexes were immunoprecipitated using antibodies against STAT3 or with normal mouse IgG as a control. Real-time PCR was used to quantify STAT3 binding. Primer sets and antibodies for ChIP assays are listed in p < 0.05. GraphPad Prism Software v7  was used for data analysis.Real-time PCR results were analyzed using ANOVA, followed by Tukey’s multiple comparison tests for individual comparisons when significant effects were detected. Gene expression data were presented as mean ± SEM. Differences were considered significant at As a first step in the present study, we set out to characterize molecular signatures of exposure to ketamine and its two active metabolites. Specifically, our study was designed to determine—as a first step—genome-wide mRNA expression profiles for HMC3 cells in response to treatment with ketamine and its two active metabolites with and without exposure to E2. Drug treatment conditions had been optimized as described in our previous study . SpecifiPrincipal component analysis (PCA) of gene expression profiles showed distinct clustering for the differing drug treatment conditions, i.e. in the presence of E2 alone or together with either ketamine or the ketamine metabolites Table 1.R,6R)-HNK and -HNK -HNK or -HNK in the same cell line -HNK or -HNK in human microglial HMC3 cells -HNK or -HNK.We next identified 237 genes that were significantly regulated by both ketamine and the two active ketamine metabolites, . The results for all of those experiments were consistent. When STAT3 was knocked down to ∼3% of baseline levels, ketamine failed to activate the expression of STAT3  was significantly altered by ketamine (FDR < 0.05) based on our RNA-seq data. “Translation elongation” was the most significant pathway associated with genes included in the STAT3 protein complex. Furthermore, EEF2 protein was found to be the most abundant protein in the STAT3 protein complex. It was detected on the basis of 13 unique peptides corresponding to 21.2% amino acid sequence coverage spanning the EEF2 protein. We subsequently verified the interaction between STAT3 and EEF2 by Co-IP . KetaminIn summary, this series of experiments suggests that an important transcription factor, STAT3, may play a significant role in response to ketamine therapy. That may be true, in part, as a result of STAT3 translocation into the nucleus in the presence of ketamine, which in turn, alters the expression of downstream genes involved in the type I interferon pathway. We also showed that STAT3 interacted with EEF2, a downstream target of mTOR signaling that is involved in the regulation of translation.2R,6R)-HNK and -HNK, also have anti-inflammatory properties.MDD is the most common psychiatric illness worldwide, with an estimated prevalence of approximately 7% in adults . EsketamThe results of the present study indicate that STAT3 might not only play an important role in transcription regulation of genes involved in the type I interferon signaling pathway but also that it may play a role in protein translation regulation for synaptic plasticity markers. These results could help to increase our understanding of ketamine’s molecular and cellular mechanisms of action in microglia.Our results have demonstrated that exposure to ketamine and two ketamine active metabolites results in similar gene expression profiles in microglia. Specifically, we found that “response to type I interferon” was the most significant pathway associated with changes in expression after treatment with ketamine and the two active ketamine metabolites that we studied . These mSex-dependent ketamine antidepressant effects have been observed in rodent models of depression . HoweverThe present study used a human microglial cell line, HMC3, to perform functional genomic studies. To our knowledge, this is currently the only commercially available human microglia cell line. Obviously, all cell lines have limitations. Therefore, our observations will have to be replicated by future studies conducted with clinical samples and/or patient-derived induced pluripotent stem cells (iPSCs) which can be differentiated into different types of central nervous system cells, i.e. iPSC-derived microglia. However, the results reported here represent a potentially important step in the process of obtaining functional insight into molecular mechanisms of action for ketamine and its active metabolites.In summary, the present study has highlighted the fact that ketamine and its two active metabolites can regulate the type I interferon pathway mediated, at least in part, through STAT3. Specifically, genes that were modulated by either ketamine or its active metabolites were enriched in the “response to type I interferon” signaling pathway. Our data also raise the possibility that STAT3, which plays a critical role in the immune response, might also play a role in the antidepressant effects of ketamine, mediated in this case, at least in part, through EEF2, a protein that plays a role in BDNF, PSD95 and SYN1 translation—all of which are genes that have been shown to contribute to the antidepressant effects of ketamine.The datasets generated for this study can be found in the NCBI, accession number GSE134782.Participated in research design: M-FH, CZ, LZ, HL, and RW. Conducted experiments: M-FH and LZ. Performed data analysis and interpretation: M-FH, CZ, LZ, HL, and RW. Wrote or contributed to the writing of the manuscript: M-FH, LZ, and RW. All authors have given final approval of the version to be published.This work was supported in part by National Institutes of Health grants R01 GM28157, R01 AA27486, U19 GM61388 (the Pharmacogenomics Research Network), K01 AA28050, and by the Mayo Clinic Center for Individualized Medicine.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Dynamic disruption and reassembly of promoter-proximal nucleosomes is a conserved hallmark of transcriptionally active chromatin. Histone H3-K56 acetylation (H3K56Ac) enhances these turnover events and promotes nucleosome assembly during S phase. Here we sequence nascent transcripts to investigate the impact of H3K56Ac on transcription throughout the yeast cell cycle. We find that H3K56Ac is a genome-wide activator of transcription. While H3K56Ac has a major impact on transcription initiation, it also appears to promote elongation and/or termination. In contrast, H3K56Ac represses promiscuous transcription that occurs immediately following replication fork passage, in this case by promoting efficient nucleosome assembly. We also detect a stepwise increase in transcription as cells transit S phase and enter G2, but this response to increased gene dosage does not require H3K56Ac. Thus, a single histone mark can exert both positive and negative impacts on transcription that are coupled to different cell cycle events. H3K56Ac promotes nucleosome turnover at promoter-proximal locations and assembly of new nucleosomes during S phase. Here the authors find that H3K56Ac is both a genome-wide activator of transcription in yeast while also repressing promiscuous transcription that occurs after replication fork passage. Nucleosomes are arranged in a regularly spaced manner, and nucleosome positioning is influenced by DNA sequence and regulated by chromatin remodeling enzymes2. While nucleosomes can be found at both genic and nongenic regions, promoters and termination regions are largely encompassed by nucleosome depleted regions (NDRs)3. NDRs are flanked by highly positioned nucleosomes (+1 and −1), where the +1 nucleosome either contains the transcription start site (TSS) or is located immediately upstream2. Interestingly these promoter-proximal nucleosomes are highly dynamic, showing rapid disassembly and reassembly through replication-independent nucleosome turnover4. One of the main features of these dynamic nucleosomes is the acetylation of lysine 56 on histone H3, and this modification has been shown to enhance the turnover of promoter-proximal nucleosomes, yielding a positive-feedback loop6. Due to its enrichment at promoters, several studies have shown a role of this modification in transcriptional activation of a select group of genes9. In addition, Weiner and his colleagues showed that H3K56Ac levels correlate with transcriptional activity10. In accordance with this role in yeast, H3K56Ac has also been shown to associate with the core transcriptional network in mammals, and has been suggested to promote pluripotency8.Early biochemical studies identified nucleosomes, the building blocks of chromatin, as physical barriers at different stages of transcription11, and unlike many other HATs, Rtt109 can only use free histones as a substrate for H3K56 acetylation12. Consequently, newly assembled nucleosomes are enriched for H3K56Ac, including nucleosomes behind replication forks, DNA repair foci, and promoter-proximal nucleosomes undergoing replication-independent nucleosome turnover14. Rtt109 does not share sequence homology with other known HATs, but the crystal structure of Rtt109 shows resemblance to the mammalian HATs, p300 and CBP15, which catalyze H3K56Ac in mammals16. Two histone chaperones, Vps75 and Asf1, are required for Rtt109 acetylation activity in vitro, influencing its substrate specificity17. When associated with Asf1, the Rtt109-Asf1 complex preferentially acetylates H3K56, whereas the Vps75–Rtt109 complex prefers to acetylate H3K919. Consistent with these in vitro studies, an asf1∆ causes larger reductions in H3K56Ac levels compared to H3K9Ac or H3K23Ac in vivo19. Several previous studies of steady state RNA levels failed to observe significant changes in transcription due to inactivation of Rtt109. We uncovered genetic interactions between Rtt109 and the nuclear exosome, suggesting that post-transcriptional events may mask the transcriptional impact of H3K56Ac loss20.The acetylation of H3K56 is catalyzed by the histone acetyltransferase (HAT) Rtt109 in budding yeast13. During replication, nucleosomes are disassembled ahead of the fork and these parental nucleosomes are segregated to the two daughter strands. Newly synthesized histones, marked with H3K56Ac, are then deposited to “fill in the gaps”21. H3K56Ac facilitates nucleosome deposition, as this mark enhances the binding of histone H3 to the key histone chaperones, chromatin assembly factor 1 (Caf1) and Rtt106, as well as promoting the subsequent binding of Caf1 to PCNA22. In the absence of H3K56Ac, nucleosome assembly is delayed, and this leads to increased genome instability13. For instance, an rtt109∆ mutant or strains harboring a H3K56R substitution derivative show increased DNA damage and sensitivity to genotoxic agents and replication stress11. Notably, persistent, genome-wide H3K56Ac also leads to genomic instability, and consequently the high, S phase levels of H3K56Ac are erased in G2/M by the Sirtuin family deacetylases, Hst3 and Hst4 , whose expression peak in late S/G2 phase24.In yeast, H3K56Ac is also incorporated during replication-coupled