The conference “Transposable Elements at the Crossroads of Evolution, Health and Disease” was hosted by Keystone Symposia in Whistler, British Columbia, Canada, on September 3–6, 2023, and was organized by Kathleen Burns, Harmit Malik and Irina Arkhipova. The central theme of the meeting was the incredible diversity of ways in which transposable elements (TEs) interact with the host, from disrupting the existing genes and pathways to creating novel gene products and expression patterns, enhancing the repertoire of host functions, and ultimately driving host evolution. The meeting was organized into six plenary sessions and two afternoon workshops with a total of 50 invited and contributed talks, two poster sessions, and a career roundtable. The topics ranged from TE roles in normal and pathological processes to restricting and harnessing TE activity based on mechanistic insights gained from genetic, structural, and biochemical studies. Transposable elements (TEs) are increasingly perceived not only as inevitable constituents of most genomes which persist due to their ability to replicate and spread, but also as active participants in shaping and re-shaping the structural and functional diversity of molecular, cellular, and developmental processes in the host species under normal and pathological conditions and in different environments. To further accelerate progress in this highly dynamic field, Keystone Symposia hosted a three-day meeting “Transposable Elements at the Crossroads of Evolution, Health and Disease” in Whistler, British Columbia, Canada, on September 3–6, 2023. The diversity of host species discussed at the meeting ranged from bacteria to fungi, plants, invertebrates, fish, and mammals. An increasing interest in harnessing the natural ability of transposons to insert into various genomic locations was also evident from presentations given by participants from the biotech industry. The dual nature of TEs as disease-causing mutagenic agents and one of the major driving forces in genome evolution was underscored by the keynote speaker, who made seminal contributions to understanding the roles played by TEs in regulatory evolution across multiple hosts.Keynote Speaker Cedric Feschotte, Barbara McClintock Professor of Molecular Biology and Genetics  energized the audience with an inspirational talk using a colorful metaphor of transposon addiction. In contrast to domestication, co-operation, and co-option, which imply that certain parts of TEs are recruited for host functions after losing connection to their original mobilizable units, the concept of directly engaging the actively transposing units into the cellular and developmental processes is relatively new. The Feschotte lab has been investigating the zebrafish retrovirus-like non-autonomous transposon which can still jump in the genome. Knockdown experiments demonstrate that, despite ongoing mobility, its expression is essential for early embryonic development, as manifested by failure to develop proper segmentation in the mesoderm following injection of antisense oligonucleotides. Investigation of the underlying molecular mechanisms of such phenomena observed in zebrafish and other experimental systems is ushering in a whole new direction of research, whereby TEs operating as mobile units become closely intertwined with organismal function to the extent that they can no longer be eliminated without damaging the host.SIRT6 overexpression and pharmacological activators of SIRT6, including one derived from brown seaweed.Session chair Kathleen Burns opened the first session with the talk given by Vera Gorbunova  who described her work on transposable elements in aging. First, her lab generated mice with knockdown of the youngest LINE-1  families, and she discussed the impact of each knockdown on mouse health and lifespan. She then discussed strategies for preventing or reversing age-related LINE-1 activation using E. Alice Lee  outlined challenges in identifying somatic retrotransposition events in human tissues, especially in non-dividing cells, such as neurons and cardiomyocytes. Her lab established PTA-HATseq, PCR-based L1-targeted sequencing combined with PTA (primary template-directed amplification), the latest whole genome amplification method, which allows genome-wide somatic L1 insertion profiling at single-nucleotide resolution in single cells. They demonstrated superior performance of PTA-HATseq, detecting ~ 2 times more non-reference insertions in single neurons than existing methods. Their data show that neurons do not show increased somatic retrotransposition with age, whereas epithelial tissues—colon, liver, skin—even postmitotic human cardiomyocytes show age-associated increases, suggesting distinct age-association across different cell/tissue types. They also applied PTA-HATseq to postmortem amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) patient brain samples. Individual neuronal nuclei were sorted by the status of cytoplasmic TDP-43 protein aggregates, hallmark pathology associated with neurodegeneration in ALS/FTD. ALS/FTD neurons showed more somatic L1 insertions than control neurons, but unexpectedly, more insertions were detected in neurons without TDP-43 pathology, requiring more work to properly interpret them.Myotis lucifugus transposon (MLT) for targeted gene therapy. These transposons are not expected to cause rearrangements and genomic instability because they do not create double-strand DNA breaks upon payload delivery. MLT has greater capacity for large cargos than other approaches, such as AAV vectors. MLT was specifically engineered to reduce insertional bias, and the whole genome integration profiles showed fewer integration “hotspot” sites and a predilection for insertion into intergenic and intronic regions compared to its un-engineered counterparts. By modifying programmable DNA binding domains, they could boost the accuracy of targeted integrations. Dr. Craig showed multiple use cases of MLT for genetic medicine. The company is working on targeted CFTR super-exon insertions to correct recessive mutations in cystic fibrosis, as well as the production of cancer-targeting CAR-T cells. Toward in vivo therapeutics, she presented data on cell-type specific delivery of lipid nanoparticles encapsulating transposase RNA and donor DNA encompassing the ABCA4 gene, with a length well beyond AAV capacity, into the mouse retina. In summary, these data suggest that MLT provides effective means for targeted gene delivery to enable genetic medicine.Nancy Craig  discussed the pros and cons of different genetic engineering strategies and showed the use and advantages of engineered mammalian DNA transposons derived from the Joshua Dubnau  explored positive feedback between endogenous retroviruses (ERVs) and TDP-43 proteinopathy underlying neurodegeneration in ALS/FTD. His lab previously reported that cytoplasmic aggregation of TDP-43 proteins triggers ERV expression. He presented recent data showing that ERV expression can also trigger TDP-43 proteinopathy . They knThis workshop consisted of short talks that captured the important roles TEs have in shaping genome-wide expression, regulating recombination, and generating Mendelian disorders.The recent advancements in long read DNA sequencing have greatly improved the assembly of genomes with high repeat content. In species such as wheat where TEs occupy over 80% of the genome, comparisons of strains can reveal extensive rearrangements. Dr. Khalil Kashkush  identified massive rearrangements between domesticated bread wheat and the wild emmer strains. Indels as large as 1 million bp suggest that such large-scale rearrangements may play a significant role in wheat speciation. By studying the polymorphic TEs within the six homologous genes of hexaploid wheat, Dr. Kashkush presented evidence that TEs inserted in exons or untranslated regions of genes correlated with altered expression .cis-regulatory activity of SAT-TE loci, some of which had characteristics of super-enhancers. The high abundance of SAT-TEs in the human genome and their cis-regulatory function suggests these dynamic loci may have significant impact on disease and development.New assemblies of the human genome published by the telomere to telomere (T2T) consortium provide an opportunity to examine the structure of regions with high repeat content. Panpan Zhang  presented identification of satellite sequences composed of tandem TEs (SAT-TEs). In the 3574 SAT-TE loci discovered by