LXRs are critical regulators of hepatic metabolism and function, but their mechanisms of action at the genome level are incompletely understood. We performed integrated analysis of genome-wide chromatin accessibility, gene expression, and transcription factor binding. We reveal distinct mechanisms of LXR transcriptional regulation of both metabolic and nonmetabolic genes in liver. We show that LXR can both activate and repress genes and that LXR binding impacts the activity of other transcription factors. The nuclear receptors liver X receptor (LXR) α and β play crucial roles in hepatic metabolism. Many genes induced in response to pharmacologic LXR agonism have been defined; however, the transcriptional consequences of loss of LXR binding to its genomic targets are less well characterized. Here, we addressed how deletion of both LXRα and LXRβ from mouse liver (LXR double knockout [DKO]) affects the transcriptional regulatory landscape by integrating changes in LXR binding, chromatin accessibility, and gene expression. Many genes involved in fatty acid metabolism showed reduced expression and chromatin accessibility at their intergenic and intronic regions in LXRDKO livers. Genes that were up-regulated with LXR deletion had increased chromatin accessibility at their promoter regions and were enriched for functions not linked to lipid metabolism. Loss of LXR binding in liver reduced the activity of a broad set of hepatic transcription factors, inferred through changes in motif accessibility. By contrast, accessibility at promoter nuclear factor Y (NF-Y) motifs was increased in the absence of LXR. Unexpectedly, we also defined a small set of LXR targets for direct ligand-dependent repression. These genes have LXR-binding sites but showed increased expression in LXRDKO liver and reduced expression in response to the LXR agonist. In summary, the binding of LXRs to the hepatic genome has broad effects on the transcriptional landscape that extend beyond its canonical function as an activator of lipid metabolic genes. Nr1h3 and Nr1h2) play important roles in hepatic lipid metabolism. LXRs are crucial for the lipogenic response to feeding as regulators of Srebf1, Fasn, and Scd1 (1Lpcat3 expression (Liver X receptor (LXR) α and LXRβ . The canonical LXR-binding site (LXRE) is a repeated nuclear receptor half-site motif (AGGTCA) separated by four nucleotides (DR4) . LXR livThe dynamics of the transcriptional landscape in response to LXR binding to the genome are largely unknown. Tools such as assay for transposase accessible chromatin (ATAC-Seq) can provide a bridge between transcription factor (TF) binding and gene expression by revealing dynamic changes in chromatin accessibility . In thisP value of <0.05 . Many classical LXR targets were down-regulated, including Srebf1 and Fasn  . In addition, lipid metabolism genes associated with PPARα were down-regulated in LXRDKO livers. Macrophage and Kupffer cell marker genes, such as Cd5l, Cd163, and Clec4f were also among the most down-regulated genes. Reduced expression of these genes likely reflects a change in the immune cell profile in LXRDKO liver, as LXR is known to be important for Kupffer cell identity . These genes were enriched for pathways including cysteine and methionine metabolism and DNA repair/p53 response .We performed RNA-Seq on livers of whole-body LXRα/β LXRDKO mice to profile transcriptional changes provoked by the absence of these transcription factors. We identified 246 up-regulated and 321 down-regulated genes using an adjusted B and C) . Down-reidentity , 14, 15.SI Appendix, Fig. S3 A and B. We ranked our ATAC-Seq peaks based on the absolute change in accessibility between LXRDKO and wild-type (WT) livers. After filtering out peaks with very weak signals, 73,597 peaks remained, of which 57.60% had passed an irreproducible discovery rate of 1e-6 . These findings broadly suggest reductions in potential enhancer activity and increases in direct promoter activity on a range of genes in LXRDKO livers.Top peaks that lost accessibility were enriched in intergenic and intronic regions B. In comSI Appendix, Fig. S3D). Genes down-regulated in the absence of LXR were more likely on average to lose accessibility in their intergenic and intronic peaks, compared to those whose expression increased or did not change . On the other hand, promoter peaks in genes up-regulated in LXRDKO liver were more likely to gain accessibility, compared to those whose expression decreased or did not change. These results agree with the enrichment of intergenic and intronic regions in the top peaks losing accessibility and the enrichment of promoters for top peaks gaining accessibility. Pathway enrichment analysis revealed that the set of genes proximal to top peaks losing accessibility in LXRDKO liver were enriched in lipid metabolism pathways, in agreement with the types of genes down-regulated . Genes proximal to top peaks gaining accessibility were enriched for pathways other than lipid metabolism . These observations support a degree of correlation between chromatin accessibility and gene expression in LXRDKO liver.Average accessibility across the gene was decreased in the genes down-regulated in the absence of LXR in comparison to up-regulated ones . Among genes with LXR-binding sites, genes that were differentially expressed between WT and LXRDKO mice tended to have a higher number of LXR-binding sites compared to genes whose expression did not change in LXRDKO liver . This suggests that only a small subset of LXR-binding sites in liver is functionally required for hepatic gene expression.To further examine changes in accessibility occurring at LXR-binding sites, we integrated our RNA-Seq and ATAC-Seq results with LXR ChIP-Seq data from livers of mice treated with vehicle  or the LXR agonist T0901317 . Our anaSI Appendix, Fig. S5C). A majority of the ATAC peaks at LXR-binding sites located at intergenic and intronic regions in LXRDKO liver showed a decrease in accessibility, but that trend was not observed for peaks located at promoter regions . Thus, the degree to which LXR-binding sites changed in accessibility in LXRDKO liver was influenced by their locations in relation to individual genes.The overall accessibility across LXR-binding sites was reduced in LXRDKO livers; 71.57% of LXR-binding sites lost some accessibility (Srebf1 locus (Srebf1 contain multiple LXR-binding sites (Srebf1c (Left). All of the LXR-binding sites at this gene lost some accessibility in the LXRDKO samples compared to controls, accompanying the down-regulation of the gene.Integrating the expression, binding, and chromatin accessibility data, LXR-binding sites associated with down-regulated genes were less accessible in LXRDKO liver than those associated with genes whose expression did not change or were up-regulated C. For inf1 locus . The regng sites D, includMethods for details) for all of the bins. We then displayed the enrichment of each of the transcription factor motifs across all bins in a heatmap .Changes in chromatin accessibility at transcription factor–binding sites may reflect a change in transcription factor activity. To analyze these trends across the genome, we ranked all of our ATAC-Seq peaks based on changes in accessibility between LXRDKO and WT samples and binned them into equal-sized bins . We performed motif enrichment analysis for known binding motifs . This result implies that loss of CTCF binding may lead to the closing of these peaks. To ensure that the motifs we identified were independently changing, we examined the peaks among the top bins that lost accessibility in LXRDKO liver with these motifs. Each family motif was present on a unique set of peaks with some overlap with other transcription factor families . This suggests that there is specificity to the reduction of motif accessibility for each of these transcription factor families, and that the reduction of accessibility of one transcription family was not completely dependent on another transcription factor family. With the exception of a modest decrease in PPARα and modest increase in Foxa2 expression in the LXR DKO samples, the expression of most of these transcription factors themselves was not different between groups, suggesting that the changes in their motif accessibility were not likely to be due to differences in transcription factor abundance .We further examined the transcription factor motifs associated with the top 1,000 ATAC peaks that lost accessibility in LXRDKO liver B. A numbSI Appendix, Fig. S7 A and B). Although this method was not as sensitive, it nevertheless provided independent validation of some of the observations shown in We further assessed changes in accessibility of some of the motifs via footprinting . This apSI Appendix, Fig. S6 C, Right). This suggests that a number of transcription factors were collectively losing accessibility in LXR target genes. As an example, the Insig2 locus has a number of LXR-binding sites that became less accessible in LXRDKO liver. Based on available liver ChIP-Seq datasets, each of the peaks associated with LXR binding was also predicted to bind to combinations of other transcription factors, including CTCF, PPAR, RXR, HNF4A, FOXA2, HNF6, and HNF1 , and included SREBP2 targets such as Hmgcr, Hmgcs1, Sqle, and Fdps. For instance, the Hmgcr promoter was on average more accessible in LXRDKO liver . As an example, Got1 was up-regulated and its promoter (with four NF-Y motifs) was on average more accessible in LXRDKO liver  E. MoreovKO liver F. The peKO liver F.Many studies on LXRs have focused on their functions as ligand-activated transcription factors, using pharmacological tools such as the potent synthetic agonists GW3965 and T0901317 , 39, 40.Srebf1, Scd1, Acaca, Fasn, and Lpcat3. ATAC peaks at these LXR-binding sites were enriched for DR1 and DR4 nuclear receptor motifs. Additionally, LXR-binding sites for these pharmacological ligand-activated genes were more likely to be located in promoter regions in comparison to pharmacological ligand-unresponsive genes down-regulated in LXRDKO liver .We next focused on genes that were regulated both by synthetic agonist and the presence or absence of LXRα/β. A majority of these genes was regulated in opposite directions by agonist treatment and LXR deletion A. We ideAcox1, Acsl5, and Fabp1 . The LXR-binding sites for these genes were enriched for the nuclear receptor DR1 motif . The DR1 motif was enriched across all intergenic and intronic LXR-binding sites regardless of their change in accessibility in LXRDKO livers. Thus, the DR4/LXRE motif is strongly associated with classical LXR targets that are up-regulated by agonist treatment and down-regulated in the LXRα/β knockout and with LXR-binding sites that lose accessibility with the loss of LXR. The data further suggest that, outside of these canonical LXR targets, LXR can bind to nuclear receptor motifs more broadly, including at DR1 motifs.The LXRE/DR4 motif was enriched among the set of intergenic and intronic ATAC peaks with putative LXR-binding sites that showed the largest loss of accessibility in LXRDKO liver . These LXR-repressed genes were involved in various cellular functions not focused on lipid metabolism. Interestingly, ATAC peaks associated with the LXR-binding sites in these repressed genes were enriched for the FOXA and C/EBP motifs. Overall accessibility of these LXR-binding sites were decreased in LXRDKO liver . As an example, Slc25a15 and Lurap1l had increased expression in LXRDKO liver and decreased expression with LXR agonist treatment. Their loci have LXR-binding sites that were on average reduced in accessibility in LXRDKO livers and have putative binding sites for FOXA2, C/EBP, and other nuclear receptors such as HNF4a and PPARα detected by ChIP-Seq  by LXR agonist (GSE149075)  .P value <0.1). Independent confirmation of the down-regulation of these predicted targets supports the conclusion that LXRs are capable of acting as ligand-dependent repressors.To further validate the ability of LXR agonists to repress gene expression through direct LXR binding, we treated WT mice with GW3965 and measured gene expression by qPCR C. Of