Leishmania tarentolae, and identified four co-purified proteins by mass spectrometry: protein phosphatase (PP1), a homolog of Wdr82, a potential PP1 regulatory protein (PNUTS) and a protein containing a J-DNA binding domain (named JBP3). Gel shift studies indicate JBP3 is a J-DNA binding protein. Reciprocal tagging, co-IP and sucrose gradient analyses indicate PP1, JGT, JBP3, Wdr82 and PNUTS form a multimeric complex in kinetoplastids, similar to the mammalian PTW/PP1 complex involved in transcription termination via PP1 mediated dephosphorylation of Pol II. Using RNAi and analysis of Pol II termination by RNA-seq and RT-PCR, we demonstrate that ablation of PNUTS, JBP3 and Wdr82 lead to defects in Pol II termination at the 3’-end of polycistronic gene arrays in Trypanosoma brucei. Mutants also contain increased antisense RNA levels upstream of transcription start sites, suggesting an additional role of the complex in regulating termination of bi-directional transcription. In addition, PNUTS loss causes derepression of silent Variant Surface Glycoprotein genes involved in host immune evasion. Our results suggest a novel mechanistic link between base J and Pol II polycistronic transcription termination in kinetoplastids.Base J, β-D-glucosyl-hydroxymethyluracil, is a modification of thymine DNA base involved in RNA Polymerase (Pol) II transcription termination in kinetoplastid protozoa. Little is understood regarding how specific thymine residues are targeted for J-modification or the mechanism of J regulated transcription termination. To identify proteins involved in J-synthesis, we expressed a tagged version of the J-glucosyltransferase (JGT) in  Trypanosoma brucei is a parasitic protozoan that causes African sleeping sickness in humans. The genome of T. brucei is organized into polycistronic gene clusters that contain multiple genes that are co-transcribed from a single promoter. We have recently described the presence of a modified DNA base J and variant of histone H3 (H3.V) at transcription termination sites within gene clusters where the loss of base J and H3.V leads to read-through transcription and the expression of downstream genes. We now identify a novel stable multimeric complex containing a J binding protein (JBP3), base J glucosyltransferase (JGT), PP1 phosphatase, PP1 interactive-regulatory protein (PNUTS) and Wdr82, which we refer to as PJW/PP1. A similar complex (PTW/PP1) has been shown to be involved in Pol II termination in humans and yeast. We demonstrate that PNUTS, JBP3 and Wdr82 mutants lead to read-through transcription in T. brucei. Our data suggest the PJW/PP1 complex regulates termination by recruitment to termination sites via JBP3-base J interactions and dephosphorylation of specific proteins (including Pol II and termination factors) by PP1. These findings significantly expand our understanding of mechanisms underlying transcription termination in eukaryotes, including organisms that utilize polycistronic transcription and novel epigenetic marks such as base J and H3.V. The studies also provide the first direct mechanistic link between J modification of DNA at termination sites and regulated Pol II termination and gene expression in kinetoplastids. Termination of RNA polymerase II (Pol II) transcription of mRNAs is a tightly regulated process where the polymerase stops RNA chain elongation and dissociates from the end of the gene or transcription unit. However, the underlying termination mechanism is not fully understood. Pol II termination of most protein-encoding genes in eukaryotes is tightly linked to the processing of the nascent transcript 3’ end (reviewed in ). This aSchizosaccharomyces pombe (S. pombe), the PP1 phosphatase Dis2 regulates termination by de-phosphorylating both the Pol II-CTD as well as Spt5 [S. cerevisiae PP1 homologue Glc7 mutant [Critical to this process is the regulation of protein phosphorylation by the major eukaryotic protein serine/threonine phosphatase, PP1. As recently demonstrated in the fission yeast 7 mutant . PP1 act7 mutant . These r7 mutant –6. One o7 mutant . PNUTS i7 mutant . This st7 mutant . In yeas7 mutant –14, and 7 mutant , 16. In 7 mutant .Trypanosoma brucei that causes human and animal African trypanosomiasis. Kinetoplastids are protozoa with unique genome arrangements where genes are organized into polycistronic transcription units (PTU) that are transcribed by Pol II. Pre-messenger RNAs (mRNA) are processed to mature mRNA by coupled 5’ RNA trans-splicing and 3’ polyadenylation [T. brucei and Leishmania major, J and H3.V are enriched at sites involved in Pol II termination [T. brucei, this includes the expression of variant surface glycoprotein (VSG) genes involved in antigenic variation during bloodstream infections [Members of the Kinetoplastida order include the parasite nylation –21. Givenylation ). In T. mination –26. Thismination –29. At Pmination , 27, 28.mination , 27, 28.fections , 27.in vitro and bind chromatin in a J-dependent manner in vivo [in vivo regardless of where it is present [It is currently unclear how base J and H3.V are involved in Pol II termination, since very little is understood regarding the mechanism of Pol II termination in kinetoplastids. Equally unclear is what regulates the specific localization of these epigenetic marks, including base J, in the genome. Base J is synthesized in a two-step pathway in which a thymidine hydroxylase (TH), JBP1 or JBP2, hydroxylates thymidine residues at specific positions in DNA to form hydroxymethyluracil (hmU) , followe in vivo –41. The  in vivo , 38. The present –44. ThisT. brucei. Ablation of JBP3, Wdr82 or PNUTS in T. brucei causes read-through transcription at termination sites. As we previously demonstrated following the removal of base J and H3.V, these defects include transcription read-through at termination sites within Pol II transcribed gene arrays and the silent Pol I transcribed VSG expression sites, leading to de-repression of genes involved in parasite pathogenesis. Furthermore, ablation of JBP3, Wdr82 or PNUTS results in expression of genes upstream of Pol II transcription start sites. Presumably this represents a previously unappreciated role of termination of antisense transcription from gene promoters in trypanosomes. Overall these findings provide a first look at mechanisms involved in Pol II transcription termination in kinetoplastids and a direct link between base J and termination.In this study, we identify a new J-binding protein  in kinetoplastids, which is present in a complex containing PP1, Wdr82 and a putative orthologue of PNUTS. To characterize the role of this (PJW/PP1) complex in transcription termination, we investigated the consequence of mutants using RNAi in Leishmania tarentolae was chosen as an experimental system, because it provides an easily grown source of high densities of parasites that synthesize base J. The proteins were cloned into the pSNSAP1 vector, carrying a C-terminal tag composed of a protein A domain, the TEV protease cleavage site, and the streptavidin binding peptide [Base J is synthesized in a two-step pathway involving the hydroxylation of specific thymidine residues in the genome by a thymidine hydroxylase, JBP1 or JBP2, followed by the addition of the glucose moiety by the glucosyltransferase, JGT. In this study, we chose to identify the proteins that co-purify with JBP1, JBP2 and JGT, in order to understand the regulation of J-biosynthesis.  peptide . Separat peptide –6. This  peptide , 47. A b peptide , 49, whi peptide  shows PNWe noticed that a region of the LtaP36.0380 protein (amino acids 101–277) has sequence similarity with the J-binding domain of JBP1 . A strucE. coli, and gel shift assays to characterize J-DNA binding activities [We have previously developed a rapid isolation procedure for His-tagged recombinant JBP1 produced in tivities , 41. We in vivo, we switched to T. brucei due to the benefits of forward and reverse genetics available in this system. However, while all other components are easily identified  (Co-immunoprecipitation studies were performed to assess the PNUTS-containing complex in BSF control)  along wicontrol) . PTP-PNUcontrol) . Similarcontrol) . In contcontrol) . Furthercontrol) , nor doecontrol) .T. brucei cells expressing epitope-tagged components were analyzed by western blot following sucrose fractionation. Analysis of T. brucei cells expressing PTP-PNUTS and HA-JBP3 or HA-Wdr82 indicate Wdr82, JBP3 and PNUTS co-migrate at <200kDa. These results indicate that JBP3, Wdr82 and PNUTS form a stable complex of <200kDa. Summation of the predicted size of the three complex components (~180 kDa) agrees with the observed mass of the PJW complex and suggests a 1:1 stoichiometry for the subunits. Taken together, data from co-immunoprecipitation, sucrose gradient analysis, and identification of PNUTS-, Wdr82-, and JBP3-associated proteins by mass spectrometry indicate that Wdr82, JBP3 and PNUTS comprise a stable PJW complex in kinetoplastids T. bruceiadj<0.05)  . In conts mapped . Interess mapped , a signis mapped . The PTUs mapped . In conts mapped . In thesT. brucei indicated that H3.V/J regulated termination at the 3’-end of PTUs, where mutants resulted in Pol II elongation into the dual strand transcription regions at cSSRs [A remaining question was whether PNUTS also regulated termination at the 3’-end of gene clusters resulting in transcription into the SSR. Our previous RNA-seq analyses in at cSSRs , 27. To Based on the initial RT-PCR analysis of the internal termination site in PNUTS, JBP3 and Wdr82 knockdowns , we expeT. brucei [T. brucei.Many promoters for RNA Pol II are bidirectional in organisms from yeast to human –67. Unid. brucei , 69. TheAnother possibility is that increased transcription within dSSRs is due to transcription initiating upstream of the divergent TSSs in the absence of PNUTS. To address this possibility, we examined transcription at Head-to-Head (HT) boundaries of PTUs. HT sites are defined where transcription of one PTU terminates and transcription of another PTU on the same DNA strand initiates. In contrast to TSSs at dSSRs, HT sites contain a termination site for the upstream PTU, indicated by H3.V and H4.V, and a TSS marked by individual peaks of histone variants, such as H2A.Z, and histone modifications , 68, 69.T. brucei, 36 are upstream of TSS [L. major has indicated that replication initiation sites occur at the genomic locations where Pol II stalls or terminates, including sites precisely downstream of base J [T. brucei replication, we analyzed whether TbPNUTS is required for proper cell-cycle progression. In T. brucei, DNA replication and segregation of kinetoplastid DNA (K) in the single mitochondrion precede those of nuclear DNA (N), so cells at different stages can be distinguished by their N and K configurations. 