Mammalian brains have evolved in stages over a long history to acquire higher functions. Recently, several transposable element (TE) families have been shown to evolve into cis-regulatory elements of brain-specific genes. However, it is not fully understood how TEs are important for gene regulatory networks. Here, we performed a single-cell level analysis using public data of scATAC-seq to discover TE-derived cis-elements that are important for specific cell types. Our results suggest that DNA elements derived from TEs, MER130 and MamRep434, can function as transcription factor-binding sites based on their internal motifs for Neurod2 and Lhx2, respectively, especially in glutamatergic neuronal progenitors. Furthermore, MER130- and MamRep434-derived cis-elements were amplified in the ancestors of Amniota and Eutheria, respectively. These results suggest that the acquisition of cis-elements with TEs occurred in different stages during evolution and may contribute to the acquisition of different functions or morphologies in the brain. A public dataset of scATAC-seq is analyzed to identify transposable element-derived cis-elements specific to certain cell types, possibly contributing to different functions or morphologies in the brain. These neurons are differentiated from neural progenitors during embryonic and adult neurogenesis4. For adult neurogenesis, neural stem cells (NSCs) are first committed to proliferative neural progenitor cells (NPCs) and then become neuronal progenitor cells and neuroblasts4. These cells then differentiate into glutamatergic excitatory neurons or GABAergic interneurons under the control of various transcription factors at different differentiation states4. In addition, transcription factors such as Neurod2 and Lhx2 play various roles in neurogenesis. For example, Neurod2 induces neuronal differentiation in NPCs and contributes to neuronal maturation7. Lhx2 has multiple spatiotemporal roles by regulating the proliferation and differentiation of NPCs, as well as area patterning and axonal wiring in postmitotic neurons8. Recent studies have shown that while adult neurogenesis occurs mainly in the subgranular zone (SGZ) or the subventricular zone (SVZ), it also could occur in other regions, such as the cortex9. In addition, environmental factors, such as stress and injury, are involved in regulating the generation and survival of newborn neurons11.In the mammalian brain, neurons are mainly responsible for the transmission and processing of information in the brain, and there are various spatiotemporal subtypes, including layer-specific excitatory neurons in the cerebral cortex13. Among them, pseudo-time analysis can characterize the different stages of neurogenesis in single-cell levels. For example, pseudo-time analysis with single-cell RNA-seq (scRNA-seq) revealed gene expression profiles during glutamatergic neurogenesis in the adult dorsal ventricular-subventricular zone (V-SVZ) and GABAergic neurogenesis in the adult ventral V-SVZ14. As for epigenetic analysis, some studies have reported dynamic changes in chromatin accessibility profiles during neurogenesis using single-cell assays for transposase accessibility by sequencing (scATAC-seq) in mammals16. scATAC-seq analyses in the brain have also revealed transcription factor motifs accessible in a cell-type-specific manner in spatiotemporally diverse neuronal subtypes 19. However, it remains to be elucidated how cell-type-specific regulatory mechanisms are acquired.Recent single-cell analyses have shown that spatiotemporally diverse neuronal subtypes have distinct genomic profiles22. TEs are DNA sequences that can change their position within a genome and occupy approximately half of the mammalian genome24. TEs are subdivided according to their evolutionary origin, metastasis mechanism, and sequence characteristics. For example, in the mouse genome, there are approximately 52 families and 790 subfamilies23. Specific types of TEs can function as gene regulatory regions by serving as the origin of transcription factor (TF) binding sites27. Most TEs that function as transcription factor-binding sites also contain transcription factor-binding motifs, suggesting that transcription factors bind to TEs in a sequence-dependent manner25. For example, recent studies reported that MER130 TEs function as cortical-specific enhancers based on their internal motifs27. TEs may also be important factors for the acquisition of novel cis-elements during evolution. Some groups suggested that TEs are involved in acquiring cis-elements, including transcription factor-binding sites that drive morphological evolution in mammals30. For example, recent studies have shown that the acquisition of AmnSINE1-derived enhancers in mammalian ancestors may lead to the acquisition of mammalian-specific brain formation32. These studies suggest that TEs may contribute to the acquisition of cell-type-specific cis-elements by inducing the binding sites of transcription factors. However, the relationship between TE-derived DNA elements and transcription factor binding at the single-cell level remains to be elucidated.Previous and recent studies have