Additionally, we describe the recently identified BLV miRNAs and antisense transcripts and their implications in BLV-induced tumorigenesis. Finally, we discuss the relevance of BLV as an experimental model for the closely related human T-lymphotropic virus HTLV-1.Bovine Leukemia Virus (BLV) is the etiological agent of enzootic bovine leukosis, a disease characterized by the neoplastic proliferation of B cells in cattle. While most European countries have introduced efficient eradication programs, BLV is still present worldwide and no treatment is available. A major feature of BLV infection is the viral latency, which enables the escape from the host immune system, the maintenance of a persistent infection and ultimately the tumoral development. BLV latency is a multifactorial phenomenon resulting in the silencing of viral genes due to genetic and epigenetic repressions of the viral promoter located in the 5ʹ Long Terminal Repeat (5ʹLTR). However, viral miRNAs and antisense transcripts are expressed from two different proviral regions, respectively the miRNA cluster and the 3ʹLTR. These latter transcripts are expressed despite the viral latency affecting the 5ʹLTR and are increasingly considered to take part in tumoral development. In the present review, we provide a summary of the experimental evidence that has enabled to characterize the molecular mechanisms regulating each of the three BLV transcriptional units, either through  Bovine Leukemia Virus (BLV) is a B-lymphotropic retrovirus naturally infecting cattle. In the majority of the cases, infected animals remain lifelong asymptomatic carriers, while ~ 30% will present a persistent lymphocytosis, characterized by an accumulation of infected B lymphocytes . In a migag, pro, pol and env as well as of the accessory genes R3, G4, Tax and Rex necessary for efficient viral replication. However, rapidly after infection, the 5ʹLTR promoter activity is repressed through genetic and epigenetic mechanisms leading to the absence of viremia and the escape from the host immune system, which certainly contribute to tumoral development  the 5ʹLTR RNAPII-dependent sense transcription, [2] the RNAPIII-dependent miRNAs transcription and [3] the 3ʹLTR RNAPII-dependent antisense transcription. Additionally, we will describe the recently identified BLV miRNAs and antisense transcripts and summarize their implication in BLV-induced tumorigenesis.Here, we will review the molecular mechanisms regulating each of the three BLV transcriptional activities, either through cis-regulatory sites including promoters, enhancers and silencers. The diverse patterns of gene expression are driven by the binding of transcription factors to promoters or enhancers which can be ubiquitous, cell-specific or induced by internal and external signals, leading to a multitude of gene expression profiles. Regarding BLV, viral gene expression is also regulated by the binding of cellular transcription factors and of the viral transactivator Tax to viral promoter and enhancer elements located in the 5ʹLTR. The 5ʹLTR  contains two canonical RNAPII core promoter elements, the CAAT and TATA boxes, responsible for transcriptional initiation at the U3/R junction of all viral sense transcripts [cis-regulatory elements bound by host cellular factors are responsible for a low basal transcriptional activity, while Tax is the unique viral protein required for the strong transactivation of the 5ʹLTR  located in the U3 region at positions nt− 164 to− 143 for TxRE1, nt− 139 to− 118 for TxRE2 and nt− 64 to − 43 for TxRE3  creb, CRE modulator (crem) and activating transcription factor 1 (atf-1) [CRE-like motifs: A CRE motif is a palindromic octanucleotide consensus sequence 5ʹ-TGACGTCA-3ʹ bound by proteins of the CREB (CRE-binding proteins)/ATF (activating transcription factors) family, encoded by three homologous genes:  (atf-1) . The com (atf-1) .To access CRE sequences and regulate gene expression, CREB needs to be phosphorylated on residue Ser133 . The fir.Concerning BLV, several laboratories have demonstrated by electrophoretic mobility shift assays (EMSAs) that the CRE-like motifs identified in the BLV 5’LTR are able to recruit the cellular transcription factors CREB , 33–35, In contrast, not all members of the CREB/ATF family have been demonstrated to positively regulate the 5’LTR promoter activity, as some of them were shown to have no effect or even to negatively regulate Tax-mediated transactivation. For example, among the other isoforms of CREM, CREMτ activates the 5’LTR promoter activity, whereas it has no effect on its Tax-mediated transactivation . HoweverThe activation of BLV gene expression by CREB/ATF family members seems to depend on the phosphorylation of the CREB/ATF factors and on the recruitment of their co-activators CBP/p300 to the BLV promoter as reported for HTLV-1 . Co-tranThese observations suggest that when a stimulus induces the cAMP pathway and/or an increase in calcium concentration, it activates the kinases PKA and/or CaMKIV, respectively, which in turn phosphorylate CREB/ATF factors. This allows their interaction with CBP and/or p300 which acetylate the histone tails See '''' Sect, E-boxes: In eukaryotic cells, E-box motifs are regulatory sequences with dual functions and able to bind numerous proteins. These proteins mainly contain a basic helix-loop-helix leucine-zipper domain and can act depending on their binding partners either as transcriptional activators (such as enhancer-binding protein 4 (AP-4), upstream stimulatory factors 1 and 2 (USF1 and USF2)) or as transcriptional repressors (such as MYC associated factor X (Max), MAX dimerization protein (Mad)) , 47.. On the contrary, the combined mutations of the 3 E-boxes result in a 2 to fourfold increase in the 5’LTR promoter activity when the corresponding reporter construct was transfected in two different cell lines (Raji or D17) [In the context of BLV, these motifs have been initially described as binding sites for the cellular factor AP-4, as the two first E-box motifs contain the consensus sequence 5’-CAGCTG-3’ corresponding to the AP-4 binding motif , 48. How or D17) , 41. Mol or D17) . In Tax- or D17) .These results suggest a complex mechanism of BLV promoter regulation depending on the cell state, the recruitment of factors and the presence of the viral transactivator Tax.cis-regulatory element recognized by transcription factors whose expression is restricted to macrophages and B cells and could thus participate to the BLV tropism for B cells.Our laboratory has identified a PU-box in the U3 region of the BLV 5ʹLTR located between the positions nt− 95 to− 83 and centered on a purine-rich motif GGAA . PU.1 anal. had suggested the presence of a ƙB binding site at a position between nt− 118 and nt− 70 [al. [Before the identification of the PU.1 binding site, Brooks et d nt− 70 . However 70 [al. , 56.al. [In mammals, following heat shock, the transcription factor heat shock factor 1 (HSF1) drives the transcriptional responses by binding to HSE . HSF1 inal.  have ideal. . Therefoal. have reported that B lymphocytes from BLV-infected cows are sensitive to glucocorticoids in vitro and in vivo [al. have assessed the effect of dexamethasone on BLV infection in cattle chronically infected with BLV [In 1979, Bloom et  in vivo . Later,  in vivo . Among t in vivo . One yea in vivo . The bas in vivo , 40, 41.with BLV . They haDerse and Casey have described a 250 bp-long element in the R/U5 region that strongly increases the activity of the BLV promoter independently of the viral transactivator Tax . The secBesides the E-boxes located in the three TxREs, our laboratory has characterized an E-box motif 5’-CAGGTG-3’ located in the R region (nt + 173 to + 178) named E-box4 , inside cis-elements located in the BLV promoter region [cis-regulatory elements.Functionally, the E-box4 motif is described as an activator of both basal and Tax-transactivated 5’LTR promoter activities, as demonstrated by transient transfection assays , 63. MorIn the U5 region of the BLV LTR, a transcriptional enhancer that contains a binding site for an interferon regulatory factor (IRF) was discovered between position nt + 250 to nt + 265 . Our labThe CCCTC binding factor (CTCF) is a cellular factor with multiple transcriptional and epigenetic regulatory roles. CTCF uses different combinations of its 11 zinc finger domains to directly bind DNA regions harboring a conserved 15 bp core palindromic motif . Mostly Transcriptional activity and establishment of viral latency are largely influenced by the genomic environment surrounding the provirus. Retroviruses integrate the host cellular genome, allowing the use of the transcriptional machinery but also subjecting the provirus to all the mechanisms regulating cellular gene expression.In the nucleus of eukaryotic cells, DNA is packaged in a structural and functional repeating unit called the nucleosome composed of a DNA segment of 146 bp wrapped around a central core octamer of histone proteins. This octamer is composed of two molecules of each canonical histone proteins H2A, H2B, H3, H4. Each nucleosome is separated from the next one by a linker DNA, forming a polynucleosome fiber further stabilized by the histone H1 binding to the linker DNA close to the core histone octamer. Each histone contains a N-terminal domain that protrudes out of the nucleosome structure and that is subjected to several post-translational modifications . BesidesIn addition to changes in nucleosome positioning, the core histones and particularly their unstructured N-terminal tails, are subjected to various reversible post-translational modifications also regulating the expression of surrounding genes. These modifications include acetylation, methylation phosphorylation, SUMOylation, ADP-ribosylation, ubiquitylation and other less represented modifications (reviewed in –79) resu. For example, the BRD-containing protein BRD4 favors transcription by binding to acetylated histones H3 or H4 and by recruiting the positive transcription elongation factor b (P-TEFb), playing a critical role in transcriptional elongation [Histone acetylation is a reversible post-translational modification widely associated to transcriptional activation and consists in the transfer of an acetyl group onto the amino termini group of a lysine residue . By doinongation , 85. Lysongation , 87 cataongation . On the ongation .Several studies have demonstrated the involvement of histone acetylation in the transcriptional regulation of BLV. First, the group of Richard Kettmann in collaboration with our laboratory has shown that HDAC inhibitors (HDACi) such as trichostatin A (TSA) and trapoxin (TPX) induce a marked increase of the BLV promoter activity in integrated reporter constructs in the D17 cell line . In addiOur laboratory has confirmed the effects of TSA and valproate on the BLV promoter and has shown that another HDACi, sodium butyrate (NaB), exerts similar activations of the BLV promoter activity in transient transfection assays and in the context of an integrated provirus . MoreoveThe effects of HDACis are in part linked to the 3 E-box motifs overlapping the CRE-like motifs located in the TxREs. Indeed, mutations in the E-box motifs decrease the responsiveness of the BLV promoter to TSA without completely abolishing this responsiveness . The obsThe group of Anne Van den Broeke has demonstrated the in vivo recruitment of HDAC1 and the known co-repressor mSin3A to the BLV promoter in the BLV-infected L267 cell line . FollowiIn line with the activating effects of HDACis, a synergism between Tax and HDACis (TSA and NaBut) was observed, which was dependent on the CRE-like motifs in the U3 region of the 5’LTR . Indeed,Factors containing an intrinsic HAT activity such as CBP/p300 have been shown to be recruited to the BLV promoter in vivo by ChIP assays using chromatin from YR2 cells treated with PMA + ionomycin . In tranIn conclusion, modulation of histone acetylation at the BLV promoter is important for BLV transcriptional regulation.. For example, H3K4me3 [Unlike histone acetylation which generally favors transcriptional activity, histone methylation has positive or negative effects on gene expression depending on the specific residues that are modified, the degree and pattern of modifications and the genomic localization of the modified histones . In hist H3K4me3  is a cha H3K4me3 . On the In opposition to lysine acetylation, methylation does not change the electronic charge of the side chain. Therefore, lysine methylation impacts gene expression mainly through effector proteins containing methyl-lysine-binding domains, such as the PHD, chromo, MBT domains