nucleosome assembly26. Indeed, early studies in both fission and budding yeast found that the mRNA synthesis rate following replication remained similar to that of G1 cells, until late S or G2 phase where a step-wise increase in mRNA synthesis eliminated this buffering phenomenon29. A similar phenomenon has also been observed in mammalian cells, and in this case the buffering is due to cell cycle dependent changes in the frequency of transcriptional bursting30. The buffering of transcription in response to gene dosage is thought to be a mechanism to couple the concentration of gene expression products to both DNA content and cell size. Interestingly, recent studies have implicated H3K56Ac in the buffering of mRNA synthesis for early replicating loci in yeast, though how this histone mark exerts such a repressive role is not clear31.During S phase, replication doubles the gene dosage, but several studies have found that transcription does not immediately increase following gene duplication32 and transient-transcriptome sequencing (TT-seq)33 to dissect the role of H3K56Ac in transcription during G1, S, and G2 phases of the cell cycle. Using these methodologies, we find that H3K56Ac globally activates gene transcription throughout the yeast cell cycle, and find that it regulates the distribution of RNA Pol II throughout gene bodies, suggesting that enhanced, replication-independent nucleosome turnover stimulates transcription. In contrast, during S-phase, H3K56Ac inhibits transcription of both coding and noncoding RNAs immediately following replication fork passage, an activity that correlates with its role in promoting efficient nucleosome assembly. Indeed, we observe a similar repressive function during S phase for components of the primary nucleosome assembly machinery, Cac1 and Rtt106. Furthermore, we find that global transcription increases ~2× in G2 phase in the presence or absence of H3K56Ac, indicating that this histone mark is not essential for buffering of gene dosage during S phase.Here, we utilize native elongating transcript sequencing (NET-seq)20. As an alternative strategy, we isolated total RNA from asynchronous cultures of isogenic wild type (WT) and rtt109∆ strains, and RNA levels were analyzed by RNA-seq. Importantly, Schizosaccharomyces pombe cells were “spiked-in” during cell harvest to provide an internal normalization control for sequencing libraries. Consistent with previous tiling array analyses, inactivation of Rtt109 led to only minor changes in the yeast transcriptome  and HTB1/HTB2 (encoding histone H2B) gene pairs are upregulated 1.5- to 1.8-fold, while levels for HHT1/HHT2 (encoding Histone H3) and HHF1/HHF2 (encoding Histone H4) were not significantly changed. Together, these results show that loss of H3K56Ac has only a minor impact on the steady state RNA transcriptome.Previously, we found that inactivation of Rtt109 had little impact on steady state RNA levels, as assayed by high-density DNA tiling arraysrtt109Δ strains. Similar to RNA-seq analyses, S. pombe cells were added during cell harvest as an external spike-in for library normalization  was employed to more directly assess transcription in both WT and ion Fig. . In thision Fig. . Notably19. To ensure that the global decreases in nascent transcription are due solely to the loss of H3K56Ac, Net-seq analyses were performed in G1-arrested cells harboring a H3K56R substitution derivative that mimics unacetylated H3-K56. In this case as well, a global decrease in nascent transcript levels was observed  also showed global decreases in nascent transcript levels  and stable unannotated transcripts (SUTs). Both CUTs and SUTs are short-RNA products transcribed by RNA Pol II, polyadenylated, and terminated by a pathway involving Nrd1–Nab3 and the TRAMP complexlls Fig. . Togethertt109∆, H3K56R, or Asf1-AA strains compared to WT. Next, we performed metagene analyses to analyze RNAPII distributions, normalizing RNAPII occupancy levels to the individual gene expression levels and plotted these values throughout ~5200 genes. This analysis confirms that loss of H3K56Ac leads to a change in RNAPII distribution, with a marked decrease in RNAPII distribution near the TSS (+1 nucleosome) and an increase in RNAPII levels over the gene body and near the TTS Fig. . Interes.05)Fig. . Togethe33. In yeast, TT-seq primarily monitors the frequency of transcription initiation events due to the relatively short length of coding regions40. Both WT and rtt109∆ cells were labeled with 2.5 mM 4-thiouridine (4tU) for 10 min, and 4tU-labeled RNAs were isolated and sequenced  monitors RNA segments synthesized during a short, 10′ pulse of 4-thio-uracilced Fig. . The resced Fig. . Omissio31. Given that we do not observe such a repressive function for H3K56Ac in G1 or asynchronous cells, we used Net-seq analysis to investigate the transcriptional impact of H3K56Ac as cells progress from G1, through S phase, and into G2. The ASF1-AA strain was arrested in G1 phase by treatment with alpha factor (αFT), and then cells were treated for 1 h with DMSO or with rapamycin to deplete the Asf1 subunit of the Rtt109 HAT complex from the nucleus. Cells were then released into a synchronous cell cycle by washing into media lacking αFT and containing or lacking rapamycin. Samples were collected for Net-seq analysis at time zero (G1), 30′ postrelease (early S phase), 60′ postrelease (late S phase), or 90′ postrelease (G2)  Fig. . Cell cyG2) Fig. .Fig. 4AsWe initially analyzed nascent transcripts for all coding genes, comparing levels in WT to those in cells depleted for Asf1 (Asf1-AA). First, we tested whether we could detect buffering of gene dosage as WT cells progressed through S phase. Consistent with buffering, WT gene expression levels in early and late S phase remained nearly identical to that of G1 cells, even though most genes had duplicated by the 60′ timepoint Fig.  and 5A–FAs expected see Fig. , depleti41 , and then we categorized them as early- or late-replicating genes based on the known timing of ARSs41 Fig. . In WT c41 Fig. . Importa41 Fig. . These l41 Fig. . Similar42. Thus, one possibility is that Asf1 depletion leads to partially assembled chromatin structures that create a permissive environment for transcription, and that slow maturation of chromatin eventually restores a normal transcription level. One prediction of this model is that disruption of the Cac1/Rtt106 nucleosome assembly machinery, which does not affect H3K56Ac levels13, should also lead to promiscuous transcription during S phase. Isogenic WT and cac1Δ rtt106Δ double mutants were arrested in G1, released into a synchronous S phase, and time points were harvested for Net-seq analyses. Remarkably, nascent transcription in the cac1Δ rtt106Δ double mutant closely mirrored the patterns observed after Asf1 depletion , and levels decreased back to WT levels by G2 phase (90′ time point). Likewise, transcription of late-replicating genes was increased (13-fold increase) during late S phase and into G2 (60′ time point). Collectively, these data suggest that disruption of nucleosome assembly behind the replication fork creates a promiscuous environment for transcription.Previous studies have demonstrated that Asf1 and H3K56Ac stimulate nucleosome assembly in vitro and promote timely nucleosome deposition in vivoion Fig. . For ins42. To directly assess the impact of H3K56Ac on the efficiency and timing of promoter nucleosome assembly during S phase, we used nascent chromatin avidin pulldown (NChAP) to probe nucleosome deposition and spacing on newly replicated DNA strands43. WT or rtt109Δ cells were pulsed labeled with the nucleotide analog, 5-ethynyl-2′-deoxyuridine (EdU), followed by a thymidine chase. Chromatin was then digested with micrococcal nuclease (MNase), and the isolated DNA fragments subjected to a click reaction that adds biotin to the newly synthesized DNA that incorporated EdU. Biotinylated DNA was then purified with streptavidin-coated magnetic beads, and the purified DNA used for preparation of sequencing libraries. Nucleosome profiles from all genes were aligned to their TSS, and two replicates of nascent profiles from either the WT or rtt109Δ strain were compared to a midlog WT standard  or BY4741 (MATa his3∆1 leu2∆0 met15∆0). The following strains for NET-seq were modified with a 3× FLAG tag on Rpb3: rtt109∆ (this study), Asf1-AA (this study), cac1∆ rtt106∆ (this study), H3K56R , rbp1-N488D (slow RNAPII) . Unless otherwise noted, cells were cultivated in YPD  at 30 °C. For S. pombe strain  , cells were cultivated in YES . For α factor arrest, cells were grown to a density of 1.5–2.5 × 107 cells/ml in YPD and arrested by 5 μg/ml αF for 1.5 h. Arrest was confirmed by microscopic observation after 90 min.Strains listed below were derivatives of either W303 . A 400 μl acid phenol (preheated to 65 °C) was added and samples were incubated at 65 °C for 60 min with brief vortexing. After centrifugation, top (aqueous) phase was transferred to a new tube, extracted once with chloroform, and the supernatant subjected to standard ethanol precipitation.53 with the addition of an ERCC spike-in mix for two biological replicates. Briefly, total RNA was fragmented and reverse transcribed with random hexamers. Following dUTP incorporation, cDNAs were end-repaired with a mix of end-repair enzymes , Klenow DNA Polymerase , and T4 PNK ) and A-tailing was performed. Adapter ligation was done using T4 DNA ligase . After UDG treatment, final PCR was performed with indexing primers. Size of the library was determined by Fragment Analyzer and the concentrations were determined by Qubit 4.0 fluorometer (Invitrogen). Sequencing of all samples was carried out on an Illumina NextSeq 500 with a read length of 150 (paired-end).Total RNA was isolated with hot acidic phenol as described above. Library preparation was performed as described54. Reads were first aligned using Bowtie256 to Saccharomyces cerevisiae rRNA, tRNA, and RDN sequences to remove contaminating reads. Reads were then aligned to a combined version of the S. cerevisiae genome  and a list of ERCC spike-ins with TopHat257 allowing up to two mismatches. The reads were separated by their respective genomes with SAMtools58, and only uniquely mapped reads were used for further analyses. For visualization in USCS genome browser, libraries were normalized by ERCC spike-in numbers. To compare RNA expression between samples, HTseq 0.9.159 was used to count the number of reads that aligned to each annotated gene. The annotation file for the S. cerevisiae genome was generated from the Xu et al. dataset35. Differential expression analysis was performed using edgeR60 or in excel using the R q value package.FASTQ files from paired-end libraries were collapsed by barcode and the Illumina adapter sequence was trimmed from the 3′ end. Files were uploaded and analyzed using the Galaxy web platform32 for two biological replicates for WT and two biological replicates for mutants  with several modifications including addition of S. pombe cells as spike-in control. Briefly, overnight cultures from single yeast colonies were diluted to an OD600 = 0.05 in 1 L of YPD. Cells were grown at 30 C until OD600 = 0.8. Rapamycin was added at a final concentration of 8 µg/mL at OD600 = 0.25 for cells with anchor-away background and cells were grown for 3 h (OD600 = 0.7–0.8). To normalize the sequencing libraries, S. pombe cells were mixed with S. cerevisiae cells at a 1:10 ratio, and the cells were harvested by filtration and cryogenic lysis. 