Dr. Zhang, LINE-1, Alu, and ERV subfamilies were commonly represented. Interestingly, haplotype variation of SAT-TEs revealed high levels of heterozygosity in some individuals. The longest SAT-TE described was a LTR12B locus expanded to 207 units covering 265 kb on the Y chromosome. ENCODE data was used to investigate Schizosaccharomyces pombe. Examination of de novo integration showed that insertions often function to increase promoter activity. Earlier, the Levin lab found that de novo insertions can adapt cells to environmental stress by increasing expression of specific groups of genes. Interestingly, the common lab strain of S. pombe contains just 13 full-length retrotransposons and Dr. Levin indicated that each is adjacent to a stress response gene including global regulators of oxidative stress and the unfolded protein response. In collaboration with Miguel Zaratiegui (Rutgers University) and Elena Hidalgo (Universitat Pompeu Fabra), Dr. Levin reported that replacing all 13 full-length retrotransposons with single LTRs resulted in sensitivity to peroxide and altered expression of many genes including some adjacent to the TEs. These data suggest that LTR-retrotransposons can provide regulatory input important for resistance to environmental stress.Session chair Henry Levin  studies LTR-retrotransposons in the fission yeast During development TEs make critical contributions to the expression of gene networks. Jean-David Larouche  studied thymus development using published human scRNA-seq data. Broad profiles of TEs were found to be expressed in plasmacytoid dendritic cells, providing an association with the stimulation of a proinflammatory microenvironment in the thymus that is favorable to self-tolerance and T-cell development .In addition to influencing gene expression, TEs have the potential to impact other aspects of DNA metabolism. Grace Lee  reported that TE abundance is associated with reduced meiotic recombination. To study the nature of this relationship, her lab examined polymorphic TEs for epigenetic marks such as H3K9me2/3 that have the potential to inhibit meiotic recombination. By developing a unique method, pooled PacBio sequencing, for mapping crossover events in two Drosophila inbred strains with different TE profiles, they found that TEs with enrichment of repressive marks were associated with reduced rates of crossovers, indicating that TEs can be modifiers of meiotic recombination.Caenorhabditis briggsae, she found that two nematode gene families, wosp proteases and krma kinases, were acquired as cargo by Mavericks and transferred between different nematode species on a global scale. This work reveals how HGT can drive genetic incompatibilities in natural populations.The contributions of TEs to their host go beyond modifying existing sequences. Sonya Widen  described her discovery that Maverick TEs can function to horizontally transfer certain cargo genes between divergent species . While hATM revealed one exonic and 12 intronic loss-of function insertions, accounting for 2.1–5.5% of the cohort. This provides an initial estimate of the TE contribution to Mendelian disease [Although it is widely understood that TE insertions in early development or in the germline can result in spontaneous cases of disease such as Factor VIII hemophilia, there has been no systematic measure of TE-generated Mendelian disease. To address this question, Boxun Zhao  profiled retroelement insertions in whole-genome sequences (WGS) for 237 patients with ataxia telangiectasia. Analysis of the disease-causing gene PEG10, a domesticated TE gene that is necessary for placentation. Elevated expression of PEG10 has been implicated in a neurodevelopmental disease, Angelman’s syndrome. Earlier, she found that a proteasome shuttle factor, UBQLN2, facilitates degradation of the gag-pol from PEG10. Importantly, mutations in UBQLN2 cause the neurodegenerative disease ALS. Dr. Whiteley now finds that a nucleocapsid-like fragment of PEG10 localizes in the nucleus and alters expression of genes involved in axon remodeling [Another study of a neurological disease was presented by Alexandra Whiteley , who studies modeling . AdditioSession chair Harmit Malik  kicked off the session with a discussion of host-virus arms races, which drive the rapid evolution of viral and antiviral proteins. He focused on one rapidly evolving primate protein involved in retroviral restriction: TRIM5α. This work, led by former postdoc Jeannette Tenthorey, used deep mutational scanning to study how mutations in the region of TRIM5α involved in binding retroviral capsids, the ‘v1’ loop, affect its ability to restrict diverse retroviruses . His talZhao Zhang  presented his lab’s recently published work on how transposable elements hijack processes in the female Drosophila gonad to integrate into oocyte DNA and the role of extrachromosomal circular DNAs in this process . Dr. ZhaD. melanogaster and the simulans clade . Centromeres are essential structures for chromosome segregation during cell division but occur in rapidly evolving genome regions rich in repeats. Combining comparative genomics and cytology, Larracuente’s lab discovered dramatic centromere reorganization involving recurrent shifts between retroelements and satellite DNAs over short evolutionary timescales. They also revealed the recent origin of telocentric chromosomes in D. sechellia, where X and 4th chromosome centromeres now sit on telomere-specific retroelements [Amanda Larracuente  discussed centromere evolutionary dynamics among four closely related Drosophila species: elements . This raCaroline Langley  gave a short talk on host-virus arms races between rapidly evolving host APOBEC3 proteins involved in restricting retroviruses and viral-encoded infectivity factor (Vif). Langley’s focus was on the so-called arms race interface between human A3G and HIV-1 Vif, originally defined by patterns of molecular evolution and recently confirmed through cryo-EM structure . They shJosien van Wolfswinkel  gave a short talk on transposable elements and regulation of planarian stem cells (neoblasts). Planarians are well studied models for tissue regeneration and the expression of PIWI proteins in neoblasts is important for regulating transposons and the regeneration process. Van Wolfswinkel discussed how the loss of the nuclear PIWI protein SMEDWI-2 has a tissue-specific effect on transposable element de-repression, leads to changes in chromatin landscapes and ultimately, the failure to properly undergo cell differentiation . Van Wol trans-acting RNA nor MERV-L-encoded proteins are required for promoting the transition from totipotency to pluripotency, shifting the attention on the changes in chromatin state that cause improper expression of two-cell-specific genes [The opening talk by Haruhiko Siomi  reinforced the paradigm-shifting concept outlined in the keynote address that TEs do not need to be converted into single-copy domesticated genes to become integral components of the host pathways and developmental processes. Studies of the murine retrovirus MERV-L revealed that it regulates a critical developmental timing window that precedes zygotic genome activation (ZGA). During the first cleavage divisions, MERV-L is transiently upregulated, while its knockdown at this stage induces a prolonged 2-cell-like state that results in developmental defects, embryo degeneration and death. Surprisingly, neither MERV-Lic genes .Arabidopsis thaliana derived from distinct geographical regions. He reported strong geographic variation in TE load between accessions, underscored the role of TEs as major contributors to structural and epigenetic variation, and discussed the principal factors contributing to TE population dynamics, including genetic polymorphisms in different components of the epigenetic silencing machinery, and the influence of environmental factors such as temperature. TE fragments within transcriptional units were shown to induce silencing at the corresponding transcribed loci [Magnus Nordborg  presented a global overview of TE dynamics in 500 sequenced natural accessions of bed loci .Gal4-driven GFP insertion into the major Drosophila melanogaster germline dual-strand piRNA cluster 42AB. Promoter inducibility was used to assess the ratio of canonical transcription to non-canonical transcription, which is dependent on the RDC complex (Rhino-Deadlock-Cutoff). The team found that establishment of piRNA clusters is a much less straightforward process than previously thought, and