1N1K content indicates that cells are in G1, 1N2K indicates cells in S phase and 2N2K indicate post-mitotic cells. Representatives of cells with these DNA contents upon DAPI staining are shown in Noncoding transcription, via defects in transcription termination, influences eukaryotic replication initiation. Transcription through origins located at 5’- and 3’-ends of Pol II transcription units leads to replication defects via dissociation of the prereplication complex (pre-ORCs) or sliding of MCM helicases –74. Of tm of TSS . Analysif base J . TherefoT. brucei genome, H3.V and base J localize within the ~14 Pol I transcribed polycistronic units located at the telomeric ends and involved in antigenic variation  (T. brucei 427 genome assembly. As shown in adj<0.1). These data indicate that transcription of (silent) telomeric VSGs in the PNUTS mutant is strongly dependent on the presence of a Pol I promoter. Interestingly, comparing the expression level of MES VSGs that are adjacent to the promoter with BES VSGs over 10 kb downstream  [22, 77–7s, BESs) . In addis, BESs) . To furtwnstream  suggestewnstream . In the wnstream  were thoThe derepression of ESAG 6 and 7 genes adjacent to the BES promoter along with the VSG ~40 kb downstream , suggestCurrent dogma in the field is that most, if not all, Pol II transcribed gene regulation is at the posttranscriptional level in kinetoplastids . This isT. brucei, validating PJW is indeed a functional module. Our data strongly suggests that the kinetoplastid PJW/PP1 complex we identified here is reminiscent of human PTW/PP1 where PNUTS is the kinetoplastid functional homologue of human PNUTS and JBP3 the homologue of Tox4. However, while we demonstrate LtPP1 is a component of the Leishmania complex, the purified module in T. brucei lacks a PP1 homologue. PP1 is the only catalytic component of the human PTW/PP1 complex and dephosporylation by PP1 is directly involved in regulating Pol II termination [T. brucei is surprising. MS analysis of the TbPNUTS complex is the result of a single purification from 12 L of PC T. brucei cells. The finding that one of the four PJW/PP1 complex purifications in Leishmania also failed to pulldown PP1 suggests PP1 may be the least stable component of the complex in T. brucei. Once we increase the yield of TbPNUTS purification, and reduce the volume of cells needed, we can perform multiple pulldown/MS analyses to address this possibility. The specific loss of PP1 and JBP3 in one of the LtJGT pulldowns, may also reflect an effect of the tandem affinity tag on LtJGT associations. Thus, future experiments should examine T. brucei complex purification using a different affinity tag or using TbJBP3 or TbWdr82 PTP-fusion proteins. We propose that PP1 is a functional component of the PJW/PP1 complex in both Leishmania and T. brucei, but association with TbPNUTS is less stable. However, further work needs to be done to determine whether PP1 is a component of the PTW complex and involved in termination in T. brucei. Therefore, we refer to the PJW complex when addressing functional analysis of the complex in T. brucei and, based on our studies in Leishmania, the PJW/PP1 complex when discussing the potential role of the complex in termination in kinetoplastids.Here we identify a new base J binding protein, JBP3, and show that it is part of a PP1-PNUTS-Wdr82 containing protein module. We named this module the PJW/PP1 module based upon the mammalian PTW/PP1 module involved in transcription termination . Mutatiomination , 61. TheT. brucei PJW complex is likely not due to the PTP-tag since co-IP analysis using tagged versions of PNUTS, JBP3 or Wdr82 fail to pull down JGT in BSF parasites. Furthermore, ablation of PNUTS, JBP3 and Wdr82 in BSF T. brucei has no significant effect on level of base J in the genome  have resulted in increased expression of procyclin [T. brucei and Leishmania, detection of increased nascent, unprocessed RNA by RT-PCR at the termination site supports the conclusion that the loss of PJW complex function leads to Pol II read-through transcription, rather than some Pol I or Pol II (re-) initiation event. The lack of identifiable Pol I promoter at these regions, and the low level of resulting transcripts where RNA Pol I transcription is known to be 5–10 times more active than transcription by RNA Pol II [T. brucei.We and others have previously shown that base J and H3.V co-localize at Pol II termination sites , 25 and  of PTUs , 26–29.  of PTUs , 27, 28.at shock . To controcyclin , 92, a gA Pol II , supportHere, we propose a model where divergent transcription at the 5’-end and readthrough transcription at the 3’-end of gene arrays is affected by the PJW/PP1 complex in kinetoplastids model in . AccordiT. brucei [Similar to the mammalian complex, we propose PNUTS is a scaffolding protein for the entire PJW/PP1 complex and regulates PP1 function via the PP1 binding RVxF motif. Only three substrates have been identified for PNUTS/PP1: the Pol II elongation factor Spt5, the CTD of Pol II and MYC , 97, 98.. brucei , 103 and. brucei . Studies. brucei . It is nT. brucei [T. brucei  and gene expression as seen here in PJW mutants, but without cell growth effects. We concluded that readthrough transcription at 3’-end and corresponding gene expression changes in the J/H3.V mutant are not lethal to the parasite. PJW mutants we analyze here lead to additional effects on transcription and gene de-repression at 5’-ends and decreased cell growth. The ability of the PJW complex to bind base J is consistent with its function at both ends of PTU’s. However, it is unclear why the effects in the J mutant are limited to the 3’-end and whether specific function of the complex at the 5’-end can explain its essential nature. While J is present at both 5’ and 3’ PTU flanking regions and involved in transcription, it is apparently not the dominant mark since H3.V had more significant effects on 3’-end transcription and gene expression [T. brucei. A significant role of H3.V in PJW complex function, as discussed above, would explain why H3.V and PJW mutants share termination defects at the 3’-end. Similarly, additional specific chromatin factors may be involved in recruitment of PJW complex to TSSs independent of base J, such as Pol II-Wdr82 interactions, as mentioned above, and modified and variant histones such as H2A.Z and H3K4Me3  or “single marker cells (SMC)”, expressing T7 RNA polymerase and the Tet repressor [T. b. brucei TREU667 and promastigote form L. tarantolae were cultured in SDM79 medium. Transfections were performed using the BioRad GenePulser II (2 pulses at 1.4 kV / 25 μF) in 0.4 cm cuvettes with 1 x 108 cells (L. tarantolae) or 3 x 107 cells (PCF T. b. brucei) in 0.5 ml cytomix . Where appropriate, the following drug concentrations were used: 25 μg/ml G418 (PCF T. b. brucei) and 50 μg/ml G418, 10 μg/ml Puromycin (L. tarantolae).Bloodstream form epressor  were useT. brucei a part of the ORF was integrated into the BamH I site of the p2T7-177 vector [For conditional PNUTS, JBP3 and Wdr82 silencing experiments in 7 vector . Sce-I lL. tarantolae, the coding regions of LtJGT, LtJBP3, and LtWdr82 lacking stop codons were amplified and cloned into the BamHI and XbaI sites of pSNSAP1 [T. brucei, the 3’ end of T. brucei genes were cloned in the Apa I and Not I sites of the pC-PTP-Neo vector [For generation of the dual (Protein A and Streptavidin Binding Protein) tagged constructs in  pSNSAP1 . The reso vector , resultio vector . PrimersTo quantify the genomic J levels, DNA was isolated and utilized in the anti-J DNA immunoblot assay as described previously . Briefly11 cells (L. tarantolae and PCF T. brucei) were harvested, 1 time washed in 1 x PBS , 2 times washed in buffer I  and 1 time washed in buffer II . Cell pellets were then adjusted to 20 ml (L. tarentolae) or 50 ml (PCF T. brucei) with buffer II with protease inhibitors  and 2 times flash frozen in liquid Nitrogen. Lysates were then sonicated  for 5 times  on ice. Extracts were cleared by centrifugation for 10 min at 21,000 x g at 4°C and incubated while rotating with 200 μl IgG Sepharose beads  for 4 hrs at 4°C. The beads were then washed with 35 ml PA-150 buffer  and 15 ml PA-150 buffer with 0.5 mM EDTA. The beads were then resuspended in 2 ml PA-150 buffer with 0.5 mM EDTA and 250 U TEV Protease (Invitrogen) while rotating for 16 hrs at 4°C. The supernatant was collected and for L. tarantolae, samples were incubated with 100 μl magnetic Streptavidin C1 Dynabeads (Invitrogen) for 4 hrs while rotating at 4°C. Beads were then washed with 50 ml PA-150 buffer, transferred into 1 ml elution buffer , incubated for 2 hrs while rotating at room temperature. Eluted protein was then TCA precipitated and subjected to MS/MS analysis. To the T. brucei supernatant samples, CaCl2 was added to a final concentration of 1.25 mM and incubated with 200 μl Anti-Protein C Affinity Matrix (Roche) for 4 hrs while rotating at 4°C. Beads were then washed with 50 ml PA-150 buffer with 1.25 mM CaCl2. Bound protein was eluted in 5 ml elution buffer , TCA precipitated and subjected to MS/MS analysis.Tandem affinity purification was performed from whole cell extracts. 2 x 108 cells in 200 μl buffer II. After the IgG Sepharose incubation, beads where washed in PA-150 buffer and boiled in Laemmli buffer. For Western analysis, 5 x 106 cell equivalents of each fraction  were analyzed on 10% PAA / SDS gels and sequentially probed with anti-HA antibodies , anti-Protein A antibodies  and anti-La antibodies .Pull-down experiments using PTP-tagged PNUTS, JBP3 and Wdr82 were performed using the first purification step of the TAP protocol on whole cell extracts from 2 x 10Leishmania tarentolae and T. brucei 927 database, using the Sequest search algorithm in Proteome Discoverer 2.2 (Thermo Fisher Scientific). Search parameters were set to allow for two missed tryptic cleavages, 20 ppm mass tolerance for precursor ions, and 0.3 Daltons mass tolerance for fragment ions. A fixed modification of carbamidomethylation on cysteine residues and a variable modification of oxidation on methionine residues were enabled to accurately match fragment ions. A fixed value peptide spectral match (PSM) validation node was used to validate each PSM at a maximum delta Cn of 0.05. True negative PSMs (decoy PSMs) were generated by searching the same raw data utilizing a decoy database, containing reversed protein sequences from the target protein database, and the exact same search parameters. All surviving PSMs were imported into ProteoIQ  for final validation. ProteoIQ used validated PSMs and decoy PSMs to generate protein identifications with a maximum false discovery rate of 1%. Each protein present at 1% FDR was also required to have at least two unique peptides and 10 PSMs to be reported. For shotgun