3× FLAG-tagged RNA Pol II was immunoprecipitated and nascent RNAs were purified using miRNAeasy mini kit (Qiagen). Following ligation of pre-adenylated DNA linker onto purified nascent RNAs, RNAs were fragmented and reverse transcribed. Resulting cDNAs were circularized using DNA Circligase (Lucigen). Final PCR was performed to obtain double stranded product to sequence. Size of the library was determined by Fragment Analyzer and the concentrations were determined by Qubit 4.0 fluorometer (Invitrogen). 3′ end sequencing of all samples was carried out on an Illumina NextSeq 500 with a read length of 75 (single end).NET-seq conditions, immunoprecipitations, isolation of nascent RNA, and library construction were carried out as previously described54. The adapter sequence was (ATCTCGTATGCCGTCTTCTGCTTG) removed and the random hexamer sequence was removed from the 5′ end. The 3′ ends of the reads were then trimmed for quality using FASTQ Quality Timmer by sliding window61 with a window size of 10 and a step size of 5. The reads were trimmed until the aggregate score was ≥21. Reads were first aligned using Bowtie256 to a combined FASTA file of S. cerevisiae and S. pombe rRNA, tRNA, and RDN sequences to remove contaminating reads. Reads were then aligned to a combined version of the S. cerevisiae genome  and the S. pombe genome  with TopHat257, allowing up to three mismatches. The reads were separated by their respective genomes with SAMtools58, and only uniquely mapped reads were used for further analyses. Libraries were normalized by scaling the uniquely mapped S. pombe reads to 100,000 reads. This scaling factor was then used to scale the uniquely mapped S. cerevisiae reads. To account for differences between sequencing run depth for various NextSeq runs, the pombe-scaled WT S. cerevisiae read counts were then scaled to 1 M reads, and this additional scaling factor was included to scale the sample reads. Finally, only the 5’ end of the sequencing read, which corresponds to the 3′ end of the nascent RNA was recorded and used for downstream analyses. TSS and TTS annotation was obtained from ref. 35. Read counts for genes and noncoding regions were obtained by summing normalized base pair reads over the region of interest. For average profiles, BAM files of biological replicates were merged and processed as above, and only genes longer than 500 bp were analyzed. Genes were scaled to 500 bp, and samples were scored in 1 bp bins using the deepTools program62. Reads were analyzed as in ref. 63. To calculate 5′ to 3′ ratios, the sum of reads from 1 to 250 bp from the TSS were divided by the sum of reads 250 bp upstream of the TTS to the TTS.NET-seq reads were processed and aligned as follows using the Galaxy web platform33 for two biological replicates. Briefly, 1.5 × 107S. cerevisiae cells were labeled with 2.5 mM of 4-thiouracil (4sU) (Sigma-Aldrich) for 10 min. Cells were harvested by centrifugation at 3000g for 2 min. Total RNA was extracted with hot acid phenol as described above. RNAs were sonicated to generate fragments of <1.5 kbp using Bioruptor Standard (Diagenode). 4sU-labeled RNA was purified from 150 μg total fragmented RNA. Labeled RNA was separated with streptavidin beads (Thermo Fisher Scientific). Strand-specific library preparation for labeled RNA was performed as described53 and as in RNA-seq section above. After library construction, size of the library was determined by fragment analyzer and the concentrations were determined by Qubit 4.0 fluorometer (Invitrogen). Sequencing of all samples was carried out on an Illumina NextSeq 500 with a read length of 150 (paired-end).TT-seq experiment was performed as described33. Briefly, FASTQ files from paired-end libraries were collapsed by barcode and the Illumina adapter sequence was trimmed from the 3′ end. Files were uploaded and analyzed using the Galaxy web platform54. Reads were first aligned using Bowtie256 to S. cerevisiae rRNA, tRNA, and RDN sequences to remove contaminating reads. Reads were then aligned to a combined version of the S. cerevisiae genome  and a list of ERCC spike-ins with TopHat257 allowing up to two mismatches. The reads were separated by their respective genomes with SAMtools58, and only uniquely mapped reads were used for further analyses. For visualization in USCS genome browser, libraries were normalized by ERCC spike-in numbers. To compare RNA expression between samples, HTseq 0.9.159 was used to count the number of reads that aligned to each annotated gene. The annotation file for the S. cerevisiae genome was generated from the Xu et al. dataset35. Differential expression analysis was performed using edgeR60 or in excel using the R q value package. All sequencing data has been submitted to GEO under the accession number: GSE125843.TT-seq data preprocessing was essentially done as described43 for two biological replicates. Briefly, cells were grown in SCD-Ura overnight at 30 °C to an OD600 = 1. Later, cell pellets were mixed with SCD-Ura + 10 μΜ ΕdU. Thymidine  was added after 5 or 20 min incubation with EdU at 30 °C and incubated for another 5 or 10 min. Purified EdU-labeled DNA was mixed with biotin azide solution in CuBr solution. After 2 h incubation at 37 °C, DNA was precipitated with sodium acetate and ethanol.EdU-thymidine pulse-chase experiment was done as described600 of 0.6–0.8. After spinning down cells, they were suspended in 70% ethanol for overnight incubation at 4 °C. Following day, cells were sonicated once at setting 3 for 5 s . After resuspending in distilled water and then 50 mM Tris (pH 8.0), cells were incubated in RNase A (10 mg/ml) at 37 °C for 3–4 h. After resuspending in 50 mM Tris (pH 7.5), cells were incubated in Proteinase K (2 mg/ml) at 50 °C for 1 h. Then, they were resuspended in FACS buffer (200 mM Tris-HCl (pH 7.5), 200 mM NaCl, 78 mM MgCl2). Cells were incubated with 1× Sytox Green before collecting in BD FACSDiva Software and analyzing in Flowjo v10.6.0.Samples were collected at certain time points at an ODa his3∆1 leu2∆0 ura3∆0 met15∆0 rbp3::RBP3-3× FLAG::NAT)32WT Rbp3-3× FLAG ((BY4741) MATrtt109∆ (BY4741) MATa his3∆1 leu2∆0 ura3∆0 met15∆0 rpb3::RBP3-3xFLAG::NAT RTT109/rtt109∆::HPH) (this study)a tor1-1 fpr1::loxP-LEU2-loxP RPL13A-2 × FKBP12:loxP BAR1∆::HISG rbp3::RBP3-3xFLAG::NAT) (this study)WT Rbp3-3× FLAG AA strain (HHY221) MATtor1-1 fpr1::loxP-LEU2-loxP RPL13A-2 × FKBP12:loxP BAR1∆::HISG rbp3::RBP3-3× FLAG::NAT Asf1-FRB:HPH) (this study)Asf1-AA (HHY221) MATa cac1∆ rtt106∆ (BY4741) MATa his3∆1 leu2∆0 ura3∆0 met15∆0 rpb3::RBP3-3× FLAG::NAT cac1∆::HPH rtt106::KanMX) (this study)ura3-1 leu2∆ trp1∆::hisG his3∆ lys2∆ met15∆ rbp1-N488D rbp3::RBP3-3× FLAG::NAT) )Rbp1-N488D RBP3-3XFLAG  AUR1c::ADH-hENT1 ∆rtt109::KanR) (this study)MATa ∆(hht1-hhf1) ∆(hht2-hhf2) leu2-3,112 ura3-62 trp1 his3 + p rbp3::RBP3-3× FLAG::NAT) (this study (H3K56R strain is courtesy of Paul Kaufman))H3K56R Rbp3-3xFLAG (S. pombe strain (h-Flag-rbp3 ade6-M216 ura4-D18 leu1) (NBRP64)WT Rbp3-3× FLAG 32: /5rApp/(N1:25252525)(N1)(N1)(N1)(N1)(N1) CTGTAGGCACCATCAAT/3ddC/DNA linker32: 5 phos/atctcgtatgccgtcttctgcttg/iSp18/cactca/iS p18/tccgacgatcattgatggtgcctacag 3RT primer oLSC00732: 5′ CAAGCAGAAGACGGCATACGA 3′Reverse primer oNTI23132: 5′-TCCGACGATCATTGATGGTGCCTACAG 3′Custom primer for NET-seq oLSC00632: 5′ agu cac uua gcg aug uac acu gac ugu g 3′Internal RNA control for NET-seq oGAB11Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Reporting Summary


Chromatin structure and dynamics are highly controlled and regulated processes that play an essential role in many aspects of cell biology. The chromatin transition stages and the factors that control this process are regulated by post-translation modifications, including phosphorylation. While the role of protein kinases in chromatin dynamics has been quite well studied, the nature and regulation of the counteracting phosphatases represent an emerging field but are still at their infancy. In this review we summarize the current literature on phosphatases involved in the regulation of chromatin structure and dynamics, with emphases on the major knowledge gaps that should require attention and more investigation. In eukaryotic cells, DNA is wrapped around proteins called histones to form a structure termed the nucleosomes, the basic unit of chromatin. The central histone octamer consist of two H2A/H2B heterodimers and two H3/H4 heterodimers and it is connected to the adjacent nucleosome core through a linker DNA, usually associated with the linker histone protein H1. Nucleosomes are organized into 10 nm chromatin fibres that adopt various secondary structures that are amenable to interdigitated packing, including a loose zig-zag. This leads to a hierarchy of globular chromatin domains that allow for effective functional compartmentalization of the genome  [At the beginning of mitosis, Condensin II starts the shortening of the mitotic chromosomes by generating 200–400 kb loops that are then subdivided into smaller 80 kb loops by Condensin I; the loops then acquire a helical arrangement [The highly dynamic chromatin structure during the cell cycle is essential for many processes that safeguard genome stability such as DNA replication and repair, gene expression and chromatin segregation. Many factors are involved in controlling chromatin organization, ranging from histone modifications to ATP-dependent chromatin remodelling complexes and non-histone chromosome proteins. The majority of these factors are susceptible to reversible post-translational modifications (PTM), including phosphorylation, that regulate their functions. In this review, we will focus on the role of protein phosphatases in the regulation of chromatin organization and dynamics involved in several aspects of chromatin biology, namely chromatin structure . In verXenopus egg extract, resulted in the loss of condensin II from the chromosomes but it did not affect the chromosomal association of condensin I or topoisomerase II. Addition of two purified PP2A complexes back to the PP2A-depleted Xenopus eggs (the PP2A core dimer (PP2A-Aα and PP2A-Cα) and the B56γ trimer (core dimer and PP2AB′/PR61/B56γ)) was able to rescue the chromosomal association of condensin II, however no rescue was obtained with the B55α trimer (core dimer and PP2A-B/PR55/B55α). Interestingly, both the PP2A dimer containing PP2Awt and the one with the PP2ACD88N mutant (which has a weak phosphatase