proposed that transposable elements (TEs) may be important in understanding gene regulatory mechanisms and their origins33. Specifically, using scATAC-seq data with labels , we first detected transcription factor motifs accessible in a cell-type-specific manner . First, we computed the bias-corrected accessibility deviations for each 7-mer with chromVAR34 (see “Methods”). Visualization of the similarity among cells with t-distributed stochastic neighbor embedding (t-SNE) showed that cells were characterized as upper/lower layer excitatory neurons, inhibitory neurons, and glial cells , we first investigated whether cell types are characterized by k-mer chromatin accessibility profiles with scATAC-seq (accessibility across peaks sharing the k-mer), using cells with known cell-type labels, each containing more than 100 cells . The variability scores of the seed k-mers in chromVAR34 were as follows: de novo motif 1: CAGATGG was 3.7, de novo motif 2: CACCCAC was 3.0, de novo motif 3: CTAATTA was 2.1, and de novo motif 4: ACAGCTG was 2.1.Then, using the k-mer accessibility profiles, we generated de novo motifs associated with high variability in chromatin accessibility across the cells with chromVAR’s assembleKmers functionz-scores calculated with chromVAR34 for each cell on t-SNE  similar to each of the de novo motifs  transcription factors, such as Neurod2, Neurog2, and Neurod136. This suggests that this motif is implicated in the differentiation of glutamatergic neurons7 . We obtained several known transcription factor candidates whose motifs are significantly (13 and gene activity levels using scATAC-seq data33 (see “Methods”). We confirmed that specific transcription factors of the candidates that regulate neural differentiation and/or neuronal activity are highly expressed in each cell population40 , Lhx2 for de novo motif 3 (accessible in excitatory neurons and astrocytes), and Tcf4 for de novo motif 4 (accessible in interneurons and inhibitory neurons) were estimated to be transcription factors that contribute the most to the de novo motif accessibility , whereas genes related to maturation, such as Mef2c44, were more active in branch    between branches  and . The results showed that transport, stress response, and blood circulation were the top enrichment terms for genes that were highly active in branch  . The marker genes of glutamatergic neuronal progenitor cells and neuroblasts, such as 34 and compared them between the branches. We found that the seed k-mers of de novo motif 1 (Neurod2-like motif) and de novo motif 3 (Lhx2-like motif) were significantly more accessible in the branch , to which Ex._neurons_CThPN-labeled cells mainly belong, compared to the branch    Fig. . These r32. To ask to what extent TEs play essential roles as cis-elements, we compared the percentage of TEs that overlap with the open chromatin regions among cell types using binarized  peak-by-cell chromatin accessibility matrix (see “Methods”). A comparison of the percentage of ATAC peaks that intersect TEs among cell types showed that TEs tended to be open chromatin significantly in excitatory neurons compared to the other cell types, such as inhibitory neurons or glial cells  and 18% (29400/163902) of the number of pseudo-bulk ATAC peaks , respectively. Then, we compared whether bulk ATAC peaks tended to overlap with H3K4me1 or H3K4me3 ChIP-seq peaks between TE-derived ATAC peaks and non-TE-derived ATAC peaks. As a result, the TE-derived ATAC peaks tended to overlap significantly with H3K4me1 when compared to non-TE-derived peaks  data of cortical tissues. From the ChIP-Atlas database51, we discovered that the class distribution of TEs overlapping with open chromatin regions was different from their distribution in the genome  was the most enriched TE subfamily in Neurod2 binding peaks and de novo motif 1 )  and MamRep434 (DNA class in Repbase51) were the most enriched TE subfamilies in Lhx2 binding peaks and de novo motif 3, respectively (q < 0.01: FDR) . As a result, we confirmed that MER130 and MamRep434 were enriched in de novo motifs 1 and 3, respectively ) Fig. . MER124 DR) Fig. . To acco34. Then we created a TE-by-cell accessibility matrix (see “Methods”). To search for TE subfamilies that were specifically accessible in branch , which was estimated as the state of neuronal progenitor cells , MER130, MamRep137 (DNA class in Repbase51), and MER102b (DNA class in Repbase51) are significantly more accessible in branch   , MamRep434 and LFSINE_Vert (SINE class in Repbase51) are significantly more accessible in branch    and de novo motif 3 (Lhx2-like motif) were detected in the consensus TE sequences for MER130 and MamRep434, respectively . The result showed that de novo motif 1 was selectively conserved among the accessible MER130 fragments when compared with the flanking regions within MER130  and confirmed that these motif sites tended to be conserved among these regions . We used ChIP-seq data of Neurod2 and Lhx2 in the neural cell type after P0 in the ChIP-Atlas database49. The results showed that the ChIP-seq peaks were concentrated in the regions of approximately 200 bp around the de novo motifs