requires maternal transmission of the cytoplasmic siRNA signal over several generations for robust establishment of non-canonical transcription and chromatin-based piRNA silencing, regardless of TE placement within any pre-existing piRNA clusters [Requirements for establishment of piRNA-based epigenetic silencing were uncovered by Alexei Aravin , whose lab studied a clusters .The short talk by Sharon Schlesinger  focused on the role of new players Smarcad1 and the histone variant H3.3 in reinforcing the familiar system of heterochromatin-based retroviral silencing in mouse ES cells based on the master regulator Trim28/KAP1, KRAB-Zn-fingers and SETDB1 H3K9 methyltransferase. She introduced a concept of “dynamic retrochromatin”, according to which the deposition of the replication-independent H3.3 helps to maintain prolonged silencing of retroviral sequences and relies on Smarcad1, an ATP-dependent nucleosome exchange factor which controls nucleosome disassembly and reassembly. Depletion of Smarcad1 led to retroviral de-repression and failure to recruit Trim28 and H3.3 to the silenced loci .https://ihec-epigenomes.org/). Spanning the best-characterized modifications of the H3 histone in 5472 ChIP-seq samples across 60 cell types, the analyzed dataset includes the marks typically associated with transcriptional activation at promoters or enhancers  as well as with repression . Initial analysis reveals that some TE families and subfamilies are disproportionally enriched in certain cell types, while others can be silenced across multiple cell types.Jeffrey Hyacinthe  in his short talk unveiled a global overview of the human epigenome as it relates to TEs, fueled by the wealth of data from eight consortia fed into the International Human Epigenome (super) Consortium (IHEC)  focused her short talk on a single antisense promoter of a specific human L1 copy, which is linked to silencing of an adjacent metastasis suppressor gene and drives expression of a colon-specific chimeric transcript joining L1 ORF0 to exons 2 and 3 of rvt) in helping bacteria, fungi and rotifers to overcome such stresses. The single-copy rvt genes represent a distinct type of reverse transcriptases (RTs) which are widespread in free-living, mostly soil-dwelling, organisms, evolve under purifying selection, and are often subject to horizontal transfers. The catalytic activity displayed by rvt proteins involves non-templated polymerization, and intact catalytic residues are required for alleviating the effects of stress.The concluding talk by session chair Irina Arkhipova  placed the emphasis on the host response to environmental stresses, such as amino acid starvation or the presence of certain antibiotic agents, and on the involvement of domesticated reverse transcriptase-like genes  presented a large body of work on the characterization of bacterial reverse transcriptases (RTs), including mobile group II intron RTs. He discussed work showing that mobile group II introns encode active RTs and elucidated group II intron retromobility mechanisms. He also presented work biochemically and structurally characterizing group II intron RTs and their use for biotechnological applications, including targetrons, the first RNA-guided gene targeting vectors, and TGIRT-seq, a high-throughput RNA-sequencing method that utilizes the advantageous biochemical properties of mobile group II intron RTs for comprehensive RNA profiling from small amounts of starting material, including tumors and blood . FunctioMiguel Branco  presented his work investigating ERV-derived enhancers in the placenta. He showed how ERVs provide functional species-specific enhancers in both mouse and human trophoblast cells, potentially contributing to species-specific biological differences affecting placental development and function . He alsoIFNAR2), which generated a broadly expressed truncated isoform that acts as a decoy receptor.Session chair Edward Chuong  presented his work on TE exonization affecting the evolution of immune gene isoforms. Long-read RNA-seq data enabled the discovery of novel or poorly characterized TE exonization events that show high levels of expression, in contrast to the general assumption that they are poorly expressed . He presP-element-like transposase fusion that is conserved in all jawed vertebrates. She described CRISPR knockout, RNA-seq, and ChIP-Seq characterization of THAP7 in human, mouse, and zebrafish cells. In zebrafish, THAP7 knockout was fatal, while in mouse, THAP7 knockouts show sex-dependent behavioral defects. Her work highlights a role for THAP7 in vertebrate development and human intellectual disability, and provides an exciting comprehensive study of the origin and function of host-transposase fusion genes.Rachel Cosby  presented her work on THAP7, a putative transcription factor derived from a Azra Lari  presented work exploring the role of B2 SINE retrotransposon activation in modulating mRNA isoform switching during gammaherpesvirus infection. Using long-read RNA-seq, she identified hundreds of host isoform-switching events dependent on virus-activated B2 SINEs and determined that these switching events were enriched for transcripts that encode innate immune factors. This work points to a mechanism that drives host-isoform switching during viral infection through the co-option of B2 SINE elements .Session chair Dixie Mager  opened the session with the talk by Kathleen Burns . She presented work demonstrating that the LINE-1 open reading frame 1 protein (ORF1p) is overexpressed in cancers and precursor lesions, while it has lower expression in normal tissues. ORF1p is highly expressed in solid tumors including ovarian, colon, uterine, gastro-esophageal, pancreatic, and breast cancers, among other malignancies. Dr. Burns described design and optimization of a single molecule array (Simoa) assay able to detect ORF1p levels in the blood, with significantly higher ORF1p in blood of cancer patients as compared to healthy controls . This biNF2, a tumor suppressor gene, as a candidate host factor necessary for L1-REPEL. Subsequent experiments revealed that NF2 appears to be necessary for the initiation, but not maintenance, of L1-REPEL. In sum, these findings suggest that NF2 plays a critical, albeit perhaps indirect, role in combatting unabated L1 retrotransposition in a hEC cell line that serves as a proxy for early stages of human development.John Moran  presented studies describing the epigenetic silencing of reporter genes integrated into the genomic DNA of a human embryonic carcinoma (hEC) cell line by human LINE-1 (L1) retrotransposition. LINE-1 reporter gene silencing  appears to be specific to retrotransposons that mobilize via TPRT (target-primed reverse transcription), and occurs either during or immediately after L1 integration. The treatment of hECs containing silenced L1-enhanced green fluorescent protein (L1-EGFP) integration events with the histone deacetylase inhibitor trichostatin A (TSA) led to a rapid activation of L1-EGFP reporter gene expression, whereas subsequent TSA removal led to the reestablishment of L1-EGFP reporter gene silencing, suggesting evidence of epigenetic memory. Dr. Moran then described how genome-wide CRISPR/Cas9-based genetic screens led to the identification of cis-interactions between enhancer and promoter regions to affect transcription and create boundary regions in between to segregate chromatin states. One example is an L1MC1-anchored loop that is human-specific that is lost upon deletion of the TE. These findings shed light on how transposable elements alter species-specific chromatin structure to control gene expression in mammals.Ting Wang  presented work on how transposable elements shape three-dimensional chromatin interactions in mammalian cells . TEs proKatherine Chiappinelli  presented work on epigenetic regulation of TEs in cancer genomes. Dr. Chiappinelli and colleagues demonstrated that the tumor suppressor P53 transcriptionally activates TEs, especially LTR and Alu elements, in cancer cell lines. Combining P53 activation with inhibition of DNA methylation increases TE transcriptional activation and hot spot mutant P53 cell lines exhibited differential TE regulation . Beyond cis-preference. Lower resolution structures of the full-length ORF2p show dynamic conformations of the N-terminal endonuclease (EN) and C-terminal domains that appear to allow ‘open’ and ‘closed ring’ states. The ORF2p structure is more similar to bacterial group II intron and other