activity) were able to rescue the localisation of condensin II suggesting that the chromosomal association of PP2A, but not its phosphatase activity, is essential for the chromosomal targeting of condensin II [Xenopus to humans. In addition, PP2A depletion also showed impairment of KIF4-chromosome binding, but not of Aurora B. KIF4 is a chromokinesin involved in mitotic chromosome assembly [PP2A (Protein Phosphatase 2A) also plays a role in condensin II binding to chromosomes; in fact, addition of Okadaic Acid (OA) (an inhibitor of Protein Phosphatase 1 (PP1) and PP2A) to chromosomes assembled using mitotic ensin II . Depletiassembly , reinforassembly .Xenopus, have been shown to be hyperphosphorylated in mitosis and these phosphorylations regulate the complex affinity for chromatin [Condensin activity is inhibited by dephosphorylation of three of its subunits, including CAP-G, by PP2A. XCAP-D2, XCAP-G, and XCAP-H, three subunits of condensin in hromatin ,14.PP2A interacts with TATA binding protein (TBP) in extracts of mitotic cells. TBP exists in cells as part of the TFIID complex and remains bound to DNA during mitosis; at these mitotic sites it recruits PP2A. Since TBP interacts with condensin as well, it promotes its dephosphorylation mediated by PP2A interaction and leads to the inactivation of condensin near these promoters to inhibit their compaction. This mechanism is part of gene bookmarking and therefore links PP2A to the preservation of the memory of gene activity through mitosis to daughter cells .Due to the different kinases and multiple phosphorylated condensin sites during mitosis, it is difficult to believe that just one phosphatase is responsible for reversing the process, thus further research on condensin dephosphorylation and the role of phosphatases in chromatin condensation would be an interesting avenue to pursue.Although the role of condensin complexes on chromosome assembly and segregation can't be argued, there must be other factors that are involved in chromosome condensation at mitotic entry, as condensin knockdown in chicken cells showed that chromosomes can still condense in the absence of condensin . This faAnother example of the involvement of PP1 in chromosome condensation/decondensation comes from studies where the PP1 targeting subunit PNUTS (PP1 nuclear targeting subunit) was shown to accelerate chromosome decondensation in a PP1-dependent manner . HoweverChromosome condensation can be induced also outside the M phase using OA and calcyculin A, two potent PP1 and PP2A inhibitors. This state of chromatin is called Premature Chromosome Condensation (PCC). This phenomenon has gained considerable attention in recent years since it has been linked to genomic rearrangements and diseases ,22. Stud1.2Telomeres are repeated DNA sequences situated at the chromosome ends that protect the DNA from deterioration and ensure genome stability. They mostly comprise of 6 to 8 base pairs stretches and the consensus sequence for vertebrates is TTAGGG. In each cell cycle, due to the end-replication problem, the telomeres get shorter and the enzyme telomerase needs to be recruited to the chromosomes to elongate and extend the TG-rich terminal repeat sequences. Studies in yeasts have shown that the action of Tel1 kinase  is crucial for the recruitment of telomerase to short telomeres and thus it is to be expected that this process is counteracted by protein phosphatases. In fact this is the case; PP1, through interaction with the targeting subunit Rif1, is essential for the suppression of TG repeat elongation via Tel1 recruitment inhibition, and this function is independent of DNA replication. Defective Rif1/PP1 interaction leads to a long-telomere phenotype although it does not compromise the binding of Rif1 to telomeres, highlighting the crucial role of the phosphatase activity . PP2A wa1.3Mitotic chromosomes are surrounded by a complex network of proteins and RNA molecules, mainly derived from the nucleolus, called the chromosome periphery. Although the function and characteristics of this layer still remain unclear, it is now known that Ki-67, a PP1-interacting subunit, is required for its formation  is formed, composed of 6 ORC proteins, Cdc6, Cdt1 and Mcm2–7, which is activated at the beginning of the S phase by several protein kinases, including CDKs and DDK (Dbf4-dependent kinase). The role of protein kinases on DNA replication has been extensively studied by several groups (reviewed in ), althou complex . Data on complex .A role for PP1 on DNA replication has also been reported. Through its regulatory subunit Rif1, PP1 stabilizes the ORC (origin recognition complex) at G1 and has a positive role in supporting origin licencing. However, at the S phase, PP1/Rif-1 complex acts as repressor of origin activation by counteracting DDK and inhibiting phosphorylation of Mcm4. While the latter has been reported in several organisms including yeast and humans , the rolDNA polymerase is the enzyme responsible for DNA replication. There are many different types of DNA polymerases and it has been reported that most of them need to be phosphorylated at specific sites to initiate replication . One of After DNA is replicated, chromatin structure needs to be re-established. Two pathways have been seen to contribute to this re-establishment: generation of parental histones that can be recycled behind the fork, and de novo deposition of newly synthetized histones. In relation to this, it has been shown that H3K27me3 recruits PRC2 to sites of DNA replication maintaining H3K27me2/3 and ensuring that the heterochromatin mark is replicated and deposited on the newly formed DNA. The phosphorylation status of the nearby H3S27 is important for PRC2 binding to chromatin ; as Repo1.5Saccharomyces cerevisiae and, while there are plenty of studies about how the checkpoints are activated, there is not much known about how they are inactivated. The DNA damage response (DDR) starts with the activation of the ATM and ATR (ATM and Rad3-related) kinases, which phosphorylate a number of proteins that will activate the DDR cascade. ATM and ATR activation are regulated by the action of several phosphatases, including PP2A, PP1, Wip1 (Wild-type p53-Induced Phosphatase 1), and PP5. PP2A has been demonstrated to interact with ATM, and inhibition of the phosphatase induced auto-phosphorylation of ATM at Ser1981, although it did not correlate with an increase of the kinase activity, suggesting that PP2A is not dispensable for ATM activation. However, ionizing radiation was able to disrupt PP2A-ATM binding and this dissociation required ATM kinase activity, suggesting that ATM auto-phosphorylation or ATM-mediated phosphorylation of PP2A could be important for complex dissociation. After IR exposure, PP2A would dissociate from ATM enabling its auto-phosphorylation and interaction with targeted protein to start DNA damage repair [DNA integrity and stability are constantly challenged by environmental agents and stressors that may cause different grades of DNA damage. The cell has mechanisms and DNA damage checkpoints essential to recover from lesions and maintain genome integrity. Four DNA damage checkpoints exist through the cell cycle and they all need to be inactivated after DNA repair to ensure cell cycle progression. DNA repair has been extensively studied in e repair . Similare repair . Furthere repair . These pe repair .1.6Chromosome segregation is facilitated by the formation of kinetochores at the centromeric regions of the chromosomes that enable microtubule attachment to pull sister chromatids apart. These proteinaceous structures are also involved in the surveillance mechanism known as the spindle assembly checkpoint (SAC); this mechanism controls that sister chromatids are properly attached to the mitotic spindle and signals when it is safe for the cell to progress to anaphase. Once more, the right balance between kinases and phosphatases is critical for the formation of kinetochores and the proper activation of SAC.Since the role of PP1 and PP2A in the SAC has been extensively studied and excellent reviews are available on the topic ,52, we wTA3) [At the core of the centromere is CENP-A, a centromere specific H3 variant that replaces histone H3 only at the centromeric domain. CENP-A deposition does not occur during S phase but only after cells have divided, in G1. CDK1 inactivation and phosphatase activity are essential for its incorporation . At lateTA3) , do not Ser68 phosphorylation has been shown to impair CENP-A binding to HJURP, an assembly factor that mediates CENP-A deposition to centromeres. During anaphase, PP1α antagonises this effect and removes the phosphate group to enable HJURP binding and proper loading of CENP-A to the centromeres . Other pWithin the SAC response, Aurora B destabilizes erroneous attachments and activates the SAC, which monitors the lack of tension. Shugoshin (in collaboration with histone H3Thr3 phosphorylation by Haspin kinase) is also important for the loading of Aurora B complex ,60 to ce22.1Drosophila PNUTs mutants increased the levels of RNAPII Ser5ph levels but not of Ser2ph [RNA polymerase II (RNAPII) possesses an evolutionary conserved C-terminal domain (CTD) made up of multiple 7 amino acid repeats that is susceptible to phosphorylation. CTD phosphorylation status, highly dynamic during the transcription cycle, is critical for many steps, as it acts as a scaffold for diverse nuclear factors. The serines mentioned in the text are numbered according to their position within the CTD repeat. Phosphorylation of Ser5 is important to recruit enzymes that cap the 5′ end of the transcript, whereas phosphorylation of Ser2 activates elongation and splicing. Subsequent dephosphorylation of these sites is needed to finish transcription and enable detachment of RNAPII of the gene. Fcp1 phosphatase specifically dephosphorylates Ser2 , while Sf Ser2ph . Ser7 onf Ser2ph . The traf Ser2ph . For an f Ser2ph .Drosophila showed that PNUTS/PP1 is important for development and loss of PNUTs affects the transcription of the majority of genes in developing larvae [PP1 is also implicated in other aspects of transcription and in fact all the three PP1 isoforms  have been found bound to promoters in HeLa cells by methylation-based DamID profiling. It seems that this binding is not direct but it is regulated by other PP1 interacting subunits (PIPs); PNUTS and NIPP1 (Nuclear inhibitor of PP1) show the highest degree of overlaps with PP1 at promoters but the majority of PP1 binding sites do not overlap with any of the major chromatin-associated subunits, suggesting that PP1 is recruited to promoters by other yet uncharacterized targeting subunits . In addig larvae . NIPP1 hg larvae ,73.2.2Chromatin remodelling and function is also controlled by changes in DNA methylation, histone variants and their post-translational modifications. These mitotically transmissible but potentially reversible modifications are