proteomics, eluted proteins were digested in-solution as previously described [For the initial JGT-S purification, purified proteins from JGT tagged cells and negative control cells were separated by SDS-PAGE gel and visualized by silver staining. Each lane of the silver-stained SDS-PAGE gel was divided into 5 large fractions before being cut down further into roughly 2mm x 2mm pieces and stored in separate tubes in preparation for digestion. Each fraction of gel was de-stained before undergoing denaturation in 10 mM dithiothreitol at 56°C for one hour. Denatured proteins were then alkylated by 55mM iodoacetamide for 45 minutes in the dark with intermittent vortexing. Sequencing grade trypsin  was then utilized to digest proteins overnight at 37°C with gentle vortexing. Peptides were extracted from each fraction separately by incubating gel pieces with a gradient of increasing concentrations of acetonitrile. All extracted peptides were concentrated, reconstituted in 0.1% formic acid in 5% acetonitrile, and passed through a 0.2um bio-inert membrane tube  to remove any residual particulate. Each fraction was injected independently into an Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Fisher Scientific) interfaced with an UltiMate 3000 RSLCnano HPLC system (Thermo Scientific). Peptides were resolved on an Acclai PepMap RSLC C18 analytical column  at a flow rate of 200nL/min using a gradient of increasing buffer B (80% acetonitrile in 0.1% formic acid) over 180 minutes. Data dependent acquisition was carried out using the Orbitrap mass analyzer collecting full scans every three seconds . The most intense ions that met mono-isotopic precursor selection requirements were selected, isolated, and fragmented using 38% collision-induced dissociation (CID). Every precursor selected for ms/ms analysis was added to the dynamic exclusion list and precursors selected twice within 10 seconds were excluded for the following 20 seconds. Fragment ions were detected using the ion trap to increase the duty cycle and achieve more ms/ms scans per experiment. Raw data for each fraction was searched separately against the appropriate databases: GeneDB escribed  and thenhttp://zhang.bioinformatics.ku.edu/I-TASSER) The initial 3D models generated by I-TASSER were of high quality, with a C-score of -0.76 and a TM-score of 0.62 (within the correct topology range of I-TASSER). TM-score is defined to assess the topological similarity of the two protein structures independent of size. A TM-score >0.5 corresponds approximately to two structures of the similar topology. The top predicted model was then aligned using TM-align from I-TASSER with the Lt JBD model 2xseA giving a TM-score of 0.727, RMSD of 1.46, and Cov score of 0.774.To analyze the putative J-DNA binding domain of JBP3, the representative region of LtJBP3 was threaded through the JBP1 JBD PDB structure to search for similar secondary structural folds using the comparative modeling program I-TASSER . (L. tarentolae JBP3 gene was amplified from genomic DNA by PCR with primers containing 5’ Nde I and 3’ BamH I restriction sites. PCR fragments were digested with Nde I and BamH I and subcloned into the pet16b expression vector. Plasmids were transformed into Escherichia coli (BL21 DE3), and bacteria were cultured in defined autoinduction media to allow growing cultures to high densities and protein expression without induction, as previously described [2PO4, Na2HPO4 and 0.05% Glucose, 0.2% Lactose and 0.6% Glycerol. Bacteria were cultured in this media in the presence of 100μg/ml ampicillin at 37°C for 2 h and then shifted to 18°C for 24 h. Cells were lysed and JBP3 purified by metal affinity as previously described for JBP1 [The escribed . Brieflyfor JBP1 , 41. TheElectrophoretic Mobility Shift Assays were carried out essentially as described previously for JBP1 and VSG-1J DNA substrate , 41, witT. brucei cell lines expressing PNUTS-PTP and JBP3-HA or Wdr82-HA and loaded onto 10 ml, 5–50% sucrose gradients. Samples were centrifuged at 38000 rpm for 18 hours using a SW41 Ti rotor (Beckman). The gradient was fractionated from top to bottom in twenty aliquots of 500ul each. Proteins from each fraction were enriched by methanol: chloroform protein precipitation and resuspended in SDS loading buffer for electrophoresis.For the sedimentation analysis of the PJW/PP1 complex, extracts were made from the BF Total RNA was isolated with Tripure Isolation Reagent (Roche). cDNA was generated from 0.5–2 μg Turbo DNase (ThermoFisher) treated total RNA with Superscript III (ThermoFisher) according to the manufacturer’s instructions with either random hexamers, oligo dT primers or strand specific oligonucleotides. Strand specific RT reactions were performed with the strand specific oligonucleotide and an antisense Asf I oligonucleotide. Equal amounts of cDNA were used in PCR reactions with Ready Go Taq Polymerase (Promega). A minus-RT control was used to ensure no contaminating genomic DNA was amplified. Primer sequences used in the analysis are available upon request.TM DNase treated as described above. Quantification of SuperscriptTM III generated cDNA was performed using an iCycler with an iQ5 multicolor real-time PCR detection system (Bio-Rad). Triplicate cDNA’s were analyzed and normalized to Asf I cDNA. qPCR oligonucleotide primer combos were designed using Integrated DNA Technologies software. cDNA reactions were diluted 10 fold and 5 μl was analyzed. A 15 μl reaction mixture contained 4.5 pmol sense and antisense primer, 7.5 μl 2X iQ SYBR green super mix (Bio-Rad Laboratories). Standard curves were prepared for each gene using 5-fold dilutions of a known quantity (100 ng/μl) of WT gDNA. The quantities were calculated using iQ5 optical detection system software. Primers sequences used are available upon request.Total RNA was isolated and TurboT. brucei RNAi cultures grown in presence or absence of tetracycline for two days using TriPure. Six mRNA-seq libraries were constructed for PNUTS RNA (triplicate samples for plus and minus tetracyclin) and four libraries for VAa RNA (duplicate samples) were constructed using Illumina TruSeq Stranded RNA LT Kit following the manufacturer’s instructions with limited modifications. The starting quantity of total RNA was adjusted to 1.3 μg, and all volumes were reduced to a third of the described quantity. High throughput sequencing was performed at the Georgia Genomics and Bioinformatics Core (GGBC) on a NextSeq500 (Illumina).For mRNA-seq, total RNA was isolated from T. brucei Lister 427 2018 version 9.0 genome assembly . Remainded from ) and theded from , using Bded from . With noded from . To ensuequences . To compT. brucei Lister 427 annotation, was within 1-kb either upstream or downstream base J peaks. J IP-seq data shown here are from previously published work [To analyze transcription defects at 3’- and 5’-end of PTUs, reads mapping to TTS (cSSRs) and TSS (dSSRs) were counted and reads per kilobase per million mapped reads (RPKM) values were generated. Similar to what we have previously done , 27, 28,hed work , 25.http://tryps.rockefeller.edu) [To examine VSG expression, trimmed reads were bowtie-aligned to the VSGnome (retrieved from ler.edu)  and diff7 / ml. Ice cold 100% ethanol was slowly added while shaking to a final concentration of 25% ethanol. Cells were then washed 2 times and resuspended in 0.5 ml PBS / 1% BSA / 1% Tween 20 with 5 μg / ml Propidium Iodide. Analysis was performed using a Cyan cytometer (DAKO).The cell cycle profile of the PNUTS RNAi cell line over time following induction was determined by staining cells with DAPI and cataloguing the nucleus/kinetoplastid (N/K) configurations of ~300 intact cells/time-point. Cells were harvested, 2 x washed in PBS, allowed to settle on glass slides and air dried for 10 min at RT. Cells were then fixed in -20°C methanol for 10 min and mounted with ProLong Gold antifade reagent with DAPI . Flow cytometry was carried out by washing the cells 2 times in PBS and resuspended at 1 x 10S1 FigL. tarentolae illustrating the presence of the conserved JBD and variable C-termini. (B) A multiple sequence alignment of the JBD of JBP1 homologues from T. brucei (Tb927.11.13640), L. major (LmjF.09.1480), T. cruzi (TcCLB.506753.120), and L. tarentolae (LtaP09.1510) and the conserved region of JBP3 is shown. The sequence alignment was generated using Maft and visualized with Jalview. Identical amino acids are indicated by highlighting; >80% agreement is highlighted in mid blue and >60% in light blue. Similar amino acids are indicated by hierarchical analysis of residue conservation shown below. (C) 3D structure prediction. Using the structure of the JBD of JBP1, JBP3 was run through I-TASSER and aligns with RMSD of 1.46, Cov score of 0.774, TM-score of 0.727. In the superposition, the thick backbones are the native JBP1 JBD structure and the thin backbone is the I-TASSER model of JBP3. Blue to red runs from N- to C-terminal.(A) Schematic representation of the structure of JBP1 and JBP3 from (TIF)Click here for additional data file.S2 Fig(A) Compositional profiling of Lt and Tb PNUTS showing the fractional amino acid composition in comparison with the compositional profile of typical ordered proteins. The compositional profile of typically disordered proteins from the DisProt database is shown for comparison below. (B) Analysis of TbPNUTS using the DISOPRED3 program for protein disorder prediction and for protein-binding site annotation within disordered regions.(TIF)Click here for additional data file.S3 FigT. brucei complex. (C) PP1-1, PP1-7 and HSP70 do not associate with PNUTS in T. brucei.(A) Endogenously PTP tagged PNUTS, JBP3, Wdr82 and CPSF73. (A and B) Co-IP experiments as described in (TIF)Click here for additional data file.S4 FigStrigomonas culicis, and Perkinselia sp. which only contain JBP-1 family members.A seed alignment of JBP-1 and JBP-3 was used to iteratively search UniProtKB  using ja(TIF)Click here for additional data file.S5 FigPNUTS RNAi cells with an endogenously WT PNUTS PTP tagged allele were used. Total protein and RNA was isolated after 2 and 3 days of RNAi induction and analyzed for the loss of PNUTS protein (protein A Western) and PNUTS mRNA (RT-PCR).(TIF)Click here for additional data file.S6 Fig(A) Gene expression changes upon RNAi ablation of PNUTS (left) and VA a (right) are plotted. Triplicate analysis of PNUTS and duplicate of VA a. On the left, red dots represent genes with greater than 3-fold change that are also significant with a Benjamini-Hochberg FDR test correction. On the right, red dots represent genes with greater than 2-fold change after VA a ablation in both replicates. (B) Gene expression changes at cSSRs (N = 193) and dSSRs (N = 197) that are within 1kb of base J matched with same number of random locations within the genome for ablation of PNUTS and VA a.