non-LTR RTs than to viral and LTR RTs. Biochemically, ORF2p is highly active and processive as both RT and DNA-directed polymerase but has limited RdRP activity. Unexpectedly, it also is efficiently primed by RNA duplexes and hairpins, including an Alu-derived RNA hairpin. In cells, ORF2p can synthesize inflammatory RNA:DNA hybrids in the cytosol, which are RT- but not EN-dependent, ruling out an origin from ‘nuclear ejection’ of TPRT intermediates. ORF2p structure explains the potencies of existing nucleoside inhibitors and the lack of inhibition by non-nucleoside RT inhibitors (NNRTI) of HIV, and provides a basis for rational design of specific LINE-1 RT inhibitors for novel therapies against cancer and autoimmune diseases.Martin Taylor  presented the structure of the LINE-1 ORF2 protein in different states. High resolution structures were determined for the ‘core’ ORF2p , by X-ray crystallography and cryo-EM in active conformations, bound to template RNA-only, and in apo form. The apo form is unstable and in a ‘thumbs up’ conformation can bind RNA, which may explain L1 The short talk by Siyu Sun  presented a comprehensive study of TEs in 214 total RNA-seq samples with 66 matched WGS data from rapid autopsies of patients with pancreatic ductal adenocarcinoma . In thisThis workshop, featuring a series of short talks, was devoted primarily to tool development in the most challenging areas of TE research, such as structural variation and methylome analysis in populations and single-cell analysis of TE transcripts and their isoforms. It was chaired by Arian Smit .Clément Goubert  presented GraffiTE, a workflow dedicated to study TE insertion polymorphisms from a wide range of data . The method introduces graph genomes to represent TE variation resulting in enhanced estimation of allele frequencies, and is applicable to models with no prior knowledge on TEs .Xiaoyu Zhuo , harnessing the high-quality data of the human pangenome reference consortium, demonstrated how PacBio CLR (continuous long reads) can be used to infer the methylation state of TEs across populations. Showing high correlation between the methylation status inferred from CLR reads and bisulfite sequencing, Dr. Zhuo showed that (i) most TE insertions are hypermethylated in the human population, (ii) Alu insertions in hypomethylated CpG islands have also low level of methylation, and (iii) there is a 3–4% increase in methylation around L1 and Alu insertions.Opening a series of talks on single-cell RNA-seq analysis of TEs, Jun Ding  introduced MATES (Multi-mapping Alignment for TE Expression quantification in Single-cell), a deep learning method to estimate a read’s multi-mapping probability based on the genomic context of each TE locus. This approach allows locus-specific quantification, showing improvement of cell clustering based on TE expression, and can be also used with single-cell ATAC-seq experiments.Tackling the same question with alternative algorithms, Matthew Bendall  presented Stellarscope, a single-cell version of TElescope, which uses Expectation-Maximisation to quantify TE expression. Notably, Dr. Bendall described an original approach using adjacency graphs to remove PCR duplicates, and demonstrated how different pooling modes available in the software help with the assignment of multi-mapping reads.Beyond their canonical expression, TEs can also be co-opted as alternative gene promoters. To identify and quantify such TE-derived transcripts across different tissues, cell types, or disease states, Bo Zhang  developed TSRdetector, which operates at single-cell resolution. Dr. Zhang’s application of TSRdetector revealed that ~ 15% of human protein coding genes show isoforms initiated by a TE.Helitron superfamily. Helitrons are particularly challenging to identify, as they do not bear typical TE signatures such as target site duplications or terminal repeats. To address this challenge, the approach searches for clusters of non-syntenic gene fragments, flanked by ATC (5′) or CTRR (3′) motifs, indicative of the Helitrons’ termini.Bimala Acharya  unveiled a method to detect genes trans-duplicated via rolling-circle elements of the Next, Anubhuti Mathur  described the company’s efforts to harness non-LTR retrotransposons for precise insertion of long sequences in the context of therapeutic genome editing. A bioinformatic search across all kingdoms of life yielded thousands of candidate elements, which were screened for activity in human cells. To further maximize efficacy, ancestral sequence reconstruction (ASR) was implemented to generate putative ancestral sequences to enhance the integration of transgene RNA. High-throughput screening of the ASR library highlighted the important role of engineered UTR sequences to increase gene writing efficiency .Cryptococcus, which can cause fatal meningitis, particularly in immunosuppressed subjects. Though mainly clonal, Cryptococcus display remarkable adaptation to heat stress, which occurs at human body temperature. In experiments at 37 °C, Cryptococcus showed increased drug resistance, driven by TEs as the primary source of mutation [In a closing talk, Asiya Gusa  presented her work on the environmental pathogenic fungi from the genus mutation . In the Session chair Orsolya Barabas  opened the session by discussing conjugative transposons carrying antibiotic resistance genes – a significant health challenge due to their linkage to the emergence of multidrug-resistant pathogens. Her talk built upon comprehensive work from her laboratory using tyrosine recombinases to annotate mobile genetic elements in prokaryotes . She desStaphylococcus aureus, which represent a significant public health problem worldwide.Phoebe Rice  continued the site-specific recombinase theme by describing structure-function studies of small and large serine recombinases and the different mechanisms by which their reaction directions are controlled. These recombinases are carried by the SCCmec mobile genetic elements conferring methicillin resistance to “MRSA” strains of Jeff Miller  switched gears to discuss diversity-generating retroelements (DGRs) in bacteria and how they accelerate protein evolution through mutagenic retrohoming . DGR-encThe session ended with two short talks on bacterial transposons from the Tn7 family. Elizabeth Kellogg  discussed cryo-EM work from her laboratory on one of the programmable CRISPR-Associated Transposons (CASTs) from this family . The CASAlba Guarné  presented the biochemical characterization and cryo-EM structure of the target-site selection complex from the prototypical Tn7 element. Building upon previous work from her laboratory describing the structure of the AAA+ ATPase adaptor protein TnsC , they weClosing remarks were delivered by Irina Arkhipova, who briefly outlined the history of the Keystone TE meetings dating back to 1991, and encouraged members of the community to attend upcoming in-person and/or hybrid TE meetings. The EMBO Workshop “The mobile genome: genetic and physiological impacts of transposable elements” took place in Heidelberg, Germany, on November 8–11, 2023. The International Congress on Transposable Elements (ICTE) in St. Malo, France will again welcome participants on April 20–23, 2024. The biannual Cold Spring Harbor meeting on Transposable Elements in Cold Spring Harbor, NY will follow on October 15-19, 2024, and the next FASEB Science Research Conference on mobile DNA is planned for the summer of 2025.The main outcome of the meeting was the establishment and maintenance of scientific interactions between researchers who would not have the time and opportunity to do so in their routine schedules. Such encounters have the potential to catalyze generation of non-trivial ideas and cross-fertilization of streamlined approaches established within narrowly specialized fields. The traditional free exchange of unpublished data is now greatly facilitated by the availability of preprints, which we opted to cite in this report for easier reference. The highly condensed three-day format minimized the available free time, but maximized the opportunities for younger researchers to have their science heard.In the future, we are expecting major advancements in the TE field to be driven by cutting-edge technologies such as determination of complete linear and spatial structures of eukaryotic chromosomes unveiling their true repetitive content, genetic manipulation of multicopy TEs validating their roles in the host biology, single-cell analysis techniques, advanced imaging and structural analysis of molecules and macromolecular complexes, and large-scale data processing with machine learning algorithms. Practical applications are expected to capitalize on TE potential as disease markers and causative agents, as well as vehicles for targeted insertion of large payloads.The post-pandemic reinvigoration of the mobile DNA field is manifested in a series of upcoming high-profile in-person and/or hybrid international meetings scheduled to occur in the coming two years on both sides of the Atlantic, relieving the travel burden to some extent. It is rewarding to see that the field is constantly attracting new organizers and new participants, helping to jump-start new collaborations and exchange ideas that will yield breakthrough discoveries in the years to come.