and others . As an eLess is known about the implication of histone methylation in BLV 5’LTR transcriptional regulation. In the latently-infected L267 cell line, the 5’LTR is associated with low levels of activating H3K4 methylation marks, in accordance with 5’LTR transcriptional repression , 93, 98.Another epigenetic mark known to be involved in the transcriptional regulation of BLV is DNA methylation. DNA methylation mainly occurs on cytosines located within CpG dinucleotides in mammalian cells. This modification is written par DNA methyltransferases (DNMTs) that catalyze the transfer of a methyl group from S-adenyl methionine to the fifth carbon of a cytosine, forming 5-methylcytosine . During . L267 is thought to represent a true latency state, while YR2 represents a mutant defective latency. Moreover, the reactivation of the L267 cell line by transduction of Tax (L267 Tax cell line) induces global demethylation of the 5ʹLTR [Concerning BLV, our laboratory has determined the DNA methylation status of the 5’LTR promoter region in two lymphoma-derived BLV latently-infected ovine B cell lines (the L267 cell line and the YR2 cell line) by bisulfite sequencing method . The L26he 5ʹLTR , 93. In The methylation status observed in the L267 Tax cell line suggests preferential demethylation of the two CRE-like motifs of the TxRE1 and TxRE2 . By gel In vitro methylation of the BLV promoter induces a decrease in basal , 108 andal. have shown the hypermethylation of the proviral promoter in both BLV-induced tumor cells and in PBMCs from BLV-infected cattle with lymphosarcoma using methylation-sensitive restriction enzymes [al. have shown, by bisulfite sequencing, the absence or poor DNA methylation of the 5ʹLTR in PBMCs of cows at different stages of the disease and of a sheep experimentally infected by BLV [The methylation status of the BLV promoter in vivo, in the context of the natural host, has been controversial. Kashmiri et  enzymes . Howeverd by BLV . These cd by BLV .Overall, these results suggest that the DNA methylation of the BLV 5ʹLTR plays an important role in the transcriptional repression of the viral promoter in the context of a fully replication-competent integrated provirus.These studies demonstrate the importance of the chromatin environment in the transcriptional regulation of the BLV promoter and its pathogenesis. Moreover, drugs targeting mechanisms of epigenetic regulation are promising in terms of therapeutic approaches.MicroRNAs (miRNAs) are a class of small single-stranded noncoding RNAs of approximately 22 nt in length that play important roles in gene expression regulation at the post-transcriptional level , 111. FiGag or Env transcripts) are not cleaved by Drosha avoiding a potential detrimental effect on BLV replication due to degradation of viral transcripts [Concerning BLV, the groups of Christopher Sullivan  and Annenscripts , 118. Thnscripts . Then, oGZMA, FOS and PPT1 are directly targeted by one BLV mature miRNA, the blv-miR-B4-3p [hbp1 and pxdn by RT-qPCR in the context of bovines naturally infected with BLV, Petersen and colleagues have observed a downregulation of pxdn compared to uninfected animals [Previous reports have demonstrated that the region now called miRNA cluster is dispensable for BLV infectivity , 122. HoiR-B4-3p . Blv-miRiR-B4-3p  and for iR-B4-3p , in B-ceiR-B4-3p ; howeveriR-B4-3p . Nonethe animals . Apart fIn contrast to the extensively analyzed regulation of RNAPII transcription, RNAPIII transcriptional regulation is considerably less understood in detail, although generally considered as simpler. Unlike RNAPII genes, RNAPIII activity does not seem to be regulated by transcription factors binding to DNA sequences upstream or downstream from the promoters elements. However, recruitment of the transcription factors essential for RNAPIII assembly is directly impacted by several factors, such as p53, Rb, Maf1 or Myc, or by epigenetic mechanisms such as histone acetylation, histone methylation and DNA CpG methylation , 134.Although most BLV miRNAs are highly expressed in both pre-leukemic and malignant BLV-infected B cells  and corrTranscription from the 3’LTR in opposite orientation of the transcription initiated from the 5’LTR promoter, hereafter called antisense transcription, is now considered as a common theme amongst retroviruses  such as cis-regulatory elements have been identified through similar approaches of reporter constructs, such as the previously identified IRF and E-box4 motifs located in the U5 and R regions, respectively [cis-regulatory elements negatively regulating antisense transcription also exist, as deletion of the U3 region in the 3ʹLTR results in an increase in antisense promoter activity. However, the precise contribution of each regulatory element located in the U3 region is still unknown [Unlike the BLV 5ʹLTR promoter, the 3’LTR is a TATA-less promoter that initiates transcription at multiple transcription start sites . Indeed,ectively . Also, Cectively . On the  unknown . Finally unknown , while t unknown . Also, Rversus cytoplasmic RNA fractions has revealed that antisense transcripts are mainly localized in the nucleus, suggesting a potential role of long non-coding RNA (lncRNA). In the HTLV-1 retrovirus, it has been shown that the antisense transcript HBZ, also mainly localized in the nucleus [surviving gene [HBZ transcript has been shown to inhibit 5ʹLTR promoter activity by directly interacting with the LTR, resulting in the displacement of the basal RNAPII transcriptional machinery [Precise function of BLV AS1-S, AS1-L and AS2 antisense transcripts is currently unknown. Although the presence of a potential open reading frame has been described for AS1-S and AS1-L , experim nucleus , supporting gene , 140. Moachinery . Due to achinery . Indeed,achinery . Similarachinery , 143.Compared to the BLV sense transcripts, viral antisense transcripts are constitutively expressed, albeit at low level, in ovine and bovine PBMCs either at the asymptomatic or leukemic stages of infection and their expression is correlated with the viral load , 142. Mocis-regulatory elements present in each BLV promoter to epigenetic modifications regulating the chromatin status of the provirus. Taking all these factors and regulations into account, we propose a model for the reactivation of the BLV 5’LTR promoter from the latency state  site [cis-regulatory elements involved in BLV transcriptional regulation. Moreover, the role of most factors identified so far has been determined using cellular models, such as reporter constructs that, although remaining highly valuable for mechanistic studies, do not recapitulate the complexity of the natural host. Therefore, future research focusing on the function of each factor should be performed in more physiologically relevant models of BLV infection such as BLV-infected cell lines and ultimately in the context of the sheep experimental model or the natural host. The ability of studying BLV infection in the context of its natural host should be further exploited to increase our understanding of BLV gene expression regulation and pathogenesis in vivo and hopefully to translate the promising findings to the closely related retrovirus HTLV-1.Compared to other well studied retroviruses such as HIV-1  and HTLVAZ) site . Therefo(See Table


PTCHD1) is a well-established susceptibility gene for autism spectrum disorder (ASD) and intellectual disability (ID). Previous studies have suggested that alterations in the dosage of PTCHD1 may contribute to the etiology of both ASD and ID. However, there has not yet been a thorough investigation regarding mechanisms that regulate PTCHD1 expression. We sought to characterize the Ptchd1 promoter in a mouse neuronal model, as well as to identify and validate cis regulatory elements. We defined specific regions of the Ptchd1 promoter essential for robust expression in P19-induced neurons. Evolutionarily-conserved putative transcription factor binding sites within these regions were subsequently identified. Using a pairwise comparison of chromatin accessibility between mouse forebrain and liver tissues, a candidate regulatory region, ~ 9.1 kbp downstream of the Ptchd1 stop codon was defined. This region harbours two ENCODE-predicted enhancer cis-regulatory elements. Further, using DNase footprint analysis, a putative YY1-binding motif was also identified. Genomic deletion of the entire 8 kbp downstream open chromatin region attenuated Ptchd1 transcription by over 60% in our neuronal model, corroborating its predicted regulatory function. This study provides mechanistic insights related to the expression of PTCHD1, and provides important context to interpret genetic and genomic variation at this locus which may influence neurodevelopment.Patched domain-containing 1 ( These features along this spectrum range from relatively mild to severely debilitating. The prevalence of ASD in developed countries has recently been estimated to be approximately 1.5%2, with a three-fold higher incidence in males3. The pathoetiology of ASD has a substantial genetic component. A recent meta-analysis of twin studies suggested that 74–93% of ASD risk is heritable4. Concomitantly, studies of ASD on a genome-wide scale have identified a multitude of ASD and intellectual disability (ID) susceptibility genes.Autism spectrum disorder (ASD) is a heterogeneous neurodevelopmental condition that is characterized by circumscribed and repetitive sensory-motor behaviours, as well as impairments in social communicationPTCHD1) (MIM: 300828)5. Studies have further implicated PTCHD1 as an ASD and ID susceptibility gene by identifying additional rare genomic PTCHD1 variants, including both microdeletion copy number variants (CNVs) and single-nucleotide variants (SNVs)6. The function of PTCHD1 protein has yet to be comprehensively elucidated, although in silico analyses predict it to be a multi-pass transmembrane protein with two putative ligand-binding exoplasmic loops, sterol-sensing domains and a C-terminal PDZ-binding domain6. Although studies suggest that PTCHD1, unlike PTCH1, does not play a role in the Sonic Hedgehog (SHH)/Smoothened (SMO) signaling pathway8, PTCHD1 has been shown to bind to cholesterol in vitro9.One specific risk factor gene for ASD and ID is Patched domain-containing 1  have also been linked to ASD and ID. The single-nucleotide polymorphism (SNP) rs7052177 is reported to be associated with ASD and ID15. This SNP is situated in the first intron of PTCHD1 within a putative binding site for the members of the signal transducer and activator of transcription (STAT) family of transcription factors STAT3, STAT5A, and STAT5B, which are all predicted to bind preferentially to the rs7052177T allele15 and therefore may conceivably affect the expression of PTCHD1. Moreover, the authors also identified a 27 bp duplication within the PTCHD1 promoter in three individuals with ASD that correlated with a 26% reduction in transcription in vitro15. Lastly, the longest allele of a microsatellite, consisting of 14 repeats of a [GCC] trinucleotide repeat, was found to be associated with ASD15. Collectively, these data suggest that the dosage of PTCHD1 may be an important variable that requires precise control during neurodevelopment.In addition to CNVs, potential expression-altering variants at the PTCHD1 may be a dosage-sensitive gene with implications for ASD and ID15, there have been no comprehensive examinations regarding the mechanisms that influence its expression. In this study, we have defined specific segments of the proximal region upstream of that regulate its transcription. Further, we identified and validated a putative regulatory region that is predicted to contain enhancer elements located downstream of Ptchd1.While it has been postulated that Ptchd1 promoter truncations, a secondary cassette encoding puromycin resistance was cloned and inserted between the f1 origin of replication and the synthetic polyadenylation signal of the luciferase reporter vector pBV-Luc   were ligated into the multiple cloning site of the modified vector   and 1% penicillin–streptomycin (PS) (Wisent) in T75 flasks . Cells were passaged every 48–72 h at semi-confluence by trypsinization (Wisent).The model system for this study is the male mouse-derived pluripotent embryonal carcinoma cell line P19Ptchd1 reporter constructs, cells were first re-seeded in 6-well culture plates . 24 h later, Lipofectamine 3000  was utilized according to the manufacturer’s instructions to first transfect linearized plasmids into P19 cells. 