referred to as epigenetics. The epigenetic landscape is crucial for chromatin structure and dynamics, and many epigenetic marks have been described for different stages of the cell cycle. Histones are modified at several sites and phosphorylations are among those modifications . AlthougHere we summarize the specific histone phosphorylation modifications that are regulated during the cell cycle , emphas2.2.1S. pombe presents several mitotic defects, including impaired chromosome segregation [S. cerevisiae does not show any mitotic impairment [As cells enter mitosis, Aurora B kinase is activated and phosphorylates Ser10 and Ser28 on histone H3. This phosphorylation starts at pericentromeric heterochromatin and it spreads along the entire length of chromosomal arms . H3S10 aregation , but thepairment , indicatFurthermore, H3T11 is also phosphorylated in mitosis by the death-associated protein (DAP)-like kinase (Dlk) and localises to the centromeres from prophase to early anaphase; this modification appears to have a role in kinetochore assembly .Histone H1 is also regulated in a cell cycle-dependent manner, and its phosphorylation significantly increases during mitosis and S phase. Several phosphorylated sites have been identified for H1 and they have been related with chromatin decondensation rather than chromatin condensation . H1 was 2.2.2An important epigenetic mark for DNA damage response (DDR) is the phosphorylation of the histone variant H2AX at Ser139 by ATM, ATR, and VRK1 (Vaccinia related kinase 1) . H2AXS13H2AS122 also seems to be phosphorylated upon DNA damage and, although it is not crucial, it facilitates survival in the presence of DNA damage .H2AX can also be phosphorylated in Tyr142 (Y142), although it is found at very low levels in cells (<1%) . Y142 isSer10 and Thr11 on H3 are also susceptible to modifications upon DNA damage; in mouse, both residues are dephosphorylated after UV radiation-induced DNA damage and re-phosphorylated after repair ,100. HowPhosphorylation of histone H4 at Ser1 by CKII also plays a role in DDR . ChIP exAnother recently identified DNA damage-related histone modification is the phosphorylation of Tyr51 on H4 by TIE-2 upon ionizing radiation. H4Y51ph is recognized by ABL1, a kinase involved in DDR, and other DDR-related proteins, indicating a positive role of this modification in DNA repair machinery .Identifying the phosphatases involved in DDR response, as well as the phosphatases that regulate chromatin structure during DNA repair will allow us to understand how DNA repair is controlled in space and time in the cell and maybe shed light on why, in some cases, DNA lesions escape the checkpoints and can pass into the next generation of cells, influencing genome stability and leading to different diseases.2.2.3c-fos, c-jun, and c-myc [Gene expression is highly regulated by chromatin organization and structure, and a large number of histone modifications have been linked to this specific process in response to several stimuli. H3S10, H3S28 and H2BS32 are phosphorylated upon specific growth factors stimulation and have been linked to the transcription of growth factor-responsive genes, including nd c-myc . Specifind c-myc , and levnd c-myc . The plend c-myc . Accordind c-myc .Phosphorylation of Thr11 and Thr6 on H3 upon androgen stimulation by PRK1 and PKCβ, respectively, is linked to regulation of androgen-associated genes by controlling H3 methylation levels. H3T11ph and H3T6ph promote removal of the repressive H3K9me mark by the Jumonji C (JmjC) domain-containing protein JMJD2C4 ,110. FurAnother histone modification that influences transcription is the phosphorylation of Tyr41 on H3 by Janus Kinase 2 (JAK2), which has been seen to disrupt HP1α binding to chromatin, leading to the transcriptional activation of JAK2-regulated genes .Drosophila identified PP1 as a suppressor of variegation, Su(var)3–6, which support the idea of PP1 as a heterochromatin regulator. Chromosomal rearrangements or transpositions can lead to euchromatic genes being moved to nearby heterochromatic regions, which might suppress the expression of genes that are usually activated. Diverse mutagenesis experiments identified several genes that enhanced the expression of variegated genes ), and some that suppressed its expression ). Su(var)3–6, which encodes for PP1, was seen as one of these genes that, once mutated, suppressed the expression of variegated genes, meaning that in normal conditions Su(var)3–6 acts as an enhancer of heterochromatin formation [Studies on position-effect variegation (PEV) in ormation .HP1 is a known repressive marker for gene transcription and heterochromatin formation, as it binds to histone H3 tri-methylated in lysine 9 (H3K9me3) and, by forming a bridge with another H3K9me3, condenses chromatin to form heterochromatin and represses gene expression . Other hPolycomb groups (PcG) proteins are also a family of proteins that epigenetically alter chromatin in order to repress gene expression, mainly of genes related to proliferation and differentiation. There are functionally two different polycomb complexes: Polycomb Repressing Complex 1 and 2 (PRC1 and PRC2). One of the main components of PRC2 is EZH2 (Enhancer of Zeste 2), a methyltransferase responsible for di−/tri-methylation of histone H3 lysine 27 (H3K27me2/3). H3K27me2/3 is a marker for gene silencing important also for stabilizing HP1 binding to chromatin . HP1 is As in HP1 chromatin binding, the nearby amino acid (H3S28) phosphorylation status is important for PRC2 binding to H3K27me2/3, and PP1 plays an important role in its regulation. In early mitosis H3S28ph is crucial for PRC2 interaction with DNA and cell proliferation. Two different phosphatases contribute to the maintenance of this epigenetic landscape. NIPP1, a PP1 regulatory subunit that inhibits PP1, can bind to phosphorylated EZH2 and block its dephosphorylation , thus keeping EZH2 bound to chromatin ,73. NIPPOther histone phosphorylations are linked to gene transcription regulation. ChIP experiments revealed a role of H2BS36ph on AMPK-mediated gene expression. Loss of H2BS36ph resulted in reduced expression and lower survival rates in response to metabolic stress. The nearby Tyr37 is also a target residue for phosphorylation by the WEE1 kinase, and it is important for the suppression of replication-dependent core histone gene transcription . H4S1 isTaken together it is apparent that histone phosphorylations are critically important for the regulation of gene expression and, although many studies have been conducted to elucidate the specific roles of the phosphorylation sites that regulate transcription, new research on this area should include the identification of the counteracting phosphatases responsible for removing the histone modifications to allow a clearer picture on how transcriptional homeostasis is maintained and regulated.2.3Transcription factors (TF) are also regulated by PTM that can affect transcription factor function by: 1) controlling the length of time TF are in the nucleus; 2) targeting them for degradation; 3) modulating its interaction with other proteins and/or with DNA and 4) modifying chromatin structure. A typical example of this modulation occurs in mitosis when most of the transcription factors are excluded from chromatin as a consequence of their phosphorylation, thus contributing to the attenuation of transcriptional activity in this stage of the cell cycle . The re-2.3.1Myc is a transcription factor crucial for the regulation of genes related with cell proliferation, differentiation, and apoptosis. Its expression is controlled at many levels, including protein phosphorylation, which decreases DNA binding during M phase to enable proper chromatin condensation and cell cycle progression . Two mai2.3.2NF-κB (Nuclear Factor κB) family of transcription factors are involved in regulation of genes related to inflammation, immune response and apoptosis. They are usually kept inactive in the cytoplasm by inhibitory proteins (IκB) until a stimulus activates the TNF receptor, which triggers the phosphorylation of IκB kinase (IKK) which in turn will target IκB for degradation, releasing NF-κB and promoting its translocation into the nucleus to control the expression of target genes. PPM1A and PPM1B, from the PPM family of phosphatases, have been shown to dephosphorylate IKK in two specific sites (S177 and S181), thus acting as a regulator of NF-κB activity . Another2.3.3Cys2His2 (C2H2) zinc finger proteins constitute the largest class of transcription factors encoded in higher eukaryotes genomes. The zinc finger modules of these proteins are usually joined by highly conserved linker sequences that are critical for DNA binding. Some studies have revealed that phosphorylation of these linker sequences by several protein kinases decreases DNA-binding affinity, interfering with gene transcription . RizkallSP1 is one zinc finger transcription factor that binds to GC-rich motifs of different promoters and regulates many cell processes including cell growth, differentiation, apoptosis, and chromatin remodelling. Its activity is also regulated by its phosphorylation status. PP2A has been identified as an important factor in the activation of SP1 by mediating the dephosphorylation of several residues, including Ser59 and Thr681 during the cell cycle, but not in mitosis . SP1 dep2.3.4+/−;PTEN+/− mice show similar lymphoma development as p53−/− animals, highlighting an important crosstalk between the two proteins. The Wu lab also demonstrated that PTEN physically interacts with p53 and regulates its transcriptional activity by modulating its DNA binding affinity [p53 is a tumour suppressor gene that acts as a transcription factor by regulating the expression of several cell cycle controls, DNA repair, and apoptotic genes. Several protein phosphatases are involved in the regulation of p53 levels and its role as a tumour suppressor, either directly or indirectly. One of the phosphatases widely known to control p53 stability is PTEN (Phosphatase and a tensin homologue deleted on chromosome 10). PTEN acts as an antagonist of the PI3K/AKT pathway, which allows the translocation of Mdm2 to the nucleus where it interacts with p53 and targets it for degradation. PTEN dephosphorylates and inhibits PI3K, blocking the translocation of Mdm2 to the nucleus and stabilizing p53 levels. In accordance, PTEN-depleted cells have decreased p53 levels and p53affinity .Other phosphatases have been associated to p53 regulation. Treatment with OA resulted in an increase of hyperphosphorylated p53, and treatment with a PP2A inhibitor, SV40 small t antigen, promoted p53-DNA binding and transcriptional activity ,138. PP22.3.5Drosophila Hox protein that determines the formation of the labial and prothoracic segments during embryogenesis. In yeasts, a homologue of the regulatory subunit (B′/PR61) of PP2A was seen to interact and dephosphorylate the homeodomain of SCR, regulating