(TIF)Click here for additional data file.S7 FigT. brucei 427 genome assembly. Only one of the two homologous chromosomes is depicted for the homologous core regions. Both chromosomes are shown for the heterozygous subtelomeric regions containing silent VSGs. The telomeric VSG expression sites are not included in this assembly. Metacyclic-form expression sites are marked with an asterisk.Upregulated genes (>3-fold) upon PNUTS RNAi are indicated in red in the (TIF)Click here for additional data file.S8 FigTranscription was measured by stranded RNA-seq. Fold changes comparing transcription levels between–and + tetracyclin induction of PNUTS RNAi were calculated in 200bp windows (100bp step) and plotted over the chromosome length. The core regions of the 11 chromosomes are shown. Forward (top strand) and reverse reads (bottom strand) were analyzed separately and plotted above and below the chromosome diagram, respectively. Blue genes are transcribed in reverse PTUs and red are forward PTUs. Asterix denote bins with greater than 2-fold change of expression following ablation.(TIF)Click here for additional data file.S9 FigNormalized reads per million estimates were derived for dSSRs and cSSRs as the average across replicates per sample. Median values are indicated by white dots. Differences between + and–Tet were measured by a Mann-Whitney U statistical test.(TIF)Click here for additional data file.S10 FigT. brucei. PPP-seq track colors: Red, reverse strand coverage; blue, forward strand coverage. RNA-seq track colors: Green, reverse strand coverage; Purple, forward strand coverage. Black arrows indicate direction of sense transcription. Red arrows indicate stimulated antisense transcription. In the case of the top strand, this ‘antisense’ transcription leads to derepression of the annotated 6430 gene (B) Confirmation of mRNA-seq transcript changes by RT-qPCR. RT-qPCR analysis was performed for the indicated genes as described in (A) Another divergent PTU region of chromosome 10 illustrating bi-directional transcription at TSSs upon TbPNUTS ablation. TSSs are denoted by PPP-seq and H2A.Z ChIP-seq enrichment in wild-type (TIF)Click here for additional data file.S11 FigT. brucei (WT 221) and PNUTS RNAi cells–and + Tet by DAPI. (B) Cell cycle analysis using flow cytometry. TbPNUTS RNAi cells were stained with Propidium Iodide (PI) and analyzed by flow cytometry.(A) Cell cycle analysis of wild-type BSF (TIF)Click here for additional data file.S12 FigqRT-PCR analysis of MES VSG 1954, MES VSG 559, BES VSG MITat 1.1 and BES VSG MiTat 1.8 expression upon TbPNUTS ablation. Error bars indicate standard deviation from at least three experiments. P values were calculated using Student’s t test. ***, p value ≤ 0.001. All RT-PCR products were confirmed by DNA sequencing.(TIF)Click here for additional data file.S13 FigT. brucei 427 BES sequences (14 BESs). Fold changes comparing plus and minus Tetracyclin were plotted over each BES as described in RNA-seq reads from the PNUTS RNAi were aligned to the (TIF)Click here for additional data file.S14 FigT. brucei cell lines for anti-J dot blot analysis. +Tet indicates samples from day 2 RNAi induction. Samples were 2-fold serially diluted. The same blots were hybridized with a radioactive tubulin probe to control for DNA loading.DNA was isolated from the indicated (TIF)Click here for additional data file.S1 TableLtJGT was purified, fractionated on SDS-PAGE and proteins identified by mass spectrometry as in (DOCX)Click here for additional data file.S2 TableLtJGT, LtJBP3 and LtWdr82 proteins were purified and proteins in the soluble fraction identified by shotgun proteomics. Proteins that were enriched at least 10-fold (based on PSMs) compared to the negative control purification are highlighted. Pep: Peptides; Cov: Coverage.(DOCX)Click here for additional data file.S3 TableT. brucei 927 database at www.TriTrypDB.org.TbPNUTS was purified and proteins in the soluble fraction identified by shotgun proteomics. List of PNUTS-PTP co-purified proteins, identified at 1% FDR by LC-MS/MS, with 10 or more PSMs are shown. Proteins that are enriched at least 40-fold compared to the negative control purification of wildtype extract and a score of at least 100 are highlighted. Included are proteins that are common contaminants of previous tandem affinity purifications . Protein annotation and accession numbers are from the (DOCX)Click here for additional data file.S4 TableDEseq analysis comparing gene expression levels of the TbPNUTS RNAi cell line at day two plus and minus Tetracyclin. mRNAs that are at least 3-fold upregulated are listed, along with available gene descriptions and P values determined by Cuffdiff. No genes are downregulated. Raw read counts and TPM (transcripts per million) for all genes are also included.(XLSX)Click here for additional data file.


The endocannabinoid system (ES) is a cell-signalling system widely distributed in biological tissues that includes endogenous ligands, receptors, and biosynthetic and hydrolysing machineries. The impairment of the ES has been associated to several pathological conditions like behavioural, neurological, or metabolic disorders and infertility, suggesting that the modulation of this system may be critical for the maintenance of health status and disease treatment. Lifestyle and environmental factors can exert long-term effects on gene expression without any change in the nucleotide sequence of DNA, affecting health maintenance and influencing both disease load and resistance. This potentially reversible “epigenetic” modulation of gene expression occurs through the chemical modification of DNA and histone protein tails or the specific production of regulatory non-coding RNA (ncRNA). Recent findings demonstrate the epigenetic modulation of the ES in biological tissues; in the same way, endocannabinoids, phytocannabinoids, and cannabinoid receptor agonists and antagonists induce widespread or gene-specific epigenetic changes with the possibility of trans-generational epigenetic inheritance in the offspring explained by the transmission of deregulated epigenetic marks in the gametes. Therefore, this review provides an update on the epigenetics of the ES, with particular attention on the emerging role in reproduction and fertility. The imps sativa ,3. Indees sativa ,5,6.In this respect, epigenetics can be defined as the overall biological processes changing gene expression without any change in the nucleotide DNA sequence . This ocTherefore, the aim of this review is to provide an update on the epigenetic modulation of the ES and the possible ES-dependent epigenetic effects on gene expression, focusing on the emerging role of the ES in male reproduction and fertility.9THC as the main biologically active constituent of marijuana plant [In 1964 Gaoni and Mechoulam identified Δna plant  giving tna plant ,18.n-6 poly unsaturated fatty acid (PUFA) arachidonic acid (ARA), and share some of the effects of Δ9THC [The main endogenous cannabinoids (“endocannabinoids”), anandamide (AEA) and 2-arachydonoylglicerol (2AG), are derivatives of the of Δ9THC . They biof Δ9THC ,2,18sn-1-diacylglycerol lipases (DAGLα and DAGLβ) for AEA and 2-AG, respectively, the key feature of endocannabinoid signalling is the modulation of endocannabinoid tone. In this respect, the fatty acid amide hydrolases (FAAH1 and FAAH2) preferentially hydrolyse AEA (and 2-AG to a lesser extent), while the monoacylglycerol lipase (MAGL) is particularly active in hydrolysing 2-AG [Apart from endocannabinoid biosynthesis, which is mediated by the N-acyl-phosphatidylethanolamine-specific phospholipase D (NAPE-PLD) and the ing 2-AG .On the basis of unexpected evidence for intracellular reservoirs and transporters of endocannabinoids, the classical “dogma” that endocannabinoids—and in particular AEA—are synthesized and released on demand via hydrolysis of cell membrane phospholipid precursors has been revisited as recently reviewed . AEA canCurrently, the ES represents a key signalling pathway involved in the modulation of most biological functions ,2,18. AsThe environmental-dependent modulation of gene expression usually occurs at transcriptional, post-transcriptional, and translational levels. In general, the main epigenetic mechanisms involve the chemical modification of DNA and histone tails with consequences on chromatin architecture and accessibility to transcriptional factors and the production of specific regulatory ncRNA .3) to cytosine located within cytosine–phosphate–guanine (CpG) islets in the gene promoter region, thus forming 5-methylcytosine (5mC). This modification changes the chromatin structure from an opened  to closed  state [The main chemical modification of DNA is the covalent transfer of the methyl group (-CHe) state . DNA mete) state . Differee) state . Classice) state .O-linked β-d-N-acetylglucosaminylation (O-GlcNAcylation) are less well known [The tight or loose interaction of DNA with basic histone and non-histone proteins deeply affects chromatin structure with consequences for gene expression. The tightly folded part of the chromatin, heterochromatin, is usually transcriptionally inactive, whereas, the loosely folded part of the chromatin, euchromatin, is the site of DNA transcription. At present, nine post-translational modifications occurring at histone protein tails have been identified. The most well-studied are: acetylation (ac), mono- (m1), di- (m2), and tri-methylation (m3) at lysine (K) residues  and the acetylation of histone H4 at lysine 16 (H4K16ac), the transcription activating H3K4me1, H3K4me3, H3K36me3, H3K79me2, and the repressive H3K27me3 and H3K9me3), phosphorylation, and ubiquitination. Crotonylation, citrullination, ADP-ribosylation, proline (P) isomerisation , and ll known ,29,30.NcRNAs virtually influence every aspect of gene expression, thus representing emerging epigenetic marks detectable in both tissues and biological fluids with upcoming relevance in the regulation of biological functions, impact on health and disease status and possible employment for the prognosis, diagnosis and treatment of diseases ,32. TheyMiRNAs (20–22 nt long) are endogenous small ncRNA classically involved in RNA interference and their exogenous counterparts are siRNAs. In most cases, they bind the 3’ untranslated region of target mRNA inhibiting their translation into protein and inducing their degradation; however in the nucleus miRNAs may target mRNA co-transcriptionally, recruiting chromatin-modifying enzymes and inducing epigenetic regulation via DNA methylation or histone tails modifications .Transfer RNAs (tRNAs) produce several fragments involved in repression of translation. Some of them, called 5’- (3’-) tRNA halves , are stress-induced and are produced in humans by the