24 h post-transfection, cells were trypsinized, re-seeded in T75 flasks containing α-MEM supplemented with 1 μg/mL puromycin  and cultured for 48–72 h. Stably-transfected cells were passaged 2–3 additional times in puromycin-supplemented α-MEM before being either used for the induction of differentiation or frozen for long-term storage. In order to eliminate potential bias through inter-clone variability, genomic location and number of sites of integration of the reporter construct, we used a polyclonal approach, rather than selecting and amplifying single transfected clones .To generate stable cell lines expressing 20 with minor modifications. Briefly, cells were trypsinized, resuspended in neural induction medium  and 6 × 104 cells per cm2 were seeded in T75 flasks for four days, with a medium change after the second day. Following induction, cells were trypsinized, resuspended in neural growth medium I [Neurocell medium (Wisent) supplemented with 1X GlutaMAX and 1X N2 supplement (Wisent)] and 9 × 104 cells per cm2 were seeded in 6-well plates coated with Matrigel (Corning) [this time point is hereafter referred to as days after induction (DAI) 0] for five days, with a medium change after 2–3 days. On DAI 5, the medium was replaced with neural differentiation medium II  for five days, with a medium change after 2–3 days. On DAI 10, the medium was replaced with neural differentiation medium III (Neurocell medium supplemented with 1X GlutaMAX and 1X W21 supplement) for up to 10 days, with medium changes every 2–3 days.Differentiation of P19 cells was carried out as described previouslyPtchd1 in our model, neurons were depolarized at DAI20 by incubation with differentiation medium III containing 55 mM potassium chloride for 6 h, followed by immediate cell lysis and downstream experimentation. For non-depolarized control neurons, fresh differentiation medium III without potassium chloride was added for the same duration.To evaluate activity-dependent induction of To isolate RNA from cells, the NucleoSpin RNA Mini Kit  was utilized according to the manufacturer’s instructions, with cells being lysed directly in the 6-well culture plate following a PBS wash. Following isolation, RNA integrity was verified by agarose gel electrophoresis. To generate cDNA, 1 μg of RNA was reverse-transcribed using the iScript cDNA Synthesis Kit .targetΔCP and EhousekeepingΔCP, with E being the efficiency of each primer set21 , and template cDNA was diluted between two- and 1000-fold in order to ensure that amplification was occurring within the linear dynamic range for the given primer pair. All qPCR primer sequences and reaction conditions are specified in Table 21 Table . For norTo evaluate luciferase activity, the Luciferase Assay System  was used according to the manufacturer’s instructions, with the 1X Reporter Lysis Buffer being used to lyse neurons directly in the 6-well culture plate at DAI 16. This terminal time point was selected because, in contrast with the parental P19 line, neurons derived from P19 clones were observed to exhibit pronounced cell death after DAI 17. Bioluminescence was quantified using the Wallac 1420 software on the VICTOR3 Multilabel Plate Reader instrument . For each biological replicate, bioluminescence values for technical replicates were averaged and normalized to total protein, which was quantified from the Bradford protein assay.PTCHD1 CDS, sequence comparisons were performed between mouse, rat, macaque and human using ECR Browser22, with evolutionary conserved regions (ECRs) recognized using a similarity percentage of 70%. Sequence alignments were synthesized by ClustalX223.To estimate the evolutionary conservation of the 1.8 kbp region upstream of the PTCHD1 promoter were inferred using the SwissRegulon database24 (expasy.org/resources/swissregulon). Similar to Guaraldo et al., candidate TFBSs were selected if they exhibited sufficient sequence conservation between the mouse, macaque and human genomes25, and possessed position-frequency matrix scores of greater than 0.8.Putative transcription factor binding sites (TFBSs) within the Ptchd1, comparative computational analyses were conducted on publicly-accessible next-generation sequencing data. For these analyses, we chose to compare DNase-seq and ChIP-seq data from mouse postnatal day 0 (P0) tissues that exhibited relatively high forebrain and low liver levels of PTCHD1 expression6. We downloaded the call sets uploaded by Prof. Stamatoyannopoulos (University of Washington) from the ENCODE portal26 (encodeproject.org/) with the following identifiers: ENCSR791AJY , ENCSR258YWW , ENCSR094TTT , ENCSR216UMD , ENCSR653AVN , and ENCSR616TJM . We set the range for the screen at a ~ 406 kbp window , including ~ 181 kbp sequence upstream and ~ 171 kbp downstream of the mouse Ptchd1-a transcript (NM_001093750.1). No other known protein coding genes lie within this region, or within the syntenic region on the human X chromosome. Additional comparisons in chromatin accessibility were made between human embryonic brain (ENCSR649KBB) and liver (ENCSR562FNN), between mouse P0 forebrain with midbrain (ENCSR767AJS) and hindbrain (ENCSR469VGZ), and between mouse P0 forebrain with liver, heart, stomach, kidney, and small intestine.To identify and characterize prospective regulatory elements proximal or distal to 27. Following DNase footprint identification, motif discovery was conducted using the HOMER suite of tools28.For DNase footprints, we used the Wellington algorithm from the pyDNase packagePtchd1 transcription, the entire genomic region was deleted by CRISPR-Cas9. Two 20 bp protospacer sequences upstream of NGG protospacer-adjacent motifs (PAM) that flank the downstream accessible region to be deleted  were identified , oligonucleotides comprising the protospacers were inserted into the linearized vector pX459 (www.addgene.org). In order to delete the downstream accessible region, P19 cells were transfected in 6-well plates with both sgRNA-encoding Cas9 vectors. 24 h post-transfection, cells were trypsinized, re-seeded in T75 flasks containing α-MEM supplemented with 1 μg/mL puromycin, and cultured for 48–72 h. Cells were then re-seeded at low density in 96-well plates  and cultured for seven days, at which point monoclonal wells were identified and subsequently expanded. Positive clones were first screened for the correct deletion by PCR, and the deletion was subsequently confirmed by Sanger sequencing  35% augmentation in Ptchd1-a expression     Fig. . In contPtchd1 promoter 5’ truncations    and Potocki-Lupski syndrome (OMIM: 610883) involve a reciprocal microdeletion and microduplication, respectively, at the locus 17p11.2 that affects the dosage-sensitive gene Retinoic acid-induced 1 (RAI1) (MIM: 607642)35. In addition, Phelan-McDermid Syndrome (OMIM: 606232) and 22q13 Duplication Syndrome involve a reciprocal microdeletion and microduplication, respectively, of the locus 22q13.3, which encodes the dosage-sensitive gene SH3 and multiple ankyrin repeat domains 3 (SHANK3) (MIM: 606230)38. Interestingly, a multitude of both inherited and de novo microdeletion CNVs affecting all or part of the PTCHD1 CDS have been associated with ASD and ID6. In addition to CNVs, a selection of common variants that do not affect the PTCHD1 CDS but are predicted to alter its expression have also been linked to the etiology of ASD15, suggesting that PTCHD1 may be a dosage-sensitive gene that is important in neurodevelopment. The present study sought to explore the sequence-specific factors at the Ptchd1 locus that affect its expression in a mouse neuronal model in vitro. Specifically, we characterized both basal and depolarization-induced Ptchd1 promoter activity, as well as identified and validated a downstream putative regulatory region.Perturbations in the expression of dosage-sensitive genes are strongly implicated in the etiology of ASD16. This cell line was selected because it was derived from a male mouse, and is therefore hemizygous for Ptchd1. This characteristic allows P19 cells to be particularly amenable to genomic modifications at this locus, as there is only one allele that needs to be modified. In addition, methods of differentiation of P19 cells and subsequent enrichment for neuronal cells have been well-established20. Despite these advantages, there are potential limitations regarding the clinical translatability of findings obtained from a mouse model. These limitations are partly mitigated due to the strong sequence homology between the human and mouse Ptchd1 CDS, proximal promoter, and annotated downstream enhancer elements. Also, a pairwise comparison between human embryonic forebrain and liver DNase-seq. data reveals a homologous open chromatin region downstream of the PTCHD1 3’ UTR in the forebrain that is absent in the liver  response element-binding protein (CREB)-binding protein (CBP), which binds exclusively to enhancers39, are located more than 2 kbp distal to TSSs13.The model system for this study is the male mouse-derived pluripotent embryonal carcinoma cell line P19ver Fig. . The conPtchd1 locus between − 177 and + 170 relative to the TSS   and GCC microsatellite  reported by Torrico et al.15 are located at regions of the PTCHD1 locus that are homologous to the annotated mouse cCRE-PLS.Candidate cis-regulatory elements with promoter-like signatures (cCRE-PLS) are characterized by both strong DNase and H3K4me3 signals, and are also located within 200 bp of an annotated TSS. Accordingly, a 347 bp cCRE-PLS is present at the Ptchd1 appear to be located between − 422 and − 147 upstream of the TSS  to elements within the Ptchd1 promoter. In addition to CHD1, this region of the promoter contains two conserved predicted binding sites for the ubiquitously-expressed transcription factor Sp1, which facilitates formation of the PIC by binding to the TATA box-binding protein (TBP)42. Furthermore, the transcriptional activator TLX2 is reportedly expressed in tissues derived from neural crest cells43 and exhibits upregulation in C1300 and SH-SY5Y neuroblastoma cell lines following stimulation with RA43, suggesting that TLX2 could directly facilitate Ptchd1 expression during differentiation. Conversely, PATZ1 contains a POZ domain, which is associated with mediating transcriptional repression44, making it unlikely that it would facilitate the robust augmentation of luciferase activity conferred by this 276 bp sequence. Interestingly, PML is a highly-pleiotropic protein that has been implicated in both transcriptional activation and repression45, and binding within this 276 bp segment could promote the expression of Ptchd1 under appropriate circumstances. Reciprocally, the slight decrease in luciferase activity that was yielded by the − 1241 to − 859 region could be explained by a conserved predicted binding motif for the transcriptional corepressor RCOR146 , we identified 56 rare SNVs in affected males within these conserved TFBSs, including one variant (MSSNG ID # REACH000589) within the RCOR1 binding motif  and human  genomes (Fig. 50, which contains two conserved TFBSs within the PTCHD1 promoter (Fig. 51. Collectively, these data suggest a mechanism whereby enhancer elements within an extruded TAD downstream of PTCHD1/Ptchd1, when accessible and in proximity to the PTCHD1/Ptchd1 promoter through YY1-mediated DNA looping, may contribute to increased PTCHD1/Ptchd1 expression. Removal of these conserved enhancers and putative YY1 binding motif through deletion of the entire downstream open chromatin region significantly attenuated Ptchd1 expression in our neuronal model (Fig. The enhancer EH38E2746819 from the region just downstream of human orm Fig. . Such DNYY1 Fig. A. Functimes Fig.  which codel Fig.  (PMID 28PTCHD1 may be a dosage-sensitive gene whose reduced expression correlates with ASD and ID15, there is a paucity of research into mechanisms that govern its expression. This study provides a higher-resolution examination of the Ptchd1 promoter, specifically elucidating a core segment containing proximal enhancers that are critical for its expression. Furthermore, we have also identified conserved putative TFBSs within the Ptchd1 promoter that may mediate its transcription. We have also identified and validated a regulatory region situated downstream of Ptchd1 that appears to contain enhancers, as well as proposing a mechanism whereby these distal enhancers may structurally interact with the core promoter. Collectively, these data will help clarify the relationship between the genomic regions surrounding PTCHD1 and their effects on its expression in neurons, serving to provide critical context for structural variations in this region that may impact neurodevelopment.While evidence has implied that Supplementary Information.