its DNA binding affinity [The Hox gene family contribute to the morphological diversification of structures of an embryo along the head-tail axis. These genes encode for homeodomain proteins that bind to DNA and regulate the transcription of target genes. Sex combs reduced (SCR) is a affinity . Howeveraffinity . The phoaffinity  so it is2.3.6Oct4 is a pluripotency transcription factor involved in embryonic stem cell self-renewal. Recent studies have shown that CDK1 indirectly regulates Oct4 binding to chromatin and therefore pluripotency-related gene expression. Upon mitotic entry, CDK1 activation promotes Aurora B-mediated phosphorylation of Oct4 at Ser229, resulting in Oct4 dissociation from chromatin. At mitotic exit, PP1 appears to be the responsible phosphatase to dephosphorylate Oct4 and enable its association with chromatin, increasing the transcription of targeted genes ,146. Liq3The plasticity and dynamics of chromatin both during cell division and interphase is fundamental for the development of an organism and the maintenance of its health. Several of these aspects are regulated by phosphorylations. Many phosphorylation sites in several chromatin regulators have been shown to alter chromatin structure and function and a good number of kinases responsible for the modifications have been identified; however, the nature of the counteracting phosphatases is far less known and studied. Nevertheless, there is evidence showing that regulation of phosphorylation by phosphatases is as important as the responsible kinases. In the last decade we have endeavoured to increase the knowledge of the phosphatase biology by understanding the principle of their assembly, regulation and mechanism of action. We still require additional reliable methods for identifying their substrates recognition and specificity. However with the development of new technologies for the identification of phosphatase substrates combined with more precise cell engineering techniques will lead to the discovery of the major phosphatases as regulators both of chromatin dynamics and epigenetics.The Authors declare no conflict of interest.Transparency document.Image 1


SATB1 plays a crucial role in expression of multiple genes throughout the thymic development and peripheral differentiation of T cells. Although SATB1 function has been subjected to intense investigation, regulation of SATB1 gene expression remains poorly understood. Analysis of RNA-seq data revealed multiple transcription start sites at the upstream regulatory region of SATB1. We further demonstrated that SATB1 gene is expressed via alternative promoters during T-helper (Th) cell differentiation. The proximal promoter “P1” is used more by the naïve and activated CD4+ T-cells whereas the middle “P2” and the distal “P3” promoters are used at a significantly higher level by polarized T-helper cells. Cytokine and TCR signaling play crucial roles toward SATB1 alternative promoter usage. Under Th2 polarization conditions, transcription factor STAT6, which operates downstream of the cytokine signaling binds to the P2 and P3 promoters. Genetic perturbation by knockout and chemical inhibition of STAT6 activation resulted in the loss of P2 and P3 promoter activity. Moreover, chemical inhibition of activation of NF-κB, a transcription factor that operates downstream of the TCR signaling, also resulted in reduced P2 and P3 promoter usage. Furthermore, usage of the P1 promoter correlated with lower SATB1 protein expression whereas P2 and P3 promoter usage correlated with higher SATB1 protein expression. Thus, the promoter switch might play a crucial role in fine-tuning of SATB1 protein expression in a cell type specific manner.SATB1 is a genome organizer protein that is expressed in a lineage specific manner in CD4 NaïSatb1-KO animals exhibit arrested thymic development  , plays aelopment . Recentlelopment .IL-5 promoter  signaling has been shown to play an important role in SATB1 gene expression during early thymocyte development  usage, differ in the sequence of the 5' UTR and splicing of the first exon that harbors them. Alternative promoters play crucial role in gene regulation in the determination of cell fate and function. APs allow diversification of transcriptional regulation enabling expression in various cell lineages and developmental stages. Use of APs results in mRNA isoforms that differ in the sequence of 5' UTRs that are crucial for post-transcriptional regulation  to studpromoter . We thenpromoter  [GSE2089promoter ] and Stapromoter  [GSE1425promoter ] for thepromoter . To stud-KO mice . Stat6-Kn levels  suggesti+ T-cells into Th1 phenotype  and Th2 polarization conditions. We observed that P2 and P3 promoter usage was higher in Th2 cells as compared to Th0 cells underlining the importance of cytokine signaling in P2 and P3 promoter expression  and consolidate the role of JAK/STAT signaling in the P2 promoter usage.To further validate the importance of Stat6 in P2 promoter usage, we established an  signals  with an  signals ] in the er usage . We perfer usage . Treatmeer usage , suggestSATB1 P2 promoter expression. However, when Jurkat cells were treated with IL-4 cytokine (in absence of PMA and Ionomycin), no increase in P2 and P3 promoter expression was observed (data not shown). The switch in promoter usage was observed only upon simultaneous activation of cytokine and TCR signaling. This observation does not rule out the role of TCR signaling in SATB1 alternative promoter usage. TCR signaling is mediated by NFAT transcription factors downstream of the CD3 signaling  cells by both transcriptional and post-transcriptional mechanisms . STAT6 also participates in the long-range intra-chromosomal interactions in Th2 cells to regulate expression of various gene loci  during T-helper cell differentiation. The usage of P2 promoter increases more than that of the P3 promoter. The P3 promoter region also harbors the start site of the divergent transcript from the SATB1-AS1 (SATB1 Anti-Sense RNA 1) gene. Bidirectional transcription is often associated with genes related to transcriptional regulation and development . However, IL-4 treatment in combination with activation using PMA and ionomycin (P+I) resulted in STAT6 activation and increase in P2 promoter activity. This suggested that either pSTAT6 alone is sufficient for increase in P2 promoter usage or the transcription factors downstream of the TCR signaling co-operate with STAT6 to drive the P2 promoter usage. Considering the role of NF-κB in regulation of cytokine signaling (SATB1 alternative promoter expression. Whether STAT6 and NF-κB interact directly at the P2 promoter remains to be studied.The Jurkat cell-line based system was also crucial in suggesting the involvement of NF-κB in ignaling , we blocignaling –45. SuchSATB1 P2 promoter expression. Interestingly, even though the expression of SATB1 is suppressed in Treg cells, the P1 promoter is active. Transcription via the P1 promoter is therefore weakly correlated with SATB1 protein expression. We extended this observation to the primary CD4+ T-cells and Jurkat cells and observed that activation of these cells by TCR-signaling alone resulted in higher P1 promoter usage and lower SATB1 protein expression as compared to the polarizing conditions. It is therefore possible that the P1 promoter is less translatable and is used when basal level of SATB1 expression is needed.SATB1 protein is expressed in T-helper cells in a lineage specific manner . Even thTranscription factor/s responsible for P1 promoter usage in activated T-cells have not yet been identified. Since NF-κB inhibition had no specific effect on P1 promoter expression, AP-1 and NF-AT transcription factors both also downstream of TCR signaling, can be studied. Also, the role of NF-κB in regulation of P2 promoter usage in T-helper cells may not be direct. Further experiments need to be performed to check the direct binding of NF-κB at the P2 promoter. A co-operative binding of STAT factors with NF-κB has been previously reported  and cannSATB1 alternative promoter usage leads to change in the sequence of the 5' untranslated region. The 5' UTR has been shown to play a role in regulation of various genes by controlling the rate of translation via secondary structure formation, presence of upstream open reading frames, and via miRNA binding . WeSATB1 gene expression via alternative promoters during peripheral differentiation of T-cells. SATB1 regulation represents a unique example with conserved mode of regulation in multiple T-helper subtypes with possible effect on protein levels. We anticipate that understanding of the unique functions of these promoters in gene regulation and the physiological consequences of their expression/repression will be a prime focus of the future studies.In summary, we have elucidated a complex mode of regulation of The datasets generated for this study can be found in NCBI SRA, PRJNA503398.This study was carried out in accordance with the recommendations of the Institutional Human Ethics Committee, IISER-Pune with written informed consent from all subjects. All subjects gave written informed consent. The protocol was approved by the Institutional Human Ethics Committee, IISER-Pune.Mice used in this study were maintained in the Central Animal Laboratory at Turku University. This study was carried out in accordance with appropriate guidelines for the care and use of laboratory animals and were approved by the “Finnish Animal Ethics Committee.” The protocol was approved by the “Finnish Animal Ethics Committee.”Stat4/Stat6 KO experiments, performed T-helper cell differentiation, and qRT-PCR analysis. RB performed Jurkat cell-line experiments. AVS assisted in Jurkat cell experiments and T-helper cell differentiation. PR assisted in RNA-Seq analysis. ZC provided expertise in Stat4/Stat6 KO mouse experiments, RL participated in experimental design, discussed and interpreted results and supported the research. SG designed the experiments, was involved in regular discussions and data interpretations, supported the research and wrote the manuscript.SK designed experiments, performed ChIP-Seq and RNA-Seq analysis, performed Jurkat cell-line experiments, T-helper cell differentiation, qRT-PCR analysis, and wrote the manuscript. AS and IP performed FACS staining, The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Peroxiredoxin 6 (Prdx6) and affects cellular homeostasis. We previously observed that dose levels of SFN are crucial in determining life or death of lens epithelial cells (LECs). Herein, we demonstrated that higher doses of SFN (>6 μM) activated death signaling by overstimulation of Nrf2/ARE (antioxidant response element)-mediated Kruppel-like factor (Klf9) repression of Prdx6 expression, which increased reactive oxygen species (ROS) load and cell death. Mechanistically, Klf9 bound to its repressive Klf9 binding elements  in the Prdx6 promoter, and repressed Prdx6 transcription. Under