endonuclease angiogenin that cleaves within the anticodon loops of mature tRNAs. Another group (17–26 nt long), usually referred as tRFs, is produced by the processing at the 5’- or 3’-end of mature or precursor tRNAs ,36.cis/trans scaffolding factors [LncRNAs are bidirectional, antisense, intronic, intergenic, or overlapping transcripts capable of modulating the transcription of neighbouring protein-coding genes with remarkable tissue specificity. They also remodel chromatin and genome architecture or stabilize RNA through the recruitment of chromatin-modifying enzymes or directly acting as  factors .Originally characterized in germ cells, piRNAs (26–31 nt long) target heterochromatic regions through the formation of a PIWI–piRNA complex which usually is associated with the repressive histone/lysine methylation marks, but may also recruit different chromatin-modifying enzymes or facilitate transcription .Lastly, circRNAs, a novel class of ncRNAs, are the result of back-splicing and usually are characterized by a covalently closed continuous loop without 5′ or 3′ polarities structure. Highly stable and widely expressed in mammalian cells, including spermatozoa , they usOnce established at embryo stage to define cell fate through the restriction of developmental potential , during CNR1 which encodes for CB1, and the hydrolysing enzyme FAAH, with subsequent alteration of endocannabinoid signalling or tone. The detected epigenetic mechanisms involve changes in DNA methylation , histone tail modifications such as acetylation, deacetylation, or methylation, and the production of specific miRNAs in different brain regions, peripheral tissues, and cell lines. Of note, epigenetic changes in the ES have been detected in several pathological situations such as Alzheimer’s disease, glioblastoma, and colorectal cancer (CRC), and the ES is the target of several ncRNAs [Recent evidence has revealed that ES undergoes epigenetic modulation by alcohol, diet, stress, smoking, exercise, or drugs ,53,54,55l ncRNAs ,62,63,64Nevertheless, phytocannabinoids, endocannabinoids, and endocannabinoid receptor agonist/antagonists all affect epigenetic mechanisms in cell lines, animal models, and humans as well ,94,95,96CNR1 in vivo and in vitro [CNR1 promoter and the expression of miR23A and miR-301a—two modulators of CB1 in the pathogenesis of colorectal cancer, thus inducing the selective expression of CB1 in rat colon. Accordingly, in Caco-2 cells CB1 is less expressed than normal colon mucosa due to the hypermethylation of DNA at CNR1 promoter; in vitro EVOO, its phenolic extract (OPE), and authentic hydroxytyrosol (HT) upregulate CB1 expression with mechanisms for OPE and HT involving the reduction of CNR1 methylation at promoter level and leading to inhibition of cell proliferation [Peripherally, the activity of ES may be epigenetically modulated by diet. In fact, extra-virgin olive oil (EVOO), a typical lipid source in the Mediterranean diet which is rich in phenolic compounds, epigenetically modulates the expression rate of in vitro . Thus, dferation .In the brain, ES is involved in the homeostatic regulation of food intake, through the interplay with peripheral nutrient-sensors and the orexigenic and anorexigenic peptides produced within the arcuate nucleus of the hypothalamus, the brain region capable of capturing and integrating environmental cues with outcomes on feeding behaviour and reproduction . To daten-6 PUFA derivatives and an ideal ratio of 5 (n-6): 1 (n-3) has been suggested in order to preserve brain functions [n6:n3 fatty acid ratio is therefore a possible risk factor to metabolic disease and might indicate an over activation of endocannabinoid signalling. Leptin, an anorexigenic peptide produced by white adipose tissue, inhibits hypothalamic ES [Ob/Ob mice, lacking leptin, over-activate the hypothalamic endocannabinoid signalling [CNR1 overexpression, with mechanisms involving chromatin remodelling at CNR1 promoter region by means of increased histone acetylation rate and increased binding of androgen receptor at CNR1 promoter as a consequence [Endocannabinoid are unctions . The evalamic ES , and Ob/gnalling  and are gnalling . Consistgnalling . Recentlgnalling . Prior osequence .FAAH gene is the only component of ES epigenetically modulated in the hypothalamus by binge-eating, a recurrent process potentially influencing the development of eating disorders. An epigenetic mechanism consisting in the reduction of H3K4ac, without any change in DNA methylation and H3K27met3 at FAAH promoter region have been reported [At present, the reported .CNR1 intron 2 is responsible for the differential transcriptional activation of CB1 in brain regions like the hippocampus and hypothalamus, with effects on the sex-specific anxiety-related behavioural profile, ethanol intake, and hypothermic response following CB1 agonism, but without any significant changes in feeding patterns [Interestingly, a highly conserved regulatory sequence in patterns .CNR1 gene in embryonic mouse hypothalamus cell line N1 [Lastly, Jiang and co-workers proposed a central adiponectin-dependent mechanism to promote the peripheral bone formation through the epigenetic regulation of the hypothalamic expression of CB1, requiring HDAC5 binding to the transcription start site 2 (TSS2) region of the  line N1 .Virtually all steps of reproduction are affected by one or more elements of the ES. In fact, this signalling system is deeply involved in the central and local control of reproduction in both sexes, with functions related to the modulation of the hypothalamus–pituitary–gonad (HPG) axis, germ cell development, successful gametogenesis, production of high-quality gametes, fertilization, embryo implantation and growth, pregnancy, and delivery ,108,109.Reproduction is a process highly sensitive to environmental factors like diet, stress, or endocrine disruptor exposure among others ,126. ThuFAAH1 in Sertoli cells, the nurse cells within the testis whose survival depends on AEA tone, FSH, and oestradiol activity [FAAH1 gene promoter contains an oestrogen-responsive element (ERE) and is notably expressed under oestradiol control in primary mouse Sertoli cells [FAAH1 requires not only the binding of ERβ to proximal ERE sequences (ERE2/3), but also the involvement of the histone demethylase LSD1, and decreased methylation of both DNA at the CpG site and H3K9 in the proximal promoter region [The first evidence of the epigenetic modulation of ES in the gonad concerns the expression of activity . The FAAli cells . Howeverr region .The ES deeply affects the development and the activity of Leydig cells , but at In spite of the lack of data on the possible epigenetic modulation of testicular somatic cells, new insights concern the possible ES-dependent modulation of spermatogenesis through epigenetic mechanisms. Germ cell development is deeply modulated by epigenetic mechanisms which first erase the epigenetic signature (DNA methylation and histone tail remodelling) in primordial germ cells (PGCs) from specifications to migration and proliferation, and subsequently resettle DNA methylation status with de novo development of epigenetic marks and gene imprinting in pro-spermatogonia ,10. Duric-Kit and Stra8 by increasing and decreasing the levels of H3K4m3 and H3K9m2, respectively, in genomic regions flanking the transcription start sites. Interestingly, the global increase in H3K4m3 occurs through the JWH-133-dependent transcriptional activation of Prdm9, the gene encoding for a zinc finger protein with HMT activity that catalyses H3K4me3 during the meiotic prophase. As a consequence, prolonged exposure to JWH-133 or administration of the specific CB2 antagonist AM630 accelerates or delays spermatogenesis onset in immature mice, pointing out the importance of correct endocannabinoid signalling for proper spermatogenesis and the deleterious effect of exogenous cannabinoids on male fertility [In detail, during the spermatogenesis in rodents, CB2 exerts a pivotal role in meiosis entry ,122 and ertility .In this respect, the modulation or the interference in the endogenous ES may affect gamete quality or impact the epigenetic mark of gametes, both critical for pregnancy and embryo development, with the  possibility of trans-generational inheritance in the offspring.-/- mice revealed the requirement of ES signalling for proper chromatin remodelling during spermiogenesis [CNR1 negatively affects the chromatin packaging, by affecting the content of TP2 mRNA and reducing histone displacement, with consequences on chromatin condensation and DNA integrity in the spermatozoa [Spermiogenesis is the process leading to the formation of spermatozoa and is characterized by round spermatid elongation, acrosome and tail formation, nuclear shaping, and DNA packaging with transcriptional silencing as a consequence. Chromatin remodelling and DNA packaging are therefore the main nuclear events in spermiogenesis, consisting of a double-step process that requires histone replacement, first by transition proteins (TP2 and TP1) and then by protamines (PRM1 and PRM2), a class of small basic proteins . The cooogenesis , and proogenesis . In factrmatozoa , which ermatozoa . Such a rmatozoa .Peg10 and Plagl1) have been reported in the sperm of JWH-133 treated animals and are maintained in placental tissue following fertilization. Thus, CB2 signalling may be critical for the integrity of the epigenome in the sperm, with the possibility of paternal epigenetic inheritance in the embryo, a process in which spermatozoa act as vectors for the delivery of epigenetic marks into the developing embryo. Consistently, two isoforms of CircNAPE-PLD (CircNAPE-PLD1 and CircNAPE-PLD2) are expressed in human and murine spermatozoa, and CircNAPE-PLD1 physically interacts with oocyte miRNAs involved in the progression of cell cycle [In line with the results reported above, recent data revealed that the chronic administration of JWH-133 reduces sperm count in mouse and affects the epigenome of spermatozoa. Interestingly, the sperm from JWH-133 treated mice maintains the ability to fertilize eggs from untreated females, but impairment of embryo growth and defects in placental size have been reported, suggesting a possible interference in paternal inheritance through epigenetic mechanisms . Accordill cycle . Therefo9THC on reproductive health status. In humans and rats, Δ9THC exposure lowers sperm concentration and alters DNA sperm methylome with substantial shifts in both hypo- and hyper-DNA methylation, with the latter predominating [9THC levels [9THC-exposed rats [9THC target genes in rat sperm substantially overlap with genes having altered methylation rate in the brain of rat offspring born to parents both exposed to Δ9THC during adolescence [The above observations point out the