The genome of mammalian sperm is largely packaged by sperm-specific proteins termed protamines. The presence of some residual nucleosomes has, however, emerged as a potential source of paternal epigenetic inheritance between generations. Sperm nucleosomes bear important regulatory histone marks and locate at gene-regulatory regions, functional elements, and intergenic regions. It is unclear whether sperm nucleosomes are retained at specific genomic locations in a deterministic manner or are randomly preserved due to inefficient exchange of histones by protamines. Recent studies indicate heterogeneity in chromatin packaging within sperm populations and an extensive reprogramming of paternal histone marks post fertilization. Obtaining single-sperm nucleosome distributions is fundamental to estimating the potential of sperm-borne nucleosomes in instructing mammalian embryonic development and in the transmission of acquired phenotypes. Current Opinion in Genetics & Development 2023, 79:102034Developmental Mechanisms, Patterning and EvolutionThis review comes from a themed issue on Haruhiko KosekiEdited by Developmental Mechanisms, Patterning and Evolution (2023)”For complete overview of the section, please refer to the article collection, “https://doi.org/10.1016/j.gde.2023.1020340959-437X/© 2023 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).Box 1The process of sperm generation, called spermatogenesis, initiates in testes at puberty in humans or few days after birth in mice. Initially, spermatogonial precursor germ cells expand through several mitotic divisions. Germ cells then progress as spermatocytes through a lengthy meiotic prophase during which parental genomes exchange genetic information via meiotic recombination. Following a reductional and an equational cell division, the resulting haploid and genetically diversified spermatids remain functionally interconnected due to incomplete cytokinesis. Such round spermatids further develop into mature sperm through a complex cell differentiation process termed spermiogenesis. During spermiogenesis, cellular structures that are required for fertilization develop, such as the flagellum and the acrosome. Further, transcription is globally shut down, the nucleus elongates and condenses, and the cytoplasm is shed. Nuclear compaction co-occurs with extensive displacement of histones from the genome and incorporation of protamines following an apical–caudal directionality. Whereas all mammals express PRM1, primates, most rodents, and some other placental mammals also express PRM2 Nucleosomes constitute the first layer of chromatin organization in eukaryotes. Through their presence, their post-translational modifications (PTMs), and the diversity of histone variants, nucleosomes regulate numerous cellular processes. In mammalian sperm, small basic proteins called protamines (PRMs) are the main constituents of chromatin. PRMs bind directly to DNA inducing circularization into toroidal structures  [1]. NonThe replacement of histones by protamines initiates in early elongating spermatids upon translation of transition proteins (TNPs) and PRMs  Figure . While TRoles of histone PTMs: Mounting evidence indicates that histone H4 acetylated at N-terminal lysine (K) residues (H4ac) is targeted for removal. While predominantly located at transcriptionally active promoters in round spermatids In vitro, protamines displace hyperacetylated nucleosomes more efficiently than unmodified ones Defective histone displacement also occurs upon dysfunction of certain DNA damage repair factors and associated PTMs such as poly(ADP-ribosyl)ation (PARylation) and serine (S)- 139 phosphorylation at histone variant H2AX (γH2AX) Rnf8 deletion co-occurs with reduced H4ac due to diminished chromatin binding of the histone acetyltransferase (HAT) MOF Interestingly, several histone PTM pathways crosstalk during spermatogenesis. For example, decreased histone ubiquitination upon Contrary to histone displacement, little is known about potential molecular machineries safeguarding nucleosomes from protamine exchange. Proteomics data indicate that most H3K27, H4K20, and H3K9 residues are modified in mature mouse sperm Roles of histone variants: PRMs are thought to bind DNA through arginine-mediated electrostatic interactions causing displacement of DNA-binding factors H2al2-deficient sperm contain less variant TH2B, H2AL2’s predominant dimerization partner Roles of TNP/PRM PTMs: PTMs of PRMs and TNPs are emerging as modulators of chromatin remodeling. To modulate their DNA-binding affinity, PRMs become phosphorylated shortly after synthesis and partially dephosphorylated upon loading onto DNA. Dephosphorylation of mouse PRM2 at S56 is required for sperm head morphogenesis and male fertility K49A sperm contain excessive amounts of histones but lower H4ac, arguing for independent removal of H4ac histones and/or a role of PRM PTMs in fine-tuning replacement of nucleosomes with diverse compositions. Importantly, dysfunction of acetylation-controlling factors frequently leads to TNP/PRM mislocalization. For instance, deletion of the Sirt1 histone deacetylase (HDAC) or the testis-specific nuclear protein in testis (Nut) results in decreased H4ac, elevated histone levels, and impaired TNP/PRM nuclear distribution Nut, Sirt1, and H2AL2 deficiencies Box 2In vitro removal of protamines with Nucleoplasmin2  greatly aids in granting homogeneous chromatin accessibility Protamines compact sperm chromatin several-fold, thereby safeguarding sperm DNA against damaging agents. Hence, studying sperm chromatin frequently requires experimental decondensation of sperm nuclei to grant accessibility to enzymes, antibodies, and other reagents. Pretreatments include permeabilization of membranes, reducing protamine disulfide bonds , and removing PRMs  are depleted in complete digestions Finally, given the large differences in chromatin composition and organization between sperm and somatic cells, it is crucial to study pure sperm samples. Contamination of somatic or germ precursor cells, combined with partial solubilization of sperm chromatin, may lead to inconclusive results. Accordingly, a recent study has challenged chromatin accessibility, nucleosome and PTM occupancies, and nuclear organization studies published to date arguing that reported results originate from contaminating cell-free DNA present in sperm samples Three decades ago, biochemical studies first suggested that certain regions of the sperm genome are predominantly packaged with histones, protamines, or both Gene promoters: Numerous studies using diverse technical procedures report nucleosomes at certain classes of gene promoters characterized by overlapping DNA hypomethylated CpG islands (CGIs) Genes involved in embryogenesis, morphogenesis, and cell fate commitment, including developmental miRNAs and noncoding RNAs, retain H3K27me3-marked nucleosomes at their DNA hypomethylated promoters and are occasionally comarked with H3K4me3 Distal-regulatory regions, enhancers, CTCF, and TADs: Several groups reported chromatin accessibility at intergenic regions in mouse and human sperm by ATAC-seq Centromeres: Centromeres are chromosomal regions enabling correct chromosome segregation during cell division. They are composed of tandem repetitive DNA  and are epigenetically specified by the H3 variant CENP-A. CENP-A locates to centromeres in spermatozoa of human, mouse, and bull, but also of Xenopus laevis and Drosophila melanogastercis-binding of satellite transcripts, among others Drosophila, depletion of CID, the CENP-A ortholog, results in failure to propagate paternal centromeres and to segregate paternal chromosomes during the first embryonic division Pericentromeric heterochromatin (PCH): centromeres are flanked by pericentromeric heterochromatic domains composed of tandem satellites (i.e. major satellites in mouse), other non-satellite repeats and abundant constitutive heterochromatic PTMs such as H3K9me3 and H4K20me3. Similarly marked nucleosomes are present at PCH in human sperm and are transmitted to the zygote to propagate constitutive heterochromatin during embryogenesis Telomeres and interspersed repetitive elements: mammalian telomeres are composed of (TTAGGG)n tandem repeats which locate at chromosome ends and preserve the genetic material during DNA replication. In human sperm, telomeres partly release nucleosome ladders upon mild MNase digestions Interspersed repetitive elements constitute major parts of mammalian genomes and have the capacity to transpose across the genome. Initial genomic studies reported nucleosome enrichments at certain repeat families in human and bull sperm Disclaimer: despite nucleosomes have been localized at loci described above, their relative abundances in sperm vary between studies. Hence, it remains unclear to what extent and in what locations nucleosomes are preferentially retained within mammalian sperm genomes , and the 10.13039/501100000781European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement ERC-AdG 695288 — Totipotency).This project was supported by the L.G.T. wrote the paper. A.H.F.M.P. supervised L.G.T and edited the paper.Nothing declared.


