Viral strategies are the leading methods for mapping neural circuits. Viral vehicles combined with genetic tools provide the possibility to visualize entire functional neural networks and monitor and manipulate neural circuit functions by high-resolution cell type- and projection-specific targeting. Optogenetics and chemogenetics drive brain research forward by exploring causal relationships among different brain regions. Viral strategies offer a fresh perspective for the analysis of the structure-function relationship of the neural circuitry. In this review, we summarize current and emerging viral strategies for targeting neural circuits and focus on adeno-associated virus (AAV) vectors. Neurons connect with each other to form complex but precise networks, namely neural circuits, which lay the foundation for many brain functions, such as cognition, emotion, learning and memory, and sensory and motor functions. Delineating the fine structure and manipulating the activity of neural circuits are essential for a holistic understanding of complicated brain functions in neuroscience research. This requires highly targeted, efficient, and accurate methods that depend to a large extent on the development and application of virus-tracing technology.In the early research on neural circuits, a series of tracers were developed: anterograde tracers, such as wheat germ agglutinin (WGA) and phaseolus vulgaris agglutinin (PHA), and retrograde tracers, such as fluorogold (FG), cholera toxin subunit B (CTB), carbocyanine, and WGA. However, these traditional tracers show no cell type selectivity and cannot carry exogenous genes, and most of them do not have the capability of transneuronal trafficking. WGA enables transsynaptic transmission but has no directionality, and gets diluted at synaptic connections, which is not conducive to accurate analysis of neural circuits.11–1014 viral genomes(vg)/mL] (Since Kristensson firstly used herpes simplex virus (HSV) as the pathfinders of viral tracers in the 1980s , various(vg)/mL] , and ach(vg)/mL] . These c(vg)/mL] .Here, we discuss the key advances of viral strategies in neural circuitry , includi13 GC/ml to 1011 GC/ml can make trans-synaptic characteristic disappear  to accomplish the replication progress. It contains a single-stranded linear DNA genome of approximately 4.7 kilobases (kb) which usually does not integrate into the host genome. The AAV genome mainly encodes two genes, Cap and Rep, and is flanked by two inverted terminal repeats . The cap open reading frame (ORF) encodes three structural proteins . These proteins assemble into an icosahedral capsid and participate in cell binding and internalization with the help of an assembly activating protein AAP; . So thatisappear . AAV1 caisappear . Howeverisappear .in vivo  DNA genome provides an extremely large insert capacity for exogenous genes of interest . The HSVVSV is a negative-strand RNA virus that belongs to the Rhabdoviridae family. The VSV glycoprotein (VSV-G) is essential for the binding of the virus to the targeted cell by combining with phosphatidylserine, which is widely expressed on the cell membrane surface and thus makes VSV possess extensive tropism . Wild-tyCanine adenovirus-2 (CAV-2) belongs to the Adenoviridae family and can be replication-defective by deleting early region 1 (E1), with a packing capacity of up to 30kb . CAV-2 pThe engineered variant AAV2-retro provides greater retrograde transport efficiency of up to two orders of magnitude than traditional AAV serotypes. It can exhibit effective retrograde access to projection neurons, and has less immunogenicity . These bPRV belongs to Alphaherpesvirinae, similar to HSV-1, and is also amenable for the insertion of large exogenous genes, but preferentially spreads retrogradely. One attenuated strain, PRV-Bartha, demonstrates complete retrograde trans-multisynaptic trafficking with reduced cytotoxicity compared to wild-type PRV . A derivRV is a negative-sense, single-stranded RNA virus that is a member of the Rhabdoviridae family, similar to VSV. Unlike viruses possessing a DNA genome, it cannot be used in combination with promoter-specific expression and intracellular genetic manipulation . RV specifically infects neuronal axon terminals, exhibiting complete trans-chemical synaptic transportation without changing neuronal metabolism, although its effective load capacity is limited  by peripheral delivery  exerts its barrier function to shield the central nervous systems (CNS) from various pathogens and toxins circulating in the blood, however, delivering any viral vectors or drugs to the CNS requires to circumvent this especial barrier function of the BBB when facing with multifocal neurological diseases. By using capsid selection method CREATE (Cre recombinase-based AAV targeted evolution), AAV-PHP.eB, a new BBB-crossing capsid variant, has been selected to enable prominent gene transduction to the CNS with the relatively low vector dose  promoter, a ubiquitous promoter, can non-selectively drive transgene expression which is prone to silencing in some tissues over time, and the chicken β-actin/CMV hybrid (CAG) promoter permits stronger and longer expression in neuronal populations ; the humEnhancers are cis-acting element that could regulate target gene transcription. By activating different sets of enhancers and other distal regulatory elements, a single genome could be transformed into a large number of highly specialized cell subclasses and tissues . EnhanceThe most commonly used recombinase-based intersectional strategy, in which AAV vectors play an extremely important role, is the combination of double viral vectors. One vector encodes site-specific DNA recombinase , and the other recombinase-dependent vector carries the gene of interest. The transgene can be expressed merely in neuron populations transduced by both vectors, which is called “intersectional targeting”. This strategy can help determine the comprehensive mapping relationship of particular neuron subpopulations receiving the projection of a specific input site or projecting to a specific output site in a targeted brain area.The Cre-LoxP system is the most widely utilized conditional expression strategy. Cre recombinase identifies special sequences (lox sites) and drives recombination by deleting coding sequences between lox sites that are in the same direction  or inducThe Cre-dependent AAV-DIO virus and retrograde-transporting virus delivering Cre recombinase (such as AAV2-retro-Cre or CAV-Cre) can be injected into the target area and efferent region, respectively, to label output-defined neuron populations in the target field . This meNotably, AAV1-Cre can also permit retrograde transport , making on/Fon, Con/Foff or Coff/Fon) using multiple-feature Boolean logic  system, a versatile single-AAV system, enables intersectional targeting refined cell popolations expressing different recombinases , followed by EnvA + RVdG  have been widely used as activity sensors to capture the dynamics of defined neuronal populations by measuring changes of Ca2 + concentration during specific behaviors. The action potential allows the opening of the voltage-gated calcium channel, thus leading to increase in cytoplasmic Ca2 + concentration that can be exhibited through fluorescence intensity of the GECIs . Thereby– pumps (NpHR) and bacteriorhodopsin-type proton pumps inhibit neurons in response to yellow or green light, respectively, which causes the inflow of Cl– ions or outflow of protons to generate hyperpolarization potential. Optogenetics have brought seminal influence on the interrogation of brain function in neuroscience research , thereby activating different G protein-coupled pathways to produce the effect of activating or inhibiting neuronal activity (Designer receptors exclusively activated by designer drugs (DREADD), the most widely used chemogenetic technology, is not activated by acetylcholine or other endogenous neurotransmitters, but is selectively activated or inhibited by the inactive clozapine analog clozapine N-oxide CNO; . Clozapiactivity . Clozapiactivity . The effactivity . Pharmacactivity . For exaactivity . Similaractivity .Many neuroscience studies require both optogenetic and chemogenetic technologies to disentangle the role of specific neural circuits in the control of particular behaviors. Optogenetics has superior temporal resolution, with an accuracy of millisecond-level temporal control . The spain vivo and activate or repress the activity of endogenous genes in an adjustable manner  and is sequestered in the cytoplasm. However, tamoxifen administration leads to the dissociation of CreERT2 and HSP90, which then induces the nuclear translocation of CreERT2. In the cell nucleus, CreERT2 recognizes loxP sites to induce recombination; thus, it can control the time window of gene expression by controlling the injection time of tamoxifen (T2) under the control of different promoters; for example, to label active neurons within a limited time window after tamoxifen administration by expressing CreERT2 through an activity-dependent IEG promoter (CreERamoxifen , but notamoxifen . The Crepromoter .This system contains two pivotal components : transcrTherefore, the CreER and Tet-On/Tet-Off systems create an “on/off” switch to express exogenous genes of interest in a transient and reversible manner at a particular time point, gain temporal specificity, and allow the time window of gene expression to be controlled.As the basic units of the nervous system, neurons are mutually intertwined to form complex neural networks. Traditional labeling techniques usually map the connections between brain areas or neuronal populations, but are difficult to achieve observation at the single-neuron level. Thus, sparse-labeling systems have been developed that enable to visualize the complete morphology, dissect the connectivity, and interrogate the function of individual cells in densely packed brain regions with tunable sparseness.Injecting a highly diluted Cre-expressing virus and simultaneously directing a high-titer virus encoding Cre-dependent fluorescent protein allow the generation of sparse but high-intensity labeling of neurons at the injection site and for subsequent reconstruction of individual neurons . SimilarIn the Supernova system, sparse labeling depends on the leaky expression of TRE and bright labeling relies on tTA/TRE positive feedback . The modTransfecting a primary neuron with vectors expressing TVA and RV-G by single-cell electroporation or the whole-cell patch clamp method, followed by infecting the TVA-expressing