detected in the consensus sequences , and the Kcnj3 gene, which has GO terms associated with components of the synaptic membrane. MamRep434-derived accessible de novo 3 motifs were located next to the genes Btbd3, Nos1, and Pbx1, which have GO terms associated with neurogenesis. These genes have been reported to have neuronal developmental functions related to the regulation of cortical layer structures developed in mammals60. Thus, in the vicinity of the TE-derived accessible motifs, there were candidate target genes that may be related to the functional evolution of the brain. The other candidate target genes located near TE-derived cis-elements are listed in Supplementary Tables We then investigated the function of genes located near the TEs. We obtained the genes near TE fragments in the genome using GREAT130 Fig. a, b. In 130 Fig. a, c. Not130 Fig. a–c. ThesWe also checked whether other TEs identified as the candidates for transcription factor binding in the putative neuronal progenitors could function as cis-elements  . Figure p < 0.01, Fisher’s exact test). These results were also similar for each cell type and differentiation process of the excitatory neurons , which is consistent with the results of Notwell et al.26. In addition, the most distantly related species in which 2.7% of the ortholog sequences of MamRep434-derived cis-elements were detected was platypus, in the same time as the origin considered in Repbase51. To determine whether the de novo motifs play an evolutionarily important role as functional domains in the MER130- and MamRep434-derived cis-elements, we also evaluated the sequence conservation of the motifs in placental mammals (see “Methods”). Among the MER130- and MamRep434-derived cis-elements in the mouse genome, motif sequences were highly conserved in mammals , and we detected some additional MER130 fragments (18 fragments) with the exact condition of the TE annotation as that found in the previous study  tended to be conserved not only among the TE subfamily accessible in the mouse genome but also among that of mammalian species Fig. . These T27. In addition, we suggest that MamRep434 may be responsible for neurogenesis-related genes, which are related to the regulation of cortical layer structures developed in Eutheria. In fact, in Eutheria, the production of neurons is more active than in other mammals, such as Metatheria66. The high conservation of motifs in the TE-derived cis-elements suggests that TE insertion contributes to the acquisition of motifs as functional domains. Furthermore, there is an association between the function of transcription factors whose binding is enriched in TEs and the function of TE-neighboring genes. The Neurod2 transcription factor, which is enriched in MER130, has been reported to be involved in the regulation of synaptic innervation and spine morphogenesis68. The Lhx2 transcription factor, enriched in MamRep434, has been reported to have several roles in the spatiotemporal regulation of neurogenesis and is considered a key factor for brain structure and neuronal identity8. For example, Lhx2 plays a role in the spatiotemporal regulation of neuron number by promoting a decrease in the number of neurons in layer V and an increase in the number of neurons in layer VI69. Interestingly, the timing of acquiring TE-derived cis-elements is associated with the function of transcription factors, which might relate to the evolutionary changes in the brain structure. This is consistent with our hypothetical scenario that TEs confer new cis-elements of transcription factors for specific cell types in multiple stages and contribute to gaining higher complexity in the brain during evolution. Our results suggest that MER130- and MamRep434-derived cis-elements have distinct functions and are acquired at different times during evolution. This may reflect the gradual evolution of the mammalian brain architecture and the acquisition of complex functions in the brain by utilizing TE-derived regulatory elements of transcription factors related to neuronal development.According to the functional analysis of the TE-neighboring genes in the mouse genome Fig. a–c and tMER130 TEs, acquired by the ancestor of Amniota, serve as binding sites for the transcription factor Neurod2 and promote glutamatergic neuronal progenitor cell-specific gene regulation. MamRep434 TEs, acquired by the ancestor of Eutheria, also serve as binding sites for the Lhx2 transcription factor and promote glutamatergic neuronal progenitor cell-specific gene regulation.33. In this study, among the cell-type labels defined by Cusanovich et al.33, we used scATAC-seq data of the labels including more than 100 cells as follows: callosal projection excitatory neurons (Ex._neurons_CPN: layer II/III excitatory neurons), subcerebral projection excitatory neurons (Ex._neurons_SCPN: layer V excitatory neurons), corticothalamic projection excitatory neurons (Ex._neurons_CThPN: layer VI excitatory neurons), SOM+_Interneurons (somatostatin positive interneurons), Inhibitory_neurons, Oligodendrocytes, Microglia, and Astrocytes. We first split the scATAC-seq data into bam files for each cell-type