After fertilization, sperm and oocyte fused and gave rise to a zygote which is the beginning of a new life. Then the embryonic development is monitored and regulated precisely from the transition of oocyte to the embryo at the early stage of embryogenesis, and this process is termed maternal‐to‐zygotic transition (MZT). MZT involves two major events that are maternal components degradation and zygotic genome activation. The epigenetic reprogramming plays crucial roles in regulating the process of MZT and supervising the normal development of early development of embryos. In recent years, benefited from the rapid development of low‐input epigenome profiling technologies, new epigenetic modifications are found to be reprogrammed dramatically and may play different roles during MZT whose dysregulation will cause an abnormal development of embryos even abortion at various stages. In this review, we summarized and discussed the important novel findings on epigenetic reprogramming and the underlying molecular mechanisms regulating MZT in mammalian embryos. Our work provided comprehensive and detailed references for the in deep understanding of epigenetic regulatory network in this key biological process and also shed light on the critical roles for epigenetic reprogramming on embryonic failure during artificial reproductive technology and nature fertilization. Graphical Abstract: The DNA methylation, histone modification, chromatin structure, and RNA modification are important epigenetic modifications that are reprogrammed during the maternal‐to‐zygote transition (MZT). Their reprogramming processes are regulated by multiple underlying molecular mechanisms that were discussed in the paper. The candidate regulators, such as the RNA methylation, BTG4, CCR4‐NOT, tut4/7, and NR5A2, are involved in the maternal mRNAs degradation and zygotic genome activation initiation during MZT. Therefore, it provided detailed and comprehensive resources for further investigation on epigenetic regulatory network during this key biological process.  At the developmental stage, both parental genomes of the zygote must be reprogrammed to accomplish the transition from terminally differentiated state to totipotency state.In addition, zygotic genome activation (ZGA) occurs gradually after fertilization, which is a process that both maternal and paternal genomes begin to be activated in a temporally controllable manner.6A modification are recently founded to be dynamically regulated during MZT.6A modification. Furthermore, the post‐translational modifications of nonhistone proteins and candidate regulators participated in the MZT were also highlighted.Epigenetic modifications which could regulate the gene expression without alterations in DNA sequence include DNA/RNA modifications, post‐translational modifications of histone, and three‐dimensional (3D) chromatin organization.2Epigenetic modifications can cause heritable changes in gene expression but do not alter the nucleotide sequence.3DNA methylation is the most widely studied form of epigenetic modification and plays an important role in regulating gene expression.3.1Nlrp14 maternal knockout  zygotes.DNA methylation in oocytes is distinct, covering approximately 40% of the genome, and is exclusively deposited in actively transcribed gene bodies. Furthermore, DNA methylation is evenly distributed and covers about 90% of the sperm genome.3.2The maternal genome was demethylated mainly in a passive manner rather than active manner.However, the predominant DNA demethylation in paternal pronuclei remains controversial. The classical model postulates that the paternal genome undergoes extensive DNA demethylation before replication and maternally inherited TET3 plays an important role in this process, which was based on the results of immunofluorescence staining.3.3In fact, DNA demethylation is accompanied by selective maintenance and de novo methylation. Some genomic regions, including ICRs and certain repetitive sequences, remain methylated during global demethylation in early embryos.4Histones are the main protein components of chromatin, and their post‐translational modifications play pivotal roles in the regulation of chromatin structure remodeling and many biological processes.4.14.1.1H3K4me3 is strongly enriched in promoter region of active genes in nongrowing oocytes (NGOs), whereas it exists as a noncanonical pattern (ncH3K4me3) that covers broad domains in promoters, gene bodies, and intergenic regions in mouse oocytes at subsequent stages.However, H3K4me3 is enriched in promoter regions in human germinal vesicle (GV) and MI oocytes, unlike the broad ncH3K4me3 in mouse oocytes. Moreover, in human 4‐cell embryos, the H3K4me3 of promoter is wider than that at other stages and about half of these promoters are activated upon ZGA.4.1.2Kmt2b with Gdf9‐Cre leads to oocyte loss, abnormal ovulation, and female sterility, accompanied by significantly decreased H3K4me3.Kmt2b causes the arrest of majority of embryos between one‐ and four‐cell stages with defective ZGA, and the dramatic decrease of H3K4me3 in these embryos.Kmt2b KO oocytes is similar to that of WT NGO oocytes, lacking the broad domains and distal peaks, which further indicates that Mll2 is required for the deposition of ncH3K4me3 but not canonical H3K4me3 during the process of oogenesis.Kmt2b has a limited influence on gene expression in GV oocytes but causes defective transcriptional repression in peri‐ovulatory oocytes.Zygote ncH3K4me3 is inherited from oocytes. It is reported that KMT2B and CXXC1‐SETD1 are required for the deposition of H3K4me3 on oocyte genome, functioning complementarily by targeting different areas of the oocyte genome.Setd1a in oocytes has no impact on fertility.Setd1b oocyte‐specific knockout reduces the amount of follicular. The ovulated MII oocytes exhibit an abnormal distribution of cytoplasmic organelles, accumulated lipid droplets, and dysregulated gene expression. Consequently, most Setd1b null embryos are arrested at the pronuclear stages with defective zona pellucida.Setd1b null oocytes.Setdb1 deletion. Meanwhile, H3K4me3 is widely deposited in MLL2 targeted regions that are across CpG‐rich sequences and hypomethylated DNA, indicating increased or misdirected KMT2B activity in certain regions.It was reported that the SETD1 histone H3K4 methyltransferase complex contributes to the generation of H3K4me3 in oocytes.Cxxc1‐maternal deletion, including the genes correlated with mRNA synthesis, translation, and degradation.Btg4) important for maternal mRNA decay further prevented the maternal mRNAs degradation in mouse CXXC1 null oocytes and zygotes.CXXC finger protein‐1  is the key subunit of the SETD1 component, which can recognize and bind to specific genomic regions. Similar to SETDB1, maternal CXXC1 is essential for oocyte maturation and embryo developmental after fertilization.4.1.3Kdm5a/5b leads to a high level of H3K4m3 at the late 2‐cell stage, as well as the downregulation of a large number of ZGA genes and developmental arrest before the blastocyst stage.In late 2‐cell stage embryos, ncH3K4me3 is replaced by canonical H3K4me3, and ZGA is indispensable for this transition.4.1.4SETD1B or CXXC1 deficiency mouse oocytes, accompanied by the unchanged abundance of DNMTs.In oocytes, H3K4me3 and DNA methylation are mostly mutually exclusive on the genome, and the deposition of these two modifications occurs in parallel.4.24.2.1H3K27me3 and H2AK119ub1, two hall markers of polycomb domains, are reprogrammed during early embryo development.After fertilization, H3K27me3 in promoter regions of developmental‐related genes is largely erased in mouse embryos and reestablished in