cells with EnvA-pseudotyped rabies viruses, allows the tracing and genetic targeting of monosynaptic inputs to a single neuron .The continuous progress of genetically modified viruses provides us with excellent specificity in how to target and access selective cell types, as well as the functional interrogation and manipulation of particular neuron subpopulations and neural circuits in a high-resolution spatially and temporally specific manner, which has remarkably changed neuroscience research. An ideal engineered viral vector must possess several pivotal properties: large payload, high-titer packaging, low immunogenicity, high cell type-specificity, and tunability in its expression dynamics, which, currently, cannot be achieved simultaneously. AAV has been widely used as the first carrier for circuit mapping and gene therapy, but its limited packing capacity is still a major hindrance. The design and/or discovery of new viral tools that allow for more specific, safer, and stronger transgene expression in targeted cell populations will be pivotal in future research on neural circuits. And using versatile viral tools to dissect how the neural network state changes during dynamical development and in various pathological conditions will also be the focus of future study. Further verification and improvement of the transduction efficiency and long-term safety of viral vectors in non-human primates will pave the way for gene intervention therapies of neurological diseases.YH, XZ, and HZ drafted the manuscript. FL, GY, and JL did figure organization. JZ, XG, and GG proposed the outline and edited the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The MLL3/4 histone methyltransferase complexes and the histone deacetylase complex MiDAC regulate the histone mark H4K20ac to calibrate gene expression of a common set of target genes. The mitotic deacetylase complex MiDAC has recently been shown to play a vital physiological role in embryonic development and neurite outgrowth. However, how MiDAC functionally intersects with other chromatin-modifying regulators is poorly understood. Here, we describe a physical interaction between the histone H3K27 demethylase UTX, a complex-specific subunit of the enhancer-associated MLL3/4 complexes, and MiDAC. We demonstrate that UTX bridges the association of the MLL3/4 complexes and MiDAC by interacting with ELMSAN1, a scaffolding subunit of MiDAC. Our data suggest that MiDAC constitutes a negative genome-wide regulator of H4K20ac, an activity which is counteracted by the MLL3/4 complexes. MiDAC and the MLL3/4 complexes co-localize at many genomic regions, which are enriched for H4K20ac and the enhancer marks H3K4me1, H3K4me2, and H3K27ac. We find that MiDAC antagonizes the recruitment of UTX and MLL4 and negatively regulates H4K20ac, and to a lesser extent H3K4me2 and H3K27ac, resulting in transcriptional attenuation of associated genes. In summary, our findings provide a paradigm how the opposing roles of chromatin-modifying components, such as MiDAC and the MLL3/4 complexes, balance the transcriptional output of specific gene expression programs. MLL3  and MLL4  are chromatin-modifying proteins that monomethylate histone H3K4 via their catalytic SET domains. MLL3 and MLL4 exist in two separate macromolecular complexes that belong to a total of six mammalian complexes of the compositionally and functionally highly conserved COMPASS family . All comLysine residues on histones can also be acetylated by histone acetyltransferases (HATs) and deacetylated by histone deacetylases (HDACs) . HistoneThe mitotic deacetylase complex MiDAC, which comprises the scaffolding subunits DNTTIP1, ELMSAN1 , and the histone deacetylases HDAC1 or HDAC2, was initially identified in a chemoproteomic screen as being specifically enriched on a HDAC-inhibitor-bound resin in cells stalled in G2/prophase of mitosis after nocodazole treatment . TRERF1 Dnttip1 KO mouse embryonic stem cells (mESCs) coincides with increased UTX and MLL4 occupancy on many genomic elements, indicating that MiDAC negatively affects the recruitment of the MLL3/4 complexes to chromatin. Whereas H3K4me1 enrichment is slightly decreased, H3K4me2 enrichment is higher at regions displaying increased UTX and MLL4 occupancy, suggesting an activity switch of MLL3/4 towards H3K4me2 in the presence of H4K20ac or absence of MiDAC function. However, in Mll3/4 double knockout (DKO) mESCs we observe an increase of H3K27me3 at regions with decreased H4K20ac and lower DNTTIP1 occupancy, indicating that H3K27me3 may be involved in suppressing H4K20ac, despite reduced MiDAC recruitment. Taken together, our study reveals for the first time a functional intersection between MiDAC and the MLL3/4 complexes in regulating H4K20ac and describes the antagonistic relationship of these chromatin-modifying complexes in their role to properly balance transcription of a specific gene expression program.Despite recent advances in our understanding of MiDAC’s structure and physiological role, it is unknown how MiDAC integrates into chromatin regulatory complexes and pathways. Applying a large-scale interactome analysis, we describe here an association of UTX with MiDAC. We show that UTX and ELMSAN1 form the interface between MiDAC and the MLL3/4 complexes. We demonstrate that the loss of MiDAC function results in a genome-wide increase of H4K20ac, suggesting that H4K20ac constitutes a MiDAC substrate in vivo. The genome-wide increase of H4K20ac in Mll3/4 DKO mESCs showed that the UTX antibody used could immunoprecipitate UTX and the MiDAC subunits DNTTIP1, ELMSAN1, and HDAC1, whereas our DNTTIP1 IP identified DNTTIP1, ELMSAN1, and HDAC1 as members of MiDAC along with UTX (ELMSAN1 TRERF1 double knockout (DKO) HEK293 cell line to establish the interaction interface between MiDAC and MLL3/4 complexes in more detail  (MLL3 (MLL3+/+ MLL4+/+) or contain a promoter deletion within the MLL4 locus (MLL3−/− MLL4−/−) . Mass spectrometry (MS) analysis detected as expected all components of the MLL3/4 complexes including all core subunits, complex-specific subunits, and the H3K4 methyltransferases MLL3 and MLL4 . Reciprowith UTX . Thus, te detail  and S3. ely lost . Interestability . Reexpreould not . It shouould not . To testMLL4+/+) . We usedMLL4−/−) . TransieMLL4−/−) . In conc ELMSAN1 .UTX-interacting proteins identified by mass spectrometry from HEK293 cells.Table S1 Dnttip1 KO mESCs   and observed higher enrichment of UTX, MLL4, H4K20ac, and H3K27ac without major changes in H3K4me1 on these sites in Dnttip1 KO mESCs  . Interesrichment . Similarrichment . To asseKO mESCs . These UWT mESCs . AlthougWT mESCs , we did KO mESCs . SimilarWT mESCs . InsteadWT mESCs and 4B. KO mESCs . InteresWT mESCs . Taken tMll3/4 double knockout (DKO) mESCs  mESCs  (DorighiO) mESCs . UnexpecKO mESCs . AltogetWT mESCs . InteresKO mESCs . LikewisKO mESCs . H3K27meKO mESCs . Thus, tomplexes , and 5C.KO mESCs . This anne marks  also alipression , whereaspression . FurtherKO mESCs  and S9. KO mESCs . OverallUTX is often mutated in the same cancer types as MLL3 and/or MLL4 linking the function of the MLL3/4 complexes directly to carcinogenesis via UTX  may prevent MiDAC and potential histone acetyltransferases that target H4K20 to access those regions. Furthermore, we cannot exclude that one or multiple H4K20 deacetylase(s) is/are activated and/or target H4K20ac more effectively in Mll3/4 DKO mESCs. A potential model of the interplay between MiDAC and the MLL3/4 complexes is described in Dnttip1 KO mESCs on regulatory elements that are also targeted by MiDAC did not result in higher H3K4me1 enrichment, but selectively appeared to be confined to increased H3K4me2 and H3K27ac enrichment  and their wild-type control mESCs were previously described by our laboratory  by the Center for Advanced Genome Engineering (CAGE) at St. Jude Children’s Research Hospital using CRISPR/Cas9–mediated gene editing (MLL3−/− MLL4+/+) were obtained from ATCC. MLL3+/+ MLL4+/+ HCT116 cells and MLL3−/− MLL4−/− HCT116 cells were generated from MLL3−/− MLL4+/+ HCT116 cells by the CAGE at St. Jude Children’s Research Hospital using CRISPR/Cas9–mediated gene editing  with 10% FBS  and 1% penicillin/streptomycin . mESC identity was authenticated by various methods including alkaline phosphatase staining and staining and cytometry analysis of the pluripotency marker FUT4 (SSEA-1). All HEK293 and HCT116 cell lines were authenticated using STR profiling and tested negative for mycoplasma contamination.boratory . Mll3/4 boratory . All mES editing . HCT116  editing . Isogeni editing . IsogeniMouse α-Actin  at 1:1,000; rabbit α-ASH2L  at 1:2,000; rabbit α-DNTTIP1  at 1:2,000; rabbit α-ELMSAN1  at 1:5,000; rabbit α-ELMSAN1  at 1:5,000; mouse α-FLAG  at 1:2,000; rabbit α-HDAC1  at 1:2,000; rabbit α-HDAC2  at 1:2,000; rabbit α-KAT2A  at 1:2,000; rabbit α-MLL3/KMT2C  at 1:5,000; rabbit α-MLL4/KMT2D (#31863 [human aa 1–181]) and #32757 [human aa 281–506]; Herz Lab at 1:5,000; rabbit α-RBBP5  at 1:2,000; rabbit α-TRERF1  at 1:2,000; rabbit α-UTX/KDM6A  at 1:2,000; α-WDR5  at 1:2,000.Rabbit α-DNTTIP1 , 5 μg per IP; rabbit IgG , 5 μg per IP; rabbit α-UTX/KDM6A , 5 μg per IP.Rabbit α-H3K4me1 , 10 μg per ChIP; mouse α-H3K4me2 , 10 μg per ChIP; rabbit α-H3K4me3 , 10 μg per ChIP; rabbit α-H3K27ac , 10 μg per ChIP; rabbit α-H3K27me3 , 30 μl per ChIP; rabbit α-H4K20ac , 10 μg per ChIP; rabbit α-DNTTIP1 , 10 μg per ChIP; rabbit α-UTX/KDM6A , 30 μl per ChIP; rabbit α-MLL4/KMT2D , 10 μg per ChIP.ELMSAN1 TRERF1 DKO clones from Flp-In T-REx HEK293 and MLL3+/+ MLL4+/+ clones and MLL3−/− MLL4−/− clones from HCT116 cells were generated using CRISPR/Cas9 technology. Briefly, 400,000 Flp-In T-REx HEK293 cells  were transiently transfected with precomplexed RNPs consisting of 100 pmol of each chemically modified sgRNA (Synthego), 70 pmol of Cas9 protein (St. Jude Protein Production Core), and 200 ng of pMaxGFP (Lonza) via nucleofection  using solution P3 and program CM130 in a small (20 μl) cuvette according to the manufacturer’s recommended protocol. 