label and performed peak calling using MACS270 with the following parameters: “- nonmodel-keep-dup all-extsize 200-shift -100” for each cell population. To create pseudo-bulk ATAC-seq peak data, we merged the peak sets for each cell population and resized the peaks to a width of 500 bp. In addition, we obtained a peak-by-cell chromatin accessibility matrix based on the fragment counts for each cell in the region corresponding to the bulk ATAC peaks.To obtain accessible regions for transcription factor binding per cell, we used scATAC-seq of the adult mouse (P56) prefrontal cortex, which is alternatively available via accession number: GSE11158633. Gene activity scores were normalized for individual cells according to size factors.Using scATAC-seq data, we also obtained gene activity scores calculated based on chromatin accessibility near each gene provided by Cusanovich et al.https://www.dropbox.com/s/cuowvm4vrf65pvq/allen_cortex.rds?dl=1, which is alternatively available via accession number: GSE7158513.To obtain gene expression levels in the cell types corresponding to scATAC-seq data, we used scRNA-seq data of the adult mouse cortex downloaded from 49 to identify transcription factor-binding sites and histone modification sites  were inputted to computeDeviations function. For k-mer annotation, we used the 7-mer annotation. In eukaryotes, the most common core motif to which transcription factors bind is 7 bp in length72; therefore, we chose the 7-mer in this study, which is also the default length for using chromVAR34 in the STREAM paper42. For motif annotation, motifs were detected in the ATAC peaks with the matchMotifs function (default parameter) in the motifmatchr package as part of chromVAR34. In this study, since the analysis was performed at the single-cell level, we used ATAC-seq as epigenomics data.We used the computeDeviations function of chromVAR34. In Figs. z-scores of each k-mer/motif were also calculated with the computeDeviations function (default parameter) of the chromVAR34.To visualize the similarity among cells for k-mer accessibility profiles in Fig. 34, we generated de novo motifs by weighting the k-mers with similar accessibility patterns to the seed k-mer with large accessibility variation across the cells. The k-mer accessibility profiles (k = 7) calculated with the computeDeviations function described in the “Calculation of k-mer/motif accessibility” were used as inputs.Using chromVAR’s assembleKmers function (the threshold of variability score = 2.0)35, which compares motifs based on the frequency of occurrence of letters at each position in the sequence, we searched the HOCOMOCO v11 database71 for known motifs that were significantly similar to the de novo motifs.We calculated the similarity of the obtained de novo motifs to known motifs in Fig. 73. We used the FindVariableFeatures function (default setting) to select genes with considerable activity variation across the cells based on scATAC-seq. Then, using the FindTransferAnchors function (default setting), the cell-type labels defined in scATAC-seq were assigned to each cell in scRNA-seq based on the similarity of gene activity/expression patterns for the selected genes.To estimate the cells of scRNA-seq data corresponding to the cell-type labels of scATAC-seq data, we transferred cell-type labels with Seurat33, we used STREAM42 for pseudo-ordering the cells based on the k-mer accessibility profiles. For pseudo-time analysis, we followed the original pipeline, and each cell was mapped to a two-dimensional tree structure called a flat tree plot calculated using STREAM in Fig. To reconstruct the differentiation process of cells labeled as neurons by Cusanovich et al.70 with the following parameter:"-nomodel-keep-dup all-extsize 200-shift -100” as in “Data sources”, and the length of each peak was aligned to 500 bp to create bulk ATAC peaks for each cell type.To investigate the overlap with TE for each cell type, we compared the overlap with TE using pseudo-bulk ATAC peaks obtained from randomly sampled cells of each cell type in Supplementary Table 74 was used to obtain the overlap regions between TF ChIP peaks and the TE subfamily. The enrichment score quantitatively evaluated the degree of overlap between the TE and ChIP peak; TEs with less overlap with ChIP peaks have a relatively low score. In cases where multiple TEs overlap with the same ChIP peak, the overlap between each TE and the peak was separately calculated in base pair (bp) units using the intersectBed tool. To prevent the enrichment score from being abnormally high due to a small number of TE fragments, we restricted the subfamilies to TEs that intersected the ChIP peak at least three times in the whole genome. To calculate statistical significance, Fisher’s exact test with the Benjamini–Hochberg correction was performed based on the numbers used to calculate the enrichment scores.To identify TE subfamilies that were enriched in transcription factor-binding sites, we calculated the enrichment of ChIP peaks for TEs in the genome in Fig. 34, and those motifs that were also included in TEs were identified as TE-derived accessible motifs. In