theIn this study, we assessed the implications of loss of LXR expression in mouse liver for gene expression, chromatin accessibility, and transcription factor activity. Unlike fork head factors, LXRs are not known to be pioneer factors that play key roles in establishing regions of open chromatin. Accordingly, the changes in chromatin accessibility we observed with loss of these nuclear receptors, especially on LXR-binding sites, were rarely dramatic; i.e., complete closing of an existing peak or opening of a new peak. A majority of the genes differentially expressed between WT and LXRDKO liver had LXR-binding sites, suggesting the change in their expression was likely to be a direct consequence of loss of LXR binding. At the same time, a majority of the genes differentially expressed between WT and LXRDKO liver did not change in WT mice treated with synthetic LXR agonist. This finding suggests that many LXR-binding sites do not transduce ligand-dependent signals or are active with basal levels of endogenous ligands. Such LXR-binding sites appear necessary to maintain expression of their target genes but do not respond to pharmacological activation, perhaps due to specific coactivator requirements. A similar disconnect between basal nuclear receptor activity and synthetic ligand response has been previously observed in macrophages . It woulCd5l and Clec4f showed reduced expression in LXRDKO liver. LXRs have also been reported to affect the capillarization of sinusoidal endothelial cells and the ability of stellate cells to contribute to fibrosis in response to injury  were generally not differentially expressed between WT and LXRDKO liver. Although LXR binding is not required for the basal expression of these genes, prior studies have shown that LXRs mediate induction of these genes in liver in response to synthetic LXR ligand or cholesterol diet challenge  were backcrossed more than 10 generation to the C57/Bl6 background. Animals were housed in a 25 °C temperature-controlled room under a 12-h light/12-h dark cycle under pathogen-free conditions. Mice had ad libitum access to water and standard chow . Mice were killed at 8 wk of age. All animal experiments were approved by the Institutional Animal Care and Research Advisory Committee of the University of California, Los Angeles.RNA from frozen tissue was extracted through TRIzol (Invitrogen) and a Qiagen RNeasy Mini Kit. Total RNA libraries were made with a KAPA Stranded kit with mRNA capture. Libraries were sequenced as single end (50 bp) on an Illumina HiSeq3000.P value <0.05. Genes were annotated using biomaRt package in R (https://www.R-project.org/)  through Enrichr  . Plots aweb tool , 60. Gen Enrichr –63.g for 10 min and the pellet was resuspended with 1 mL cold resuspension buffer (RSB) . Approximately 50,000 nuclei from these samples were removed and centrifuged at 4 °C at 500 × g for 5 min. Supernatant was removed and the transposase reaction was performed immediately as described  within the same TF family were combined. Footprinting was done with Hmm-based identification of transcription factor footprints (HINT)-ATAC using the bam files and the JASPAR motif database as input , LXR vehicle ChIP-Seq data (GSM864670), and LXR T09 peaks (GSM864669). Differentially expressed genes in response to LXR agonist treatment were obtained from GSE149075. Hepatic SREBP-2 peaks were obtained from GSE28082. The ChIP-Atlas was used to provide a summarized ChIP-Seq experiment from mouse liver or hepatocytes or liver-derived cell lines .SI Appendix, Table S1.Nine-week-old mice on mixed background 129 × 1/SvJ and C57BL/6 were gavaged with 40 mg/kg GW3965  first 17Supplementary File


Advances achieved with molecular biology and genomics technologies have permitted investigators to discover epigenetic mechanisms, such as DNA methylation and histone posttranslational modifications, which are critical for gene expression in almost all tissues and in brain health and disease. These advances have influenced much interest in understanding the dysregulation of epigenetic mechanisms in neurodegenerative disorders. Although these disorders diverge in their fundamental causes and pathophysiology, several involve the dysregulation of histone methylation-mediated gene expression. Interestingly, epigenetic remodeling via histone methylation in specific brain regions has been suggested to play a critical function in the neurobiology of psychiatric disorders, including that related to neurodegenerative diseases. Prominently, epigenetic dysregulation currently brings considerable interest as an essential player in neurodegenerative disorders, such as Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), Amyotrophic lateral sclerosis (ALS) and drugs of abuse, including alcohol abuse disorder, where it may facilitate connections between genetic and environmental risk factors or directly influence disease-specific pathological factors. We have discussed the current state of histone methylation, therapeutic strategies, and future perspectives for these disorders. While not somatically heritable, the enzymes responsible for histone methylation regulation, such as histone methyltransferases and demethylases in neurons, are dynamic and reversible. They have become promising potential therapeutic targets to treat or prevent several neurodegenerative disorders. These findings, along with clinical data, may provide links between molecular-level changes and behavioral differences and provide novel avenues through which the epigenome may be targeted early on in people at risk for neurodegenerative disorders. Neurodegenerative (ND) disorders are among the leading bases of disability and death worldwide ,2,3. TheIn the past decade and a half, ND has been the topic of an immense number of investigations. Much progress has been made, but for patients with ND disorder, these ailments remain overwhelming and deadly, emphasizing an urgent need to develop more effective treatments. Recent progress suggests that epigenetic modifications are highly relevant targets, and thus, they represent a potential site for pharmaceutical intervention. Aberrant epigenetic modifications related to ND disorders are starting to emerge and emphasize the great need to assess recent advancements in epigenetic mechanisms in ND disease research. We and others have recently discussed other mechanisms involved in severe ND disorders, including Alzheimer’s disease (AD), Huntington’s disease (HD), Parkinson’s disease (PD), and Amyotrophic lateral sclerosis (ALS) ,8,9. TheThe term “epigenetic” denotes modification of gene expression without directly altering the DNA sequence . These cHistones are a highly conserved set of nuclear proteins that assemble in an octamer composed of pairs of H2A, H2B, H3, and H4. Nuclear DNA is highly condensed and wrapped around these nuclear proteins, nucleosomes, in the form of chromatin to accommodate chromosomes in the nuclei . The hisHistone methylation usually occurs at the arginine or lysine N-terminal region, which leads to the activation or suppression of gene expression. Each arginine residue in histones can be subjected to monomethylation and symmetric/asymmetric dimethylation. Likewise, each lysine residue can be mono-, di- and tri-methylated, and also nearby residues can form different methylation combinations ,21,22,232+ dioxygenase Jumonji-C (JmjC) domain (KDM2 family proteins) was identified as a specific H3K36me1/2 demethylase (KDM2A and B), which does not demethylate H3K36me3 [The amine oxidase containing lysine-specific demethylase 1 (LSD1/KDM1A) and lysine-specific demethylase 2 (LSD2/KDM1B)  remove mH3K36me3 . The KDMH3K36me3 . The KDMH3K36me3 ,58,59. IH3K36me3 ,61. KDM5H3K36me3 ,64,65,66H3K36me3 ,67. KDM6H3K36me3 ,69,70,71H3K36me3 ,77,78,79H3K36me3 ,79,80. KH3K36me3 ,82,83. RH3K36me3  and may H3K36me3  and/or eAD is the most established neurodegenerative disorder in the world. Emerging findings indicate that epigenetic dysregulation of gene expression may play a significant role in aging and ND ,87,88,89HD is a late-onset, autosomal progressive neurodegenerative disorder caused by the trinucleotide CAG repeat in the coding for glutamine (Q) in exon 1 of the Huntingtin (Htt) gene , leadingEnhanced H3K9me2 has been found in murine models of HD ,111,112,Htt WT and targeted inactivation of both copies of Htt (dKO) genotypes suggested that the loss of Htt triggered a significant reduction in the total number of H3K27me3-marked promoters [Besides, studies have also shown a functional interaction of the Htt gene with protein arginine methyltransferase 5 (PRMT5), an enzyme mediating the dimethylation of arginine (R) of essential cellular proteins, including histones and spliceosomal proteins . Htt hasromoters . These fromoters , such asromoters ,124.The additional function of PRC2 in HD was supported by H3K27me3 ChIP-sequencing in neuronal chromatin obtained from the HD postmortem PFC and non-neurologic controls. The findings suggest the loss of neuronal PRC2-H3K27me3 sites and the upregulation of some PRC2 target genes associated mainly with Hox gene clusters and developmentally related proteins in the HD-affected human brain ,127,128.BDNF gene locus [Htt (Q150) knock-in mice [Notably, the diminished enrichment of H3K4me3, a mark of active gene transcription, has been associated with impaired transcription of target genes  in both human HD postmortem brains as well as the cortex and striatum of R6/2 mice . Reducedne locus . These one locus ,133. Fur-in mice . Notably-in mice ), and th-in mice . A simil-in mice ,135 was -in mice , furtherPD is the second most predominant neurodegenerative disorder in the world after AD . The mosSNCA promoter region in postmortem brain samples from patients with PD and matched controls [SNCA promoter reduced α-synuclein levels in neuronal cell lines and PD-derived induced pluripotent stem cell lines (iPSCs) [SNCA promoter in the neuronal nuclei (NeuN) of positive neurons of substantia nigra (SN) tissue samples from PD patients. Interestingly, significant reductions in H3K4me3 and H3K27me3 marks were observed in SH–SY5Y cells treated with the neurotoxin 6-hydroxydopamine (6-OHDA) [SNCA gene, directly regulates the accessibility of transcription factors that can access gene regulatory regions. Finally, there are still undiscovered histone methylation marks that may regulate synaptic and pathological abnormalities found in PD. Those may also have a considerable impact on regulating chromatin structure and overall effects on gene transcription in PD and should be of future interest.A mounting number of studies overwhelmingly suggest the critical function of the histone modification and PD-associated α-synuclein coding gene SNCA expression , meaningcontrols . Similar (iPSCs) . Signifi(6-OHDA) . In the (6-OHDA) ,148, sig(6-OHDA) . In cont(6-OHDA) , indicatALS is a fatal ND distinguished by the selective loss of both upper and lower motor neurons . There ir 43 kD (TDP-43)-based pathology [C9orf72 exposed the likelihood that epigenetic changes might also involve ALS pathology. The studies of epigenetic changes in ALS are still in infancy and much remains to be studied. Because other epigenetic modifications in ALS have been recently reviewed [Few epigenetic changes were reported related to ALS before discovering noncoding GGGGCC hexanucleotide repeat (HRE) in the gene C9ORF72 that is strongly associated with ALS disease ,157, whiathology , the obsreviewed ,159, we C9orf72 mRNA in the frontal cortices and cerebella of c9FTD/ALS patients is associated with increased binding of C9orf72 to H3K9me3, H3K27me3, and H4K20me3 marks [C9orf72 binding to trimethylated histones , but also enhanced C9orf72 gene expression [R521C mouse model of ALS, overexpression of PRMT1 rescues neurite growth associated with oxidative stress [R521C mouse model of ALS [The reduced e3 marks , associapression . In yeaspression , whereaspression ,163. In e stress  through l of ALS . Consistl of ALS . TDP-43 l of ALS . It is, l of ALS . There wl of ALS . Similarl of ALS , suggestCollectively, the findings discussed above  suggest Because alcohol is a neurotoxic agent, its use has