the condition of higher dose of SFN, excessive Nrf2 abundance caused death signaling by enforcing Klf9 activation through ARE (5-RTGAYnnnGC-3) in Klf9 promoter that suppress antioxidant genes such as Prdx6 via a Klf9-dependent fashion. Klf9-depletion showed that Klf9 independently caused ROS reduction and subsequent cell survival, demonstrating that Klf9 upregulation caused cell death. Our work revealed the molecular mechanism of dose-dependent altered activity of SFN in LECs, and demonstrated that SFN activity was linked to levels of Nrf2/Klf9/Prdx6 axis. We proposed that in the development of therapeutic interventions for aging/oxidative disorders, combinations of Klf9-ShRNA and Nrf2 inducers may prove to be a promising strategy.Sulforaphane (SFN), an activator of transcription factor Nrf2 (NFE2-related factor), modulates antioxidant defense by Nrf2-mediated regulation of antioxidant genes like  Substantial experimental evidence indicates that etiopathobiology of age-related disorders is associated with increased levels of reactive oxygen species (ROS) and reduced expression and activity of antioxidant pathway, leading to molecular damage that result in pathobiology and disease. To cope with internal and external oxidative stresses, cells have evolved defensive mechanisms that integrate the coordinated stimulation of a group of phase I and phase II antioxidant enzymes, such as NAD(p)H quinone oxidoreductase NQO1), Thioredoxin reductases (Txnrds) Glutathione peroxidase (GPx), Catalase (Cat), Superoxide dismutases (SODs) and peroxiredoxin 6 (Prdx6). These enzymes are upregulated by Nrf2 (NFE2-related factor 2), a universal transregulator of antioxidant response genes, to revive cellular redox homeostasis , Thiored,4. The NNrf2, but surprisingly caused a dramatic reduction in expression of its target antioxidant gene Prdx6, such as Prdx6 resulting in cell death. The Nrf2/ARE pathway has a cytoprotective role in maintaining redox homeostasis [Recent evidence reveals that oxidative stress is a major culprit in aging disorders, and that the ability to prevent or blunt oxidative stress is a key determinant of aging diseases; thus, strategies should be aimed at limiting oxidative load or enhancing the cellular host defense system. Boosting or reactivating the defense system by means of Nrf2-mediated antioxidant pathway appears to be a reasonable and practical approach to reducing oxidative-induced injurious signaling. Work by our lab and others has shown that naturally occurring compounds such as sulforaphane (SFN), curcumin and α-lipoic acid have the ability to reactivate Nrf2/ARE-mediated antioxidant pathway and provide cytoprotection against pathobiology and cell damage induced by aging and oxidative stress ,11,12,13eostasis ,22,23,24eostasis . These sKlf9) gene promoter and transactivates its expression, which suppresses some of the antioxidant genes by binding to their repressive sites (5′-CA/GCCC-3′) [Nrf2 is a major sensor of oxidative stress in the cells and a master regulator of antioxidant defense pathway ,27,28. BGCCC-3′) ,31 and cGCCC-3′) .Klf9 promoter [Prdx6 gene promoter might bear Klf9 binding elements. In silico analysis of a Prdx6 promoter revealed that, indeed, the Prdx6 promoter has several Klf9 binding sites of single GC boxes (but not tandem repeats), suggesting that Klf9 may act as repressor for Prdx6 gene transcription in excessive oxidative load (current study).A ubiquitously expressed member of an evolutionarily conserved transcriptional regulator KLF family, Klf9 is also designated as BTEB1 (basic transcription element binding protein 1). Klf9 can act as either a repressor or an activator of gene transcription. This activity of Klf9 is related to the number of binding sites, GC-boxes, present in gene promoter and cell types . Studiespromoter ,41,42. B2+-independent phospholipase A2 activities, belongs to the Prdx family. The six members of the Prdx family are classified based on the number of cysteine (Cys) residues. Prdx6 contains 1-Cys at position 47 (Cys47), while Prdx1-5 have 2-Cys residues. Prdx6 is a cytosolic protein and is abundantly expressed in lung, eye lens, keratinocytes, skin and brain [Prdx6 is transcriptionally regulated by Nrf2, and dysregulation of Nrf2 during aging causes reduction in Prdx6 expression that leads to increased ROS-induced cell death [The multitasking protective protein Prdx6, with its glutathione (GSH) peroxidase and acidic Cand brain ,44,45,46nd brain ,49,50,51nd brain ,52,53,54nd brain ,56,57,58ll death ,59,60, pll death  and athell death .Prdx6 and dramatic increase in ROS levels with aberrant expression and nuclear accumulation of Nrf2. This accumulation of Nrf2 led to its binding to ARE present in the promoter of unfavorable target Klf9 and enhanced its expression and nuclear abundance. We found that Klf9 bound to its Repressive Klf9 Binding Elements (RKBE) of the Prdx6 promoter and repressed the Prdx6 expression that in turn resulted in increased ROS-induced cell injury, suggesting an advent of dominant Klf9-mediated repressive signaling during high doses of SFN. Klf9 depletion experiments showed that Klf9 dramatically reduced ROS levels and cell injury. Aberrant Klf9 expression induced by higher doses of SFN was a major culprit in acceleration of ROS generation and cell death, while lower doses of SFN did not affect Klf9 expression, and led to SFN-induced Nrf2/ARE-mediated cellular protection.SFN is a naturally occurring organosulfur found in cruciferous vegetables, with high levels detected in broccoli and broccoli sprouts. Recent evidence shows SFN′s diversified activities, ranging from cell survival and cytoprotection to cytotoxicity and growth inhibition; these activities are determined by concentrations of SFN and cellular background ,65,66,672 environment at 37 °C as described previously [hLECs were derived from 12 infants who underwent surgery for retinopathy of prematurity  (a kind eviously . To examIntracellular ROS level was measured by use of fluorescent dye dichlorofluorescin diacetate (H2DCFDA), a nonpolar compound that is converted into a polar derivative (dichlorofluorescein) by cellular esterase after incorporation into cells ,52,72,73® Deep Red Oxidative Stress Reagent, Catalog No. C10422) and described in previous protocol [3) cells were seeded in 96 well plate and 24 h later treated with different concentrations of SFN as indicated. After 48 h, CellROX deep red reagent was added with a final concentration of 5 µM and cells were incubated at 37 °C for 30 min. Media containing CellROX deep red reagent were removed and fixed with 3.7% formaldehyde. After 15 min, fluorescence signal were measured at Ex 640 nm/ Em 665 nm with Spectra Max Gemini EM .ROS level were measured according to the company′s protocol  was performed as described earlier ,75,76. T® LC480 Sequence detector system (Roche Diagnostic Corporation). PCR conditions of 10 min (min) hot start at 95 °C, followed by 45 cycles of 10 s (sec) at 95 °C, 30 sec at 60 °C and 10 sec at 72 °C. The primer Sequence was: Nuclear factor (erythroid-derived 2)-like 2 (Nrf2), Forward Primer: 5′-TGC TTT ATA GCG TGC AAA CCT CGC-3′; Reverse Primer: 5′-ATC CAT GTC CCT TGA CAG CAC AGA-3′; Klf9, Forward Primer: 5′-CTGGTTGCTGGGACTGTAGC-3′; Reverse Primer: 5′-GTTTTCCAGCTCCCAAACAG-3′; Prdx6, Forward Primer: 5′-GCATCCGTTTCCACGACT-3′ and Reverse Primer: 5′-TGCACACTGGGGTAAAGTCC-3′; β-actin, Forward Primer: 5′-CCAACCGCGAGAAGATGA-3′ and Reverse Primer: 5′-CCAGAGGCGTACAGGGATAG-3′.Total RNA from the cultured hLECs untreated and/or treated with SFN  were isolated using the single-step guanidine thiocyanate/phenol/chloroform extraction method . To examine the levels of Nrf2, Klf9 and Prdx6, 0.5 to 5 micrograms of total RNA was converted to cDNA using Superscript II RNAase H-reverse-transcriptase. Real-time quantitative PCR was performed with SYBR Green Master Mix  in a RocheThe relative quantity of the mRNA was obtained using the comparative threshold cycle (CT) method. The expression levels of target genes were normalized to the levels of β-actin as an endogenous control in each group.6) were cultured in 100-mm plates. hLECs were treated with DMSO control or different concentrations of SFN  for 24 h. The cells were washed gently with chilled phosphate-buffered saline (pH 7.4). Cells were collected by centrifugation using a micro-centrifuge and resuspended in 5 pellet volumes of cytoplasmic extract buffer ((10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid , 10 mM KCl, 0.1 mM EDTA (Ethylenediaminetetraacetic acid), 0.4% (v/v) Nonidet P-40, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM DTT (Dithiothreitol) and Protease inhibitor). After a short incubation on ice and centrifugation (4 °C) at 10000 rpm for 10 min, the cytoplasmic extract was transfer in fresh tube from the pellet. Following careful washing with cytoplasmic extract without detergent (Nonidet P-40), the fragile nuclei were resuspended in nuclear extract buffer ((20 mM HEPES (adjusted pH at 7.9), 0.4 M NaCl, 1 mM EDTA, 10% (v/v) glycerol, 1 mM DTT, 0.5 mM PMSF and Protease Inhibitor) and incubated for 2 h at 4 °C with continuous vortexing. Finally, the extract was spun at 14,000 rpm for 15 min to pellet the nuclei. After centrifugation, the nuclear extract was transferred and aliquoted in fresh tubes, and individual aliquots were stored at −70 °C to avoid repeated freezing and thawing of the preparation.Nuclear extract was prepared following as described earlier ,77. Brie6) were cultured in 100-mm plates. The cells were washed gently with chilled phosphate-buffered saline (pH 7.4). Cells were collected by centrifugation using a micro-centrifuge and resuspended in three pellet volumes of RIPA buffer * 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate , 0.5 mM phenylmethylsulfonyl fluoride (PMSF), and Protease inhibitor). After 30 min of incubation on ice and centrifugation (4 °C) at 10,000 rpm for 10 min, the total cell lysates was transfer in fresh tube from the pellet. Protein level was estimated according to the Bradford protein assay and/or Pierce™ BCA Protein assay methods and extract was used for experiment as required.Total cell lysates of LECs were prepared in ice-cold radioimmune precipitation buffer (RIPA buffer) . Briefly®, Cambridge, MA, USA), Anti-Prdx6 antibody  or β-actin /Lamin B1  as internal control to monitor those protein expressions. After secondary antibody , protein bands were visualized by incubating the membrane with luminol reagent  and images were recorded with a FUJIFILM-LAS-4000 luminescent image analyzer .Total cell lysates, cytosol and nuclear extract of LECs were prepared and protein blot analysis was performed as described previously ,52,78,79Nrf2 activation assay was performed according to manufacturer′s protocol . In brief, 10 µg of nuclear extract (up to 10 µL diluted with complete lysis buffer) prepared from SFN treated hLECs added to the strips well, following the addition of 40 µL complete binding buffer contains 20 pmol of the wild-type and/or mutated consensus oligonucleotide to each sample well. For blank well, 10 µL of complete lysis buffer were used. The plate was incubated for 1 h at room temperature (RT) with mild agitation. 