possible risk for epigenome integrity of spermatozoa following marijuana use. Spermatozoa contain a complete ES devoted to the control of sperm physiology; acrosome reaction, acquisition of motility, spermatozoa–oocyte interaction all require the physiological activity of the endogenous ES ,106. Claminating . In partC levels . Alteredsed rats . Intereslescence .9THC exposure-dependent changes in the DNA methylation of rat sperm [Discs-Large Associated Protein 2 (DLGAP2), a gene involved in synapse organization and neuronal signalling [9THC exposure in adult rats differently methylated DLGAP2 gene in spermatozoa [Consistently with the above observation, Δat sperm  do not sat sperm . In addignalling ,81. SimiLastly, IBN Lahmar Andaloussi et al. recently reported the behavioural and epigenetic effects of stress in male rats whose fathers were exposed to cannabinoids during adolescence  in the pTaken together, the use of cannabis for recreational use may represent a serious risk for both the fertility of marijuana smokers and the health of the offspring.CNR1, and FAAH genes encoding the CB1 receptor and FAAH hydrolysing enzyme may play a relevant role both in physiological processes regulating  fertility and reproduction as well as in disease pathogenesis and progression including cancer. Interestingly, it has been documented that external epigenetic cues such as alcohol induce DNA methylation changes in the mouse model of foetal alcohol spectrum disorder, and the lack of a functional CNR1 gene protects against ethanol-induced impairments of DNMT1, DNMT3A, and DNA methylation [The ES is an almost ubiquitous cell signalling system regulating several processes inside cells that are not yet completely understood. From its discovery in 1990s, it was clear that this system could be modulated by both extrinsic (cannabis and its derivatives) and intrinsic (the endogenous ligands) signals. Subsequent studies pointed out that the ES is much more complex than was thought since it is able to cross-talk with many other transduction cell signalling pathways, therefore regulating key biological processes such as cell proliferation and differentiation, synaptic plasticity, gametogenesis, and fertility. From the data herein reported, it emerges that epigenetic modifications of the ES by means of DNA methylation, histone acetylation/deacetylation at the hylation . This suhylation , also su


Monomethylation on lysine 4 of histone H3 (H3K4me1) is commonly associated with distal enhancers, but H3K4me1 is also present at promoter regions proximal to transcription start sites. To assess a possible role for H3K4me1 in dictating gene regulatory states at promoters, we examined H3K4me1 peak density around promoters in human and mouse germ cells using an analytic strategy that allowed us to assess relationships between different epigenetic marks on a promoter-by-promoter basis. We found that H3K4me1 exhibits either a bimodal pattern at active promoters, where it flanks H3K4me3, or a unimodal pattern at poised promoters, where it coincides with both H3K4me3 and H3K27me3. This pattern is correlated with gene expression level, but is more strongly linked to a poised chromatin state, defined by the simultaneous presence of H3K4me3 and H3K27me3, than to transcriptional activity. The pattern is especially prominent in germ cells, but is also present in other cell types, including embryonic stem cells and differentiated somatic cells. We propose that H3K4me1 is a key feature of the poised epigenetic state, and suggest possible roles for this mark in epigenetic memory. Post-translational modifications on histone tails are closely correlated to transcriptional states. For example, trimethylation of histone H3 lysine 4 (H3K4me3) marks active gene promoters, while H3K9me3 marks regions subject to long-term repression . One sucAt the same time, not all H3K4me1-enriched regions correspond to enhancers. H3K4me1 marks also exist at promoters, which implies that the H3K4me1 modification may have a context-dependent role in regulating transcription. Several studies have addressed questions about the role of H3K4me1 at promoters and highlighted the transcriptional features that may distinguish the functions of H3K4me1 at promoters compared to enhancers. H3K4me1 at promoters in the absence of H3K4me3 was associated with gene repression, while at active gene promoters H3K4me1 appeared to flank H3K4me3 in one study examining skeletal muscle cells . AnotherCell type is another aspect of regulatory context that may play a role in H3K4me1 function. Different modes of epigenetic regulation can take on larger or smaller roles in certain cell types. For example, embryonic stem cells (ESCs) and germ cells exhibit specialized regulatory mechanisms to maintain pluripotency and reprogramming potential. An epigenetic feature that is especially prominent in ESCs and germ cells is poised chromatin, which is defined by the simultaneous presence of two histone modifications, H3K4me3 and H3K27me3, at transcriptionally repressed promoters . H3K4me3Here, we report distinct patterns of H3K4me1 that predict transcriptional regulatory states at promoters in germ cells and ESCs. We used an alternative approach to ChIP-seq data analysis in which we summarized chromatin signal around promoters based on ChIP-seq peak density instead of signal density. This approach allowed us to quantitatively interrogate histone modification patterns at a promoter-by-promoter level, in contrast to more commonly-used approaches that require pooling of multiple promoters to obtain average ChIP signals for a given mark. We used our approach to ask if patterns of H3K4me1 deposition near promoters could convey information about the regulatory state of the promoter. We examined ChIP-seq data for H3K4me1, H3K4me3, H3K27me3, and H3K27ac in mouse and human male germ cells, and found that H3K4me1 peak density around the transcription start sites (TSS) exhibits either a broad bimodal profile or a narrower unimodal profile centered at the TSS. We then examined the position of the H3K4me1 marks relative to H3K4me3, and found that unimodal H3K4me1 directly at the TSS predicts a poised state of chromatin, while bimodal H3K4me1 flanking the TSS predicts an active state. The bimodal distribution is not explained by nucleosome clearing, and the unimodal distribution is not explained only by low expression at these promoters. We conclude that unimodal H3K4me1 centered on the TSS is a characteristic feature of the poised epigenetic state in ESCs and germ cells.These studies were carried out in accordance with the Declaration of Helsinki. The protocol was approved by Yale University’s Human Subjects Institutional Review Board. Written informed consent was obtained from all subjects.This study was carried out in accordance with the principles of the Basel Declaration and recommendations of Yale University’s Institutional Animal Care and Use Committee (IACUC). All procedures involving mice were approved by the Yale IACUC.Human testis samples were obtained from adult male patients undergoing vasectomy reversals at the Infertility Clinic of St. Louis. All men whose tissue was used in this study had a prior history of fertility demonstrated by at least one living child. Epididymal sperm quality and abundance proximal to the vasectomy site was assessed at the time of biopsy, and abundant, motile, morphologically normal sperm were confirmed for each patient. Testis biopsy samples were minced, dissociated using collagenase and trypsin, and then filtered to obtain a single-cell suspension as described . Mouse t4 and 5 × 106 cells were used as starting material, depending on the number obtained from sample isolation and sorting. Pachytene spermatocytes and round spermatids were treated identically. For human spermatogenic cells, fixed cells frozen in lysis buffer  were thawed on ice. For mouse spermatogenic cells, fixed cells frozen in PBS were thawed on ice, then washed once in cold PBS and resuspended in 100 ul lysis buffer. Once in lysis buffer, cells were incubated on ice for 5 min. 200 ul ChIP dilution buffer  was then added to each sample. Samples were sonicated in aliquots of 150 ul in 0.5 ml Eppendorf tubes at 4C using a BioRuptor (Diagenode) for 35 cycles on High setting, 30 s on/30 s off. Aliquots of the same sample were then re-pooled and spun down at 12,000 × g for 5 min, and the supernatant moved to a fresh tube. Chromatin from each sample was then split into two separate tubes (150 ul in each), and 700 ul dilution buffer, 50 ul lysis buffer, and 100 proteinase inhibitor cocktail  were added to each tube. 50 ul of each sample was set aside as input. The remainder of the ChIP was performed as previously described (RRID:AB_306847) or 1.0 ug of antibody to H3K27ac .H3K4me3 and H3K27me3 data was previously published and is available on GEO (GSE68507). For H3K4me1 and H3K27ac ChIP, between 5 × 10escribed , except ChIP libraries were prepared using a TruSeq ChIP sample prep kit (Illumina), according to the manufacturer’s instructions, except that size selection was performed after (instead of before) PCR amplification. All libraries were sequenced on an Illumina HiSeq2500 with 40-base-pair single-end reads.RRID:SCR_005534) with the following parameters: -q 20 -p 80. ChIP-seq data was aligned to either the mouse (mm10) or human (hg19) genome using Bowtie2 in –end-to-end –fast mode with default settings (RRID:SCR_005476). Peaks were called using MACS2 with the following parameters: narrowPeak, q = 0.1 (H3K4me3); broadPeak, q = 0.2 (H3K4me1 from mouse PS and RS); broadPeak, q = 0.1  (RRID:SCR_013291). q-values for peak calling were selected based on reconciliation of peak boundaries with ChIP signal visualized on the UCSC genome browser.Image analysis and base calling were done with the standard Illumina pipeline for HiSeq2500. Data was quality-filtered using fastq_quality_filter from the FASTX toolkit (RRID:SCR_016582) with the following parameters: –bias -b 40 -t 8. Ensembl transcripts (cDNA and ncDNA) from mm10 or hg19 were used to create reference indexes for each species  and custom R scripts. For each TSS, its distance to the nearest H3K4me1 and H3K4me3 was calculated, and the two distance values per TSS were used to generate a heatmap based on 2d bin counts using geom_bin2d in ggplot2.Density plots were generated using custom R scripts. First, the center of each peak ascertained by MACS2 was obtained and the distance from the center of the peak to the nearest TSS was calculated. The distance values were used as input to calculate a density distribution using a Gaussian smoothing function with bandwidth of 15, and the resulting probability function was plotted in R. Heatmaps were generated using the ggplot2 package in R (RRID:SCR_006646). To obtain H3K4me1 peaks at poised promoters, a