Phaeodactylum tricornutum, which have been shown to be important for transcriptional activation in animal and plant species. To further investigate their evolutionary implications, we utilized chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) and explored their genome-wide distribution in P. tricornutum. Our study aimed to determine their role in transcriptional regulation of genes and transposable elements (TEs) and their co-occurrence with other histone marks. Our results revealed that H3K27ac and H3K36me3 were predominantly localized in promoters and genic regions indicating a high conservation pattern with studies of the same marks in plants and animals. Furthermore, we report the diversity of genes encoding H3 lysine 36 (H3K36) trimethylation–specific methyltransferase in microalgae leveraging diverse sequencing resources including the Marine Microbial Eukaryote Transcriptome Sequencing Project database (MMETSP). Our study expands the repertoire of epigenetic marks in a model microalga and provides valuable insights into the evolutionary context of epigenetic-mediated gene regulation. These findings shed light on the intricate interplay between histone modifications and gene expression in microalgae, contributing to our understanding of the broader epigenetic landscape in eukaryotic organisms.Histone methylation and acetylation play a crucial role in response to developmental cues and environmental changes. Previously, we employed mass spectrometry to identify histone modifications such as H3K27ac and H3K36me3 in the model diatom  Diatoms, a group of unicellular photosynthetic organisms belonging to the stramenopile lineage, are characterized by their remarkable diversity, over 100,000 extant species known to date . Their dPhaeodactylum tricornutum and Thalassiosira pseudonana [In the context of diatoms evolution, epigenetic mechanisms have likely played a role in shaping the diversity and adaptability of these organisms. Epigenetic studies in diatoms have only recently emerged within the past decade, following the release of two model diatom genomes, eudonana ,7,8. Cureudonana . In eukaeudonana ,10.Histone modifications play a critical role in regulating gene expression by modifying the structure of chromatin, thus affecting the accessibility of DNA to the transcriptional machinery. Additionally, these modifications can also recruit enzyme complexes to the chromatin, further influencing gene expression ,12,13. HP. tricornutum was released in 2015, which identified 62 post-translational modifications (PTMs) on histones using mass spectrometry, including mono-, di-, and tri-methylation, acetylation, and mono-ubiquitination of lysines. Combining genome-wide mapping of six key histone marks  with DNA methylation profiling revealed different chromatin states and gene expression patterns, extending the histone code to Stramenopiles. This epigenetic code displayed both unique and broadly conserved chromatin features, reflecting the chimeric nature of P. tricornutum genome [P. tricornutum [P. tricornutum’s phylogenetic position. Such insights can provide valuable understanding of the underlying mechanisms that drive adaptation and survival in response to environmental changes. The first genome-wide characterization of histone modifications in m genome ,20,21. 5m genome ,23,24. Tcornutum . The ideThe dynamic regulation of histone modifications by a variety of histone modifying enzymes is essential for proper chromatin function and cellular homeostasis. The process of acetylation of ε-amino group in lysine on histone proteins is a crucial mechanism for the activation of transcription . This moSchizosaccharomyces pombe and Saccharomyces cerevisiae, Set2 is the sole lysine methyltransferase that mediates all forms of H3K36 methylation, and couples H3K36me3 with transcriptional elongation [Arabidopsis thaliana, the homolog of SETD2, termed SDG8, has been identified as a critical enzyme involved in the di- and trimethylation of H3K36, while in Drosophila melanogaster, dSet2/HYPB is primarily responsible for H3K36me3 [A. thaliana, SDG8 was shown to exhibit specific regulatory functions in modulating histone methylation at H3K36 within gene bodies, particularly for genes associated with photosynthesis and metabolism that are responsive to light and/or carbon cues [In contrast, different histone methyltransferases (HMTs) are responsible for methylating different lysine or arginine residues on histones, and the effects of methylation can vary depending on the specific residue and degree of methylation. For example, the addition of the methyl group to the lysine residue at position 36 of histone H3 (H3K36) is catalyzed by histone methyltransferases, such as Set2 in yeast and “SET domain-containing protein 2” (SETD2) in humans . H3K36meongation . SETD2, ongation . The ortH3K36me3 ,38. In Abon cues ,40. P. tricornutum have revealed a comprehensive repertoire of histone-modifying enzymes, highlighting the remarkable conservation of these writers of histone modification marks in diatoms and their ancient evolutionary origins [Previous genomic sequencing studies of  origins ,41, inclP. tricornutum. Additionally, we sought to broaden our understanding of the H3K36me3 diversity in diatoms by analyzing the occurrence and evolution of H3K36me3-specific methyltransferase from other species using the Marine Microbial Eukaryote Transcriptome Sequencing Project database (MMETSP) [P. tricornutum [In this study, we used chromatin immunoprecipitation coupled with deep sequencing to investigate the distribution pattern and functional implications of H3K27ac and H3K36me3 in the model diatom (MMETSP) . Furthercornutum ,43,44 anCaenorhabditis elegans, lacks an SRI domain, which may suggest that additional mechanisms are primarily responsible for recruiting Set2 to chromatin [P. tricornutum and T. pseudonana, in addition to transcriptome libraries such as the MMETSP database [Shionodiscus, Synedra, Pleurosigma, Navicula, Haslea, Guinardia, and Minidiscus, that were sequenced by Genoscope. A maximum likelihood phylogenetic tree was then constructed to understand the evolutionary divergence of SETD2 among unicellular eukaryotes   [P. tricornutum.The H3K27ac modification covered nearly 13.24% of the f 747 bp . Most H3f 747 bp B,D,E. We < 0.01) F, which  < 0.01) , which m < 0.01) ,43  . In cont < 0.01) . Hollist < 0.01) . This fiP. tricornutum genome , forming approximately on average 2-kb-long blocks (p < 0.01). These results are consistent with the model that Set2 is recruited by the transcription elongation apparatus and that it methylates local nucleosomes during active transcription [p < 0.05)  G. Notabl < 0.05) , suggest < 0.05) . Upon fu < 0.05) H. It is S. cerevisiae, which has been shown to play a role in regulating levels of H3K27ac and H3K56ac modifications, potentially preventing inappropriate transcriptional initiation by controlling nucleosome remodeling and histone exchange [P. tricornutum. In addition, DNA methylation and three repressive histone marks, namely H3K27me3, H3K9me2, and H3K9me3, indicate a chromatin state that efficiently represses gene expression. Notably, this is a unique pattern that have not been observed in animals or plants. However, when these repressive histone marks are associated with H3K27ac and/or H3K36me3, they transition to an active chromatin state that can have a significant impact on the increase of global transcript levels, observed in few genomic locations (around 200 genes). By uncovering new patterns and relationships among various histone modifications, we can gain a more comprehensive understanding of how chromatin states contribute to gene expression and cellular differentiation.To fully understand the function of a particular histone modification, it is essential to consider its interaction with other modifications, as per the ‘histone code hypothesis’. This hypothesis suggests that various histone modifications cooperate in a sequential, cooperative, antagonistic, or redundant way to establish and maintain a chromatin structure that is conducive to gene expression . To inveexchange . It is wexchange . This hiTo gain insights into the functional categories enriched in H3K27ac marked genes, we further performed KEGG pathway enrichment analysis on H3K27ac/H3K36me3-associated genes . We obseAmong the genes specifically targeted by H3K36me3, there are 144 loci involved in various metabolic pathways, including glucose metabolism, amino acid metabolism and TCA cycle. Recent studies have suggested that H3K36me3 acts as lipopolysaccharide-sensitive epigenetic mark that senses multiple upstream metabolic pathways to control the availability of S-adenosylmethionine (SAM), allowing direct communication between the metabolic state and the chromatin state . This enP. tricornutum, H3K27ac and H3K36me3 are mainly located in genic regions and are associated with high-level of gene expression. Genes marked with these histone modifications have the highest expression levels and frequently co-occur with other permissive histone marks, such as H3K4me3 and H3K9/14ac. Additionally, KEGG pathway analysis revealed that the genes marked with H3K27ac and H3K36me3 are involved in various metabolic pathways that enable cells to adapt to environmental changes. Further investigations in P. tricornutum and other emerging unicellular models will certainly provide new insights and deepen our comprehension of the regulatory roles of these histone modifications in modulating gene expression and epigenetic regulation mechanisms in response to changing environments. Both marks are now available on the epigenome browser PhaeoEpiview [Taken together, our findings suggest that the genes encoding H3 lysine 36 (H3K36) trimethylation-specific methyltransferase in diatoms exhibit both functional and structural similarities, providing a potential avenue to explore the evolution of Set2/SETD2 proteins in regulating gene expression and chromatin structure in microalgae. In oEpiview , which rhttp://itol.embl.de/ (accessed on 27 February 2023)).A representative set of published reference sequences of H3K36me3-specific methyltransferase was selected, and a multiple sequence alignment (MSA) was generated using the MUSCLE alignment tool in the Geneious 11.0.5 software, then a maximum likelihood tree was constructed with default parameters. Next, a profile Hidden Markov Model (HMM) was built using HMMER 3.3.2  to captuPhaeodactylum tricornutum Bohlin Clone Pt1 8.6 (CCMP2561) cells were obtained from the culture collection of the Provasoli-Guillard Marine Algae for Culture of Marine Phytoplankton . Constantly shaken (100 rpm) cultures were cultivated at 19 °C, 70 µmol photons m−2 s−1 and with a 12 h light/12 h dark photoperiod in Enhanced Artificial Sea Water (EASW) [2, 5 mM 2- mercaptothanol, 10 mM Tris-HCl pH8) and kept on ice for 20 min. After centrifugation , pellets were resuspended in 1 mL of Extraction Buffer II , 5 mM 2-MERCAPTOETHANOL) and centrifuged . This step was repeated until the pellet became almost white. Pellet was resuspended in 300 μL of Extraction Buffer III , 0.15% Triton X-100, 2 mM MgCl2, 5 mM 2-MERCAPTOETHANOL 10 mM Tris HCl pH 8). The 300 μL resuspended pellet were added to a clean Eppendorf tube with 300 μL buffer III and centrifuged . After removal of the supernatant, the chromatin pellet was resuspended in 300 μL of Nuclei Lysis Buffer , 1% SDS). Each ChIP-seq experiment was conducted in two independent biological replicates. Then chromatin immunoprecipitation was performed as described previously [Chromatin isolation was performed as described previously  with a feviously .Chromatin Immunoprecipitation (ChIP) was conducted as described previously . DNA conWe performed ChIP-qPCR validation using monoclonal antibodies against H3K37ac and H3K36me3. Primers designed on randomly selected genes and used for ChIP-qPCR validation were listed in Western blots were performed on nuclear extracts prepared from 50 mL cultures containing approximately 1 million cells/mL. Extracted nuclei were lysed in 20 μL Nuclei Lysis Buffer , 1% SDS), as described previously . The proChIP-seq peaks were annotated with gene annotation from lifted genome annotation . The inthttps://doi.org/10.5281/zenodo.5127899 (accessed on 23 July 2021)) with a read quality (Phred score) cutoff of 20 and a stringency value of 3bp. Using the 25 to 25 telomere assembly published in 2021 as a reference genome, the filtered reads were mapped using Bowtie2 2.4.5 [Pair-end sequencing of H3K27ac and H3K36me3 ChIP and input samples was performed on Illumina NovaSeq platform with read length of 2 × 150 bp. Previously published ChIP sequencing for H3K9me2, H3K9me3, H3K4me2, H3K27me3, H3K9/K14Ac and H3K4me3 were retrieved from NCBI’s Gene Expression Omnibus accessions GSE68513 and GSE139676 ,25. Raw e2 2.4.5 . We thene2 2.4.5 . Two bioe2 2.4.5 .https://PhaeoEpiView.univ-nantes.fr (accessed on 23 May 2023)). All graphs were created using ggplot2 v3.4.0 [To identify regions that were significantly enriched, we used MACS2 v2.2.7.1 on the c2 v3.4.0 .We performed KEGG analysis on a set of genes that were enriched in epigenetic regulatory marks, using the DAVID 2021 software  . Our tar