5 d post transfection, cells were single cell sorted by FACS to enrich for GFP-positive (transfected) cells, clonally selected and verified for the desired targeted modification via targeted deep sequencing. Targeted amplicons were generated using gene specific primers with partial Illumina adapter overhangs and sequenced as previously described  by a HPLC system (Thermo EASY-nLC 1000). Buffer A was 0.2% formic acid and Buffer B was 0.2% formic acid in 70% acetonitrile. The peptides were eluted by increasing Buffer B from 12% to 70% over a 60–90-min gradient. The peptides were ionized by electrospray ionization and detected by a Thermo LTQ Orbitrap Elite mass spectrometer. The mass spectrometer was operated in data-dependent mode. For each duty cycle, a high-resolution survey scan in the Orbitrap and 20 low-resolution MS/MS scans were acquired in the ion trap.P-value which was derived by the G-test  . The date G-test .2, 5 mM NaCl, 5 mM MgCl2, 250 mM sucrose, 1 mM DTT, and 10 mM sodium butyrate) supplemented with 0.1% NP-40 on ice for 5 min. Nuclei were pelleted by centrifugation  and washed twice in ice-cold hypotonic lysis buffer w/o NP-40. Nuclei were resuspended in 5X nuclei pellet volumes of ice-cold 0.2 M sulfuric acid and mixed on a rotation wheel for 120 min at 4°C. Insolubilized nuclear debris was pelleted by centrifugation . Supernatant was transferred to a fresh low-protein binding Eppendorf tube and histone proteins were precipitated by adding ice-cold trichloroacetic acid (TCA) to the final concentration of 20% (vol/vol) followed by a 60-min incubation on ice. Precipitated histone proteins were pelleted by centrifugation , washed three times with acetone (−20°C), and resuspended in MS-grade water.Histones were acid extracted as described previously . In brieExtracted histones were prepared for LC–MS/MS analysis using the hybrid chemical derivatization method as described previously . In brieThe resulting peptide mixtures were analyzed using nano-flow liquid chromatography tandem mass spectrometry (LC–MS/MS) on a Q-Exactive HF mass spectrometer coupled to an Ultimate 3000 nano-UPLC  in data-dependent acquisition mode. A ∼200 ng peptide aliquot was used per one sample per one injection. Peptides were loaded automatically on a trap column  before C18 reversed-phase chromatography on the analytical column . Peptides were separated at a flowrate of 0.250 μl per minute by a linear gradient from 1% buffer B  to 25% buffer B over 40 min followed by a linear gradient to 40% B in 20 min, then to 85% B in 5 min. After 5 min at 85% buffer B, the gradient was reduced to 1% buffer B over 2 min and then allowed to equilibrate for 8 min. Full mass range spectra were at 60,000 resolution (at m/z 400), and product ions spectra were collected in a “top 15” data-dependent scan cycle at 15,000 resolution.RAW MS data were analyzed using EpiProfile 2.0 software . The rephttps://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) (10.14806/ej.17.1.200), to remove low quality reads, remove potential adapters and quality trim reads’ 3′ ends. Quality score cutoff was set to Q20. Next, the remaining reads were mapped to the mouse reference genome (mm10) with bwa aln, followed by bwa samse (Dnttip1 KO mESCs) were identified as those with overlapping FDR50 peaks present in all replicates at a given genomic region. Otherwise, for ChIP-seq targets without replicates, only the high confidence peaks were considered. Finally, the reproducible peaks from the same immunoprecipitation target  were merged into the collection of reference peaks, here further referred to as all reproducible peaks.Following the procedure described before , raw seqgalore/)  and cutawa samse  (v0.7.12wa samse  (v1.2). wa samse  (v1.11) wa samse  (v2.24.0wa samse  (v4), anwa samse  was usedDnttip1 KO mESCs and H4K20ac, H3K4me1 and DNTTIP1 in Mll3/4 DKO mESCs. Those scaling factors were computed by first calculating the median of single base-pair resolution enrichment signal from the bigwig track files, corrected previously for sequencing depth (see above), which was accomplished with the pybigwig tool (available online at: https://github.com/deeptools/pyBigWig), over all reproducible peaks. Next, the median signal from each sample was multiplied by 1/1,000,000, and converted to integer, which value imitated the spike-in read counts for the purpose of the scaling factor calculation and is further referred to as pseudo-spike reads. Next, similar to the approach used by the authors of DESeq2 and edgeR  > 1, for increased binding, or log2(fold-change) < −1 for decreased binding. For some experiments without available biological replicates , the identification of differentially bound regions was based on the fold-change value, using a log2(fold-change) > 1 threshold to identify increased binding and log2(fold-change) < −1 for decreased binding.To perform statistical testing between experimental groups, the number of fragments for each reference peak was counted with intersect command from pybedtools  (v0.8.1)nd edgeR  the scalFollowing the approach used previously , genomichttps://www.bioinformatics.babraham.ac.uk/projects/trim_galore/), and then aligned to the mouse reference genome (mm10) using STAR , using the pre-ranked list of genes, where the per gene metric was an equivalent of the log2(fold-change) values, derived from differential gene expression analyses. GSEApy’s prerank function was used with standard parameters, except –min-size and –max-size flags, whose values were set to 5 and 5,000, respectively. The GSEA analysis was run with the collection of gene sets from the KEGG database (P-value was lower than 0.05.GSEA was conducted with GSEApy  (v0.10.4database , downloadatabase . The abodatabase  (Bayá etdatabase , we consDnttip1 KO mESCs were obtained from GSE131062. RNA-seq data from WT and Mll3/4 DKO mESCs were obtained from GSE98063. DNTTIP1 ChIP-seq data in WT and Dnttip1 KO mESCs were used from GSE131062. The accession number for the remaining ChIP-seq datasets reported in this article is GEO: GSE190323.RNA-seq data from WT and


This Special Issue highlights the advantages of using combined approaches to explore chromatin molecular complexes. The published findings provide insights into a range of molecular mechanisms which chromatin complexes employ in functionally interconnecting the world of nucleic acids and proteins. The main topic in this Special Issue is the activation of transcription. The authors investigated transcription activation at different levels—from straightforward nucleic acid structures to intricate cooperation pathways of high-order nucleoprotein complexes.Special DNA arrangements into G-quadruplexes affect the initial phases of DNA transcription. Recent structural investigations of the Ferré-D’Amaré laboratory revealed the structural basis of G-quadruplex unfolding via helicases. The structural data suggest that the helicase rearranges its structure and pulls on the single-stranded DNA tail A. The heThe Brazdova laboratory published immunofluorescence studies within this Special Issue, suggesting that G-quadruplexes are located in GC-rich euchromatin regions and outside the fibrillarin-positive compartments of nucleoli. When comparing several human and mouse cell lines, Komůrková et al. showed that G-quadruplexes occur most frequently in human embryonic stem cells which are more transcription-permissive than differentiated cells .Moreover, the high degree of colocalization between G-quadruplexes and transcription factories suggests that G-quadruplexes and associated transcription factors cooperate during transcription initiation. In another independent study using ChIP-seq analysis with a G4 antibody, Lago et al. proposed that G-quadruplexes cooperate with associated transcription factors to determine cell-specific transcriptional programs . Thus, GRecently, the Cramer laboratory reported cryo-EM structural studies combined with electromobility shift assays showing that histone H1 binding to nucleosome arrays requires sufficiently long DNA linkers to enable H1 binding to arrayed nucleosomes. If DNA linkers are short, H1 cannot bind and structurally stabilize nucleosomes. Thus, transcription is enabled through the structural arrangement of the histones B 6]..6].Another mechanism of transcription activation through histone phosphorylation has been suggested in this Special Issue. Saha et al. showed that the phosphorylated H1.4 histone variant remains associated with active promoters and may thus contribute to transcription activation . Based oDrosophila strains have revealed that BAP170 plays an essential role in enhancer-dependent transcriptional activation mediated by PBAP chromatin remodelers [Two studies within the Special Issue have been dedicated to chromatin remodeling factors and their roles in transcription C. In themodelers .The second publication regarding chromatin remodeling factors focused on the interaction between the SWI/SNF chromatin remodeling complex and transcription factor CTCF. Valletta et al.  used an The high occurrence of peptides from BRG1—the major ATPase subunit of the SWI/SNF complex during CTCF immunoprecipitation—has been detected. After the subsequent validation of selected subunits of the SWI/SNF complex via Western blot and ChIP-seq analyses, quantitative binding studies showed that the BRK domain of BRG1 directly interacts with CTCF at the region of zinc finger motifs 4–8 . The pubPrevious proteomic and bioinformatic studies revealed mutations of the SWI/SNF complex in over 20% of human cancers . The impWithin this Special Issue, Morrison and Thakur reviewed distinct chromatin factors and molecular complexes that constitute (i) euchromatin as an open chromatin structure associated with active transcription, (ii) heterochromatin as a less accessible chromatin associated with silencing and (iii) centromeric chromatin as the site of spindle binding in chromosome segregation . In the review section devoted to euchromatin, it was described that even though euchromatin forms only 8% of chromatin, euchromatin regulates the transcription of genes differentially and tightly via chromatin remodelers, histone modifications, histone variants and chaperones ,13.In the concluding remarks dedicated to heterochromatin, Morrison and Thakur asked the appealing question of what the recent research focus on the phase separation properties of heterochromatin adds to our knowledge of the molecular machinery of compaction, spatial arrangements and the function of heterochromatin.Finally, in the centromeric chromatin section, it was described how highly specific chromatin-associated proteins participate in performing chromosome segregation. Centromeric chromatin is presented as a chromatin containing one of the least-conserved histone variants.In summary, the articles published within this Special Issue contribute to our understanding of the fundamental mechanisms underlying the regulation of gene transcription and chromatin remodeling which could be employed in the localization, regulation and prevention of transcription, especially for diseases with peculiar transcription patterns.