been associated with neurodegeneration ,173,174 GIPC1, BCL2L1, and UBE1) expressions were observed in the PFC and HP of postmortem alcoholic brain tissues [Investigation on histone methylation in AUD is in infancy, and studies are beginning to explore the function of each different histone methylation regulated gene expression. As discussed earlier, histone methylation could regulate gene repression  or activation . Postmortem brains of alcoholic individuals  exhibite tissues . H3K27me tissues . Consist tissues . These lMag, Mbp, Mobp, and Plp) and H3K36me levels was found in AIE adolescent PFC [Arc gene, enabling increased negative elongation factor (NELF) binding to the Arc promoter was observed in AIE adult rats. These events inhibited Arc expression in the amygdala. Inhibition of Kdm6b or Arc eRNA expression in the CeA significantly increased H3K27me3 occupancy at the Arc SARE site and recapitulated the molecular and chromatin phenotypes of AIE [Smyd3 (di- and trimethylates H3K4), Setdb1 (trimethylates H3K9) and Setd6 (mono-methylates H2AZK8) gene expression. This paradigm also increased Setd7 (mono-methylates H3K4), Setd3 (methylates H3K4 and H3K36), Ash1l (methylates H3K36) [Similar observations were also observed in animal studies decreased H3K27me3 at the Mt1 gene promoter and increased global H3K4me3 levels at the Mt2 gene promoter in the cerebral cortex were observed when mice were exposed to acute alcohol for 6 h . Increascent PFC . A recencent PFC . In the cent PFC . Consists of AIE . Alcohols H3K36) . These fAlcohol exposure during early development has been shown to induce neuronal loss at different stages of brain development, leading to neuronal dysfunction at the adult stage. In addition to histone acetylation and DNA methylation changes, histone methylation changes have been shown in many developmental alcohol studies. GD 7 acute ethanol exposure increased H3K9me2, decreased H3K27me3 and altered G9a, Setdb1, Kdm1a, Kdm4c, Uhrf1 and Ezh2 mRNA levels . ReducedIn summary, with the available findings, it is clear that suppressive chromatin mediated by various histone marks regulated by a dynamic balance between specific HMT and HMD significantly contributes to the neurodegeneration and progression of disease pathology . Given t


Transposable elements (TEs) are self-replicating DNA elements that constitute major fractions of eukaryote genomes. Their ability to transpose can modify the genome structure with potentially deleterious effects. To repress TE activity, host cells have developed numerous strategies, including epigenetic pathways, such as DNA methylation or histone modifications. Although TE neo-insertions are mostly deleterious or neutral, they can become advantageous for the host under specific circumstances. The phenomenon leading to the appropriation of TE-derived sequences by the host is known as TE exaptation or co-option. TE exaptation can be of different natures, through the production of coding or non-coding DNA sequences with ultimately an adaptive benefit for the host. In this review, we first give new insights into the silencing pathways controlling TE activity. We then discuss a model to explain how, under specific environmental conditions, TEs are unleashed, leading to a TE burst and neo-insertions, with potential benefits for the host. Finally, we review our current knowledge of coding and non-coding TE exaptation by providing several examples in various organisms and describing a method to identify TE co-option events. In eukaryotic cells, the chromatin fiber is precisely packaged and organized in subnuclear compartments that undergo dynamic spatial rearrangements depending on developmental transitions and environmental constraints ,2,3,4. AArabidopsis thaliana up to 85% in maize  operating as an integrated system in the control of gene action” ,11, mostin maize ,13,14; ir genome . Altogetr genome .TEs are classified in two major classes depending on their mode of transposition. Class I TEs (or retrotransposons) mobilize via an RNA intermediate in a “copy-and-paste” strategy, while class II TEs (or DNA transposons) mobilize via a DNA intermediate in a “cut-and-paste” strategy . Each clDepending on the insertion site, neo-insertions of TEs can differently impact the host genome. They can first be neutral, for instance, if they happen in non-regulatory intergenic regions. They can also be deleterious, by disrupting the sequence of protein-coding genes, or provoking chromosomal rearrangement ,23,24,25In this review, we will first describe the recent progress made in the field of TE silencing, with a focus on plants. In the second part, we will attempt to explain how harmful, parasitic TEs can turn into beneficial genetic elements that contribute to evolutionary innovation of the host genome. Finally, we will describe different approaches that exist to study host-TE interaction, and to bring to light genuine events of TE exaptation.DNA methylation, histone modifications and histone variants and non-coding RNA (ncRNA), like small interfering RNA (siRNA), chromatin remodelers and other epigenetic factors, play important roles in many chromatin-related processes, including genome organization, DNA replication and repair, regulation of gene expression and TE silencing ,28,29. ANeurospora crassa, plants and most animals . F. FNeurosnd SUVH6 A.A. thaliana also showed that several classes of TEs are reactivated in epigenetic mutants showing DNA hypomethylation [DNMT mutants [The role of 5mC in TE silencing was first demonstrated in maize . More rehylation ,44,45,46 mutants ,48,49.Although de novo and maintenance of 5mC pathways co-exist throughout the plant life cycle, each pathway plays an important role at specific developmental stages or in response to a specific environmental stimulus that can lead to a burst of TEs. Young TEs, capable of transposition, are rapidly targeted by the RNA-DEPENDENT RNA POLYMERASE 6 (RDR6)-dependent RdDM, or non-canonical RdDM ,51. The Remarkably, a similar scenario was recently described in tomato, in which young TEs undergoing retrotransposition and increasing in copy number are first targeted by PTGS, then by RdDM, and eventually by the CTM3/KYP and MET1 pathways promoting a robust TGS . Then, tA. thaliana [A. thaliana, it was suggested that, conversely, 5mC inhibited RdDM in constitutive heterochromatin by blocking RNA Pol IV and Pol V activities. Indeed, loss of this 5mC resulted in the spreading of RdDM across the genome, diluting RdDM factors and then compromising their efficiency, which is defined as the dilution model [A major question is how epigenetic factors cooperate to establish the appropriate pattern of 5mC at TE locations. Previous studies reported an intricate relationship between the RdDM and the 5mC maintenance pathways at the heterochromatic regions. In plants with a decreased 5mC in these regions, an activation of RdDM was observed, suggesting an inhibition of RdDM by heterochromatin in thaliana ,51,54,55thaliana ,57,58. Aon model ,57,58. TA. thaliana investigated the reestablishment of 5mC in the progeny of epigenetic mutants impaired in non-CG methylation [A study in hylation . The authylation .A. thaliana shoot apical meristem (SAM), in which TEs are more expressed than in surrounding cells [Finally, 5mC dynamic at TEs was investigated in ng cells . In SAM ng cells .Post-translational modifications (PTMs) of histones affect nucleosome structure, which impacts DNA accessibility to other epigenetic players and transcription factors (TFs) regulating gene expression ,62,63. HA. thaliana HDACs, HDA6 is essential for TE silencing, and 5mC hypomethylation was reported in hda6 mutant [Among the 6 mutant ,69,70  showing an enrichment of H3K27me3 in wt plants, despite the presence of mCG. To explain this discrepancy, it was proposed that H3K27me3 accumulation at EVD was due to the low density of mCG at this location, a feature that was also observed in other species or specific conditions , an enrichment of H3K27me3 at TEs losing m5C was observed [ddm1, this enrichment was dependent on the PRC2-component SET-domain protein CURLY LEAF (CLF) [ddm1 clf double mutant did not show a global increase in TE expression, but instead a partial rescue of ddm1 silencing defects that is most likely attributed to DNA hypermethylation. Again, EVD behaved as an outlier, showing increased transcription and transposition rates in ddm1 clf [EVD, CLF-mediated H3K27me3 deposition can act as a backup silencing system in ddm1 mutant plants.In tissues with low 5mC, like the seed endosperm in plants or during the embryonic development in mammals where DNA demethylation waves are observed, TEs are targeted by H3K27me3 to potentially repress them ,82. Furtobserved ,83. In dA. thaliana plants impaired in the two Jumonji H3K27me3 histone demethylases EARLY FLOWERING 6 (ELF6) and RELATIVE OF EARLY FLOWERING 6 (REF6) showed transgenerational epigenetic defects associated with a loss of 5mC and gain of H3K27me3 at heterochromatic TEs [mariner TEs carry silencer elements that are similar to the Polycomb/Trithorax response elements (PRE/TREs), allowing the recruitment of PRC2 to dampen TE expression [A. thaliana TEs carry DNA motifs that are potentially recognized by PRC2 [In parallel, a study analyzing atic TEs . Of notepression . Likewis by PRC2 . Further by PRC2 . To fullParamecium tetraurelia or Tetrahymena thermophila, in which PRC2 catalyzes the deposition of both H3K9me3 and H3K27me3 on TEs [Cyanidioschizon merolae, where 50% of TEs are covered by H3K27me3, suggesting that this repressive mark might be involved in TE silencing [Marchantia polymorpha, a representative species of liverworts, which diverged from other embryophyte lineages in the early Paleozoic, about 500 Mya [M. polymorpha genome, TEs are associated with different epigenetic signatures. While a large proportion of TEs that are mostly located on the highly condensed sex chromosome V are predominantly marked by 5mC, H3K9 methylation and H3K27me1, about 20% of TEs, mostly autosomal, are instead targeted by H3K27me3 and display either a significant reduction or a complete depletion in 5mC level [M. polymorpha. Targeting of TEs by H3K27me3 would be an ancient silencing mechanism that would have evolved towards dedicated 5mC- and H3K9-methylation-mediated TE silencing pathways throughout the evolution of other embryophyte lineages -like1 (EZL1) induces TE silencing defects [ezl1 mutant, it is difficult to precisely determine the role of H3K27me3 in TE repression. Similarly, whether PRC2-mediated H3K27me3 is required for TE silencing remains to be tested in liverwort.In other organisms with a low genomic 5mC level, H3K27me3 enrichment at TEs is also observed: such as in the ciliates 3 on TEs ,87, or iilencing . Further 500 Mya . DependimC level . Thus, tussed in ). In P.  defects ,87. NeveChromatin remodelers are protein factors that catalyze a broad range of reactions impacting chromatin organization and structure, like nucleosome sliding across the DNA or changing the conformation of the histone octamer . All chrA. thaliana AtDDM1 is an SNF2 chromatin remodeler belonging to the mammalian lymphoid-specific helicase (LSH) subfamily that has been studied for decades [The  decades . This es decades ,94,95.h1 mutant shows a complex 5mC pattern: decrease in CHH methylation at short euchromatic TEs and increased 5mC levels at long heterochromatic TEs [h1 mutation on TE silencing is modest, but in h1 met1, mutant synergistic effects can be observed at a subset of TEs showing a substantial upregulation in comparison to respective single mutants [ddm1, h2a.w mutants develop like wt plants, with a minor impact on TE silencing, and a slight decrease of non-CG methylation at pericentromeric TEs [h2a.w, and heterochromatic H1 level is increased. This correlates with a mild reduction in heterochromatin accessibility in h2a.w compared to wt plants, a molecular phenotype that is abolished in h1 h2a.w double mutant, displaying chromatin accessibility greater than wt plants. It was proposed that H2A.W would regulate H1 occupancy in constitutive heterochromatin by competing with H1 for linker DNA binding in order