Arabidopsis thaliana, 11,565 in Oryza sativa, and 32,397 in Zea mays for their characteristic features and explored the associated genomic and epigenomic features. We found lncRNAs were distributed throughout the chromosomes and the Helitron family of transposable elements (TEs) enriched, while the terminal inverted repeat depleted in lncRNA transcribing regions. Our analyses determined that lncRNA transcribing regions show rare or weak signals for most epigenetic marks except for H3K9me2 and cytosine methylation in all three plant species. LncRNAs showed preferential localization in the nucleus and cytoplasm; however, the distribution ratio in the cytoplasm and nucleus varies among the studied plant species. We identified several conserved endogenous target mimic sites in the lncRNAs among the studied plants. We found 233, 301, and 273 unique miRNAs, potentially targeting the lncRNAs of A. thaliana, O. sativa, and Z. mays, respectively. Our study has revealed that miRNAs, which interact with lncRNAs, target genes that are involved in a diverse array of biological and molecular processes. The miRNA-targeted lncRNAs displayed a strong affinity for several transcription factors, including ERF and BBR-BPC, mutually present in all three plants, advocating their conserved functions. Overall, the present study showed that plant lncRNAs exhibit conserved genomic and epigenomic characteristics and potentially govern the growth and development of plants.Over the past decade, long non-coding RNA (lncRNA), which lacks protein-coding potential, has emerged as an essential regulator of the genome. The present study examined 13,599 lncRNAs in  It has been firmly established that these small RNA molecules play pivotal roles in various regulatory processes such as transcription, post-transcription, and translation3.Non-coding RNAs (ncRNAs) have been known to exist in higher eukaryotes for a long time, with most attention focused on small RNAs such as micro-RNA (miRNA) and small interfering RNA (siRNA). With increasing interest and focus on small RNAs, the list of these special classes of small regulatory RNAs keeps growing, e.g., Piwi-interacting RNA, repeat-associated siRNA, trans-acting siRNA, natural antisense transcript siRNA, heterochromatic siRNA, small scan RNA, and reveals their distinct functions in the regulation of biological processes in different organisms4. This has initiated to unearth a new class of ncRNA, long non-coding RNA (lncRNA). These transcripts are > 200 nucleotides in length and are known to modulate the biological activities throughout the realms of plants and animals7. The lncRNA has gained worldwide attention from researchers, which drives lncRNA identification across the kingdom5. The systematic examination of lncRNAs in plants, animals, and mammals has demonstrated that they play an essential role at the molecular level and contribute to processes such as transcription regulation, miRNA sponge, precursors of miRNAs and phasiRNAs, regulation of alternative splicing, and molecular cargos for protein transportation9. Despite influencing a wide range of biological processes at the molecular level, little is known about the mechanistic details of lncRNA function. However, several well-studied lncRNAs in plants and mammals have provided imperative clues about their functioning and mode of action13.Advanced sequencing technology and sensitivity have expedited the detection of novel transcripts, predominantly derived from the non-protein-coding region of the genome15, response to external stimuli16, role in stress response18, hormone signalling19, nutrient uptake and homeostasis20, transcriptional regulation21, epigenetic regulation15 etc. Following are some well-known examples of lncRNAs biological function in plants. In rice, a lncRNA named long-day-specific male-fertility-associated RNA (LDMAR) is required for normal pollen development of plants grown under long-day conditions. A single nucleotide mutation causes changes in LDMAR's structure, leading to reduced transcription, causing premature cell death in anthers and photoperiod-sensitive male sterility14. A study in Sea buckthorns revealed two lncRNA, LNC1 and LNC2, acting as miRNA target mimics to influence anthocyanin content via SPL9 and MYB114 regulation and revealed their role in anthocyanin content and fruit ripening22. A group of well-characterized antisense lncRNAs transcribing from the floral-repressor locus (FLC), called COOLAIR adopts various conformational structures governing the FLC transcriptional output in response to warm and cold conditions16. In cotton, lncRNA973 has been shown to enhance salt tolerance by regulating the expression of several salt stress-related genes18. In wheat, lncR9A, lncR117 and lncR616 were shown to control the level of CDS1 by modulating the expression of tae-miR398 and improving the cold resistance mechanism in winter wheat21. Another study in rice revealed the role of lncRNA, TCONS_00021861 YUCCA7 gene by modulating the level of miR528-3p, which leads to an increased level of IAA and confers drought tolerance17. In Z. mays, researchers identify GIBBERELLIN-RESPONSIVE lncRNA (GARR2) derived from a Gypsy LTR retrotransposon19. GARR2 editing showed GA-induced effects, altering GA-related genes and affect on primary auxin response. GARR2 interacted with ZmUPL1, a HECT ubiquitin-protein ligase. GARR2 influenced ZmUPL1 levels in GA response, revealing lncRNA roles in GA-modulated plant height19. Franco-Zorrilla and colleagues showed that in A. thaliana, lncRNA INDUCED BY PHOSPHATE STARVATION1 (IPS1) governs the Pi homeostasis by modulating the expression of PHO2 through sequestering the miR-39920. In A. thaliana, winter cold triggers epigenetic repression of FLOWERING LOCUS C (FLC), via cold-induced histone modification involving a lncRNA, COLD ASSISTED INTRONIC NONCODING RNA (COLDAIR), which interacts and recruits PRC to FLC15.In plants, the multifaceted function of lncRNA showed their involvement in growth and development24. This knowledge gap has spurred us to systematically analyze plant lncRNAs to determine their conserved features, which might help us understand their biological significance. In the present study, we aimed to determine the general characteristics of lncRNAs in A. thaliana, O. sativa, and Z. mays and explore their genomic and epigenomic-associated features. We examined the subcellular localization of lncRNAs and studied the interaction network of transcription factors (TFs) and lncRNA. Furthermore, we systematically analyzed the association of transposable elements (TEs) with lncRNAs in plants. Finally, we investigated the lncRNA-miRNA-mRNA interactome network to explore the role of lncRNA in biological and cellular processes. Our study will provide novel insights into the characteristics and conserved features associated with plant lncRNAs.The growing list of lncRNAs across different plant species vouches for their functions in plant growth, development, and stress response, necessitating the understanding of features associated with lncRNAsA. thaliana, O. sativa, and Z. mays were retrieved from the public repository PLncDB V2.0, containing an extensive catalogue of plant lncRNAs23. A total of 13,599, 11,565, and 32,397 lncRNAs were obtained for A. thaliana, O. sativa, and Z. mays, respectively. We analyzed lncRNA distribution along the chromosome to determine whether lncRNAs were transcribed from any preferential region. The distribution pattern revealed that lncRNAs are distributed throughout the chromosomes, chromosomal arms, telomeric and centromeric regions in studied plants  of small transcripts (< 1 Kb), followed by Z. mays (66.3%) and O. sativa (59%) and both O. sativa and Z. mays contain lncRNA transcripts > 10 Kb approximately twice that of A. thaliana.The lncRNAs of nts Fig. . Furthernts Fig. , while tA. thaliana, O. sativa, and Z. mays, respectively, which means that A. thaliana had the most mono-exonic lncRNAs and Z. mays had the most multi-exonic lncRNA transcripts . Comparing the expression profiles of lncRNAs in the four subcellular compartments, we found that there is no typical pattern in the expression profile of lncRNAs localized in different subcellular fractions in A. thaliana, O. sativa, and Z. mays into 36, 28, and 38 different epigenetic states, respectively, based on various epigenetic and genomic features25. Using the PCSD database web tool, we investigated the epigenetic and genomic features associated with lncRNA in three studied plant species. In A. thaliana, epigenetic states 21, 29, and 30 predominantly overlapped with lncRNA transcribing regions, which are mainly enriched for genomic features such as the promoter and intergenic regions of the genome  family also showed enrichment in lncRNA transcribing regions but not in A. thaliana and O. sativa  and Z. mays (> 20x) compared to the A. thaliana positively link the TEs number with lncRNAs overlapping with TEs. Interestingly, the overall TE percentage distribution in lncRNA transcribing regions seems very uniform (~ 6–8%), even though the density of TEs varies among the studied plant  in A. thaliana, O. sativa, and Z. mays, respectively  regulate organism response to stress by facilitating chromatin remodeling along the promoter of the fbp1 + gene and promote the association of the transcription factor Atf1 with its regulatory elements44.The analysed lncRNAs showed enrichment in epigenetic states previously identified as hotspots for other ncRNAsA. thaliana showed enrichment in ESs 21, 29, and 30  in several lncRNAs, supporting our findings55.Franco-Zorrilla and colleagues first defined the target mimic in 2007 in gene regulation. They showed that Induced by Phosphate Starvation1 (IPS1), a lncRNA binds to ath-miR399 with interrupted pairing at the cleavage site of ath-miR399 in nts Fig. . A studyA. thaliana, O. sativa, and Z. mays, respectively , a master regulator of flowering initiation, whereas MAF4 Antisense (MAS) positively regulates the FLC expression69. The circadian-regulated long non-coding RNA (FLORE), a natural antisense of CDF5, is known to repress several CYCLING DOF FACTOR genes and negatively regulate FLOWERING LOCUS T (FT) and consequently activate the FT to promote photoperiodic flowering70.In tc. Fig. A. LncRNAZ. mays showed processes such as DNA repair, lignin metabolic and catabolic processes, and DNA biosynthesis enrichment  factors (Upf1/SMG6) to DNA double-strand breaks and subsequently helps in DSB repair72. Another lncRNA LINP1, was reported to direct the NHEJ-mediated DNA repair by interacting with the NHEJ factor Ku70/Ku80 (Ku) and Ku complexes73. An extensive study in Moso bamboo (Phyllostachys edulis) from different tissues and treatments highlighted the lncRNAs role in the secondary cell wall biosynthesis pathway74.Enrichment analysis of lncRNA-miRNA targeted genes in ent Fig. B. In pla75. The molecular function of identified lncRNA-miRNA-mRNA highlights their role in kinase and transferase activity  to GAGA motifs for the transcriptional repression of downstream target genes80. It has been shown that BPCs directly recruit PRC2 and catalyze the trimethylation of histone H3 at Lys27 at target genes81. A systematic analysis of multiple BPC genes revealed their pleiotropic effects on