post‐implantation embryos.For H2AK119ub1, some groups found that the newly established H2AK119ub1 in paternal pronuclei is mainly enriched in gene desert regions in zygotes. In maternal pronuclei, the distribution of H2AK119ub1 on maternal genome largely resembles that in MII oocytes, which indicated the inheritance from oocytes. Sperm H2AK119ub1 is largely erased after fertilization. Moreover, the H2AK119ub1 domains on maternal genome are more than that on paternal genome in zygotes.4.2.2The establishment of H3K27me3 is catalyzed by PRC2 during mouse oocyte growth.USP16 deletion prevents the ZGA and causes nearly half of the embryos arrested at the 2‐cell stage, which may be attributed to the retention of H2AK119ub1 at the TSSs of early zygotic genes in oocytes. It seems that the removal of maternal H2AK119ub1 regulated by USP16 may be a prerequisite for proper ZGA at the 2‐cell stage embryos.H2AK119ub1 is co‐established with H3K27me3 during oocyte growth, which is mediated by PRC1 and participates in transcriptional repression.PRC2 depletion does not affect H2AK119ub1 in oocytes but disrupts the deposition of H2AK119ub1 at some H3K27me3 imprinting loci in morulae.PRC1 deficiency not only severely impairs the formation of H2AK119ub1 but also causes gene‐selective loss of H3K27me3 in FGOs. More importantly, this H3K27me3 loss is inherited by embryos, leading to defective H3K27me3‐dependent imprinting and placental enlargement.Interestingly, there are some interactions between the formations of H3K27me3 and H2AK119ub1. The removal of H3K27me3 upon 4.2.3Kdm6b mRNA injection affects the paternal allele‐biased expression of putative H3K27me3‐dependent imprinted genes rather than the canonical imprinted genes.After fertilization, the H3K27me3 on promoter is largely erased in a ZGA‐independent (minor or major) manner.4.2.4Ring1a/1b knockout mouse FGOs.H2AK119ub1 mostly deposited in the promoters of transcriptionally silenced genes that modified by H3K27me3.4.34.3.1After fertilization, the parental H3K9me3 undergoes global reprogramming in mouse embryos.4.3.2KDM4A ablation oocytes. Moreover, embryos derived from these oocytes show defective ZGA and preimplantation lethality.SUV39H2.In oocytes, SETDB1 mediates the deposition of H3K9me2/3 and is essential for meiotic progression and preimplantation development.4.3.3In zygotes, the bias deposition of H3K9 methylation in the maternal pronuclei contributes to preventing TET‐mediated 5mC oxidation on maternal genome, which is mediated by EHMT2 and SETDB1.4.44.4.1H3K27ac exists as a noncanonical broad pattern (ncH3K27ac) in GV stage and becomes very weak in MII mouse oocytes.4.4.2+‐SIRT1‐mediated deacetylation facilitate the erasure of H3K27ac and the timely silencing of minor ZGA genes.Broad ncH3K27ac domains are mainly located in gene dense regions and strongly correlated with ncH3K4me3 in mouse zygotes.+ supplement promotes the removal of H3K27ac and improves the blastocyst rate of human intracytoplasmic sperm injection (ICSI) embryos. It seems that the NAD+‐SIRT1‐mediated deacetylation also plays an important role in human embryo development.In human, broad H3K27ac domains are highly correlated with broad H3K4me3 domains before ZGA. Moreover, HDACs are required for the transition of broad H3K27ac domains to narrow peaks at the ZGA stage.5In eukaryotic cells, the genomic DNA is packaged into a hierarchical structure in nucleus, which is essential for proper regulation of gene expression.5.1Studies using Hi‐C technology have revealed that the entire genome can be divided into two compartments at a solution of 1Mb, called compartment A/B.5.2TADs are smaller units of 3D nuclear organization with two features: self‐association and insulation from regions outside the domains.5.3Lamina‐associated domains (LADs) are genomic regions that interact with the nuclear lamina (NL), which are associated with the chromosome organize inside the nucleus and gene repression.5.4Domains that are self‐interacting, marked by noncanonical H3K27me3 and cohesion‐independent, are termed polycomb‐associating domains (PADs).6N6‐methyladenosine (m6A) is the most abundant modification on RNA and is reversible in eukaryotic cells.6A is essential for maternal RNA degradation and early embryo development.6A level decreased from GV to 2‐cell stage but increased gradually after ZGA by immunofluorescence staining.6A decreased continuously during mouse MZT, but the number of m6A marked transcripts increased after fertilization  and 2‐cell marker genes (like Dux and Zscan4).6A methyltransferase METTL3 is important for both oocyte and embryo development. In mouse oocytes, knockout of Mettl3 not only affects mRNAs degradation but also disrupts mRNAs translation efficiency, which lead to a low maturation rate.6A on ZGA transcripts and MERVL, and regulating the timely degradation of these transcripts.6A binding protein, YTHDF2 is abundant in oocytes and mediates RNA degradation process through recognizing and destabilizing m6A‐modified mRNAs.YTHDF2 deficiency oocytes can develop to MII stage, the derived embryos can develop beyond 2‐cell stage with the repressed maternal mRNAs decay.6A reader, IGF2BP2 is also required for early embryonic development.IGFBP2 causes developmental arrest of majority of embryos at the 2‐cell stage and decreases both transcriptional and translational activity of transcriptions during ZGA.6A‐dependent manner remains to be explored.n Figure .156 In7Besides histone proteins, various studies have demonstrated that post‐translational modification of nonhistone proteins plays an important role in regulating the proper MZT.7.1Wee1b (Wee2) caused by homozygous mutation of Wee1b is also found to be responsible for the occurrence of fertilization failure.Protein phosphorylation is an extensively studied post‐translational modification that involves in many biological processes in eukaryotes.Besides, ERK1/2‐mediated phosphorylation is also essential for oocytes oocyte meiotic maturation and MZT.7.2Rnf114 results in abnormal protein metabolism in oocytes, and most maternal KO embryos fail to develop beyond the 2‐cell stage with major ZGA defects.Tab1 or Cbx5, can partially rescue the abnormal development of maternal Rnf114 KO embryos.Protein ubiquitination, an important post‐translational modification, is involved in ubiquitin‐proteasome pathway.8MZT involves maternal material decay and zygotic genomic activation, which is crucial for early embryo development.8.1In oocytes, numerous mRNAs are translationally repressed and stably accumulated in cytoplasm and will be recruited for translation during oocyte meiotic maturation and early embryonic development.8.1.1ZAR1 deficiency causes delayed GVBD and PB1 emission in oocytes, with the disruption of MARDO and the premature loss of mRNAs that localized in MARDO.ZAR1 null oocytes are blocked at 1‐cell stage embryos, resulting in the infertility of ZAR1 maternal KO female mice.Zar2 is the homolog of Zar1, which is dispensable for the development of oocyte and female fertility. However, the double deletion of both ZAR1 and ZAR2 leads to a severer phenotype than ZAR1 deletion, which indicating that ZAR2 seems to enhance the function of ZAR1. ZAR1/2 knockout oocytes are primarily arrested at MI stage with impaired mitotic resumption and emission of PB1 and spindle assembly defects, implicating maternal mRNA instability and impaired translation of important oocyte proteins. Moreover, ZAR1/2 maternal deficiency embryos also fail to develop beyond the 2‐cell stage.Recently, Cheng et al. reported that the maternal mRNAs are stored in a mitochondria‐associated membranelles compartment (MARDO) in mammalian full‐grown oocytes. RNA‐binding proteins like ZAR1, MSY2, DDX6, LSM14B, and 4E‐T are localized in MARDO. Moreover, ZAR1 is essential for the assembly and coalescence of the MARDO around mitochondria. 