to optimize heterochromatin accessibility of epigenetic factors such as DNA methyltransferases [ddm1 remain obscure, and cannot be attributed to a loss of H2A.W. One hypothesis is that these epigenetic defects would result from major alterations of the chromatin structure occurring in ddm1 that would be inherited upon cell division, with concomitantly an inefficient maintenance of 5mC and H3K9me2, together with an invasion of H3K27me3 at heterochromatic TEs.It was initially proposed that DDM1 facilitated DNA methyltransferase activities by evicting the linker histone H1. The atic TEs ,96,97. T mutants . Importa mutants ,99. InstIt is remarkable to note that in humans, the deposition of the heterochromatic histone variant macroH2A by LSH1 is sufficient to repress transcription in a 5mC- and H3K9me3-independent manner . Thus, ASlDDM1 homologs reallocates the RdDM pathway to heterochromatin, leading to mCHH and siRNA redistribution [A. thaliana DDM1, the fact that DDM1 mediates H2A.Z deposition in this model plant will most likely impose the requirement to investigate this aspect for future DDM1-related studies in crops.Several studies have investigated the role of DDM1 orthologs in crops, such as tomato, rice or maize. In tomato, disrupting the two ribution . In riceribution . Finallyribution . Thus, tTy3/gypsy TEs upon TE gene exaptation [A. thaliana PMD proteins were first identified as cellular factors required for SAM and root apical meristem (RAM) organization, genome stability and cell division, hence the name MAINTENANCE OF MERISTEMS (MAIN) for the PMD founding member, and MAIN-LIKE 1 (MAIL1) for its closest homolog [main and mail1 mutants, with the notable exception of a slight increase in CHG methylation at pericentromeric regions and in chromosome arm TEs [main drm1 drm2 cmt3 quadruple mutant showed exacerbation of TE silencing defects, with a number being specifically derepressed in this mutant background [The plant mobile domain (PMD) is a protein domain of unknown function only found in angiosperms, and presumably deriving from aptation ,103,104  homolog ,106. Lat homolog ,108. GenDrosophila and Schizosaccharomyces pombe, two species largely depleted in 5mC [A. thaliana, the identification of a hypomorphic smc4 mutant revealed that SMC4 was required for TE silencing [Structural maintenance of chromosomes (SMC) proteins compose the core unit of condensin and cohesin ATPase complexes that play an essential role in higher-order chromatin organization, chromosome condensation and sister chromatid cohesion by performing topological entrapment of DNA . The rold in 5mC ,111,112.ilencing   is a plant-specific protein identified in a screen for mutants that released transcriptional gene silencing of a transgenic locus  Figure F. MOM1 itin foci . The protin foci ,116. CMMtin foci ,118. Bestin foci . AlthougA. thaliana, through the identification of atmorc1 and atmorc6 mutants showing upregulation of several TEs and decondensation of constitutive heterochromatic chromocenters [A. thaliana MORC proteins are recruited to constitutive heterochromatin have been extensively studied. AtMORC6 interacts with several RdDM components, and artificial tethering of AtMORC6 to chromatin is sufficient to recruit the RdDM pathway [MICRORCHIDIA (MORC) proteins are gyrase, HSP90, histidine kinase, MutL (GHKL)-type ATPases conserved in prokaryotes and eukaryotes ,120. A cocenters   that acts as a transcriptional repressor [sln and mbd5 mbd6 mutants. Moreover, artificial chromatin tethering of SLN was sufficient to promote gene silencing. Besides, SLN, MBD5 and MBD6 interact with several HEAT SHOCK PROTEIN 70 (HSP70) chaperones [Lately, a study described the involvement of the two epressor   located in its LTR [ONSEN activation is subsequently followed by transposition in several heat-stressed RdDM mutants, demonstrating the importance of this epigenetic pathway to regulate TE mobilization [ONSEN insertions in two abscisic acid (ABA)-responsive genes improved ABA-related stress tolerance, suggesting TE-driven positive events of adaptation upon environmental changes [In natura, environmentally challenged organisms are prone to undergo massive TE reactivation, also known as TE burst, which, upon mobilization, will generate genomic structural variations (SVs) ,139,140. its LTR ,142. ONSlization . Althoug changes . Thus, t changes .TE-induced SVs have been well documented during the process of plant domestication, in which these events are selected and maintained under high selective pressure by humans, as they provide selective advantages in relation to desired agronomic traits ,145,146 . TE inseAt the macroevolution scale, the phenomenon leading to the appropriation of TE-derived sequences by the host is defined as TE exaptation—also known as co-option or TE domestication . ExaptatAt the molecular level, TE exaptation can be of various natures, through the co-option of coding or non-coding (nc)DNA/RNA sequences (from TE genes or TE-derived regulatory elements) . NowadayDrosophila in 1992 that Miller and colleagues identified the first P element-derived ETE genes; the authors suggested that TE exaptation was an evolutionary process potentially favoring genetic innovation and creating new biological functions for the host organism [One of the most striking beneficial contributions of TEs to the host genome is illustrated by the exaptation of TE-derived protein-coding sequences, which is a phenomenon leading to new functional host genes that can be referred to as ETE genes. It is in organism . Since torganism . ConsideTEs have diverse, attractive and sophisticated molecular tools, making them a dynamic reservoir for potential ETE genes with new cellular functions. Their genomes encode transposases, integrases and reverse transcriptases, as well as structural and envelope proteins that can be co-opted by the host during evolution . NotablyFAR-RED IMPAIRED RESPONSE1 (FAR1) and FAR-RED ELONGATED HYPOCOTYLS3 (FHY3). FAR1, FHY3 and homologs are TFs deriving from Mutator-like (MULE) transposases [A. thaliana, FAR1 and FHY3 act cooperatively downstream of the photoreceptor phytochrome A to specifically modulate far-red light-responsive gene expression [CIRCADIAN CLOCK ASSOCIATED1 (CCA1), which is a central gene controlling the circadian clock [In plants, the first and by far the most thoroughly characterized ETE genes are sposases . In A. tpression . These ppression ,166,167.pression . FBS arean clock ,166. Conan clock . AltogetA. thaliana [FAR1/FHY3 genes in several plant species. A significant expansion of an FAR1/FHY3 gene family would correlate with the prostrate-to-erect growth switch specifically observed in the domesticated Rosa wichuraiana “Basye’s Thornless” (BT) species, with potential links to molecular processes related to light signaling, shade perception and flowering time [FAR1/FHY3 genes observed in the wild species Medicago ruthenica, in comparison to two related species, M. truncatula and M. sativa, could contribute to the higher tolerance of M. ruthenica to various abiotic stresses including drought, making it a valuable alternative as a legume forage crop [Camellia sinensis), the steady-state level of FAR1/FHY3 mRNAs is highly modulated in response to various abiotic stresses such as high salt, high/low temperature, polyethylene glycol (PEG)-mimicked drought or abscisic acid (ABA) treatment [A. thaliana showing that mutant plants knocked out for genes belonging to the FAR1/FHY3 family display changes of their phenotypical traits when challenged by several abiotic stresses [There are fourteen genes belonging to the FAR1/FHY3 family in thaliana . FAR1/FHthaliana . Comparaing time . The larage crop . In the reatment . Altogetstresses . Thus, FDAYSLEEPER, the founding member of the SLEEPER gene family, was first discovered in a yeast one-hybrid screen aimed at identifying Arabidopsis proteins bound to the promoter of Ku70, a gene involved in DNA repair, through the recognition of the DNA Kubox1 motif [DAYSLEEPER encodes a protein carrying the canonical transposase domains of hAT DNA transposons  that are a multiblock hAT dimerization domain required for self-interaction, and a BED-type ZF domain interacting with DNA [hAT transposases [DAYSLEEPER has also lost the flanking 8-bp genomic target-site duplications (TSDs), hallmarks of hAT integration, and the terminal inverted repeats (TIRs) that are required for transposition [daysleeper null mutants showed dramatic developmental phenotype, failing to develop normal organs, plants overexpressing DAYSLEEPER were capable of developing despite morphological defects [DAYSLEEPER-overexpressing plants revealed several upregulated genes, although none of them carried a Kubox1 motif in their promoter. DAYSLEEPER is highly expressed in SAM and RAM, reproductive organs and seeds [Arabidopsis protoplasts revealed a cytoplasmic localization in multi-vesicular bodies and potentially late endosomes [x1 motif . DAYSLEEwith DNA . Howeversposases . DAYSLEEposition . While d defects . Gene exnd seeds . Subcellnd seeds . Besidesndosomes . ConsideA. thaliana, the DAYSLEEPER closest homolog was called CYTOSLEEPER because the protein is cytoplasmic, which is consistent with the absence of an NLS. Unlike DAYSLEEPER loss-of-function, cytosleeper mutants are undistinguishable from wt plants [SLEEPER-like genes using the conserved “sleeperdomains” identified several candidates in the angiosperm sister species Amborella trichopoda as well as in distant species such as the water lily Nuphar variegata. In Oryza sativa, several SLEEPER-like genes were identified and named RICESLEEPERs. Remarkably, functional studies carried out in rice strongly suggested that RICESLEEPER1 and RICESLEEPER2 genes were both crucial for plant growth, as only heterozygote mutant plants could be retrieved [DAYSLEEPER and relatives in plants.In t plants . Seekingetrieved . AltogetFAR1/FHY3 or DAYSLEEPER genes that were identified by classical genetic approaches, the MUSTANG (MUG) genes were discovered by in silico analyses seeking for MULEs TE-derived mudrA-like ETE genes conserved in several angiosperms including A. thaliana and O. sativa [A. thaliana genome encodes eight MUG homologs gathering into two distinct clades: MUGA (MUG1 to MUG4) and MUGB (MUG5 to MUG8) [MUGA and MUGB would have arisen from two independent exaptation events [MUG genes act redundantly to ensure proper plant development. Although mug1, mug2 and mug3 simple mutants are phenotypically undistinguishable from wt plants, high-order mutant combinations such as mug1 mug2 or mug1 mug2 mug3 can seriously impact plant growth, flower development and fertility [mug7 mug8 double mutant displays severe growth delays [far1/fhy3 mutants, phenomic studies showed that the growth of several mug mutants is impacted by phosphate starvation or various abiotic stresses [Unlike the . sativa . The A. to MUG8) . It has n events . MUG genertility ,180. Simstresses .P instability factor (PIF)/Harbinger superfamily is composed of class II TIR DNA TEs encoding two distinct proteins: a superfamily-specific transposase with a DDE triad for endonuclease activity, and a DBD protein with a conserved SANT/myb/trihelix motif [PIF/Harbinger-derived ETE gene was Harbinger Transposase-Derived 1 (HARB1), identified in humans and several mammals, chicken, frogs and fish [Harbinger-derived transposase and DBD proteins occurs concomitantly, in pairs. Thus, HARB1 was found to interact with the co-opted DBD protein nuclear apoptosis-inducing factor 1 (NAIF1), and functional homologies in the molecular mechanisms involving Harbinger transposase/DBD proteins and their domesticated counterparts have been proposed [Originally identified in maize but also present in animals, the ix motif . The firand fish . Remarkaproposed . To datePIF/Harbinger TEs is frequent in Drosophila [Malus domestica (apple), MdHARB1 transcription is induced by heat stress, and transgenic plants overexpressing MdHARB1 are more resistant to heat [Exaptation of osophila , and HAR to heat .A. thaliana, several co-opted Harbinger proteins have been related to various epigenetic pathways. The tendency of Harbinger TEs to be