vegetative and reproductive development. Thus, the BPC TF family is an integral part of several biological processes essential for plant growth and development, which complements our enrichment analyses78.LncRNAs can bind to transcriptional regulatory proteins, which can regulate gene expression. Therefore, targeting lncRNAs that can bind to transcription factors through miRNA cleavage can directly influence the transcriptional landscape of organisms. Our analyses revealed BBR-BPC and ERF association with lncRNA in all three studied plants suggesting their potential and conserved role in governing the biological processes in plants. BASIC PENTACYSTEINE (BPC), also called BARLEY B-RECOMBINANT (BBR) family, encodes GAGA-motif binding factors that govern several biological processes in plants82. A study on spinach has identified two lncRNAs, namely MSTRG.16566.1 and MSTRG.16121.1, as potential endogenous target mimics for miR172, which target three genes encoding AP2/ERF83. This study indirectly links the ERF with lncRNA. A total of 122 ERFs have been found in A. thaliana, while 139 and 166 ERFs homologs have been reported in O. sativa and Z. mays, respectively84. The ERF family of TF acts as an essential regulator in many biological and physiological processes, such as the establishment of the floral meristem, plant morphogenesis, responsive mechanisms to hormone stress signaling, coordination of stress signaling in response to wound repair mechanisms, signal transduction, and metabolite regulation85. Additionally, we found several other TFs, such as B3, bHLH, bZIP, NAC, C2H2, and WRKY, that interact with lncRNA and are known to regulate vegetative and reproductive growth, responses to the broad spectrum of stresses, phytohormonal regulation, defense signaling, etc.89.The ERF family, a prominent plant-specific transcription factor family, governs multiple developmental and physiological processesDespite lacking protein-coding capability, lncRNAs have become crucial players in plant gene regulation and cellular processes. Our analysis has revealed that the complexity of lncRNA transcripts increases with the complexity of genomes. The subcellular localization of lncRNAs predominantly in the cytoplasm and nucleus reflects their apparent site of action. The analysis of lncRNA-miRNA-mRNA networks and the functional annotation of Gene Ontology provide a deeper understanding of the biological processes associated with lncRNAs in different plants. We observed the conservation of several miRNAs targeted by endogenous target mimics of lncRNAs, indicating a conserved mechanism in plants for controlling gene expression by fine-tuning miRNA activity. Further, we found that lncRNAs exhibit a strong affinity for several transcription factors essential for plant growth and development across the three studied plant species. The binding sites of these known transcription factors in the lncRNAs provide valuable insights for deciphering their associated functions and narrowing down the approach required for functional characterization, thereby uncovering their potential roles.LncRNAs overlapping with protein-coding genes poses a challenge when manipulating the lncRNAs in vivo without perturbing the genes on the opposite strand. However, having this information beforehand enables us to choose the appropriate approach for functional characterization. Understanding the functional significance of lncRNAs in plants holds immense potential for crop improvement, stress tolerance, and sustainable agriculture. Unraveling their intricate regulatory networks and deciphering their roles will offer valuable insights into plant biology and create new opportunities for manipulating plant traits to address the challenges of food security and environmental sustainability. This study contributes to the characterization of lncRNAs and establishes the groundwork for future investigations into their specific roles.A. thaliana, O. sativa, and Z. mays were downloaded from PLncDB V2.0, a comprehensive plant lncRNA database23. We used TBtools interactive toolkit to visualize the distribution of lncRNAs across chromosomes90. The coordinates of lncRNA transcripts and chromosomes were used as inputs. Using the GTF and sequence files of lncRNAs, we determined the median transcript length, exon length, splice variants, and GC content of lncRNAs.The lncRNAs and their associated data for 91. LncLocator utilizes k-mer and high-level abstraction features to construct a classifier that predicts subcellular localizations91. For the lncRNAs that showed localization in more than one cellular compartment, we considered the localization with the highest score for a subcellular compartment.To comprehend the biological roles of lncRNAs, we determined their localization in different subcellular organelles. We used lncLocator, an ensemble classifier for determining the subcellular localizations of lncRNAs25. For the association between lncRNAs and epigenetic states, we mapped lncRNA genes onto different epigenetic states, which included 36, 38, and 26 chromatin states dispersed across the genome of A. thaliana, O. sativa, and Z. mays, respectively, using the PCSD web tool25. The lncRNA distribution in different epigenetic states were plotted using a pie chart.To analyze the epigenetic signatures and conserved features associated with lncRNAs, we used the epigenetic dataset of the Plant Chromatin State Database (PCSD)92. For TEs, we used the Atlas of Plant Transposable Elements database (APTEdb), which has uniform annotation criteria for plant TE classification and categorizes them into LTR, LINE, SINE, TIR, MITE, Helitron, and other remaining categories26. We downloaded the TEs annotation data for A. thaliana, O. sativa, and Z. mays from APTEdb in GFF3 format for association analysis26.To investigate the relationship between lncRNAs and TEs, we determined the overlap between the lncRNA transcribing regions and TEs using the bedtools intersect intervals27. This tool identifies a motif in the target sequence complementary to the miRNA. However, this complementarity is disrupted by a bulge around the supposed cleavage site, a key feature for target mimic activity. We used mature miRNA and lncRNA fasta files as input files. The mature miRNA sequences of studied plant species were downloaded from miRBase (https://mirbase.org/).To determine the endogenous target mimics in the lncRNAs, we used the psMimic v1.1 tool with default parameters93. The cladogram data were generated using the Neighbour-joining tree method without distance corrections and visualized using iTOL v694. To identify conserved motifs, we used MEME with default parameters95. We uploaded the output .xml files generated by motif scans through the MEME suite to the iTOL tree for visualization.To study the phylogenetic analysis of miRNA and lncRNA sequences that possess the endogenous target mimic, we performed multiple sequence alignments separately for miRNA stem-loop and lncRNA sequences using T-Coffee96. We also determined the potential target genes for miRNAs interacting with lncRNAs using the psRNATarget at default parameters and an expectation value of 3. The genes targeted by lncRNA-associated miRNA were used to perform GO enrichment analysis using ShinyGO v0.7697.To identify miRNA targets in the lncRNAs, we used the psRNATarget. We identified miRNA–lncRNA interactome using the default parameters, except for a more stringent cutoff threshold for the expectation value (Expectation = 3)98. We used PlantTFDB v5.0 to identify TF binding sites with a threshold P-value ≤ 1e−8. The interaction networks of TFs and lncRNAs were developed using Gephi 0.9.199.We extracted the lncRNA sequences targeted and cleaved by miRNAs (determined by psRNATarget) and used them to identify the TF binding sites. To determine potential TFs binding to lncRNAs, we used the Binding Site Prediction tool of the Plant Transcriptional Regulatory MapWe have summarize the primary toolsets employed for various analyses within the current study, aiming to provide an overarching framework that encapsulates our study. Fig. .Supplementary Figures.Supplementary Table S1.Supplementary Information 3.Supplementary Information 4.Supplementary Information 5.Supplementary Information 6.


Arabidopsis thaliana as the model organism to study histone modifications and their upstream regulations. Similarities among histones and key histone modifiers such as the Polycomb group (PcG) and Trithorax group (TrxG) in Drosophila, Human, and Arabidopsis are examined. Furthermore, prolonged cold-induced vernalization system has been well-studied and revealed the relationship between the controllable environment input , its chromatin modifications of FLOWERING LOCUS C (FLC), following gene expression, and the corresponding phenotypes. Such evidence suggests that research on Arabidopsis can bring insights into incomplete signalling pathways outside of the histone box, which can be achieved through viable reverse genetic screenings based on the phenotypes instead of direct monitoring of histone modifications among individual mutants. The potential upstream regulators in Arabidopsis can provide cues or directions for animal research based on the similarities between them.Histones are subjected to extensive covalent modifications that affect inter-nucleosomal interactions as well as alter chromatin structure and DNA accessibility. Through switching the corresponding histone modifications, the level of transcription and diverse downstream biological processes can be regulated. Although animal systems are widely used in studying histone modifications, the signalling processes that occur outside the nucleus prior to histone modifications have not been well understood due to the limitations including non viable mutants, partial lethality, and infertility of survivors. Here, we review the benefits of using  Eukaryotic nuclear DNA is packed into chromatin, a condensed structure whose basic unit is the nucleosomes . The corDrosophila and mice are the most chosen model organisms for histone modification studies because of their well-studied genetic frameworks and the conserved molecular pathways that are relevant to human diseases [Drosophila and mice cannot cover all epigenetic pathways. As an example, in the Mouse Genome Informatics (http://www.informatics.jax.org/) database, 189 epigenetics-related genes can be found. By matching the gene symbols with records in Mutant Mouse Resource & Research Centers (MMRRC), a national mutant mice repository, only 35 knock-out strains are found to be available [Histone modifications have attracted significant attention in biomedical research due to their involvement in cancer onset, neurological diseases, and embryonic development . Becausediseases ,21. Howediseases . As an adiseases . The avavailable . The mutvailable . The avavailable ,32. The vailable . Due to vailable .Arabidopsis thaliana information database, 131 genes are annotated with epigenetics-related functions and all of their corresponding knock-out mutants are available, which implies that they are not infertile or lethal [Arabidopsis thaliana generated via Agrobacterium-mediated T-DNA mutagenesis , covering almost all Arabidopsis thaliana genes [Arabidopsis thaliana stock centres worldwide, such as Arabidopsis Biological Resource Center (ABRC) (https://abrc.osu.edu), Nottingham Arabidopsis Stock Centre (NASC) (https://arabidopsis.info) and GABI-Kat (https://www.gabi-kat.de). Additionally, plants are immobile and so are more plastic in post-embryonic development to react to changing environmental conditions [Arabidopsis, making it a good candidate to investigate cues of epigenetic memory [Various studies have shown that the mechanisms of histone modifications and their molecular components are highly conserved evolutionarily across higher eukaryotes including plants, suggesting that plants research has the potential to unravel epigenetic pathways that are yet not completely understood and complement research conducted by animal models . Accordir lethal . Moreovena genes . These cnditions , which tc memory ,47.Arabidopsis thaliana enables the study of cold-induced environmental epigenetics of FLOWERING LOCUS C (FLC) for the following reasons: (1) competence to flower via vernalization is determined by chromatin modifications of FLC expression, (2) cold exposure can be quantitatively controlled, and (3) mutants of chromatin control in vernalization process can be easily identified by monitoring flowering time. Polycomb Repressive Complex 2 (PRC2) and Trithorax group (TrxG) complexes are found in both plants and animals and confer the balancing of FLC expression via quantitative depositions of H3K27me3 and H3K4me3 on FLC chromatin. During cold exposure, FLC expression is gradually reduced via increased Histone 3 lysine 27 trimethylation (H3K27me3) deposition on FLC chromatin by PRC2, and the decrease of Histone 3 lysine 4 trimethylation (H3K4me3) deposition was also monitored [FLC silencing is stably maintained even when the plant grows in warm conditions via PRC1. Because FLC expression in Arabidopsis is the only determinant to promote flowering in spring, the flowering time is correlated with the duration of cold treatment, deposition of H3K27me3 on FLC chromatin, and the level of FLC expression. This example demonstrates the potential of using Arabidopsis as a controllable system to explore mechanisms and components transducing signals from the environmental stimuli to the responses inside the nucleus.The research using plant models has contributed to understanding the process that upstream environment inputs alter histone modifications and gene expression patterns . For exaonitored ,51–53. AHistone proteins are highly conserved in eukaryotes, with structural constraint likely resulting from necessary assembly into the histone octamers and functional constraint from compact binding on DNA . Among tArabidopsis thaliana histone subunits H3.1, H3.3, and H4 with those of other species indicates a high level of conservation across organisms , H3.3 (98% identity and 100% similarity) or H4 (100% identity and 100% similarity). However, it is important to note that sequence similarities may not always represent the same function as shown by previous research which compares Arabidopsis UVR8 receptor sequence with RCC1 protein family in animals [The sequence alignment of the N-terminal tails in  animals .Table 1Drosophila, and Arabidopsis [Drosophila, overexpression of H3K36M in the eye decreases the H3K36me2, suggesting its dominant-negative effect on lysine methyltransferases [The basic C-terminal domain (CTD) of histone proteins is critical for chromatin binding and the N-terminal tail of histones modulates nucleosome structure and function ,80. Covabidopsis ,85–88. Sbidopsis ,89,90. Isferases . In Arabsferases .Drosophila (H3.3A and H3.3B) and human (H3f3a and H3f3b), and the knock-out mutants of one of either genes show decreased viability, partial lethality or infertility of the survivors [HTR4, HTR5, and HTR8 encode H3.3. Although the triple knockout is lethal, HTR4-HTR5-double-knockout-HTR8-RNAi lines could survive [Another example is the histone H3.3, which is required for reprogramming events during development across species and is associated with genes that are actively transcribing in both animals and plants . Histoneurvivors ,94,95. Kurvivors . In Arab survive . Researc survive . The viaDrosophila as the repressors of homoeotic (Hox) genes during embryonic development [Other than homologies in histone proteins and modification types, the shared or similar modifying mechanisms and conserved modifiers between Arabidopsis and animal models are also of great importance. Polycomb group (PcG) proteins were originally identified in elopment . PcG homelopment ,99. Althelopment ,101, intelopment . In highelopment ,106. PcGelopment . PRC2 caelopment .Drosophila PRC2 core comprises four subunits: Enhancer of zeste (E(Z)), Extra sex comb (ESC/ESCL), Suppressor of zeste 12 (SU(Z)12), and NURF55 [Drosophila, mice, and human, which are represented in the same colour of Drosophila [Drosophila, PRC2 interacts with DNA-binding proteins to recognize and catalyse H3K27 methylation at Polycomb Response Elements (PREs) loci and DNA fragments with transcriptional factor (TF) binding site, with the latter contributes to the PRC2 recruitment [Drosophila demonstrates that PREs may not be a necessary condition for H3K27me3 modification [FLC chromatin. Besides, R-loop, a DNA-RNA hybrid structure, is also suggested to promote PRC2 recruitment in Drosophila, mammalian, and Arabidopsis through similar mechanisms [PRC2 establishes H3K27 methylation in various organisms through the cooperation of the highly conserved core complex ,113. Thed NURF55  ’s binding to PRC2 is essential for high levels of H3K27me3 [PRC2 core complex can gain functional specificity by associating with partner proteins that can regulate PRC2 methyltransferase activity and/or its recruitment to the specific sites of the genome . This gaH3K27me3 . Pcl, JaH3K27me3 . In mammH3K27me3 . SimilarH3K27me3 ,147. In H3K27me3 . SimilarH3K27me3 ,148. AraH3K27me3 . ArabidoH3K27me3 .Drosophila are the E3 (Sce/dRING and Psc/Su(z)2) monoubiquitin ligase module and associated Pc, Ph, and SCM [Hox clusters and Pc specifically binds to H3K27me3 [FLC, is executed by Arabidopsis LHP1, an H3K27me3-binding factor [PRC1 is less evolutionarily conserved between animals and plants because of the divergence events early in animal evolution . The cor and SCM . Ph and H3K27me3 ,159. SCMH3K27me3 . Most ofH3K27me3 , which aH3K27me3 . The preg factor . Evidencg factor .Drosophila as the transcription activator of Hox gene, which has the opposite to transcription-suppressive PcG proteins [Drosophila, MLLs in humans, SET1A/B and MLLs in mice, and ATXs and SDGs in Arabidopsis [Drosophila, BRG1 and hBRM in humans, and BRM and SYD in Arabidopsis [Trithorax group (TrxG) proteins function to establish, maintain, and transmit the gene expression patterns mainly through methylating lysine 4 of histone H3 (H3K4) and remodelling nucleosomes ,164. Trxproteins . Similarproteins , and theproteins . The firproteins ,173,174.bidopsis ,175,176 bidopsis ,177,178.bidopsis . Collectbidopsis . A majorbidopsis ,179.FigDrosophila) component modules (Drosophila’s homologs are SET1/2 (dSet), WDR5 (Wds), Ash2 (Ash2), RbBP5 (Rbbp5), and Dpy30 (Dpy30), respectively. In mice, they are SET1/2 (SET1A/B), WDR5 (WDR5), Ash2 (ASH2L), RbBP5 (RBBP5), and Dpy30 (DPY30) [COMPASS complexes, also known as SET1A/B, mediate H3K4me3 as an activation mark at non-transcribing regions of the gene, including 5’ regions and promoters in animals and plants . COMPASS modules , which i (DPY30) . Arabido (DPY30) . The hom (DPY30) , while WDrosophila are HCF1, dCxxC1, and Wdr82 while in Arabidopsis are HOS1, MAS bZIP28 and bZIP60.COMPASS also acquires certain functions by interacting with associate factors . In bothSaccharomyces cerevisiae, and similar complexes were characterized later in Drosophila [SWI/SNF functions to mediate ATP-dependent chromatin remodelling, one of the basic mechanisms required for the compaction of the nucleosome . SWI/SNFosophila ,205. Conosophila . The catosophila . In humaosophila . BAFs arosophila ,208. Altosophila .Drosophila, PcG proteins collectively act as repressors of Hox genes, while the gene activation is mediated by TrxG proteins [HOX genes [AGAMOUS (AG) gene through H3K27me3 while TrxG protein ATX1 acts to keep high-level AG transcription, suggesting the antagonist function of ATX1 [Initially discovered in proteins . Later wproteins . Trx assproteins . TrxG prOX genes ,215. On OX genes . With th of ATX1 .More recent studies on the epigenetic components of cellular memory and plasticity have revealed that control of chromatin status is not limited to the modifications performed by the multiple complexes mentioned in the previous section. The chromatin structure is also shaped by the histone sliding and the incorporation of histone variants  mediated by four subfamilies of ATP-dependent chromatin-remodelling enzymes: imitation switch (ISWI), chromodomain helicase DNA-binding (CHD), SWI/SNF, and INO80 ,219. MorIdentification of the upstream regulatory components using mutant can be challenging in some animal models due to lethality and/or sterility. For example, loss-of-function mutations on genes encoding Polycomb proteins frequently cause lethality in mammalian development ,234. HomArabidopsis thaliana is a powerful model to identify upstream regulators of histone modifications. Moreover, the vernalization system in Arabidopsis brings advantages to reveal the relationship between the controllable environment input  correlate phenotypic change, chromatin modifications, and following gene expression. FLC locus is the key to conferring the cold-induced chromatin modification via the balance of the chromatin modification between PRC2 and TrxG, resulting in corresponding flowering time phenotypes [FLC together with the deposition of H3K4me3 mediated by TrxG, leading to the consequent suppression of flowering and delayed flowering time [VIN3, is gradually induced, and FLC expression is gradually reduced via increased H3K27me3 deposition on FLC chromatin. After vernalization, regardless of FLC activation by FRI, PRC2-mediated FLC silencing is stably maintained even when the plant grows in warm conditions because of the function of PRC1 [vin3 mutant is vernalization insensitive, resulting in delayed flowering after vernalization [FLC, COOLAIR [COLDAIR [COLDWRAP [FLC. Interestingly, the plant homeodomain (PHD) finger domain of VIN3 is found to be critical for PRC2 recruitment in human cells [Considering these aspects, enotypes ,237,238.ing time  . Therefore, a mutant of the upstream negative regulator of VIN3 should exhibit: (1) rapid induction of the VIN3-GUS reporter and thus native VIN3, (2) rapid flowering without cold exposure depending on the chromatin status of FLC, and (3) vernalization-insensitive flowering with the loss of VIN3 in a double mutant. This experiment identified a mutant that meets the three criteria, the causative locus encodes a methyltransferase, SET DOMAIN GROUP7 (SDG7), and the mutant has a missense mutation in a highly conserved proline residue (P199L) in SET domain-containing proteins (sdg7–1) [sdg7 mutants without cold exposure. Increased deposition of H3K27me3 on FLC chromatin, resulting in decreased FLC expression, suggesting that SDG7 is an upstream regulator of VIN3  . Furthermembrane , but no  unclear . ArabidoArabidopsis thaliana can serve as exceptional experimental subjects to study the components of epigenetic signalling induced by environmental cues. It will also be a powerful model that opens up more opportunities for the understanding of histone modifications, especially to understand how upstream regulators located outside of the nucleus work to transduce environmental signals. Because of the high similarity of histone proteins, chromatin modification modules, the key protein complexes and associated components in animals and plants, the upstream signalling and its components could be conserved at the molecular level. In addition, findings in animal models have already provided numerous key inspirations guiding the studies in plant, i.e., the discovery of Polycomb and Trithorax proteins which are first identified in Drosophila [Arabidopsis thaliana, vice versa, have great potential to provide cues for animal studies. Arabidopsis has made a significant contribution to the field of small RNA and related epigenetic mechanisms, such as the finding of the Argonaute gene and RNA-directed DNA methylation [The involvements of epigenetic regulation, especially histone modifications in the response to environmental changes, have been revealed, but there are missing pieces that demonstrate how the extracellular signals are transduced into the nucleus. osophila . The finhylation . The chrhylation . These ehylation ,258. Inthylation . These phylation . Investihylation ,258. In hylation ,260.The ease of cultivation provides another reason for applying plants as the model organisms for epigenetic research such as the short generation time of fewer than 2 months, the smaller size that makes it easier to handle larger scale samples in growth facilities, and the potential to maintain epigenomes between generations on account of the self-pollinations . FurtherClick here for additional data file.