Chromatin is spatially and temporally regulated through a series of orchestrated processes resulting in the formation of 3D chromatin structures such as topologically associating domains (TADs), loops and Polycomb Bodies. These structures are closely linked to transcriptional regulation, with loss of control of these processes a frequent feature of cancer and developmental syndromes. One such oncogenic disruption of the 3D genome is through recurrent dysregulation of Polycomb Group Complex (PcG) functions either through genetic mutations, amplification or deletion of genes that encode for PcG proteins. PcG complexes are evolutionarily conserved epigenetic complexes. They are key for early development and are essential transcriptional repressors. PcG complexes include PRC1, PRC2 and PR-DUB which are responsible for the control of the histone modifications H2AK119ub1 and H3K27me3. The spatial distribution of the complexes within the nuclear environment, and their associated modifications have profound effects on the regulation of gene transcription and the 3D genome. Nevertheless, how PcG complexes regulate 3D chromatin organization is still poorly understood. Here we glean insights into the role of PcG complexes in 3D genome regulation and compaction, how these processes go awry during tumorigenesis and the therapeutic implications that result from our insights into these mechanisms. Cell fate regulation is a critical developmental process, governed carefully by transcriptional control. This results in the ability of a given cell to activate and repress specific gene sets, culminating in a great diversity of cell types and transcriptional phenotypes. While it has long been appreciated that the 3D structure of chromatin is critical to the determination of cell fate and maintenance of cellular identity, technological advances of the last decade have facilitated a renaissance in the molecular dissection of genome architecture . MoreoveForemost among those protein complexes that control genome architecture and transcription are the Polycomb group complexes (PcG). These are a family of highly conserved histone modifying multi-subunit complexes that are essential for development . MoreoveThe ever-expanding repertoire of genomics techniques to study the regulation of genome architecture has allowed a deep molecular understanding of the modes of PcG-mediated compaction . In thisvia histone-modifying activities. PRC2 catalyses methylation at lysine 27 of histone H3 (H3K27me1/2/3) through its catalytic core of EZH1/2, SUZ12, EED, and RBBP4/7 (via the E3-ligases RING1A/B which heterodimerize with one of the six PcG RING finger domain (PCGF1–6) paralogs (The PcG machinery can be divided into three major complexes: Polycomb Repressive Complex 1 and 2 (PRC1 and PRC2) and Polycomb Repressive Deubiquitinase complex (PR-DUB). Traditionally, PcG complexes are associated with maintenance of gene repression, mainly  RBBP4/7 . PRC1 deparalogs . In mammparalogs . H2AK119paralogs . BAP1 foparalogs .These PcG complexes are responsible for two distinct forms of chromatin structural features ; either The distal form of ‘PcG mediated loops’, more commonly termed PcG bodies, involves mid/long-range loops from one PcG bound region to another. These can be observed at distances of up to 100 MB  Schoenf. This tyThere is huge diversity in the proteomic composition of the PcG complexes in mammals  with speWhile the catalytic products of these complexes are clearly correlated with transcriptional repression of nearby genes, the precise contribution of each histone modification in maintaining or initiating repression is still incompletely understood . CatalytThe PcG complex cores act as the scaffold upon which auxiliary subunits bind to confer their distinct functions, including proteins that are involved in recruitment such as CBXs for cPRC1, KDM2B or MAX/MGA for vPRC1, PCL1-3 or JARID2 for PRC2 and FOXK1/2 for PR-DUB  Farcas . ImportaWhile the precise function of these intergenic, diffuse histone modifications is yet to be fully understood, work to date suggests that they can contribute to the maintenance of proximal PcG compacted domains. Whether this is through a direct role of the modifications, or their established antagonisms with other epigenetic modifications such as H3K36me2, DNA methylation or RNA molecules remains to be fully elucidated.The biochemical diversity of PcG provides these complexes with tools to allosterically modulate the activities of their catalytic cores. These rheostats within the PcG complexes fine tune histone modifying activity to maintain an equilibrium of H2AK119ub1 and H3K27me3 at specific regions in the genome. PRC2 is a prime example of this, with a positive regulator of allosteric activity within each of its two subcomplexes, PALI1/2 within PRC2.1 and JARID2 within PRC2.2  Sanulli. Each ofin trans and prevents its spreading from nucleation sites at CpG islands  and the Polyhomeotic (PHC1-3) subunits  are recrfunction .The CBX subunits of cPRC1 are primarily involved in cPRC1 recruitment to chromatin. However, the CBX2 subunit has unique properties in proximal chromatin compaction that seemingly cause phase separation of PcG target loci  Plys et. In DrosPCGF2 and PCGF4 double knockout in ESC fails to induce major gene expression changes  forms containing PCGF1/3/5/6 . In fact changes . The samhenotype . Notwithhenotype . TogetheOgt exhibit failure to repress PcG targets akin to Ph mutants   . Biochemparalog) . Therefoparalog) . In mammparalog) . HoweverThe role of PRC2 in directly controlling compaction is less well defined than PRC1. Although, emerging biochemical data suggests two distinct modes through which PRC2 can contribute to chromatin compaction. These are through i) SUZ12-mediated dimerisation and ii) direct nucleosomal compaction by EZH1 . While iin vitro. This divergent role of EZH1 in compaction is strikingly similar to the divergent evolution of PRC1 subcomplexes with cPRC1 having weaker enzymatic potency than vPRC1 but instead maintaining functions in chromatin compaction  vPRC1 is recruited to chromatin through DNA binding at CpG islands or transcription factor binding sites . ii) TheEZH2 occur in several cancers including T-cell Acute Lymphoblastic Leukemia (T-ALL) and Acute Myeloid Leukemia (AML)  (Loss-of-function (LOF) mutations of ia (AML) . The SUZ (MPNST) . These P (MPNST)  and ther (MPNST) .BAP1 in mesothelioma, uveal melanoma, and renal clear cell carcinomas   . Intrigusubunits . TIP60 isubunits . These asubunits . This bisubunits , such asXD locus .SMARCA2 and SMARCA4 occurs in several cancer types such as small cell carcinoma of the ovary hyper-calcaemic Type (SCCOHT)  . This ne(SCCOHT) . The mecASXL1, DNMT3A, BCOR and BCORL1  . TherefoDuring tumorigenesis it has been shown that PRC1 complexes are recruited to both transcriptionally repressed and active genes, as well as at active enhancers. Interestingly, PRC1 is co-recruited with oncogenic transcription factors (TFs) to active enhancers in estrogen receptor positive (ER+), triple-negative breast cancer (TNBC), leukemia, hepatocellular carcinoma cell lines and Ewing sarcomas . This suWhile H3K27me3 and H2AK119ub1 are particularly enriched at repressed promoters (in proximity to sites of stable PcG binding and CpG islands) a large proportion of these modifications are distributed at low “blanket”-like levels throughout the intergenic genome . These dBAP1 null cells, broad accumulation of H2AK119ub1 causes reductions in H3K36me2   . These m41/A677) . These m41/A677) . Importa41/A677) . This co) (PRDM1 . Therefo) (PRDM1 . Patient) (PRDM1 .H3F3A and H3F3B) include lysine 36 to methionine (H3K36M) found in chondroblastomas, lysine 27 to methionine (H3K27M) found in pediatric gliomas, and glycine 34 to tryptophan/leucine (H3G34W/L) mutations found in giant cell tumors of the bone (EZH2 GOF (gain-of-function) mutations through removal or reduction in H3K36 methylation levels. These reductions in H3K36me2 cause widespread accumulation of PcG domains via diffuse spreading of H3K27me3 in turn promoting hyper-repression of distal regulatory elements  mutations and EZH2 GOF mutations  and EZHIP overexpression in ependymoma, while featuring increased levels of H3K27me3 at tumor suppressor genes such as CDKN2A, have broadly reduced levels of H3K27me3 and H3K27me2. This is caused by the inability to spread these modifications from PcG nucleation sites at promoters  translocation that places the NSD2 gene under the control of the IgH immunoglobulin promoter  inhibitors: Several compounds that reduce BMI1 protein levels have been described. High-throughput screenings designed to identify compounds that reduced BMI1 transcript levels identified PTC209 , Cohen-Gibson (EED), SOTOS (NSD1), Bohring-Opitz (ASXL1), Bainbridge-Roper (ASXL3) and Tatton-Brown-Raman (DNMT3A) syndromes all feature syndrome-defining mutations in chromatin regulators that control the PcG-H3K36me2 domain axis (A curious commonality between the genes that regulate PcG domains is that they are often recurrently mutated in neurodevelopmental syndromes. Weavers (ain axis . This feain axis .


In this review, we explored and summarized the current views about totipotency from various angles, including genetic and epigenetic aspects. This will hopefully formulate a panoramic view of totipotency from the available research works until now. It can also help delineate the scaffold and formulate new hypotheses on totipotency for future research works.Totipotency represents the highest developmental potency. By definition, totipotent stem cells are capable of giving rise to all embryonic and extraembryonic cell types. In mammalian embryos, totipotency occurs around the zygotic genome activation period, which is around the 2-cell stage in mouse embryo or the 4-to 8-cell stage in human embryo. Currently, with the development of  The very first demonstration of totipotent cells in the embryo was from the works of Tarkowski, in which a single blastomere isolated from a 2-cell (2C) stage embryo was proven to be able to generate a whole adult mouse . By defiThe transcription network regulating totipotency undergoes dynamic wiring from 1C to 4C stages and expands the zygotic genome activation (ZGA) period. Shortly after fertilization, maternal RNAs and proteins stored in the oocyte start to be degraded, a process known as maternal decay; at the same time, the zygotic genome begins to be transcribed, known as ZGA . ZGA usuin vitro totipotent-like stem cell culture systems have significantly enabled the dissection of the totipotency regulation. Currently, different in vitro totipotency models share similarities but also bear substantial differences compared to in vivo totipotency states. Under serum/leukemia inhibitory factor (LIF) culture conditions, around 1%–2% of mouse embryonic stem cells (mESCs) were found to exhibit transcription features such as the 2C embryos and were known as 2C-like embryonic stem cells (2CLCs)  were also discovered to exist in the heterogeneous human naïve embryonic stem cells, which transcriptionally resemble the 8-cell human embryos  were first established by Liu and Deng laboratories , resembling mouse totipotent 2C embryo stage cells at transcriptional, epigenetic, and metabolic levels  . 