inserted in gene-rich regions of the genome, as well as the dual nature of their transposase module could have predisposed them to exaptation as components of chromatin-related processes. Besides, considering that epigenetic pathways repress TEs, it was proposed that co-option of TE genes to be reallocated to these cellular processes could reflect an adaptation to evolutionary conflicts in the never-ending fight between the host and invasive elements [In elements .alp1 and alp2 mutants on TE silencing is unknown. Nevertheless, these functional studies clearly showed the importance of ALP1 and ALP2, most likely by controlling PRC2 activities during important developmental transitions.The transposase ANTAGONIST OF LIKE HETEROCHROMATIN PROTEIN 1 (ALP1) and the divergent DBD protein ALP2 interact together to antagonize PRC2-mediated H3K27me3 deposition and gene silencing ,185. ImpAnother example of dual exaptation involves the transposase Harbinger-derived protein 1 (HDP1) and the DBD protein HDP2. The two proteins interact together as well as with several components of the increased DNA methylation 1 (IDM1) histone acetyltransferase complex, including the methyl-DNA-binding protein MBD7, to prevent DNA hypermethylation and TE silencing . HDP2 DBhhp1 or mbd1/2/4 mutants did not show abnormal growth, the sant quadruple mutant displayed a late flowering phenotype similar to the hda6 mutant. Further analyses revealed that SANT and HDA6 co-regulate histone acetylation and are required for the proper expression of a common subset of genes, such as the floral repressor FLOWERING LOCUS C. It is unknown whether HDA6 targeting at these misregulated genes required the DBD of SANT proteins. Besides, it will be important to precisely delineate the role of HHP1 and MBDs in this pathway. Considering that these proteins are most likely required for the indirect interaction of HDA6 and SANT proteins, it would be relevant to test whether the hhp1 mbd1/2/4 quadruple mutant displays a late flowering phenotype.Finally, the co-opted transposase HDA6-associated Harbinger transposon-derived protein 1 (HHP1) was recently identified as a new HDA6 partner, forming a complex together with the four Harbinger-derived SANT-domain-containing proteins SANT1, SANT2, SANT3 and SANT4, and the MBD1, MBD2 and MBD4 . Then, sTE exonization is a molecular process leading to the creation of new exons from mutated introns composed of TE sequences . TE exonSETMAR which is composed of a Tcl-Mariner transposase inserted downstream of a SET histone methyl transferase gene [Tcl-Mariner terminal sequence (Hsmar1 ITRs) that is still present in thousands of copies in the human genome. A modest overexpression of SETMAR leads to the misregulation of 1500 genes, with an enrichment for the Hsmar1 ITRs in upregulated loci. Furthermore, these genes may be involved in cancer [SETMAR gene occurred in the anthropoid primate lineage. The three SETMAR protein domains, i.e., its SET, catalytic transposase and DNA-binding domains are under a strong purifying selection [A striking example of host gene-transposase fusion (HTF) is the primate gene ase gene ,190. Then cancer . The exaelection . All theHTF gene structure revealed that transposase capture occurred through alternative splicing events. All known eukaryotic DNA transposon superfamilies contribute to HTF formation, with a predominance of Tc1/mariner-, hAT- and P element/Kolobok-derived transposases fused to host domains involved in transcriptional regulation. While host gene/transposase-derived DBD fusions are prevalent, HTFs can also involve whole transposase sequences. For instance, the vesper bats KRABINER protein derives from an HTF event between the host transcriptional repressor Krüppel- associated box (KRAB) and a full-length Mlmar1 mariner transposase. KRABINER is involved in transcriptional repression in a DNA-sequence-specific manner, and binds genome-wide hundreds of cognate TE-derived CREs, controlling a large network of genes [Recently, an elegant analysis using comparative genomics surveyed the occurrence of HTFs in available tetrapod genomes . The autof genes . The facof genes .Alu elements are particularly prone to TE exonization, through the use of cryptic splice sites residing within their sequences [In humans, intronic SINE equences ,195. Thoequences ,197,198.equences .A. thaliana and plants with more complex genomes.Advances in sequencing technologies have made it possible to investigate the transcriptional landscape of TEs at the genomic scale. Five prime cap (5ʹ-Cap) capture coupled to nanopore-based long-read sequencing of mRNA was carried out in locust that has a complex genome highly enriched in TEs, to characterize full-length transcripts in their native form and evaluate the propensity for TE exonization. Among the 60,908 representative RNA transcripts, 51.88% of them contained at least one TE-derived sequence, mostly in the first or last exons, but to a lesser extent, in internal exons as well . It willA. thaliana, 7.8% of expressed genes were found to contain a region with close similarity to a known TE sequence [In angiosperms, there are a substantial proportion of genes carrying TE fragments. For example, in rice, more than 10% of transcripts were reported to contain TEs or TE-derived sequences, and TEs contribute to about 2% of rice protein coding regions . Similarsequence . Recentlsequence . This ilAs mentioned above, TEs have a propensity to capture and transpose entire host genes or fragments through the process of TE-mediated exon shuffling, which can result in gene duplication or rearrangement. This phenomenon is named transduction or transduplication, depending on the involvement of class I or class II TEs, respectively . AlthougBs1 created a novel chimeric gene composed of three host gene fragments fused to the Bs1 gag domain. This chimeric gene is expressed at the protein level, and its function could be involved in reproductive development [Katydid-At1, an element of the terminal-repeat retrotransposons in miniature (TRIM) family of non-autonomous LTR retrotransposons that was involved in the transduction of a gene potentially involved in nonsense-mediated mRNA decay (NMD) [Class I TE-mediated transduction involves transcriptional readthrough from the retrotransposon promoter to the adjacent host gene sequence and its subsequent incorporation into the TE sequence during reverse transcription . There aelopment . Anotheray (NMD) .L1 and SVA (SINE/VNTR/Alu) elements also contribute to transduction events of adjacent host sequences [L1 TEs would be involved in the generation of ten thousand genes through the process of transduction, many of which are transcribed and some of them acquired new cellular functions [In humans, LINE-1 equences ,209. It unctions .Pack-TYPE TEs that are predominantly derived from MULE, CACTA and Harbinger TEs in A. thaliana. Pack-TEs share many features with host genes. They are usually not highly repeated, and enriched in euchromatic instead of pericentromeric regions. Pack-TE-mediated transduplication events generate genes that are under purifying selection [Pack-TE to transduplicate a whole gene. The second feature is that, unlike regular duplicated genes, the alignment of transduplicated gene fragments with their original counterpart reveals a sharp boundary at the breakpoint of duplication. The third feature is that transduplicated gene fragments are often flanked by inverted or direct repeats. The presence of TSD flanking the TE DNA repeats is the unambiguous evidence that the entire structure belongs to a single TE. Furthermore, each transduplication event should be associated with specific flanking TSD. However, TSD are not always trivial to spot, making the identification of this phenomenon difficult. Thus, it is probable that the number of transduplicated genes may be strongly underestimated, as it is only recently that transduplication events may have been identified with confidence. As a consequence, it remains a great challenge to identify this type of gene [In plants, Class II TE-mediated transduplication involves election ,212. Thielection . The fir of gene .As mentioned above, the MUGB MUG7 and MUG8 proteins carry a PB1 domain of unknown function, usually not associated with TEs, that was likely acquired via transduplication before the exaptation of MUGB TE precursor ,180. TheRecently, the first report of a real-time mobilization of non-autonomous Pack-CACTA has been highlighted . In thisFinally, the epigenetic state of over a thousand transduplicated genes was analyzed in the maize genome . RemarkaTE mobilization can potentially lead to the production of TE-derived non-coding (nc)DNA sequences with positive selection signatures for host adaptation. They can be TE-promoter-derived CREs used as alternative promoters, as well as enhancers, insulators or repressor DNA elements regulating the transcription of host genes by the recognition of specific TFs . BecauseA. thaliana [A. thaliana and A. lyrata, two sister species that diverged 10 million years ago, showed that 16% and 24% of genes harbor a TE insertion in a 500 bp window of the coding region [A. lyrata genome [miniature mPing and MuDr TEs, respectively, preferentially insert into the promoters of genes, which can potentially upregulate adjacent genes [TEs do not mobilize randomly in the genomes, and genomic distribution varies according to TE families, as exemplified in thaliana ,217. Furthaliana . In humathaliana ,219. Comg region . This dia genome . In ricent genes ,222. In nt genes . These rnt genes . It is int genes . Likewisnt genes . As thesA. thaliana, while the neo-insertions of VANDAL21, ATENSPM3 and ATCOPIA93 (EVD) TEs occur preferentially in or nearby genes, only VANDAL21 TEs are significantly more often inserted in the promoters or 5′UTRs of transcriptionally active genes, enriched in H3K4me3 and H3K36me3 active marks. Conversely, ATENSPM3 and ATCOPIA93 TEs are more often inserted throughout the whole body of genes that are transcriptionally repressed by H3K27me3 deposition, and enriched in H2A.Z variant [A. thaliana, it would be interesting to determine if VANDAL21 neo-inserted TEs harbor active epigenetic mark and tend to be more expressed in comparison to their ATENSPM3 and ATCOPIA93 counterparts, which would display a developmental-specific expression pattern, as they are inserted in or nearby PcG-repressed genes. In a similar approach, Zhang and colleagues analyzed the genome-wide distribution and chromatin landscape of TE neo-insertions in several plant and animal genomes [Specific epigenetic chromatin states are most likely important factors influencing TE neo-insertions. In  variant . Accordi variant . It is u genomes . They no genomes .Citrus sinensis (orange), where the LTR-retrotransposon Rider controls the expression of Ruby, a gene encoding a MYB TF required for anthocyanin production. Remarkably, different fruit pigmentations among orange varieties correlate with allelic variation of the cold-responsive Ruby locus carrying or not Rider in its promoter [Rider insertion is recent, it should not be considered a TE exaptation. It could, however, be seen as a preliminary step to a future TE exaptation event. Similar cases have been highlighted in other crops, as in maize and apple [A. thaliana as well, it was suggested that an unmethylated ATCOPIA93-derived solo-LTR had been evolutionarily co-opted to cis-regulate the disease resistance gene RPP4, conferring plant immunity advantage [EPCOT3 that was positively selected in A. thaliana, but not in A. lyrata, to regulate the expression of CYP82C2 through the binding of WRKY33, a pathogen-responsive TF [CYP82C2, which is a gene involved in the production of metabolites for pathogen defense, originated from the duplication of the iron-stress response gene CYP82C4 in the two Arabidopsis species. They proposed that CYP82C2 underwent neo-functionalization upon EPCOT3 insertion in its promoter, and subsequent rewiring of WRKY33 regulon, leading to a positive effect on fitness [A. lyrata CYP82C2 transcription remain elusive, purifying selection analyses suggest that it might be involved in pathogen defense.As TE