100 µL primary antibody (1:1000 in 1× antibody binding buffer) added after three washes with 1× washing buffer and incubated at RT for 1 h without agitation. 100 µL of diluted anti-rabbit HRP (horseradish peroxidase)- conjugated antibody (1:1000 dilution in 1× antibody binding buffer) after three washing was added and incubated for 1 h at RT. 100 µL of developing solution was added to wells after four washing and incubated at RT in dark for 2 to 10 min. Finally, by addition of 100 µL of stop solution, optical density (O.D.) was recorded at absorbance 450 nm.® Express  and ChIP-IT® qPCR analysis kit  following the manufacturer′s protocol and as described earlier [ChIP was performed using the ChIP-IT earlier ,7. The fKlf9 ARE3 (ChIP-Primer):Forward: 5′-CGCTAGAGTTACGAAACAGGG-3′; Reverse: 5′-GAAAGGCCATCCGTTCATGC-3′Klf9 ARE4 (ChIP-Primer):Forward: 5′-GCGCCAGCACCCGGCCGAACC-3′; Reverse: 5′-GCTGGTGTTGCTGTCCCTGG-3′Prdx6 RKBE1 (ChIP-Primer):Forward: 5′-GCTGTGCGAAGCCGCCGCA-3′; Reverse: 5′-GAAGCTTGAGGATGCGCCA-3Prdx6 RKBE2 (ChIP-Primer):Forward: 5′-GTAGTCGAGCAGTCACTCCA-3′; Reverse: 5′-GAAGGAAGAGGAACGCGGCAG-3Prdx6 RKBE3 (ChIP-Primer):Forward: 5′-GGTTCATAACAAACAGAAAGG-3′; Reverse: 5′-AGCCCAGCTACGATGAACTG-3Prdx6 RKBE4 (ChIP-Primer):Forward: 5′-GTCTGTCACCGGTTTCCCTT-3′; Reverse: 5′-GAGACCTACTGTGTGCAGGT-3Prdx6 RKBE5 (ChIP-Primer):Forward: 5′-CAGAGCACCTACCGTGAGCT-3′; Reverse: 5′-GAAGGGAAACCGGTGACAGA-3Klf9 gene ranging from −5856 to +71bp was isolated from mouse genomic DNA by using an Advantage® Genomic PCR Kit . A construct of −5856 to +71 bp was constructed by ligating this DNA fragment into CAT reporter plasmid (p), basic pCAT vector (Promega) using MluI and XhoI sites. The plasmid was amplified and sequenced. Primers used for isolating the genomic DNA fragment were as follows: Forward Primer (MluI site): 5′-AAAAACGCGTGGTCATCGTAGGAAAGATGTGG-3′ and Reverse Primer (XhoI site) 1: 5′-AAAACTCGAGCCTACGAGACACTTCTTCCC-3′.5′-Flanking region of mouse ® Genomic PCR Kit . PCR product was cleaned and verified by sequencing as described previously [SacI and XhoI sites. Primers were as follows: Sense; 5′-GACAGAGTTGAGCTCCACACAG-3′; and antisense; 5′-CACGTCCTCGAGAAGCAGAC-3′.The 5′-flanking region spanning from −1559 to +30 bp was isolated from human genomic DNA by using an Advantageeviously . A const™ lightning site-directed mutagenesis kit , following the company′s protocol. Briefly, amino acid exchanges at the Klf9 sites (RKBE 1 and 3) mutants  were generated by point mutations in the human promoter of Prdx6-CAT plasmid. Nrf2/ARE (3 and 4) sites mutants (T to G) generated by point mutation in the mouse Klf9 promoter of Klf9-CAT reporter plasmid. The following complementary primers were used (changed nucleotides are in red boldface type and underlined):PCR-based site-directed mutagenesis was carried out using the QuikChangeKlf9 ARE site3 mutant :GGAACCTGCCTCCTC-3′Forward primer: 5′-CTGTCCTCAAACTTTGAGGACAG-3′Reverse primer: 5′-GAGGAGGCAGGTTCKlf9 ARE site4 mutant :CTCTCCACTCGCAC-3′Forward primer: 5′-CGATTCCTGCAAAGTCGGACTTTGCAGGAATCG-3′Reverse primer: 5′-GTGCGAGTGGAGAPrdx6 RKBE1 mutant : TTCCTGCAGAGTCAAACC-3′Forward primer: 5′-CCCTAAAGCGCGTACAAGTACGCGCTTTAGGG-3Reverse primer: 5′-GGTTTGACTCTGCAGGPrdx6 RKBE3 mutant :TTCCATACTTCTGGGTC-3′Forward primer: 5′-CTCTGACATAAGGTCAAGACCTTATGTCAGAG-3Reverse primer: 5′-GACCCAGAAGTATGGCopGFP control lentiviral particle  and BTEB1 (Klf9)/GFP ShRNA  were purchased from Santa Cruz Biotechnology and hLECs were infected following the Company′s protocol. Briefly, hLECs were cultured in 12 well plate in complete medium. After 24 h, media were removed and then substituted with 1 mL medium containing polybrene  at a final concentration of 5 µg/mL. Cells were infected by adding the Sh-Control and Sh-Klf9 lentiviral particles to the culture, mixed by swirling and incubated overnight. At 24 h post infection, polybrene-containing medium were removed and fresh complete medium (without polybrene) were added. Infected cells were split and incubated in complete medium for a further 24–48 h. For stable selection, the infectants were treated with puromycin dihydrochloride (sc-108071) selection marker. These stably infected hLECs with LV Sh-Control or LV Sh-Klf9 were used for the current studies.p value of <0.05 and 0.001 for two or more independent experiments.For all quantitative data collected, statistical analysis was conducted by Student′s t test and /or one-way ANOVA when appropriate, and was presented as mean ± S.D. of the indicated number of experiments. A significant difference between control and treatment group was defined as a We recently showed  that SFNPrdx6 mRNA expression, were increased in response to 6 μM of SFN treatment, but Klf9 levels were not upregulated  as shown in Klf9 and Prdx6 promoters for putative binding sites of Nrf2 and Klf9, respectively.We found a dramatic inverse correlation between expression of s of SFN A, which Klf9 promoter, spanning from 5′ −5856 to +1 bp (TSS), contained four well-conserved antioxidant response elements  [Prdx6 promoter, ranging from 5′-1559 to transcription start site (+1) bp, showed that it had five conserved Repressive Klf9 Binding Elements   ,26, a knGCCC-3′) B in the d others .Nrf2 is not a bona fide regulator of Klf9, as are antioxidant genes. However, it has been shown that Klf9 gene is transcriptionally regulated by Nrf2 during higher levels of oxidative stress [Klf9 promoter as predicted ; (iii) pCAT-Klf9 mutant at site 4  (iv) pCAT-Klf9 mutant at both sites (ARE3-mut + ARE4-mut). To examine the functional consequences of SFN-induced changes on Klf9 transcription, we transfected hLECs with a pCAT-Klf9 WT reporter plasmid  or its mutant plasmids (RGGAYnnnGC) as noted above, along with a GFP plasmid as indicated in Klf9 promoter showed robust loss in transcription activity compared to wild-type promoter′s transcriptional activity in response to higher doses of SFN (Klf9 promoter played a part in activation of Klf9 transcription in response to higher doses of SFN. It was interesting to observe that SFN failed to activate Klf9 transcription at lower concentrations (data not shown) at which SFN activates Nrf2-mediated antioxidant pathway [Prdx6 promoter; however, exploring this phenomenon would require a separate line of investigation. Furthermore, to determine whether upregulation of Klf9 is activated only by SFN, or whether other Nrf2 activators at high doses may have the same effect, we treated transfectants containing wild-type pCAT-Klf9 and its ARE mutant at both sites for 6 h with a high dose of tBOOH (200 μM), a well-known activator of Nrf2 [Klf9 transcriptional activity, in contrast to mutant plasmid . As shown in Prdx6 promoter at RKBEs (5′-CA/GCCC-3′), and the increased enrichment of Klf9 to the sequences was SFN concentration-dependent, while no enrichment of Klf9 occurred at 6 µM of SFN. Moreover, no amplicon was detected with control IgG, validating the specificity of Klf9 antibody used in the experiment. Thus, the experiment revealed that Klf9 interacted with all RKBEs of Prdx6 promoter in vivo as predicted through bioinformatics tool, and the progressive increase in the binding of Klf9/RKBE was SFN dose-dependent, as indicated in In silico analyses spotted out the presence of five RKBEs (a single GC boxes) ,33 in Prnt genes . Based oes [Klf9  in respoPrdx6 promoter fused to reporter gene CAT, and treated cells with the same increasing doses of SFN as shown in Prdx6 promoter linked to CAT reporter gene plasmid having Klf9 sites (Prdx6 promoter plasmids were transfected into hLECs. 48h later transfectants were treated with different concentrations of SFN (for 24h) or tBOOH (6h) as shown in Prdx6 promoter  or both sites (RKBE1-mut plus RKBE3-mut) and provided a similar pattern of Prdx6 transcription as observed in SFN experiments in cells treated with toxic dose of tBOOH (200μM for 6h)  in Prdx6 repression. Collectively, data revealed that RKBE negatively regulated Prdx6 expression through repressing its transcription in cells facing SFN-induced toxicity. This also suggests that the advent of Klf9-mediated dominant repressive signaling in hLECs in response to high doses of SFN that surpasses Nrf2-mediated activation of Prdx6. Results unveiled the molecular mechanism involved in SFN-mediated cytotoxicity, which is linked to attenuation of Prdx6 by Nrf2/Klf9 axis as shown in To examine the functionality of the dose-dependent SFN-induced increased Klf9 binding to RKBE on Prdx6 transcription, we performed transactivation assay in hLECs containing f9 sites A, and mupromoter  were funPrdx6 in response to high doses of SFN. Recently, several reports have noted that SFN application can halt or abate several diseases and their complications [Klf9, we used a Klf9 knockdown strategy to examine if Klf9-depleted cells can resist increasing doses of SFN. Our goal was to provide a proof of concept for a potential basis for combination therapy (SFN plus Klf9-ShRNA). As described in Materials and Methods, we stably infected hLECs using lentiviral (LV) Sh-Klf9 or LV Sh-control -mediated antioxidant pathway is the major cellular defense against several internal and external environmental stresses. Oxidative stress and many compounds with electrophilic properties, such as heavy metals, tBOOH and naturally occurring phytochemicals such as SFN, have been reported to yield therapeutic benefits by modulating Nrf2-ARE pathway ,96,97,98egulated . Howeverll death ,103,104.ll death ,107,108. protein . We thoupromoter , but exppromoter  demonstrpromoter B coupledpromoter , emphasie system ,77,78,79e system . Prdx6 ie system ,57,58,79e system . Prdx6 lπ in human, mouse and rat LECs in a concentration-dependent manner [Prdx6 mRNA and protein in hLECs in response to high doses of SFN  [Klf9 has been identified as a new target gene for Nrf2 [Klf9 regulatory region bearing active ARE3 and ARE4   and actie (NADH) ,120. Abefor Nrf2 ,121,122.and ARE4  has lessnt genes . We obseKlf9 acted as repressor of Prdx6 expression. We found that Prdx6 genes bear five Klf9 repressive binding sites , and these sites were not repeated GC boxes, but were single GC box sequences, demonstrating that Klf9 acted as repressor for Prdx6 by binding to RKBE 1, 2, 3, 4 and 5 present in its gene promoter during toxic doses of SFN . We propose that a therapy combining SFN with a Klf9 ShRNA would be an ideal approach to abating or treating disorders linked to aging and oxidative stressors.In conclusion, our work revealed, for the first time, the novel molecular mechanism underlying how SFN-induced Nrf2-mediated protective signaling goes awry in hLECs in response to increased doses of Nrf2 activator, SFN. We showed that during increased doses of SFN, Klf9-mediated repression of