2CLCs p (2CLCs) . The som (2CLCs) . In this (2CLCs) . Since t embryos . In addiratories , which cratories . Transcrratories . Recentlratories . TBLCs sratories . Howeverratories , questioc levels . Excitinin vitro . In geneHere, we explored and summarized the current understanding on regulation of totipotent stem cells and totipotency in early embryos from various genetic and epigenetic perspectives, such as transcriptional regulation, epigenetic regulation, cell cycle, DNA damage, and RNA metabolism. From the current emerging evidence, the panoramic regulation of the totipotency state will surface more.Specific cell states are shaped by various pathways, and among all, gene expression plays essential roles. The emergence of totipotency is accompanied by the transcription network sustaining ZGA. Both cis- and trans-genomic elements and coding and non-coding genes are actively engaged in ZGA and the establishment of the totipotent state.in vitro and in vivo totipotency models.Transcription factors work by recognizing specific DNA sequences and recruiting RNA polymerase for specific gene transcription. Totipotent stem cells have their own unique transcriptional regulatory network. Currently, the understanding of the totipotent transcriptional regulatory network is just beginning. A few positive and negative transcription regulators have been identified from the Dux, a double homeodomain transcription factor, was the most significantly studied, which was initially found to reprogram pluripotent mESCs toward a 2C-like state  were transcriptionally activated. The chromatin landscape was also reorganized to resemble that of mouse 2C embryos   . On the ed Nelfa . In addi embryos . Recentl2C stage . Thus, mZscan4 family members are also highly expressed transcription factors in mouse and human totipotent cells, among which Zscan4d is the most highly transcribed in 2C embryos, and Zscan4c is a major transcript in mESCs (Zscan4c interacted with 2C-specific transposable element MT2 (LTR of MERVL) through the SCAN domain, which interacted with the GBAF chromatin remodeling complex to promote the enhancer activity of MT2  were more poised to transition to a 2C-like state  genome is critical for gene regulation and is highly dynamic during embryo development. The DNA in the nucleus is hierarchically organized into chromosome territories, compartments, TADs, and chromatin loops. In general, dramatic reprogramming of the 3D chromatin architecture occurs after fertilization. Also, substantial chromatin compaction occurs during the 1-cell to 2-cell transition, resulting in the formation of large chromatin blocks that accumulate near the nuclear envelope . A higheDot1l can induce 2C-like transcription state with collapsed chromocenters and reduced H3K79me3, representing the remodeled pericentromeric heterochromatin. As a result, MERVL loci were released from the heterochromatic environment and become activated  interactions and TAD insulation  Figure . While tctivated .Dux loci can dissociate from the nucleolar surface and get activated, promoting the transition to 2CLCs  and cohesin . CTCF caDux loci . The traDux loci . CTCF al exhibited a prolonged G1 phase, while cells with lower MERVL expression (2C-OUT) exhibited a prolonged G2/M phase  to stabilize Dux mRNA and induce 2C-like states , a metabolite of vitamin A, displays important roles during embryonic development. Several studies reported that RA drove a NELFA-mediated 2C-like state under S/LIF culture conditions  through Gm12794c . Not onlGm12794c . HoweverGm12794c . In the Gm12794c , Nanog epression , and phopression . Chemicain vitro .in vitro totipotent stem cell culture system has been a long-term goal of the stem cell and early developmental biology field. Currently, various in vitro culture systems have been identified or established, including 2CLCs (The establishment of a stable ng 2CLCs , EPSCs (ng 2CLCs , TBLCs (ng 2CLCs , TLSCs (ng 2CLCs , ciTotiSng 2CLCs , and TPSng 2CLCs . Cells fIn vitro totipotent state is a dynamic state, and the cells induced to reprogram the 2C-like state were found to spontaneously roll back to the original pluripotent state (Dppa2 and Dppa4 formed a negative feedback loop involving Dux and Dux repressors LINE-1 (Dux upstream of the TSS unit and downregulated Dux expression in the presence of double-stranded DNA breaks (Duxbl1 expression and 2CL state were induced by the treatment of RA (Duxbl1 can directly downregulate Zscan4 and endogenous Duxbl1 (in vitro totipotency obtained so far. Moreover, early totipotent embryos and totipotent-like cells exhibit low metabolism and DNA damage activation. These properties argue against a self-sustainable totipotency state which can be stably maintained in the long term.nt state . It is ts LINE-1 . OtherwiA breaks . In addint of RA ; howevers Duxbl1 . These sDux-induced 2CLC system, Dux seems to play a very important role during the 2C exit process. Apart from RNA metabolism, biological processes negatively regulating the 2C state may be involved in the exit from totipotency and embryonic development after 2C. RNA splicing, RNA decay (Prdm14a, a pluripotency transcription factor, acted as a barrier in the transition of mESCs to 2CLCs (Myc can prevent the transition to the 2C-like state by actively maintaining the pluripotent transcriptome (Otx2, a member of the OTX family highly expressed in mouse blastocysts and ESCs, negatively regulated Dux, Zscan4c, MERVL, and other ZGA genes in mESCs (TRIM66/DAX1 in mESCs served as a negative regulator of the 2C-like state through repressing Dux transcription (Using transcriptome profiling of the NA decay , and rRNNA decay  were allNA decay . Prdm14ato 2CLCs . Myc cancriptome . Otx2, ain mESCs . Highly cription . These fin vitro 2C-like state also mimics the developmental process from 2C to morula and blastocyst stages. Understanding whether 2C embryos also exhibit similar 2C exit mechanisms as in vitro 2C-like cells and whether the totipotency state is intrinsically associated with a dynamically wired and unstable transcription state has great implications for us to understand the transcriptional properties in early embryo development.Therefore, the exit from the in vitro greatly helped us chart the map of embryonic development. Given the knowledge about totipotency obtained so far, large gaps still exist in fully understanding the regulatory and functional properties of the totipotency state. Totipotency represents the highest developmental potential in early embryogenesis and thus potentially can contribute toward the widest applications of stem cells such as reproduction, development, and tissue/organ regeneration. It is evident that further understanding of totipotency regulation and optimizing chemically defined culture conditions will further increase the efficiency of totipotency reprogramming and create more functional in vitro totipotency models for basic research and regenerative medicine (The establishment of a totipotent-like system medicine .


The complexity of genetic circuits has not seen a significant increase over the last decades, even with the rapid development of synthetic biology tools. One of the bottlenecks is the limited number of orthogonal transcription factor–operator pairs. Researchers have tried to use aptamer–ligand pairs as genetic parts to regulate transcription. However, most aptamers selected using traditional methods cannot be directly applied in gene circuits for transcriptional regulation. To that end, we report a new method called CIVT-SELEX to select DNA aptamers that can not only bind to macromolecule ligands but also undergo significant conformational changes, thus affecting transcription. The single-stranded DNA library with affinity to our example ligand human thrombin protein is first selected and enriched. Then, these ssDNAs are inserted into a genetic circuit and tested in the in vitro transcription screening to obtain the ones with significant inhibitory effects on downstream gene transcription when thrombins are present. These aptamer–thrombin pairs can inhibit the transcription of downstream genes, demonstrating the feasibility and robustness of their use as genetic parts in both linear DNAs and plasmids. We believe that this method can be applied to select aptamers of any target ligands and vastly expand the genetic part library for transcriptional regulation. The development of synthetic biology has made it possible to design dynamic and complex functions in living organisms, so that we are now entering a more advanced stage of “engineering life” . The reaThrombin aptamers are the most commonly used model to demonstrate the application of aptamers ,12. As bIn this work, we attempted to use complementary double-stranded aptamers as gene regulatory elements to regulate transcription. As a proof of concept, we inserted the widely used human thrombin aptamer sequence into different locations near the promoter to explore the feasibility of using aptamers as gene regulatory elements. However, this promoting effect is weak and the specificity is poor, and results showed that the aptamers obtained in vitro selection were not suitable to be directly used as gene elements due to their weakened ability to bind to their ligands in the double-stranded form. Then, we developed CIVT-SELEX (Capture and In Vitro Transcription-SELEX) to select thrombin aptamers suitable to be the gene elements for transcriptional regulation. The preliminary selection of Capture-SELEX ensured that the enriched aptamer sequences had a strong binding affinity with the target ligand while reducing the diversity of the library. This selective library was then cloned and re-screened in the process of TX-TL transcription to obtain three aptamer candidates with a significant inhibitory effect on downstream gene transcription in the presence of thrombin. These three aptamer candidates, in the form of both linear and plasmids, were able to specifically respond to 0.5 μM of human thrombin protein in the system during transcription, inhibiting the transcriptional output signal of downstream genes, preliminarily demonstrating the feasibility and robustness of their use as gene regulatory elements. We believe that CITV-SELEX can be applied to select other aptamer–ligand pairs, which can then be used as gene regulatory elements for constructing more complex biological circuits.Our goal was to use aptamer–ligand pairs as gene regulatory elements to regulate transcription in plasmids. We first tried to insert the well-studied thrombin aptamer 29mer into different locations near the promoter to explore the feasibility of using aptamers as gene regulatory elements  13]. Th. Th13]. In order to obtain aptamers that responded to low concentrations of ligands at the transcriptional level, we used CIVT-SELEX (Capture and In Vitro Transcription-SELEX) to select thrombin aptamers . We firsThe oligonucleotide library for Capture-SELEX contained two restriction sites, 40 nt and 10 nt random nucleotides, and a docking sequence (DS) that was complementary to the capture oligo (CO) sequence A. The 3′E. coli competent cells. Cell cultures were plated on multiple plates and grown overnight. Then, we picked every single colony on plates and amplified the region containing promoter–aptamer–reporter as linear DNAs (pJ23151-aptamer-3WJdB). A total of 832 linear DNAs were successfully amplified and purified. To reduce the workload, every two pieces of DNA were co-purified, and together these formed a test group for subsequent experiments . Each test group was transcribed directly in the cell-free system with or without human thrombin protein, and then fluorescence changes were compared. After preliminary experiments, test groups with significant fluorescence changes were selected, and this time individual linear DNAs were transcribed again in the cell-free system, and we compared the fluorescence change with or without adding 0.5 μM human thrombin . Aptamers were also truncated to 40mer and 51mer for the CD experiment to investigate the core binding site sequences. The CD spectra D showed Aptamers have various molecular applications due to their advantages of simple preparation, low cost, small size, and good versatility, and in recent years, studies on aptamers as genetic regulators to control gene transcription and translation have increased. Meanwhile, the cell-free transcription–translation system (TX-TL) has great potential as a general platform for the rapid screening of aptamers and prototype circuit design. By combining these two, the aptamer-based regulation can be tested and optimized in a cell-free system before application to the complex in vivo environment. In this work, we used the CIVT-SELEX to select aptamers that could not only specifically bind to human thrombins at lower concentrations but also repress the transcription of the downstream gene when thrombin was present. Although there were studies using thrombin aptamers as gene elements to successfully regulate downstream genes, the plasmid DNAs used in these studies needed to be artificially synthesized, which limited their applications in vivo. Therefore, it was necessary to develop a method that allowed DNA containing aptamer sequences to self-replicate in organisms while being able to regulate downstream genes. Our CIVT-SELEX method is beneficial to both small molecule target ligands that are difficult to anchor and macromolecules that are not toxic to cell-free systems. In parallel research, our group is using CIVT-SELEX to select aptamers that can bind to UDP and insulin and achieve transcriptional level regulation. The results from these studies should further demonstrate the universality of this method and its potential of creating more ligand–aptamer pairs for transcriptional regulation.In our CIVT-SELEX, we added a target ligand to screen for aptamers during transcription, and when the transcriptional output signal changed after the addition of the ligand, this change was believed to be caused by the recognition response of ligands and aptamers; it was inferred that ligands can bind to aptamer sequences during transcription and affect transcription. As our method was based on Capture-SELEX, which has been shown to be able to select aptamers that can bind to ligands and undergo conformational changes upon binding, our candidate aptamers were expected to be able to bind to the ligand. In our in vitro transcription screening, only a few of these candidate aptamers exhibited transcription inhibition. Our control experiments also suggested that this inhibition upon binding with ligands was aptamer-specific.At the same time, considering the termination mechanism of Rho-independent terminators, a strong secondary toehold structure is able to stop transcription. Therefore, we believe if the aptamer-ligand complex is as strong as a terminator, it should be able to regulate transcription. A possible molecular mechanism of this transcriptional regulation is detailed as follows. During bacterial transcription, RNA polymerase binds to promoter DNA, melts ~14 bp around the transcription start site, and forms a single-stranded “transcription bubble” within a catalytically active RNAP-DNA open complex. With the insertion of the aptamer and the presence of the ligand, the single-stranded aptamer region is exposed due to the transcription bubble and therefore has the opportunity to bind to the ligand, causing a conformational change to the aptamer region, thereby blocking the movement of RNAP and the transcription of downstream genes . For theThe protocol used in this work was based on the one described by Stoltenburg et al.  with somThe ssDNA library was immobilized onto modified magnetic beads via hybridization between the two complementary docking sequences. We used 200 μL of streptavidin magnetic beads to bind approximately 320 pmol of CO per round of screening. These were annealed in an equimolar ratio in a washing buffer  system (1 mg magnetic beads can bind >400 pmol of biotinylated Capture Oligo DNA). Annealing program: denaturation at 95 °C for 10 min, −0.1 ℃ per second, × 350 times until 60 °C for 5 min, incubation at 4 °C. 200 μL of magnetic beads were adsorbed on a magnetic stand to remove the supernatant, and the magnetic beads were repeatedly washed three times with 200 μL of WB. The annealed ssDNA Library-CO complexes were incubated with magnetic beads overnight on a rotator at 35 rpm/min, after the magnetic bead mixture was repeatedly washed 4 times with 200 μL WB.2, 1 M DTT) containing a certain concentration of human thrombin target ligand to the washed ssDNA Library–CO–magnetic bead complex. We mixed and placed it on a rotator at 35 r/min, incubated it at 4 °C for 60 min, then placed it on a magnetic stand, collected the supernatant, and used the above-mentioned equal volume of Binding Buffer containing the same concentration of ligand for elution to retain the supernatant. We repeated the process twice and numbered the respective elutions 1, 2, and 3. The collected supernatant contained oligonucleotides with a strong affinity for human thrombin. These oligonucleotides bound the human thrombin by folding into a specific three-dimensional structure, inducing the release of ssDNA from the DNA-bead complexes. Finally, we washed the magnetic beads with 100 μL SSC Buffer , and incubated them at 95 °C for 10 min. We repeated this 3 times and numbered the respective batches HOT 1, 2, and 3.Human thrombin was used as a target for the selection of aptamers during the Capture-SELEX process. A stock solution of human thrombin at 50 μM was prepared and aliquoted before storage at −20 °C. We added 100 μL of Binding Buffer  with 1/10 and absolute ethanol with 2.5 times of the mixed liquid volume. All were mixed and placed in a −20 °C refrigerator for 60 min, and then centrifuged at 12,000 rpm in a refrigerated centrifuge at 4 °C for 30 min to precipitate DNA, and the centrifuge tube was opened and placed at room temperature until the ethanol was completely evaporated. Then, the precipitated ssDNA was amplified by ExTaq enzyme, the forward primer (5′-CGCAACCAGGTCTCTAAGC-3′), and the reverse primer (5′-CACAGGCCGGTCTCACATT-3′) with a phosphorylation label at the 3′ end. PCR amplification conditions were the following: 30 sec at 95 °C, 8 cycles of 10 sec at 95 °C, 30 sec at 58 °C, 5 sec at 72 °C and elongation step of 5 min at 72 °C.We mixed batches 1, 2, and 3 of the elution obtained by target competitive binding in each round of screening into a 1.5 mL centrifuge tube, and added 3 M CHThe product of PCR amplification was ethanol-precipitated again, and Lambda Exonuclease was used to digest dsDNA in a water bath at 37 °C for 30 min to obtain the ssDNA library for next round.During the positive screening process, the target ligand concentration in the system was continuously reduced from 1~0.25 μM, and the next steps were the same as described above.The ssDNA library enriched after 8 rounds of positive screening was used as the initial library for negative screening. The difference in the screening process was that the ssDNA Library–CO–magnetic bead complex was placed in the cell-free system before the addition of the target ligand for incubation. It was incubated at room temperature for 60 min to remove non-specific sequences bound to the cell-free system, then the system was replaced with Binding Buffer and the magnetic beads were washed. The elution rate of each round was calculated by drawing a standard curve of absolute quantification by qPCR. All the next steps were the same as described above, and the negative screening ligand concentration was 0.25 μM.E. coli DH5α, and single colonies were picked for colony PCR. One thousand single colonies were picked on ten successfully transformed plates for colony PCR. Based on agarose gel electrophoresis results, there were 832 linear DNAs for transcription that successfully connected and contained sequences with promoter, dsDNA aptamer, output signal 3WJdB, and terminator. The 832 successfully amplified linear DNAs were recovered in groups of two and numbered as AN-N. Their concentration was determined for subsequent mixed detection, and then 50 nM linear DNA were directly transcribed in the cell-free system with or without 0.5 μM human thrombin and their fluorescence changes were compared.The ssDNA library after Capture-SELEX was amplified into dsDNA with correct restriction sites and recognition sites, and then the dsDNA was directly inserted into the vector fragment containing the backbone, promoter J23151, 3WJdB, and terminator by Golden Gate Assembly, then transformed in The components with significant fluorescence changes were again tested for the individual transcription process, and the real-time fluorescence changes were compared. We selected the mixed aptamer candidates with obvious fluorescence changes after the preliminary mixed detection, and found the corresponding two single colony bacteria liquids of each group according to the number to carry out colony PCR and product recovery again. We obtained the linear DNA for transcription corresponding to the two aptamer sequences in the candidate and renumber ASN-N, and transcribed all single linear DNA for transcription in the cell-free system according to the above method.The ssDNA library screened by Capture-SELEX was amplified by forward and reverse primers to obtain a dsDNA aptamer library. Parts of the vector backbone, promoter (pT7/pJ23151/pTet/pLac), RBS(BCD2), and terminator (T500) were amplified from plasmids by PCR or synthesized directly by the company. The dsDNA aptamer library was ligated with vector backbone, T7 promoter, 3WJdB, and terminator (the mole ratio of parts and target sequences were 1:1) to construct a circular vector by Golden Gate Assembly, and linear DNAs were amplified from the single colony by colony PCR. The TXTL reaction mix was performed at 37 °C for 4 h for transcription and at 29 °C for 9 h for transcription and translation. The cell-free system was prepared according to previous work [Sample preparation: 5 μM solution of each group ssDNA samples were prepared in Binding Buffer and annealed at 95 °C for 10 min, and placed on ice immediately for 5 min to maintain the loose structure. Human thrombin protein was added to a final concentration of 0.5 μM and cooled down to room temperature to allow the structure to refold, then spun and mixed for 1 h to allow the target ligand to bind to the ssDNA aptamer. Additionally, 0.5 μM human thrombin was prepared in Binding Buffer. CD spectra were measured by Jasco J-1500 CD spectrometer using a fused quartz cuvette with 1 mm of light path length. The data were collected at 25 °C in the range of 220–320 nm with 1 nm bandwidth and data pith. Spectra were collected with 3 accumulations and at a 100 nm/min scanning speed. Baseline correction was achieved with Binding Buffer, and buffer signals were subtracted. The data were plotted using GraphPad Prism. To smooth the curves, spectra were shown used the LOWESS function in GraphPad Prism default settings.The binding energetics of AS2-3 to thrombin was obtained with the Nano ITC from Waters company at 25 °C. An amount of 1500 μL of 1 μM aptamer candidates AS2-3 was mixed in 1× Binding Buffer at 95 °C for 10 min, then immediately placed on ice to cool. We transferred 100 μL 1× Binding Buffer containing 10 μM human thrombin into the syringe. Each injection titration was performed in a volume of 2 μL with 25 consecutive injections at 300 s intervals with stirring at 300 rpm.In summary, we developed a method, CIVT-SELEX, which combined Capture-SELEX with in vitro transcription screening to select aptamers for human thrombin proteins. The single-stranded DNA library from Capture-SELEX was amplified and screened in the in vitro transcription screening to obtain aptamers with significant inhibitory effects on downstream gene transcription when thrombins were present. These aptamer–thrombin pairs can inhibit the transcription of downstream genes, demonstrating the feasibility and robustness of their use as biological parts in both linear DNAs and plasmids. We believe that CIVT-SELEX can be applied to select aptamers of other target ligands, which can be used as biological parts for regulation in gene circuits.