promoters often carry stress-responsive elements, TE-derived ncDNAs might to some extent be positively selected to temporally regulate the expression of adjacent genes upon environmental stimuli or stresses . In planpromoter . As thisnd apple ,233. In dvantage . In addinsive TF . The aut fitness . AlthougDuring transposition bursts and subsequent genomic rearrangements, TEs can spread their own promoter-borne CREs, as well as host CREs through the processes of transduction or transduplication (as described in MER20 DNA transposons in an evolutionary process that occurred more than 100 million years ago [RLTR13 endogenous retroviruses (ERVs) rewired hundreds of placenta-specific enhancers in mice, suggesting a role for ERVs in the swift evolution of placenta morphology between mammal lineages [One of the most outstanding examples of TE-derived CRE network is the elaboration of the mammalian placenta, involving ears ago . More relineages . It is nlineages . Other slineages ,239. Thelineages .CRE networks promoting TFBS rewiring have been described [In plants as well, several examples of TE-driven escribed . Among tescribed ,241,242.cis and trans regulatory functions. TE-derived lncRNAs and small RNAs acting in cis have been well documented, especially in the RdDM or PTGS processes that were discussed above. TE-derived ncRNAs can also act in trans to regulate the expression of host genes at the transcriptional or post-transcriptional level. Besides, when embedded in a transcribed host mRNA, they can contribute to a plethora of mechanisms governing the mRNA outcome [TEs are a major source of long non-coding (lnc)RNAs and small RNAs with  outcome ,244,245.SINE are well-studied TEs producing ncRNAs that have been involved in virtually all molecular processes regulating gene expression and organizing chromatin and nucleolus structure [SINE TEs, such as Alu elements (Alus), are predominantly found in introns and 3′UTR of RNA Pol-II-transcribed genes [SINE sequences can promote back-splicing events to form exonic circular RNAs [Alus can form inverted-repeat (IRAlu) structures on mRNAs with regulatory functions. These 3′UTR IRAlus can influence host mRNA metabolism by controlling its subcellular localization, translation and stability through the recruitment of specific RNA-binding proteins [Alus may be involved in alternative polyadenylation (APA) site usage to produce various mRNA isoforms [Drosophila pogo DNA transposon was observed; pogo insertion in the 3′ UTR of the CG11699 gene results in a shorter 3′UTR, elevated transcript levels and increased resistance to xenobiotic stress, suggesting an adaptive effect of the insertion [In mammals, the RNA Pol-III-transcribed tructure ,246,247.isoforms . Remarkansertion .There is compelling evidence, both in animals and plants, that TEs are a major source of lncRNAs, which are transcripts with a size greater than 200 bp and low protein-coding potential. In vertebrates, TEs account for up to 80% of lncRNA compositions. Comparative analyses performed in 40 plant species revealed more than 14,000 overlaps between TE and lncRNAs. While vertebrate lncRNAs appear to be lineage-specific, plant lncRNAs are generally poorly conserved between the same family species ,244. TheAs mentioned above, TE-derived small RNAs have been widely described in plants, especially in the process of siRNA-mediated heterochromatinization trans the host gene OsDRM2, which is involved in TE silencing, invoking the evolutionary arms race phenomenon between the TE and its host [A. thaliana describing Athila Ty3/Gypsy TE-derived siRNAs targeting UBP1b, which encodes a factor repressing Athila translation [WRKY45 allelic variants, WRKY45-1 and WRKY45-2, which differ from each other by the presence or absence of two intronic WANDERER-type MITE TEs in opposite orientation, respectively [WRKY45-1 allele correlates with the accumulation of MITE-derived TE-siR815, which downregulates SL1 expression by RdDM, conferring higher sensitivity to Xanthomonas oryzae (Xoo) infection. Conversely, WRKY45-2 expression confers improved resistance. The negative effect of TE-siR815 was confirmed by creating transgenic plants overexpressing WRKY45-1 with deleted TE-siR815 sequence that showed enhanced resistance, similar to WRKY45-2-overexpressing plants [MITE (sMITE) TE is integrated in the 3’-UTR of the Ghd2 gene, which promotes Ghd2 translational repression in a siRNA-dependent manner interfering with panicle development and grain yield [Ghd2 is important for agronomical purposes, as it controls grain number, plant height and heading date. Comparative analyses revealed that sMITE is present in O. rufipogon, a wild relative of cultivated Asian rice [MITE-driven translation repression could be a widespread mechanism [The interaction between TE-derived small RNAs and host genome is well documented in rice. First, the TE-related microRNA280 (miRNA820) downregulates in its host . This isnslation . A seconectively . The expg plants . Third, in yield . Ghd2 isian rice , which sechanism .Triticum aestivum L.), a recent study showed that class II MITE and Mariner TEs are important sources of miRNAs with a potential role in plant immunity [Blumeria graminis f.sp tritici. Tae_miR1436-1 is conserved in the triticum lineage, but not in other plant species [In maize, captures of gene fragments by TEs can lead to evolutionary conflicts . SiRNA cimmunity . Particu species . WhetherXist is most likely the most-studied lncRNA, with a major role in the process of X-chromosome inactivation in female eutherian mammals [Briefly, in animals, TE-derived lncRNAs have been related to several developmental processes. The maintenance of pluripotent embryonic stem cell program involves ERV-lncRNAs and LINE RNAs in humans. The mouse and human SINEUP lncRNAs have been involved in brain development by positively regulating the translation of factors essential for brain development. Finally, the LINE- and SINE-derived  mammals ,243. In  mammals ,245.Considering that overall, plant lncRNAs are involved in many biological processes, such as chromatin topology, transcriptional regulation, alternative splicing and small RNA buffering, all together regulating fundamental processes of plant development such as flowering time or stress perception ,245, it A stepwise approach must be implemented to define true exapted TE events. Some steps, such as identification, functional validation and conservation among close relative species are requisite and common to both types of exapted sequences (coding and non-coding ETE). Others, for instance, expression analyses when referring to ETE genes or exaptation of TE-derived RNAs, are more specific to one type of event ,258.Constant improvements in sequencing technologies together with drastic reduction of sequencing costs have allowed large-scale genomic projects aimed at determining the genome of hundreds of species. It is nevertheless important to mention that careful genomic annotations, with a special attention to DNA repeats, are a prerequisite for the identification of TE-derived sequences. Indeed, identifying an exapted TE among a pool of “regular” TEs can be seen as looking for a needle in a haystack. Thus, meticulous computational analyses must be undertaken.A. thaliana [MUSTANG and FAR1/FHY3 diversification [For the identification of ETE genes, one can integrate a suite of common properties including loss of mobility—absence of flanking terminal repeats such as TIRs or LTRs, sequence similarity with functional proteins (like transposases), low copy number, a conserved genomic location among sister/cousin species (synteny), evidence of active transcription and purifying selection of key codons . Using sthaliana . A partification .A. thaliana. Based on a k-mer strategy, the authors suggested that half of the genome would originate from TEs, which is significantly more than previously anticipated [To identify ETE non-coding elements acting as CREs, it is important to first annotate TE-derived sequences in the genome. A recent study described a new approach to find old and degenerated TEs in icipated . Then, ticipated . Baud anicipated . Wet labicipated ,262,263.cis-regulatory activity of the TE and the endogenous gene expression difficult. Another approach to validate the role of a specific non-coding ETE sequence on host gene regulation is to carry out a loss of function experiment. Recent development of precise genome editing technologies such as CRISPR-Cas9 system allows one to answer this question [It is subsequently important to perform functional experiments when evaluating gene regulatory roles of TEs. For this purpose, in vivo enhancer assays, such as luciferase-based reporter experiments, can validate candidates . Howeverquestion ,265,266.Microsynteny analyses are an essential step to define true ETE events. They rely on analyzing the genomic environment of TE-derived sequences among sister and more distant cousin species of a specific lineage to highlight evolutionary conservation. However, these analyses can be tricky when dealing with old events. Indeed, synteny can be interrupted by genomic rearrangements, making it difficult to determine the conservation of exapted events among distantly related species. This demonstrates the difficulty in drawing a conclusion based on a single feature. Furthermore, evidence of purifying selection events for conserved key codons within orthologous ETE genes is another essential feature reinforcing the confidence in exaptation events. Nevertheless, it is likely that many ETE sequences have escaped detection due to lack of solid evidence of conservation among closely related species.Although not sufficient by itself, evidence of active transcription is a good clue of TE exaptation if combined with other features described above. Nevertheless, in some cases, it might be difficult to detect signs of expression. Indeed, it is important to bear in mind that the ETE sequences could be expressed at a low level or display spatiotemporal expression patterns, as they are often cell-lineage- or tissue-specific . The newThe best way to highlight the impact of ETE events on host genome is to combine different approaches, such as computational genomics, transcriptomics (RNA-seq), genome editing and multiple chromatin-related analyses allowing the genome-wide mapping of TE-derived CREs or TF-like ETE proteins . Besides, cell biology and biochemical studies are essential complementary approaches to fully picture the functional role of ETE sequences. Although they might not be genuine ETEs, it is most likely that TEs and other DNA repeats overall contribute to 3D genome organization. Chromosome conformation capture (3C) analysis, such as Hi-C, studies subnuclear chromatin organization and compartmentalization, which can give new insights into the role of TEs in these processes. Mammalian class I TEs contribute to the species-specific subgenomic binding of the insulator protein CCCTC-binding factor (CTCF), which differentially impacts DNA looping and gene expression at the species level ,271. LikA thaliana line depicting chimeric TE/gene fusion event [As already mentioned, the fact that ETE sequences derive from highly repeated TEs can make their identification strenuous. For instance, upon a recent exaptation event, it might be difficult to differentiate the functional ETE gene from homologous copies that did not enter positive selection. Long read sequencing technologies, such as Pacific Biosciences (PacBio) or Oxford Nanopore sequencing, can identify whole TE or TE-derived sequences in complex genomes, as described recently in a genomically instable on event . Such teon event , giving In the future, the advent of large-scale genome sequencing combined with long read sequencing technologies will undoubtedly contribute to the identification of new TE-derived sequences with potential selective advantage for the host. Besides, pursuing our understanding of molecular processes involved in the regulation of epigenetic pathways and TE silencing will also be crucial. Altogether, these studies will undoubtedly contribute to unravelling the complex interplay between TEs and their host.