8.1.2Btg4 does not affect the maturation of oocytes, embryos derived from Btg4−/− females are arrested at the 1‐ or 2‐cell stage with M‐decay defect.Pabpn1l also causes the embryo arrest at the 1‐ or 2‐cell stage.Btg4 causes zygotic cleavage failure and impairs the degradation of maternal transcripts in human embryos.Mos, which encodes the upstream kinase of ERK1/2, also disrupts maternal mRNA clearance in human oocytes, and causes female infertility, characterized by human early embryonic arrest.It is reported that maternal‐factor BTG4 is essential for M‐decay in mouse oocytes and zygotes. Although the knockout of CNOT6L occurs earlier and in a wider range than that triggered by BTG4‐CCR4‐NOTCNOT7/8.CNOT6L−/− oocytes. Consequently, majority of zygotes derived from these oocytes failed to develop beyond the 4‐cell stage.CNOT6L guided maternal mRNA decay has been demonstrated to be necessary for accurate MZT in human. The biallelic mutation in Zfp36l2 perturbs the removal of massive ARE‐containing transcripts in human zygotes and is potentially responsible for the occurrence recurrent preimplantation embryo developmental arrest.6A modification is important for maternal mRNAs degradation during MZT.6A reader YTHDF2 mediates the mRNAs degradation by recruiting CCR4–NOT complex (binds CNOT1 subunit) in mammalian cells.Another RNA binding protein ZFP36L2 can bind ARE‐harboring maternal mRNAs (A/U‐rich element) and recruit the CCR4‐NOT complex (binds CNOT6L subunit) to promote the degradation of these mRNAs during meiotic maturation in mouse oocytes. The degradation mediated by ZFP36L2‐CCR4‐NOT8.1.3197 Moreover, it is found that the loss of BTG4, CNOT7, or TUT4/7 in zygote impaired several Z‐decay mRNAs degradation.Tut4/7.Ago2 is reported to be highly expressed in mouse oocytes and early embryos. The deletion of Ago2 in mouse zygotes impairs the development of early embryos and the degradation of over half of the maternal mRNAs.Z‐decay transcripts tend to have longer 3′‐UTRs and higher translational activity than M‐decay transcripts in matured oocytes.8.2To date, the key factors required for ZGA initiation in mammal embryos are largely unknown. Pioneer factors are a class of transcript factors (TFs), which can bind near chromatin directly and subsequently initiate gene expression.Recently, maternally inherited orphan nuclear receptor NR5A2 is reported to be an important pioneer TF for genome‐wide gene activation during ZGA in mouse embryos.Mlx or Rfx1 leads to significant downregulation of minor or major ZGA genes, respectively.Rfx1 causes embryo lethality before the morula stage.Another report indicated that MLX and RFX1 were important for the nucleosome‐depleted regions formation in promoters of minor or major ZGA genes in the paternal pronuclei of the mouse ICSI embryos.Dux4 in human induced pluripotent stem cells or Dux in mESCs can activate the transcription of cleavage‐specific genes and ERVL‐family retrotransposons.Dux homozygous knockout mice can survival to adulthood, which further indicates that DUX does not play a major role in ZGA initiation in mouse embryos.Dux silencing at DNA level as well as the degradation at RNA and protein levels are necessary for the exit of 2‐cell state and embryo development.Besides, DUX and its human ortholog DUX4 are found to be expressed before major ZGA in mouse or human embryos, respectively.9In this review, we comprehensively summarized the current knowledge of epigenetic reprogramming during MZT. During MZT, embryos undergo temporally controlled maternal decay and zygote genome activation, with dramatic global epigenetic reprogramming.CXXC1‐depleted mouse oocytes, H3K27me3/H2AK119ub1 is increased mainly in gene body regions where DNA methylation was significantly decreased.Accumulative evidences indicated that different types of epigenetic modifications could work with each other. It has been found that H3K4me3 is positively correlated with DNA methylation by repelling DNA methyltransferases, whereas H3K9 methylation in the maternal pronuclei protects maternal genome from TET3‐mediated DNA demethylation.Furthermore, the relationship between epigenome reprogramming and ZGA remains unclear. Several reports proposed that the clearance of H3K27me3 and the establishment of higher order chromatin conformation can occur without transcriptional activation of the zygotic genome.Xiangpeng Dai and Yurong Chen conceived the topic and Yurong Chen wrote the manuscript with part help from Luyao Wang and Fucheng Guo. Xiaoling Zhang and Xiangpeng Dai edited the manuscript. All authors read and approved the final manuscript.The authors declare that they have no conflicts of interest.Not applicable.


All the cells of an organism contain the same genome. However, each cell expresses only a minor fraction of its potential and, in particular, the genes encoding the proteins necessary for basal metabolism and the proteins responsible for its specific phenotype. The ability to use only the right and necessary genes involved in specific functions depends on the structural organization of the nuclear chromatin, which in turn depends on the epigenetic history of each cell, which is stored in the form of a collection of DNA and protein modifications. Among these modifications, DNA methylation and many kinds of post-translational modifications of histones play a key role in organizing the complex indexing of usable genes. In addition, non-canonical histone proteins , the synthesis of which is not directly linked with DNA replication, are used to mark specific regions of the genome. Here, we will discuss the role of the H3.3 histone variant, with particular attention to its loading into chromatin in the mammalian nervous system, both in physiological and pathological conditions. Indeed, chromatin modifications that mark cell memory seem to be of special importance for the cells involved in the complex processes of learning and memory. Since Gurdon’s experiments , based oMost importantly, the chromatin structure differs at the level of different genes that, depending on their structural organization, can be transcriptionally repressed or active, and this is why different cells are able to express different and specific genes, as well as a family of common genes involved in basic metabolism. Thus, specific properties and the behavior of somatic cells do not depend on changes in the genotype but on the specific arrangement of chromatin, according to the concept of “epigenetics” proposed by Waddington many decades ago , which iThe chromatin structure is highly dynamic and changes in different cells during development and differentiation; it also changes in terminally differentiated cells in response to specific inducing factors, such as, for example, thyroid or steroid hormones . At leasHerein, we will mainly focus on the latter mechanism  are synthesized exclusively during the S phase of the cell cycle when DNA is replicated. The corresponding genes are highly repeated and very often clustered; moreover, they do not contain introns, and the corresponding mRNAs are not poly-adenylated ,20,21. ADrosophila fruit fly, the effects of a diet containing low amounts of branched-chain amino acids (BCAAs) were analyzed. In particular, the authors found that total histone H3 abundance decreased in flies fed a low BCAA diet, while H3 mRNA increased [Many years ago, our group showed that two histone variants, i.e., the linker histone H1.0 and the core histone H3.3, are specifically expressed in the rat brain during brain maturation . Interesncreased . This obncreased .H3.3A and H3.3B , which are located on different chromosomes. While the distribution of exons and introns, as well as promoters and other regulatory regions, are different in the two genes, the corresponding proteins are identical [It is also worth noting that two genes encoding the H3.3 histone are present in mammals: dentical  and alsodentical . In partdentical , althougdentical .Actually, the H3.3 protein is not so different with respect to the canonical H3.1 and H3.2 isoforms: indeed, it differs by only five and four amino acids, respectively, from them ,36,37. HDrosophila cells, H3.3 is the only H3 species deposited in chromatin in a replication-independent way, and they suggested that this event might be responsible for the activation of genes previously silenced because of histone PTMs [The H3.3 variant is indeed very often bound to active chromatin and regulates transcription. In 2002, Ahmad and Henikoff reported that in one PTMs . As a coone PTMs ,45,46.It was also reported that some chromatin remodeling complexes, such as the SWItch/Sucrose Non-Fermentable (SWI/SNF) complex and, in particular, its subunit “T-rich interactive domain-containing protein 1A” (ARAD1A) are required for maintaining the H3.3 histone at the level of regulatory sequences, among which are the so-called super-enhancers . On the Drosophila. CHD1 is indeed involved in the loading of H3.3 in the fly brain, where it seems to contribute to the regulation of genes that control the homeostasis of hunger and satiety signals [Drosophila male germline maturation [Recently, a brain-specific