LINC00607 to be highly enriched in human endothelial cells. LINC00607 was induced in response to hypoxia, arteriosclerosis regression in non-human primates, post-atherosclerotic cultured endothelial cells from patients and also in response to propranolol used to induce regression of human arteriovenous malformations. siRNA knockdown or CRISPR/Cas9 knockout of LINC00607 attenuated VEGF-A-induced angiogenic sprouting. LINC00607 knockout in endothelial cells also integrated less into newly formed vascular networks in an in vivo assay in SCID mice. Overexpression of LINC00607 in CRISPR knockout cells restored normal endothelial function. RNA- and ATAC-Seq after LINC00607 knockout revealed changes in the transcription of endothelial gene sets linked to the endothelial phenotype and in chromatin accessibility around ERG-binding sites. Mechanistically, LINC00607 interacted with the SWI/SNF chromatin remodeling protein BRG1. CRISPR/Cas9-mediated knockout of BRG1 in HUVEC followed by CUT&RUN revealed that BRG1 is required to secure a stable chromatin state, mainly on ERG-binding sites. In conclusion, LINC00607 is an endothelial-enriched lncRNA that maintains ERG target gene transcription by interacting with the chromatin remodeler BRG1 to ultimately mediate angiogenesis.Long non-coding RNAs (lncRNAs) can act as regulatory RNAs which, by altering the expression of target genes, impact on the cellular phenotype and cardiovascular disease development. Endothelial lncRNAs and their vascular functions are largely undefined. Deep RNA-Seq and FANTOM5 CAGE analysis revealed the lncRNA The online version contains supplementary material available at 10.1007/s00395-023-00978-3. Endothelial cells form the selectively permeable monolayer between the vessel and the blood. Resting endothelium provides an anti-coagulant and anti-inflammatory surface and contributes to the control of local vascular tone. It also facilitates the vascular response to inflammation, shear stress and hypoxia , 67. In Recent studies suggest that long non-coding RNAs (lncRNAs) are essential in the regulation of cardiovascular gene programs , 57, 65.EVF2 directly inhibits the ATPase and chromatin remodeling activity . After centrifugation  , w(v1.0.0) , a collev1.36.0)  package (v3.3.5) . Motif ev4.11.1)  by provihttps://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE199878The RNA-Seq and ATAC-Seq datasets have been deposited and are available at NCBI GEO with the accession number GSE199878: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE201824BRG1 CUT&RUN datasets have been deposited and are available at NCBI GEO with the accession number GSE201824: t test, of unpaired samples by unpaired t-test and if not normally distributed by Mann–Whitney test. P values of < 0.05 was considered as significant. Unless otherwise indicated, n indicates the number of individual experiments.Unless otherwise indicated, data are given as means ± standard deviation (SD). Calculations were performed with Prism 8.0 or BiAS.10.12. The latter was also used to test for normal distribution (Shapiro–Wilk) and similarity of variance. In case of multiple testing, Bonferroni correction was applied. For multiple group comparisons ANOVA followed by post hoc testing was performed. Individual statistics of dependent samples were performed by unpaired LINC00607 is one of the most endothelial-enriched lncRNAs .A screen for the top-expressed endothelial lncRNAs in the FANTOM5 CAGE -ENCODE database revealed that NAs Fig. A. ParticECs Fig. B. Additieus Fig. A, which eus Fig. C. RT-qPCLINC00607 expression was altered in various cardiovascular diseases. Endothelial cells isolated and cultured from plaque-containing arteries, regarded as post-atherosclerotic, showed significant upregulation of LINC00607 in atherosclerotic-derived ECs compared to plaque-free-derived ECs  was strongly induced in Macaca fascicularis samples undergoing atherosclerosis regression after a high fat diet  upstream, revealed binding motifs for multiple transcription factors. In particular, ARNT  and HIF1A (Hypoxia Inducible Factor 1 Alpha) were identified multiple times and in close proximity to the transcription start site, indicative of transcriptional regulation by hypoxia (FLINC00607 expression was significantly increased when HUVEC were cultured under hypoxic conditions (1% oxygen) , a process in which SMADs play a central role [LINC00607 . Successful knockout was confirmed on the levels of both the DNA  was performed . Comparison of ATAC-Seq and RNA-Seq for the multiple VEGF-signaling genes revealed a similar effect of LINC00607 knockout on chromatin accessibility of gene-linked enhancers  and genEpiRegio . DNA-motout Fig. B. Intereout Fig. C. ERG anion Fig. D. Even tged Fig. E. These LINC00607 as an interaction partner of BRG1  [LINC00607 knockout overlapping with BRG1 CUT&RUN binding sites were shared with genes ERG binds close to   from HUV to Fig. E.Fig. 5LVWF (von Willebrand factor), SGK1 (Serum/Glucocorticoid Regulated Kinase 1), TSPAN12 (Tetraspanin 12) and KDR (Kinase Insert Domain Receptor)  Fig. H. SGK1, out Fig. A. Of theway Fig. E, which ite Fig. H which iLINC00607 to be specifically expressed in EC and to be important for vascular sprouting and ERG-dependent gene expression through BRG1. Although already constitutively highly expressed in EC, LINC00607 itself was upregulated by hypoxia and EndMT. Through RNA- and ATAC-Seq we identified LINC00607 as a lncRNA important for central pathways of endothelial cells, in particular for VEGF signaling. After LentiCRISPR-mediated knockout of LINC00607, endothelial cells exhibited an impaired response to VEGF-A in respect to vascular sprouting and a reduced ability to integrate into the vascular network of SCID mice. Mechanistically, the trans-acting lncRNA interacts with the chromatin remodeling protein BRG1 in order to maintain chromatin states for ERG-dependent transcription. Thereby, LINC00607 preserves endothelial gene expression patterns, which are essential for angiogenesis. As exemplified by lncRNA SAIL (scaffold attachment factor B interacting lncRNA), which has been reported to impair the transcription of fibrosis-related genes in human cardiac fibroblasts [In the present study, we identified roblasts , such cacis at nearby genes, or in trans genome wide [LINC00607 restored endothelial function after LINC00607 knockout, demonstrating that LINC00607 acts in trans rather than in cis, because the effect of locus-disruption by CRISPR/Cas9 gene editing was overruled by the plasmid-based overexpression of LINC00607.In terms of transcriptional control, lncRNAs can either act in ome wide . OverexpLINC00607 interacts with the chromatin remodeling protein BRG1. Several lncRNAs have been linked to BRG1, influencing its activity in the cardiovascular system and other tissues. For example, EVF2 has been shown to inhibit the ATPase activity of BRG1 [MALAT1 promoting BRG1 interaction with HDAC9 [SMANTIS guides BRG1 to specific genes related to endothelial lineage specification [LINC00607, BRG1 and ERG overlap arguing that the LINC00607 could potentially facilitate BRG1 binding to genes linked to the endothelial phenotype. Recent studies highlight the importance of constant SWI/SNF remodeling to maintain a stable open chromatin state [LINC00607 provides a specific link for ERG securing BRG1 binding to genes maintaining the endothelial phenotype. This specific context would explain why LINC00607 is so highly expressed in endothelial cells.Importantly,  of BRG1 . Additioth HDAC9 . lncRNAsfication . Our prein state , 62. OurLINC00607 knockout, and genes with binding sites for the ERG transcription factor. ERG belongs to the ETS transcription factor family, which act as key regulators of the majority of endothelial genes, as the ETS recognition motif can be found in promotors of many endothelial genes [LINC00607 for the expression control of ERG-regulated genes through its interaction with BRG1. In this context, it is interesting to note that SWI/SNF is required to maintain open chromatin [LINC00607 as one link between BRG1-mediated stabilization of chromatin states and ERG target gene expression in healthy endothelium.Indeed, we uncovered a large overlap between genes with altered chromatin state and differential expression after al genes . This shhromatin , 62. BRGLINC00607 was increased by hypoxia, EndMT, and endothelial dysfunction as induced by TNFα and high glucose [LINC00607 matches the transcription factor binding motifs identified in the promotor analysis. Hypoxia signalling through VEGF is an important trigger for angiogenic specification of endothelial cells [LINC00607 under hypoxic conditions, the pro-angiogenic endothelial phenotype could potentially be secured by tightening the interaction of LINC00607 with BRG1. Specifically to HIF1-controlled LINC00607 expression, we found that DMOG-dependent upregulation and acriflavine-mediated HIF inhibition altered the expression of the lncRNA. This in particular illustrates the close interaction of hypoxia-signaling, angiogenesis and LINC00607.The endothelial expression of  glucose . Under tal cells  as well al cells . ThroughLINC00607 points towards a role for LINC00607 in expression control beyond the endothelial phenotype. Potentially LINC00607 guides BRG1 to genes involved in EndMT. In line with this, LINC00607 is also expressed in certain malignant cells. In this context, LINC00607 was upregulated in doxorubicin-resistant thyroid cancer cells [LINC00607 was described to be required for tumor proliferation of osteosarcoma cells [LINC00607 guides BRG1 to ERG target genes and during endothelial dysfunction to pro-angiogenic genes to maintain an open and accessible chromatin state for ERG. Since genome-wide association studies identified many risk loci globally in non-coding regions, transcriptome-wide association studies could be important in future to identify gene-trait associations [LINC00607 as a candidate gene in other disease-relevant tissues.The fact that EndMT induction by TGF-β2 and IL-1β also increased the expression of er cells . Furtherma cells  and was ma cells . Linkingciations  and therLINC00607 is involved in securing endothelial BRG1 and ERG-dependent target gene expression, as well as appropriate responses to stress and cardiovascular diseases. The function of LINC00607 in cardiovascular diseases with hypoxia signaling needs more investigation as LINC00607 might be a potential therapeutic target or clinical marker.Taken together, these findings suggest that Supplementary file1 (PDF 9341 KB)Supplementary file2 (DOCX 18 KB)Below is the link to the electronic supplementary material.


Epigenetics are the heritable changes in gene expression patterns which occur without altering DNA sequence. These changes are reversible and do not change the sequence of the DNA but can alter the way in which the DNA sequences are read. Epigenetic modifications are induced by DNA methylation, histone modification, and RNA-mediated mechanisms which alter the gene expression, primarily at the transcriptional level. Such alterations do control genome activity through transcriptional silencing of transposable elements thereby contributing toward genome stability. Plants being sessile in nature are highly susceptible to the extremes of changing environmental conditions. This increases the likelihood of epigenetic modifications within the composite network of genes that affect the developmental changes of a plant species. Genetic and epigenetic reprogramming enhances the growth and development, imparts phenotypic plasticity, and also ensures flowering under stress conditions without changing the genotype for several generations. Epigenetic modifications hold an immense significance during the development of male and female gametophytes, fertilization, embryogenesis, fruit formation, and seed germination. In this review, we focus on the mechanism of epigenetic modifications and their dynamic role in maintaining the genomic integrity during plant development and reproduction. Epigenetics is the study of mitotically and/or meiotically heritable changes in gene function that cannot be explained by changes in the DNA sequence . EpigenePlants being sessile in nature are invariably affected by changing environmental conditions. However, they have the ability to adapt their biological processes according to the changing environments. They interact with their environment through consistent adjustments at the molecular level by modifying the patterns of gene expression . EpigeneDifferent biological pathways such as phytohormone signaling, photoperiodism, and vernalization in combination with the environmental signals (epigenetic changes) regulate flowering time by integrating internal state of development of plants . This nePhenotypic plasticity within the plant species plays an important role in adaptation to different environmental conditions allowing different cultivars to adjust and grow. Plasticity maintains the homeostasis within changing environmental conditions that allows better gene expression to adapt to different biotic or abiotic constrains  . Genetic3 group (methyl group) at