Genome-wide profiling of interactions between genome and various functional proteins is critical for understanding regulatory processes involved in development and diseases. Conventional assays require a large number of cells and high-quality data on tissue samples are scarce. Here we optimized a low-input chromatin immunoprecipitation followed by sequencing (ChIP-seq) technology for profiling RNA polymerase II (Pol II), transcription factor (TF), and enzyme binding at the genome scale. The new approach produces high-quality binding profiles using 1,000–50,000 cells. We used the approach to examine the binding of Pol II and two TFs (EGR1 and MEF2C) in cerebellum and prefrontal cortex of mouse brain and found that their binding profiles are highly reflective of the functional differences between the two brain regions. Our analysis reveals the potential for linking genome-wide TF or Pol II profiles with neuroanatomical origins of brain cells. Protein-DNA interactions are widely and critically involved in the regulations of gene transcription and expression . RNA polin vivo genome-wide binding of proteins. Although ChIP-seq has been generating reliable and high-quality data on histone modifications (i.e. the interaction between a modification histone and the genome) (7 cells per assay) for probing genomic binding of proteins that are not histones and the results tend to have lower reproducibility compared to histone ChIP-seq (Chromatin immunoprecipitation coupled with sequencing (ChIP-seq) is a simple and direct approach to profile  genome) , ChIP-seChIP-seq . In receChIP-seq ,19–26. HChIP-seq .n)  .5–108). Our approach, referred to as native MOWChIP-seq or nMOWChIP-seq, combines MNase digestion of native chromatin with a microfluidics-based low-input ChIP-seq technology -digestion-based native MOWChIP-seq as a general tool for low-input profiling of protein binding to genome. MNase digestion was previously applied in native ChIP to probe mostly histone modifications  and alsoChIP-seq ,17) that2. Cells were subcultured every 3 d to maintain exponential growth.GM12878 cells were obtained from Coriell Institute for Medical Research. Cells were cultured in RPMI-1640 medium  with 15% fetal bovine serum  and 1% pen-strep (Invitrogen) at 37°C, 5% CO2 followed by cervical dislocation. Mouse brains were rapidly dissected, frozen on dry ice and stored at −80°C. This study was approved by the Institutional Animal Care and Use Committee (IACUC) at Virginia Tech.C57BL/6J mice were purchased from Jackson Laboratory and maintained in the animal facility with 12-h light/12-h dark cycles and food and water ad libitum. 8-week old male mice were sacrificed by compressed CO2, 3 mM Mg(Ac)2, 0.1 mM EDTA, 10 mM tris-HCl, and 0.1% Triton X-100, with 30 μl of PIC , 3 μl of 100 mM PMSF, and 3 μl of 1 M dithiothreitol added before use]. Tissue was homogenized in the grinder set  by slowly douncing 15 times with pestle A and 25 times with pestle B. Homogenate was filtered through a 40 μm cell strainer into a 15 ml tube and centrifuged at 1000g for 10 min. The supernatant was removed and the pellet was resuspended in 500 μl nuclei extraction buffer and transferred to a 1.5 ml tube. 750 μl of 50% iodixanol, 7.5 μl of PIC, 0.75 μl of 100 mM PMSF and 0.75 μl of 1M dithiothreitol were added and mixed by pipetting. The mixture was centrifuged at 10,000g for 20 min and the supernatant was removed. If mixed nuclei (without separation of neurons and glia) were used for nMOWChIP directly, the nuclei pellet was resuspended in 200 μl of Dulbecco's phosphate-buffered saline (DPBS). If nuclei labeling and FACS sorting were conducted, 500 μl of 2% normal goat serum  in DPBS was added to the nuclei pellet and incubated for 10 min before resuspending. Anti-NeuN antibody conjugated with Alexa 488  was diluted with DPBS to 2 ng/μl. 8 μl of anti-NeuN was added to each 500 μl of nuclei suspension and incubated at 4°C for 1 h on a rotator. The labeled nuclei were then sorted using FACS . 8 μl of non-labeled nuclei were saved as unstained control prior to addition of anti-NeuN antobody. The concentration of nuclei suspension after FACS was typically low (∼1.2 × 105/ml). The nuclei were re-concentrated by adding 200 μl of 1.8 M sucrose, 5 μl of 1M CaCl2 and 3 μl of 1M Mg(Ac)2 to 1 ml of the nuclei suspension. The mixture was incubated on ice for 15 min and centrifuged at 1800g for 15 min. Supernatant was removed and the nuclei pellet was resuspended in DPBS to generate a suspension containing 4 × 106 nuclei/ml.A mouse brain was put on ice and PFC and cerebellum were dissected for nuclei isolation. The following steps were performed on ice and centrifugation performed at 4°C. Tissue was placed in 3 ml of ice-cold nuclei extraction buffer  were added, mixed by vortexing and incubated at room temperature for 10 min. 10 μl of 100 mM CaCl2 and 2.5 μl 100U MNase  were added, mixed by vortexing and incubated at room temperature for 10 min. 22 μl of 0.5 M EDTA was then added, mixed by vortexing and incubated on ice for 10 min. The solution was centrifuged at 16,100g for 5 min at 4°C. Supernatant containing fragmented chromatin was collected into a new 1.5 ml tube and placed on ice for use. Volumes equal to 100,000 and 50,000 cells/nuclei were portioned from the final chromatin (∼220 μl) for assays. For assays using 10,000 cells/nuclei or less, 100 μl of suspension containing 40,000 cells/nuclei was used in the first step and the same procedure as above was followed.This protocol is scalable in volume and can digest cell/nuclei suspension with concentration up to 4 × 105 cells/nuclei, while 0.5 μg of antibody was added for each assay using 5 × 104 or fewer cells/nuclei. We used the following antibodies in this work: anti-Pol II-total , anti-Pol II-S5 , anti-Pol II-S5 4H8 , anti-EGR1 , anti-MEF2C , anti-HDAC2 . The suspension was incubated on a rotator at 4°C for 2 h. The beads were then washed with IP buffer twice and resuspended in 5 μl of IP buffer for loading into the device chamber.5 μl of protein A Dynabeads  were used in each MOWChIP assay. The beads were washed twice with IP buffer  sodium deoxycholate, 0.1% SDS, 1% (v/v) Triton X-100) and resuspended in 150 μl of IP buffer. 1 μg of Pol II or TF antibody was added into the bead suspension for each assay using 10The microfluidics-based MOWChIP-seq process, including microfluidic device fabrication and operation, was conducted following our published protocol .After nMOWChIP, IP beads were rinsed once with IP buffer and resuspended in 200 μl of DNA elution buffer . 2 μl of 20 mg/ml proteinase K was added and the suspension was incubated at 65°C for 1 h. DNA was then extracted and purified by phenol-chloroform extraction and ethanol precipitation. DNA pellet was resuspended in 8 μl of low EDTA TE buffer. Input DNA was purified with the same process, by digesting chromatin solution directly using proteinase K, followed by the same extraction and purification process.Libraries were constructed using Accel-NGS 2S Plus DNA Library kit (Swift Biosciences) following the manufacturer's instructions. 1 × EvaGreen dye (Biotium) was added to the amplification mixture to monitor the PCR amplification. Library was eluted to 7 μl of low EDTA TE buffer. Library fragment size was examined using TapeStation (Agilent) and the concentration was quantified with KAPA Library Quantification kit (Kapa Biosystems). We also examined the enrichment of the libraries using qPCR and primers listed in dba.count command with default parameters to process MACS2 peaks and uniquely mapped reads and calculate consensus peaks and affinity score. Consensus peaks between technical replicates are identified by bedtools intersect with the additional setting of -f 0.5. Overlap between consensus peaks are plotted using Intervene  with default settings. Reads that passed the quality check were mapped to reference genomes hg38 (human) or mm10 (mouse) with bowtie (1.1.2) . Uniquel(2.11.2) . Peak ca(2.11.2)  with def(2.11.2) . Pearsonntervene .–15. Thresholds were applied to both the EGR1 and Pol II sets, and AUC was calculated with the R package ROC. Subsampling of EGR1 bam files to between 25% to 95% of original reads was performed using samtools. Five separate, but consistent, seeds were used at each subsampling percentage. Matthew's correlation coefficient (MCC) was calculated in R (mltools) at each percentage and each seed using a MACS2 q-value cutoff of 0.05 for both EGR1 and Pol II peaks.ROC curves were created by calculating the true positive rate (TPR) and false positive rate (FPR) at 100 different peak-calling thresholds from 0.99 and 10RNA Pol II, TF or enzyme binding has been conventionally studied after crosslinking using reagent such as formaldehyde that firmly immobilizes the protein to the genomic DNA ,50. HoweWe then applied nMOWChIP-seq to profile protein binding to the genome. MNase digestion was previously applied in native ChIP to probe mostly histone modifications  and alsoUsing GM12878, a lymphoblastoid and ENCODE tier 1 cell line, we showed that our method could generate high quality ChIP-seq data with input as little as 1,000 cells per assay for Pol II-S5 ab5131) Figure . Cells w31 FigureWe also determined that the binding of Pol II was stable enough to be preserved at −80°C, a unique property not observed in histone modifications or TFs. From our testing, MNase-digested chromatin can be frozen at −80°C or on dry ice for 2 d without substantial degradation in data quality for Pol II. We digested 40,000 cells and stored the chromatin on dry ice for 2 d. Volumes equal to 10,000 cells were then portioned for MOWChIP-seq and the data is shown as ‘10,000-st’ .PTGES3 and NACA  . We condand NACA . Similarand NACA .We profiled a transcription factor, early growth response protein 1 (EGR1), using as few as 5,000 GM12878 cells Figure . PearsonFinally, we applied nMOWChIP-seq to examine the binding of histone deacetylase HDAC2 in GM12878 cells Figure . At leasACTG1 and MDM2 as examples) with Pol II binding over the entire gene body showed that Pol II-total profile captured the initial binding of unphosphorylated Pol II at TSS, which was absent in Pol II-S5 profile in comparison  and all Pol II regardless of its phosphorylation status . Our data showed the distinction Figure . The twon Figure . We examTo determine whether Pol II-total or Pol II-S5 was more effective in predicting TF binding sites, we analyzed the overlap of either Pol II-total peaks or Pol II-S5 peaks with EGR1 peaks at varying peak-calling thresholds, shown as receiver operating characteristic (ROC) curves that display the true positive rate (TPR) versus the false positive rate (FPR) Figure . To quanWe applied nMOWChIP-seq to profile Pol II binding in mouse PFC and cerebellum. PFC have roles in cognitive functions, decision-making and short-term memory ,53, whilP < 10–5). Differentially bound peaks for Pol II-total are listed in We mapped Pol II-total binding in B6 mouse brain using nuclei extracted from PFC and cerebellum with 50,000 nuclei used per assay Figure . The genPlk2, which is a gene known to participate in rodent brain development and cell proliferation  development  refers to the ratio of Pol II read density between promoter proximal region (−30 to +300 bp of TSS) and gene body (+300 bp of TSS to TES), with higher PI indicating more Pol II binding near TSS ,56. Pol feration , and Npad memory , both shd memory . The datr folate . Furtherr folate . 