function of the chromodomain-helicase-DNA binding protein 1 (CHD1) which is a member of the SWI/SNF family of chromatin remodeling complexes was reported in  signals . On the turation .Further work is required to understand how H3.3, which, as mentioned above, is not so different from the canonical H3 species, can stimulate transcription. Evidently, its sequence should contain features that are able to attract, directly or indirectly, the transcriptional apparatus to the genes to which it is bound. Interestingly, for example, the H3.3 amino-terminal tail contains a serine residue (S31) that is not present in the other H3 species . Moreover, this serine can be phosphorylated, and it has been suggested that this might represent a feature determinant for preferential transcription . It shouOne possible link between H3.3 modification and gene expression was suggested by Martire and co-workers . Using mAs mentioned above, all the cells of the same organism have the same genome. However, thanks to rearrangements in the structural organization of chromatin, each cell expresses only the genes necessary for basal metabolism, as well as the genes involved in its tissue-specific functions. The cell-specific overall organization of chromatin, in turn, derives from the specific history of that cell. In other words, we can state that the nuclear organization of the genome is a form of cell memory, since, at any moment, it reflects the experiences that a cell has had in its life. This epigenetic memory is built up with the specific combination and location of the different histone variants and with their specific post-translational modifications (PTMs) ,60, thusInterestingly, many chromatin-specific marks should be transmitted to the daughter cells during each cycle of DNA replication and cell division ; if the As for other cells, epigenetic marks, such as histone acetylation and DNA methylation, accumulate in neuronal chromatin during cellular responses to received internal and/or environmental stimuli ,77,78,79It is also worth noting that, recently, a new kind of histone PTM was described: modification by monoamine neurotransmitters, such as dopamine and serotonin ,86,87,88In addition to PTMs, loading of the H3.3 histone variant onto chromatin seems to be required for neuronal differentiation and, in particular, for activation of mature neuron-specific genes ,95. For Now, if we accept the principle that nuclear chromatin organization confers to each cell a specific phenotype because it represents the epigenetic memory of the history of that cell , then weIn addition to a variety of histone PTMs, the H3.3 histone is also enriched on active chromatin related to memory storing , and it As mentioned above, the H3.3 histone also has a role to play at certain heterochromatic regions, such as telomeres and pericentric repeats, where it is trimethylated at the level of its lysine 9 (H3.3K9me3), a modification bound to its ability to stabilize heterochromatin . RecentlThe importance of the H3.3 histone in brain development was confirmed with the discovery and analysis of different neurodevelopmental disorders. For example, mutations in the H3.3A/H3.3B genes have been indicated as new candidates for microcephaly, and intellectual disability, as well as in a novel neurodevelopmental disorder now known as Bryant-Li-Bhoj syndrome ,106,107.Drosophila models of HD [Some studies have also shown that PTMs of histones can contribute to the pathogenesis of neurodegenerative diseases, such as Parkinson’s disease (PD) and Huntington’s disease (HD). In an experimental model of PD, exposure of dopaminergic cells (N27 cells) from the rat midbrain to pesticides, i.e., neurotoxic substances, induced hyperacetylation of the core histones H3/H4 and the subsequent death of dopaminergic neurons. The inhibition of hyperacetylation was neuroprotective, preventing N27 cell apoptosis . Notablyls of HD . Alteratls of HD . Howeverls of HD .Interestingly, it was also suggested that mutant huntingtin, which accumulates in HD, can interact with HAT enzymes, inducing a decrease in their activity ,117. ThiNotably, alterations of histone sequences and PTMs play a role in cancer ,120. As Interestingly, a variety of somatic mutations have been discovered in histone genes ,125. It Now, cancer cells are clearly characterized by modification of their metabolism: in particular, they show an increase in lipid metabolism, thanks to which they can produce higher amounts of membranes in order to proliferate. Synthesis of higher amounts of lipids, in turn, requires reduced nicotinamide adenine dinucleotide phosphate (NADPH). Indeed, it was recently demonstrated the connection between breast cancer metastasis and the H3.3-driven upregulation of NADK enzyme that, in turn, favors NAPDH production, thus sustaining proliferation . AnotherA mutation of both H3.3 and H3.1 histones has been found in diffuse intrinsic pontine glioma (DIPG), a very aggressive tumor with a median overall survival of 8–11 months. This kind of tumor belongs to the category of diffuse midline gliomas (DMG) and primarily occurs in children. The lysine to methionine mutation at position 27 (K27M) of the H3.3 histone, in particular, is often associated with TP53 and ATRX mutations . These lOne of the suggested H3.3 interactors is polycomb repressive complex 2 (PRC2), a methyltransferase involved in the control of gene expression and, more precisely, in the silencing of the promoters of specific genes by the trimethylation of lysine 27 of the H3 histones. In particular, in vitro studies showed binding between PRC2 and the mutated H3.3 ,136. TheAnother frequent mutation found in histone H3.3 in gliomas is the glycine to arginine  variation at position 34 (G34R/V). It has been suggested that this mutation affects the activity of the enzymes that modify the status of lysine 36 methylation, but a clear mechanism deriving from the original defect is not yet known. Differently from the K27M mutation, which can affect both the H3.3 and H3.1 histones, the G34R mutation is specific to the variant H3.3 .As already mentioned, ATRX is a partner of the DAXX chaperone and forms with it a complex that deposits H3.3 on chromatin. A mutation to the components of this complex is frequent in different cancers and is associated with the pathway called alternative lengthening of telomeres (ALT) .In many pediatric high-grade gliomas, ATRX is mutated, and, in the majority of cases, the evidenced mutation is associated with either the H3.3 K27M or H3.3 G34R/V mutation .Moreover, the G34R/V mutation causes a reduced capacity of DNA repair in patients suffering from pHGG (high-grade glioma), leading to an enhanced susceptibility to DNA damage, also from radiotherapy .In the alveolar rhabdomyosarcoma, H3.3 is overexpressed both in vivo and in cell lines: in this latter system, knockdown of the H3F3A gene reduces the ability of the cell to migrate, suggesting that the H3.3 variant regulates genes involved in motility, like melanoma cell adhesion molecule  .Mutations of the H3.3 histone were also described in bone and cartilage cancers, such as the giant cell tumor (GCT) of bone . One H3.In conclusion, all the cells of an organism are able to store memory of their past experiences in the form of specific modifications to chromatin structural organization. On the other hand, nerve cells not only store information related to the steps that allowed their development and differentiation, but they also store information related to events that determine macroscopic phenomena known as learning and memory. However, at the molecular level, the basal mechanisms that allow cognitive acquisition are very similar to the ones used by all cells to remember their history. Indeed, DNA methylation, histone PTMs of many kinds, and histone variant loading into chromatin constitute a collection of marks that is able to index the different genes to be transcribed. All these events, as discussed, are of central importance both for the general and specific structural organization of genes, and they also have an effect because of the existing interactions among different marks, as well as the binding of further factors (the readers), which are able to recognize the marks. Most importantly, long-term potentiation relies on these kinds of events, and it is induced mostly in the periphery of the nerve cells, as it depends on the signals that arrive through a variety of mechanisms, such as wiring (synaptic) and volume transmission  . Therefo