the fifth carbon position on cytosine residue generating 5-methyl cytosine in a sequence-specific manner. The methyl group acts as a platform for various protein complexes to attach and modify the chromatin scaffolds causing altered gene expression   . METHYLTT, or C) . DNA metT, or C) . DependiT, or C)   . There aT, or C) . In the T, or C) . CytosinT, or C) .de novo via DRM2 at all sequence contexts, and its role in CHH methylation is more prominent because CHH methylation depends only on de novo methylation. The RdDM pathway regulates the DRM2 activity. The RdDM pathway contains two plant-specific DNA-dependent RNA polymerases. The large subunit of these polymerases consists of proteins, i.e., NRPD1 and NRPE1, and functions specifically in transcriptional gene silencing and de novo DNA methylation. Single-stranded RNAs are produced through DNA-dependent RNA polymerase IV (Pol IV) in DNA repeat sites and in transposon regions. Pol IV transcription is facilitated by chromatin remodeling protein CLASSYs  . Single-stranded RNA is converted to double-stranded RNAs via RNA-dependent RNA polymerase (RDR2). Double-stranded RNA is then cleaved by Dicer-like protein (DCL3) into 24-nt siRNA. This 24-nt siRNA binds to ARGONAUTE proteins . Loading of siRNA to ARGONAUTE proteins require KOW CONTAINING TRANSCRIPTION FACTOR 1 (KTF1)/SPT5-like protein. KTF1 (RNA-binding protein) binds to noncoding RNA transcripts produced from Pol V forming RNA-directed DNA methylation effector complex. Pol V generates single-stranded RNA transcripts in intergenic noncoding (IGN) regions. RNA transcripts generation via Pol V requires DRD1, DMS3, RDM1 and RDM4. RDM1 binds to single-stranded methylated DNA and leads to the recruitment of Pol V to chromatin regions. To catalyze new DNA methylation, de novo DNA methyltransferases DRM2 are directed to specific chromatin regions via effector complex (Arabidopsis and named as FACTOR of DNA METHYLATION 1–5 (FDM1-5) and INVOLVED IN DE NOVO (IDN2/RDM12). They belong to the SGS3-like plant-specific protein family, and their rice homolog is X1. They have an important role in transcriptional gene silencing like the SGS3 protein family ], extra sex combs (Esc), suppressor of zeste 12 [Su(z)12], and the histone-binding nucleosome-remodeling factor 55 kDa . One component of PRC2, i.e., E(z), specifically belongs to the SET  domain family and is responsible for histone H3 tri-methylation at Lys27 , viz., CURLY LEAF (CLF), SWINGER (SWN), and MEDEA (MEA); three homologs of Su(z), viz., EMBRYONIC FLOWER 2 (EMF2), VERNALIZATION 2 (VRN2), and FERTILIZATION-INDEPENDENT SEED 2 (FIS2); and one homolog of Esc, viz., COPY SUPPRESSOR OF IRA 1–5 (MSI1-5). FIS2 regulates mega-gametogenesis and endosperm development in plants during postfertilization events and EMF, and VRN polycomb repressive complexes regulate the development of sporophyte and phase transition, i.e., vegetative to reproductive in plants , JUMONJI 30 (JMJ30), JUMONJI 32 (JMJ32), EARLY FLOWERING 6 (ELF6), and RELATIVE OF EARLY FLOWERING 6 (REF6), demethylate H3K27 and depress genes temporally or spatially for processes like flowering, signaling of hormones, and circadian clock control (The overexpression of antisense of the histone deacetylase gene (AtHD1) induces histone acetylation activity in venility . In planilencing . H3K27menogaster . In planat Lys27 . In casen plants . Histone control .Arabidopsis respond dynamically to dehydration stress  symmetric dimethylation (H4R3sme2) and causes FLC expression and a number of stress-responsive genes to be downregulated. As a result, its mutant characteristics include salt hypersensitivity, late flowering, and stunted development (Arabidopsis (HDA19). AGAMOUS (AG) and SEPALATA3 (SEP3), the C class and E class genes, respectively, are negatively regulated by the transcription repressor complex. Deacetylation of H4K16 in regulatory areas of AG and SEP3 mediates gene suppression , interacts directly with HDA6. The complex represses the expression of three flowering repressors: FLD, MADS AFFECTING FLOWERING 4 (MAF4), and MAF5 by removing the acetyl and methyl groups from histone 3 at their loci , and the lncRNAs gets transformed to double-stranded RNAs (dsRNAs) through RDR2 , and to control siRNA-dependent heterochromatin, there is a requirement of functional AtHDA6 , and it transposes into siRNA-defective mutants  in their chromosomes, and multicellular eukaryotes have TEs as a key component of their genome. The majority of transposable elements are silenced epigenetically, although certain transposable elements have active transcription in epigenetic regulation mutants. Furthermore, environmental stress can trigger TE transcription, a mechanism that occurs across the evolutionary spectrum from bacteria to mammals . McClint mutants . LTR of  mutants . As a re mutants . All theson Tam3 . Transposon Tam3 . It has son Tam3 . The actson Tam3 .Arabidopsis thaliana, one egg cell and one central cell containing two nuclei are produced during the process of female gametogenesis, and several accessory cells are also produced. The egg cell fuses with one sperm cell during double fertilization process forming an embryo, and the second sperm cell fuses with the central cell leading to the formation of an endosperm (triploid). A helix hairpin DNA glycosylase, DEMETER (DME) causes hypomethylation in the endosperm by removing methylated cytosine residues. Demethylation via DME activates expression of transposons through the RNAi pathway that introduces transcripts of transposons and produces additional siRNAs guiding DNA methylation. The siRNAs expression in the endosperm development indicates genome imprinting, and siRNAs production guides DNA methylation in egg cell reinforcing transposons silencing in the germ cells. Transposons silencing occurs in the germ cells, but mild activity of transposons in endosperm have no major effects as the endosperm is not inherited to next generation  was also found to show late flowering characteristics  gene, DCL1 gene, HUA-ENHANCER 1 (HEN1) gene, and HASTY gene   Figure . The miRnduction . miR156 nduction . The levnduction . The expnduction   is a peptide ligand that inhibits stomatal development, resulting in an excess of stomatal lineage cells. Malfunction of ROS1 results in promoter hypermethylation and suppression of the gene encoding EPF2, resulting in stomatal cell development , a chromatin-remodeling plant homeodomain (PHD) finger protein that increases acetylation levels, is induced by cold exposure. This protein is essential for FLC repression and flowering enhancement. Because FLC expression is not lowered by cold treatment, the mutant lines for VIN3 do not respond to vernalization and so remain in a vegetative state for longer durations  is characterized by anomalies in the development of flowers, leading to alteration in the auxin/cytokinin ratio  overexpression retards floral initiation through upregulation of expression of FLC  , a cold-inducible factor that is highly expressed in flowers (Rosa hybrida Samantha). ET is involved in the regulation of petal cell expansion during the opening of a rose flower  . HDA6 upthaliana  . Transcriptional gene silencing (TGS) is another permanent defense mechanism against DNA viruses via RdDM . ALKBH9B, an Arabidopsis protein having demethylase activity, removes m6A from ssRNA molecules and accumulates in the cytoplasm of the siRNA bodies. This process suggests the role of m6A demethylase in mRNA silencing   . Differeigestion . After iigestion .Hyaloperonospora arabidopsidis is similarly increased in DNA hypomethylation mutants like nrpe1 and diminished in DNA hypermethylation mutants like ros1  mutant does not have altered resistance to Pst DC3000 or fungal infections, unlike POL V mutants, indicating that POL V can regulate plant immune responses independently of canonical RdDM  encodes a transcription factor HD-ZIP IV which is required for the biogenesis of small RNAs and the other 21-nt phasiRNA . This trdent way .Arabidopsis genome, and their role in regulating chromatin, DNA, and crossover frequency has been studied  in the anthers and the megaspore mother cells (MMCs) in the ovaries are generated in the floral meristem as a consequence of two meiotic divisions followed by a series of mitotic divisions, resulting in haploid male and female gametophytes, respectively . The malArabidopsis that the methylation level of PMCs in a normal environment (CG and CHG) was greater than in an adverse situation (CHH)  . SymmetrThe transition from the mitotic to meiotic phase is accompanied by a decrease in restrictive chromatin (H3K27me1 and H3K27me3) and an increase in permissive chromatin (H3K4me3) . The vegDuring mitotic and meiotic cell division, the centromeric histone H3 (CENH3) variation is critical for the assembly and function of kinetochores. The inclusion of CENH3 into centromeric nucleosomes is the first step in kinetochore formation. The amount of CENH3 deposited on the centromeres changes depending on the stage of the cell cycle. CENH3 is also required for vegetative cell division and the removal of additional DNA . HypometArabidopsis  are more methylated than maternal genomes . Endospeing them .Solanum lycopersicum is mediated by DML2. The expression of DNA demethylase DME-LIKE 2 (DML2) increases rapidly in ripening fruits. In S. lycopersicum, DML2 targets both ripening-induced and ripening-repressed genes, implying that active DNA demethylation is necessary for both ripening-induced gene activation and ripening-repressed gene suppression  mutant of Prunus persica, contributed toward increasing molecular work at the level of genes, thereby renewing the field of dormancy. Deletion in tandemly repeated sequences of MADS-box genes, i.e., DORMANCY-ASSOCIATED MADS-box gene (DAM1-6) leads to nondormant phenotype of evg mutant. DAM genes are expressed in buds and get affected by photoperiod and chilling temperatures, thereby affecting the developmental stages of plants (Rubus idaeus, Prunus mume, Pyrus pyrifolia, and Actinidia deliciosa, DAM-like genes have been found to have dormancy-dependent expressions. In transgenic plants, the heterologous expression of these genes has regulatory roles in flowering and dormancy. In Arabidopsis, the expression of DAM1 and of SVP1 and SVP3 in Actinidia deliciosa led to delay in the time of flowering (In perennial plants, epigenetic modifications regulate seasonal dormancy cycles. Meristem and bud growth are controlled by photoperiod, temperature, etc. Transcriptomic studies have revealed that bud dormancy events in plants such as o stress . Charactf plants . The conf plants . In manylowering  (Table 3via thorough study at the molecular level will be helpful. FLC controls flowering time in Arabidopsis that involves many genes in FLC expression via chromatin modifications. Further studies have to be undertaken to know how these diverse epigenetic modifications interact with one another to regulate the expression of FLC. CRISPR-based systems can be useful in altering the expression of genes for research applications as well as crop improvement efforts. The generation of genetically viable agricultural varieties that can endure a warmer world relies heavily on information generated from the plants' epigenomic profiles subjected to environmental challenges. Understanding the dynamic management of histone methylation and how histone methylation controls plant growth would be expanded through biochemical and genetic studies. Identification of intrinsic histone demethylases in plants, particularly lysine and/or arginine demethylases, and elucidation of their functions in regulating plant development and genome stability would require biochemical and genetic evidence. The roles of regulation of H3K27me3 in plants are limited and more in-depth knowledge will enable new researchers to enhance productivity of crops under limiting environmental conditions.Plants being sessile in nature are invariably affected by changing environmental conditions. However, they have the ability to adapt their biological processes according to the changing environments. They interact with their environment through consistent adjustments at the molecular levels. Epigenetic mechanisms contribute to these adjustments. These changes within the plant species modify the gene expression and help different plant species to withstand the extremes of different biotic or abiotic constrains. Such changes are induced due to DNA methylation, histone modification, e.g. acetylation/deacetylation, methylation/demethylation, ubiquitination, phosphorylation, and sRNA/modifications, which work in tandem with respect to environmental signals, transposon silencing, and hormone signaling to control the expression of genes in plants. These alterations help to maintain the survival of plants and maximize the chances of sexual reproduction under stress conditions. The role of DNA methylation in plant improvement is the preferred mechanism to investigate gene function and manipulate plants for creating novel varieties having capability to survive under stress conditions. Therefore, improved knowledge of epigenetic mechanisms