36% of r folate . This war folate . We idenr folate . Among telopment , and Slce in CNS . Shank3 ormation , and Flrelopment .Kalrn  plays important roles in nerve growth (Gria1(Glutamate receptor 1) which is involved in synaptic transmission  protein family (P < 10–5). These regions were further analyzed for GO term enrichment analysis using GREAT and linked to walking behavior, motor coordination, and cerebellum morphology and development in the case of EGR1, cerebellar development and limb coordination in the case of MEF2C  binding using nuclei extracted from mouse PFC and cerebellum. Chromatin from 100,000 nuclei were used in each assay, yielding high quality data that revealed differential binding between the two regions of brain Figure . We picke growth . Higher smission . Cacna1adisorder  and exprntiation . Zic1 ann family , whose ln family . Higher n family . We obsen family . Such con family . We furtn family . In totaC Figure . In contChIP-seq profiling of proteins bound to the genome is generally much more challenging than that of modified histones. Histone ChIP-seq can be conducted with crosslinking and sonication or under native ChIP condition. Due to the robust interaction between histones and genome, native ChIP-seq for histone modifications can have very high efficiency. In contrast, previous ChIP-seq of protein bindings often involves crosslinking and sonication that immobilizes the protein to the interacting DNA sequence before breaking chromatin into fragments. Crosslinking is often considered necessary when TF, Pol II and enzyme interaction with the genome is studied, due to perceived needs and benefits for preservation of such interactions by crosslinking. However, crosslinking and sonication potentially cause epitope masking  and damaIn this work, by conducting nMOWChIP-seq without crosslinking and sonication, we demonstrate a low-input technology that works with as few as 1,000–50,000 cells per assay for profiling a wide range of protein-genome interactions. We show that nMOWChIP-seq method effectively preserves the interaction between Pol II/TFs/enzyme and the genome. In our approach, the required number of cells depends on the robustness of the protein binding to the genome under native ChIP conditions, the number of binding sites, and the quality of the antibody. Compared to the state-of-the-art ChIP-seq data taken using millions of cells per assay, our datasets generally show very high signal-to-noise ratio and low background, which are characterized by high FRiP values. Although side-by-side comparison is difficult due to the lack of matching cell type, input quantity and antibody, our method appears to be at least comparable to CUT&RUN ,35 in teCompared to histone modification data, ChIP-seq data on Pol II, TFs, enzyme in tissues are very scarce. We applied nMOWChIP-seq to profile Pol II, and key TFs EGR1 and MEF2C in a brain-region-specific manner in cerebellum and prefrontal cortex of mouse brain. We found that Pol II and TF profiles are highly characteristic of the brain regions. The Pol II binding profiles had 4,218 differential peaks between cerebellum and PFC, while EGR1/MEF2C profiles had > 1,000 differential peaks between the two brain regions. The peaks with their intensity high in PFC and low in cerebellum were highly enriched in functions including cognition, learning or memory, while the peaks that were high in cerebellum and low in PFC were enriched in GO terms including walking behavior, motor coordination and cerebellar cortex formation. The fact that the binding profiles of these functional molecules are highly characteristic of the functions of various brain regions indicate that Pol II and the key TFs are critically involved in the molecular dynamics associated with the spatial configuration of brain functions. Our results suggest the possibility of deciphering genome-wide non-histone molecular binding profiles to establish the connections between cells and their functions. In the case of brain cells, the approach potentially allows us to establish the neuroanatomical origin of a brain tissue.The ChIP-seq data sets are deposited in the Gene Expression Omnibus (GEO) repository with the following accession number GSE172224.lqac030_Supplemental_FilesClick here for additional data file.


The spatiotemporal organization of chromatin is regulated at different levels in the nucleus. Epigenetic modifications such as DNA methylation and histone modifications are involved in chromatin regulation and play fundamental roles in genome function. While the one-dimensional epigenomic landscape in many cell types has been revealed by chromatin immunoprecipitation and sequencing, the dynamic changes of chromatin modifications and their relevance to chromatin organization and genome function remain elusive. Live-cell probes to visualize chromatin and its modifications have become powerful tools to monitor dynamic chromatin regulation. Bulk chromatin can be visualized by both small fluorescent dyes and fluorescent proteins, and specific endogenous genomic loci have been detected by adapting genome-editing tools. To track chromatin modifications in living cells, various types of probes have been developed. Protein domains that bind weakly to specific modifications, such as chromodomains for histone methylation, can be repeated to create a tighter binding probe that can then be tagged with a fluorescent protein. It has also been demonstrated that antigen-binding fragments and single-chain variable fragments from modification-specific antibodies can serve as binding probes without disturbing cell division, development and differentiation. These modification-binding modules are used in modification sensors based on fluorescence/Förster resonance energy transfer to measure the intramolecular conformational changes triggered by modifications. Other probes can be created using a bivalent binding system, such as fluorescence complementation or luciferase chemiluminescence. Live-cell chromatin modification imaging using these probes will address dynamic chromatin regulation and will be useful for assaying and screening effective epigenome drugs in cells and organisms. In eukaryotic nuclei, DNA wraps around histone proteins to form a nucleosome, a basic structural unit of chromatin . Each nuA mechanism that regulates chromatin organization and dynamics involves the post-translational modification of histones, such as phosphorylation, acetylation, methylation and ubiquitination. The best-characterized histone modifications are acetylation and methylation of lysine residues on the N-terminal tails of histones H3 and H4. Lysine acetylation is correlated with active transcription, whereas lysine methylation is associated with either transcription activation or repression, depending on the site and the degree of methylation from one to three ,2. The pThere has been a great effort to elucidate how histone modifications regulate gene expression and how specific modifications are regulated during development and differentiation. As many modifying enzymes can be responsible for modifications at different sites on histones and non-histone proteins, and a modification can be added by multiple enzymes, it is not straightforward to interpret a result by loss-of-function experiments of an enzyme as a function of the specific modification. Therefore, it has been challenging to reveal the biological function of a specific modification.In higher eukaryotes, DNA modifications play an important role in gene regulation . The mosChromatin modifications are involved in the spatiotemporal organization of the genome at different levels, including the physical and biochemical properties that affect the flexibility and compaction of nucleosome arrays, higher-order chromatin structures and subnuclear localization . The orgChromatin can be visualized in bulk by using DNA-staining dyes and by expressing histones tagged with a fluorescent protein (FP). By bulk labeling, the distribution of chromatin can be observed, and densely stained regions essentially represent condensed heterochromatin. A popular DNA staining dye, Hoechst 33342, binds preferentially to AT-rich sequences, and so, it highlights AT-rich heterochromatin better than histone–FP. Hoechst 33342 is cell-permeable and convenient to use in many cell types without genetic manipulation . A drawbTo label nucleosomes, histone H2B–FP has been widely used to detect nuclei and chromosomes in living cells . Once stBesides the incorporation of histone-FP, nucleosomes can also be labeled with nucleosome-binding molecules. A number of cellular and virus proteins are known to bind to the acidic patch of H2A–H2B in a nucleosome . N-termiTo visualize specific genomic loci in living cells, several systems have been developed. As a fluorescent focus requires detectable brightness over background signals, the fluorescent reporter needs to be concentrated at the locus. Fluorescent reporter–operator systems are based on the insertion of an array of bacterial operator sequences  to which a corresponding FP-tagged repressor protein  binds . The insInstead of inserting exogenous DNA sequences and expressing corresponding binding proteins, endogenous genomic loci can now be visualized using artificial DNA binding proteins derived from genome editing systems, including zinc finger (ZF) proteins , transcrTo detect the distribution of chromatin modifications and their dynamics in living cells, several types of probes have been developed .Histone modifications are recognized by their reader proteins through specific binding domains, such as chromodomains (CDs) and plant homeodomains (PHDs) for methylated lysine residues and bromodomains for acetylated lysine residues . TherefoConnecting two different binding domains with weak affinities has also been used to develop unique probes. By fusing Polycomb CD that binds to H3K27me3 and TAF3 PHD that binds to H3K4me3 at the N-terminus and C-terminus of an FP, bivalent nucleosomes that have both modifications were visualized . A probeFor visualizing DNA methylation, an FP-tagged methyl-CpG-binding domain (MBD) of the MBD1 protein is widely used ,55. TranModifications at specific genome loci have also been detected using a combination of a sequence-specific DNA binding module  and a modification binder (MBD or HP1 CD) tagged with the N-terminus and C-terminus of Venus, respectively . Only whin vivo imaging by establishing stable cell lines and transgenic animals  it is not straightforward to deliver IgG molecules into cells, (2) the loaded immunoglobulin G (IgG) is too big to pass through nuclear pores and (3) the binding affinity (at the picomolar to nanomolar order for the dissociation constant) may be too strong and block the access of endogenous binding proteins to the target modification. In contrast to the whole IgG, fluorescently labeled antigen-binding fragments (Fabs) have been shown to be suitable for detecting endogenous histone modifications in living cells and embryos ,60–62. U animals  63,64].,64.in viIn living cells, the probes described above are present at least in two fractions, bound and unbound to the target modifications. The location of modifications is thus evaluated by the enrichment of probes over the diffuse background fluorescence. If the affinity of the probe is relatively low or the target modification is less abundant, the signal at the modification sites can be ambiguous because the unbound molecules that diffuse freely in the nucleus are present at high levels. Increasing the probe affinity can reduce the background from free molecules, but this may block the binding of endogenous proteins. It is therefore ideal if the probe becomes fluorescent only when binding to the target. A FP complementation strategy can be used for this purpose to reconstitute FPs at the site of modifications ,69,70, bA FRET sensor usually consists of a modification site and a modification-binding domain in between a donor and an acceptor FP . The verin vivo imaging, a split-Renilla luciferase (Rluc) complementation system has been applied for histone methylation [in vivo. A degron blockade methylation sensor was also developed for luciferase-mediated methylation sensing [For hylation . In this sensing . In thisBy using these modification-specific live-cell probes, the distribution and changes of modifications on DNA, histones and other chromatin proteins can be tracked in living cells. One of the applications of live-cell chromatin modification imaging is to reveal the role and function of modifications. There has been a long-standing question on chromatin modifications—are they regulators or just passive by-products of genome function? Especially in the field of transcriptional regulation, it has been elusive whether active marks of chromatin (e.g. acetylated histones) can activate transcription or are added as a consequence of transcription ,84. To din vivo analysis [Another major application of live-cell modification imaging is to monitor the effect of drugs. As epigenome drugs are expected to be a new class of agents against cancers and other diseases , a conveanalysis .We anticipate that the use of these probes described above will be broadly applicable and shed light on the intricate connections between chromatin modifications and genome functions, as well as for screening effective epigenome drugs.