list of H3K4me1 peaks with overlaps with poised H3K4me3 was generated again with -wa option on BEDTools.To obtain a list of poised promoter regions, a list of H3K4me3 peaks that overlap H3K27me3 peaks were generated with BEDTools using -wa option  and GSE145225 (H3K4me1 and H3K27ac ChIP-seq). Other public datasets used in this study are from mouse myeloid cells (GSE85072), fetal liver (GSE119201), and adult kidney (GSE31039).All germ cell datasets are available on GEO under accession numbers 1.Custom R and Python scripts used for this study are available on Githuby axis shows the probability that the nearest peak is centered a given distance away from the TSS (We set out to examine H3K4me1 distribution in germ cells and embryonic stem cells (ESCs), cell types that share many regulatory mechanisms related to reprogramming and pluripotency. To evaluate the distribution of H3K4me1 at promoters relative to other histone modifications, we called peaks from ChIP-seq data for H3K4me1 and H3K27ac (this study), and from H3K4me3 and H3K27me3  from mou the TSS . H3K4me3 the TSS .We considered several possible explanations for the unique distribution of H3K4me1 peaks around promoters. First, most H3K4me1 signal might be coming from enhancers, as has been previously described. If so, peak distribution would be centered away from the TSS, but we would expect to see a broad distribution of H3K4me1 peaks extending away from the TSS in both directions, rather than the accumulation in density we observe in the 300–1000 bp range. Second, the bimodal profile could result from nucleosome clearing at active promoters, similar to the pattern commonly seen in metagene plots of H3K4me3 signal . This exTo determine whether the pattern of H3K4me1 distribution around the TSS was unique to germ cells, we applied the same data-processing protocol to published ChIP-seq data from mouse embryonic stem cells (mESCs) and human embryonic stem cells (hESCs) . The mixp < 0.05, Kruskal-Wallis test). The same trend is present in human round spermatids and hESCs, although the distribution differences were not statistically significant. Lack of statistical significance in the latter two cell types may be due to increased noise in the datasets, although we cannot exclude a true biological difference in histone mark distributions.We next asked whether the H3K4me1 distribution around the TSS was related to gene expression level. For each TSS in the mouse genome, we found the distance to the nearest H3K4me1 peak, then grouped the TSS into four categories based on expression level as measured by RNA-seq  see sec. The difBecause H3K4me1 is an intermediate molecular state between unmethylated H3K4 and H3K4me3, we considered the possibility that H3K4me1 distribution at promoters is purely a byproduct of mechanisms that directly regulate H3K4me3. In this scenario, H3K4me1 would be expected to mark genes in transition between expression states: either gain of H3K4me3 along with gain of transcription, or loss of H3K4me3 along with loss of transcription.We therefore examined how the H3K4me1 pattern relates to the H3K4me3 pattern around promoter regions. We calculated the distance to both the nearest H3K4me1 peak and the nearest H3K4me3 peak for each TSS . We visuBased on our finding that transcription is related to the shape of the H3K4me1 distribution, we hypothesized that the two patterns observed in our joint analysis might represent poised and active promoters. Active promoters are H3K4me3 positive, H3K27me3 negative, and transcriptionally active, while poised promoters are marked by both H3K4me3 and H3K27me3 and are transcriptionally repressed. We sorted the TSS into “poised” and “active” groups based on whether or not the H3K4me3 region nearest a given TSS overlapped with H3K27me3 signals. When poised and active TSS were plotted separately, we found that the pattern observed in our first heatmap resolved into separate bimodal and unimodal patterns based on whether the TSS is active or poised, respectively . These tIn all three cell types from mouse, heatmaps of TSS reflected a distribution in which H3K4me1 directly occupied promoter regions of poised genes and separated to border the H3K4me3 signal in active promoters. A similar phenomenon was observed for human germ cells and ESCs. While H3K4me1 marks did not always directly overlap the TSS of poised genes in human round spermatids and ESCs, the marks localized closer to the TSS for poised genes compared to active genes .n ≥ 167 for all categories and datasets). We found that the unimodal, promoter-centered H3K4me1 distribution marked poised promoters regardless of expression level, while a bimodal H3K4me1 distribution marked epigenetically ‘active’ (H3K4me3-only) promoters regardless of expression level . Then we calculated the distances between the TSS and the nearest H3K4me1 peak and grouped the TSS based on whether the nearest H3K4me1 peaks overlapped with poised peaks. TSS where the nearest H3K4me1 peak overlapped with a poised peak were classified as poised TSS. We then defined epigenetically ‘active’ TSS as TSS where the nearest H3K4me1 peak did not overlap a poised peak, and the nearest H3K4me3 peak was within 500bp of the TSS . Then, each of the two categories of TSS were further classified into four groups based on expression levels, using the same thresholds for transcription level that we used in our previous analysis. As expected, poised promoters were more likely to be silent (tpm = 0) compared to epigenetically active genes, and when expressed their transcript levels were significantly lower than expressed epigenetically active genes . Howeveron level . We concWe identified two distinct H3K4me1 distribution patterns at promoters in mouse and human germ cells and ESCs by examining the density of H3K4me1 peaks around transcription start sites. We found a unimodal distribution of peaks at one set of promoters, corresponding to H3K4me1 occupying the promoter region directly at the TSS. A different set of promoters is associated with a bimodal peak density that corresponds to H3K4me1-enriched regions flanking the TSS. These distributions corresponded to alternative regulatory states. Unimodal H3K4me1 signal was correlated with lower transcript expression and the presence of poised  chromatin, while the bimodal H3K4me1 pattern was linked to high expression level and the absence of poising. By examining promoters where chromatin state and expression level were discordant, we found that H3K4me1 patterns were more strongly associated with the chromatin state dictated by H3K4me3 and H3K27me3 than with the expression level of the promoter.in vitro and in adult mouse pancreatic islet and liver cells, as well as a handful of other cell types  under accession number The studies involving human participants were reviewed and approved by the Yale University Human Subjects Institutional Review Board. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by the Yale University Institutional Animal Care and Use Committee.SB wrote the code, designed and performed the analysis, interpreted the data, and wrote the manuscript. BL performed the ChIP experiments, contributed to data interpretation, and contributed to writing the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The Adenosine Monophosphate-activated Protein Kinase (AMPK) and the Mechanistic Target of Rapamycin (mTOR) are two evolutionarily conserved kinases that together regulate nearly every aspect of cellular and systemic metabolism. These two kinases sense cellular energy and nutrient levels that in turn are determined by environmental nutrient availability. Because AMPK and mTOR are kinases, the large majority of studies remained focused on downstream substrate phosphorylation by these two proteins, and how AMPK and mTOR regulate signaling and metabolism in normal and disease physiology through phosphorylation of their substrates. Compared to the wealth of information known about the signaling and metabolic pathways modulated by these two kinases, much less is known about how the transcription of AMPK and mTOR pathway genes themselves are regulated, and the extent to which AMPK and mTOR regulate gene expression to cause durable changes in phenotype. Acute modification of cellular systems can be achieved through phosphorylation, however, induction of chronic changes requires modulation of gene expression. In this review we will assemble evidence from published studies on transcriptional regulation by AMPK and mTOR and discuss about the putative transcription factors that regulate expression of AMPK and mTOR complex genes. Both pathways can act in isolation or synergistically (Adenosine Monophosphate-activated Protein Kinase (AMPK) is a serine-threonine kinase that exist as a heterotrimer of catalytic α and regulatory β and γ subunits . Mammalsstically . The intstically , which aThe mTOR kinase exists is two distinct complexes – mTORC1 and mTORC2. The C1 complex is composed of five proteins – mTOR kinase, RAPTOR (regulatory-associated protein of mTOR), mLST8 , PRAS40 (proline-rich AKT substrate 40 kDa), and Deptor (Dep-domain containing mTOR-interacting protein). The C2 complex is composed of six proteins – mTOR kinase, RICTOR (rapamycin-insensitive companion of mTOR), mSIN1 , Protor-1 (protein observed with Rictor-1), mLST8 and Deptor . In someExcellent reviews have been written on the signaling mechanisms regulated by AMPK and mTOR . TherefoPrkaa1 (α1), and Prkaa2 (α2), Prkab1 (β1), Prkab2 (β2), Prkag1 (γ1), Prkag2 (γ2), and Prkag3 (γ3) , and Dmel/SNF4Ag (FlyBase). C. elegans express two catalytic α subunits (Aak1 and Aak2), two β subunits (Aakb1 and Aakb2), and five putative γ subunits (Aakg1-5) (WormBase version Ws238). In the budding yeast AMPK subunits are encoded by five genes - Snf1a (α subunit), Sip1, Sip2, and Gal83b (β subunits) and Snf4g (γ subunit) (Arabidopsis the SNF1-related kinase 1 (Snrk1), a homolog of yeast Snf1 and mammalian Prka are transcribed by two typical α subunits (Kin10 and Kin11), two β subunits, one γ subunit along with two atypical subunits - β3 and βγ (Stk11 (Lkb1) and Camkk2. Their homologs in lower eukaryotes include Par4 in C elegans, Dmel/Lkb1 in D. melanogaster, Pak1, Tos3, and Elm1 in S. cerevisiae, and Grik1 and Grik2 in A. thaliana  . The rookaryotes . With onkaryotes . AMPK sur plants , and theMPK gene . The Drosubunit) . In Arab3 and βγ . In addithaliana .Mtor, Raptor, Deptor, Lst8, and Akt1s1 (PRAS40). The mTORC2 complex which is composed of six proteins are encoded by Mtor, Rictor, Sin1, Lst8, Prr5l (PROTOR2), and Deptor. Akin to mammals, S. Cerevisiae also have TORC1 and TORC2 complexes (Raptor (Kog1), mLST8 (Lst8), Rictor (Avo3), and mSin1 (Avo1), although additional components are specific to yeast or mammals  encodes all five mTORC1/C2 genes, all plants encode the Tor, Raptor, Lst8 genes. Other unicellular organisms such as the diatom Phaeodactylum tricornutum and the intestinal parasite Giardia intestinalis encode only mTORC1 genes, while the free-living ciliates such as Tetrahymena thermophila encodes only TORC2 genes. Breaking the rule among lower organisms, the free-living unicellular protist Naegleria gruberi and the human parasite Leishmania major express genes for both TORC1 and C2 complex. The malarial parasite Plasmodium falciparum seems to be an exception that does not encode any mTOR complex genes  and RAPTOR. Extensive literature exists on this area , and thecis regulatory elements proximal to transcription start sites of the AMPK and mTOR pathway genes.Protein phosphorylation by kinases is one of the most widely studied post-translational modifications (PTM) . While s1 and/or TRANFAC2  are located. We would like to recognize that although in most TF binding site (TFBS) distribution studies, the most relevant TFBSs are generally located ±2 Kb from the TF start site, more complex regulatory circuits may be constructed which are beyond the scope of this review. ChIP-seq has been instrumental in the examination of TF-binding sites , Camkk2 in the AMPK pathway, and Tsc1, Tsc2, Rptor, Rictor, Mtor, Mlst8, Akt1s1 (PRAS40), Mapkap1 (SIN1), Prr5 (PROTOR), Rheb, Rraga (RAGA), Rragb (RAGB), Ragc (RAGC), and Ragd (RAGD) in the mTOR pathway. This analysis revealed that Prkab1, Prkab2, Prkag1, and the Stk11 (LKB1) binding partners Cab39 and Strada in the AMPK pathway  of assigned TFs were Prkag1, Prkab1, Strada, Rraga and Tsc2.The potential TF binding sites (TFBSs) can be predicted using the known and inferred motifs, represented as position-specific scoring matrices (PSSMs) derived from the various binding models such as protein-binding microarrays (PBMs), high-throughput SELEX (HT-SELEX), and manually curated models including JASPARTRANFAC2 . Due to ng sites . Caveats pathway , and Mto pathway  have the pathway , and Akt pathway  are tranA simple explanation of these results is that transcriptional regulation of some genes of the AMPK and mTOR pathways are more flexible and more adaptive with several TFs able to initiate transcription under various cues, while regulation of other genes with just one or two TFs is tight and critically depend on the abundance of these TFs. An alternative explanation is that one or two TFs are sufficient for optimal transcription of some genes, while other genes require cooperative interaction of multiple TFs for optimal transcription. It is also possible that the one or two TFs that we identified for some genes in the 2 Kb upstream and 1 Kb downstream region of the TSS are actually insufficient and require coordination of co-activators and distant enhancers. Another intriguing observation that is apparent from It is now well-established that both metabolic enzymes and metabolites synthesized in intermediary metabolism directly or indirectly impact gene expression. DNA itself and DNA-binding histones are modified and regulated by several mechanisms that uses metabolites generated in intermediary metabolism . These iin vitro, an experimental condition that reduces AMPK activity in most if not all normal cells. In several cancer cells, however, high basal AMPK activity is insensitive to such manipulation of glucose levels  came from Many TFs require post-translational modifications (PTM) for activity, nuclear/cytoplasmic translocation, interaction with binding partners including other TFs, stability and DNA binding . These iSaccharomyces to mammalian cells. In the budding yeast Saccharomyces cerevisiae, glucose suppresses expression of a set of genes that are used to catabolize carbon sources other than glucose in glucose-limited growth conditions. Derepression/activation of glucose-repressed genes was found to rely on Snf1-dependent phosphorylation of multiple serine residues on the zinc finger TF Mig1  in endogenous FoxO3 to enhance its transcriptional activity -mediated transcription either directly in the nucleus or indirectly by regulating nuclear translocation of TFs . It is wcription . In mammcription . WhetherSince its discovery as a cytosolic metabolic kinase that purified with and inhibited Acetyl Co-A carboxylase (the rate limiting enzyme for long chain fatty acid synthesis) , we haveAS and BD wrote the manuscript. KC mined the ENCODE data. All authors contributed to the article and approved the submitted version.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.


Correction to: Mol Cancerhttps://doi.org/10.1186/s12943-019-1127-7After the publication of this work , the autThe figure legends:Fig 1. The classification of HDAC family. Specific domains, subcellular localization and targeting inhibitors (HDACis) for the HDAC family. Some HDACis are HDAC isoform-selective inhibitors. RGFP966 is the selective inhibitor for HDAC3. Citarinostat and SLK-23bb are the selective inhibitors for HDAC6. Divalproex sodium is a selective inhibitor for SIRT1. Number 0 stands for the preclinical trial. Number 1, 2, 3 and 4 stands for phase I/II/III/IV clinical trials, respectively.Fig 2. Schematic representation of the main HDACs and HDAC-related TFs involved in hematopoietic lineage commitment. Generally, HDACs (marked in red) regulate hematopoietic development by interplaying with specific TFs (marked in blue) at different stages. HDAC1/2 are widely involved in the development of hematopoietic lineages, while other HDACs, such as HDAC8, promote the differentiation of HSCs to progenitor cells. HDAC3 is mainly involved in the development of lymphoid lineages. HDAC5 mainly participates in the regulation of erythrocyte differentiation, while HDAC7, HDAC4 and HDAC11 are involved in the development of macrophages, dendritic cells and neutrophils, respectively.Fig 3. A model highlights component transformation in transcriptional complex is critical for leukemic transformation. In normal myeloid differentiation, DNA-bound AML1 interacts with p300, MOZ, pCAF and nuclear receptor coactivators (CoA). This association results in an increase in histone acetylation, chromatin remodeling and transcriptional activation. While in tAML, the AML1/ETO fusion recruits the NCoR/SMRT–Sin3a–HDAC complex, which leads to a histone acetylation reduction, chromatin organization inhibition and transcriptional repression, thus blocking myeloid differentiation.Fig 4. A model of CBP/P300 and HDAC component patterns determines the transcriptional function of TF in erythroleukemia cell differentiation. HDAC1 interacts with the nuclear co-repressor mSin3A and associates with EKLF in undifferentiated EBHX11L cells. This co-repressor complex mediates β-globin gene transcriptional repression. During the differentiation of EBHX11L cells to a primitive erythroid phenotype, the formation of EKLF-P300/CBP-SWI/SNF complexes acetylates both EKLF and histones. P300/CBP-mediated EKLF acetylation decreases EKLF interaction with mSin3A-containing complexes. This leads to a conversion of EKLF from a transcriptional repressor to activator and promotes cellular differentiation.Fig 5. A model of class II HDAC interaction patterns in erythroid differentiation. pERK phosphorylates the nuclear localization domain of HDAC5 and facilitates HDAC5-mediated shuttle of GATA1 and EKLF from the cytoplasm to the nucleus. Once the nuclear remodeling shuttle erythroid (NuRSERY) complex of pERK- HDAC5-GATA1-EKLF enters the nucleus, HDAC5 indirectly modulates the deacetylation of GATA1 and EKLF by recruiting HDAC3 to the complex. With erythroid maturation, HDAC5, GATA1 and EKLF remains associated but the levels of pERK sharply decreased, which leads to the shuttle of the complex from the cytoplasm to nucleus and dissociation of HDAC3.Fig 6. A model of TF modification affects the recruitment of HDAC to the promoter. HDAC1 can be recruited by Ikaros to repress the demethylase of KDM58 in normal cells. In B-ALL, Casein kinase 2 (CK2)-mediated phosphorylation of Ikaros decreases HDAC1 recruitment to the KDM58 gene, which contributes to KDM58 expression and leukemogenesis.Fig 7. Abnormal gene expression of HDACs in human different hematological malignancies. The most significantly upregulated HDAC genes are marked in red. The most prominently downregulated HDAC genes are marked in blue. CML: chronic myelocytic leukemia, AML: acute myelocytic leukemia, AML MLL: acute myelocytic leukemia with mixed lineage leukemia, CLL: chronic lymphocytic leukemia, ALL: acute lymphoblastic leukemia, MDS: myelodysplastic syndromes. The data are analyzed based on the TCGA database.Fig 8. Sensitivity and resistance mechanisms of hematological malignancies to HDACis. The major anti-tumor actions of HDACi include HDACi-induced DNA damage, cell cycle arrest, differentiation, apoptosis, ROS and activated autophagy. The potential resistant mechanisms to HDACi include ABC transport-mediated drug efflux, increased DNA repair capacity, CDK/CDKI overexpression-induced normal cell cycle, strongly activated autophagy, multiple pathways-mediated apoptosis inhibition and PlsEtn overexpression-mediated inhibition of ROS damage.In order to improving literature evidence, additional five references are cited as supplementaries.Identification of proteins related to epigenetic regulation in the malignant transformation of aberrant karyotypic human embryonic stem cells by quantitative proteomics.PLoS One 2014, 9:e85823. (as a complement to reference1 and 2)1. Sun Y, Yang Y, Zeng S, Tan Y, Lu G, Lin G: Unexpected role for p19INK4d in posttranscriptional regulation of GATA1 and modulation of human terminal erythropoiesis.Blood 2017, 129:226-237. (as a complement to reference 45)2. Han X, Zhang J, Peng Y, Peng M, Chen X, Chen H, Song J, Hu X, Ye M, Li J, et al: Deubiquitylase USP7 regulates human terminal erythroid differentiation by stabilizing GATA1.Haematologica 2019, 104:2178-2187. (as a complement to reference 45)3. Liang L, Peng Y, Zhang J, Zhang Y, Roy M, Han X, Xiao X, Sun S, Liu H, Nie L, et al: All-Trans Retinoic Acid Induces CD4+CD25+FOXP3+ Regulatory T Cells by Increasing FOXP3 Demethylation in Systemic Sclerosis CD4+ T Cells.J Immunol Res 2018, 2018:8658156. (as a complement to reference 59 and 60)4. Sun X, Xiao Y, Zeng Z, Shi Y, Tang B, Long H, Kanekura T, Wang J, Wu H, Zhao M, et al: Propranolol Attenuates Surgical Stress-Induced Elevation of the Regulatory T Cell Response in Patients Undergoing Radical Mastectomy.J Immunol 2016, 196:3460-3469. (as a complement to reference 61)5. Zhou L, Li Y, Li X, Chen G, Liang H, Wu Y, Tong J, Ouyang W: The institution name: ‘1. The Xiangya Hospital, Central South University, Changsha 410005, Hunan, China’ change to the new one: ‘1. Hepatobiliary and Enteric Surgery Research Center, Xiangya Hospital, Central South University, Changsha 410008, Hunan, China’