this analysis, we used bulk ATAC-seq data obtained by merging the scATAC-seq peaks (described in “Data sources”).The motifs in the ATAC peaks were detected using chromVAR’s matchMotifs function (default parameter)74 was used to obtain the ATAC peaks where the entire motif was detected. Subsequently, as “number of ATAC peaks with motifs in the TE subfamily” in  and used them to compute the robust z-score.We also calculated the enrichment of accessible motifs for TEs in the genome in Fig. 34. We used the getAnnotations function of chromVAR to obtain annotations where ATAC peaks intersect TE regions by more than 1 bp in the genome. Then, the annotation and peak-by-cell accessibility matrix (described in “Data sources”) were input to compute the deviation function as in “Calculation of k-mer/motif accessibility” to calculate the deviation scores of the TE subfamily. In this analysis, to exclude cases in which TEs were assessed as abnormally accessible, we restricted our analysis to the TE subfamily, in which more than 100 neuron-labeled cells overlapped with at least one ATAC peak. All neuron-labeled cells in the scATAC-seq data were used for the calculation. The Mann–Whitney U test with Benjamini–Hochberg correction was performed to test for statistical significance.Using the TE subfamily as annotations instead of the k-mer/motif, we calculated the TE subfamily accessibility scores for each cell with chromVAR75 and visualized with Jalview76. The genomic regions where TEs were detected as the anti-sense strands were converted to the sense sequences to align them with other sequences.Multiple alignments were performed for TE fragments overlapping ATAC peaks in the genome. TE fragments of MER130 or MamRep434 overlapping with the pseudo-bulk ATAC peaks by more than 1 bp were collected from the alignment results between the mouse genome mm9) and the consensus TE sequence obtained from RepeatMasker . In Fig.  and the 49. In this experiment, we converted ChIP-seq peaks mapped to the TEs in the genome into the consensus TE sequences. ChIP-seq data used in this experiment was indicated in the Data Sources section ("Data sources”). The regions in the consensus TE sequence corresponding to the ChIP peak regions  of deeptools77 was performed with the bam file of ChIP-seq reads mapped using bowtie278 as the input, and the ratio of featureReadCount / totalReadCount was compared for each IP and input file in Supplementary Fig. To account for ChIP-seq inputs, we also evaluated the IP/input ratio for the proportion of reads mapped to the sites of TE-derived de novo motifs detected with chromVAR34 to consensus TE sequences. In Fig. 54.To detect sites in the consensus TE sequence corresponding to TE-derived accessible motifs in the genome, we mapped the sites of TE-derived accessible motifs detected with chromVAR53 was used to detect individual motifs in consensus TE sequences. In this study, de novo motifs generated with chromVAR34 were used as inputs for motif detection in consensus TE sequences.FIMO55 was used to associate genomic regions with neighboring genes . Gene Ontology analysis of gene lists was performed using g:Profiler56. g:Profiler outputs GO terms that are significantly enriched (q < 0.05) in the input genes list.GREAT79. Using ATAC peaks in the mouse genome as input, we estimated the acquisition time of the cis-elements by checking for the presence of orthologous sequences in other species (minimum ratio of bases that must remap: 0.5). Among the species for which orthologs were identified, each ATAC peak was assigned to the species most distantly related to the mouse in the reference phylogenetic tree83 and used to estimate the time of sequence acquisition. The following species were searched as orthologs: Rat (rn5), Guinea pig (cavPor3), Rabbit (oryCun2), Human (hg19), Cow (BosTau7), Dog(CanFarm2), Horse (EquCab2), Tenrec (echTel1), Elephant (loxAfr3), Opossum (monDom5), Platypus (ornAna1), Chicken , Turkey , Lizard (anoCar2), X. tropicalis (xenTro3), Zebrafish (danRer7), Medaka (oryLat2). For the TE-derived cis-elements, the ATAC peaks that overlapped with the TE annotation of RepeatMasker were used as input in Fig. The orthologous sequences of the ATAC peak were searched using the liftOver tool using the cross-species chain data obtained from the UCSC Genome Browser database84. Average sequence conservation scores were obtained for each TE-derived accessible motif set. As a control, 7-bp sequences from the intersection of the ATAC peaks and the TE regions were randomly obtained 500 times with the shuffleBed tool (with the -noOverlapping option) of the BEDTools package74. At this time, only sequences that did not overlap with the motif detection sites were used as controls.Sequence conservation scores  in placental mammal genomes per bp were obtained from the UCSC Genome Browser databaseThe type of statistical tests used are described in the figure legends or the main text. To ensure reproducibility, we provided the source code on GitHub (described in the “Code availability” section).Further information on research design is available in the Peer Review FileSupplementary InformationDescription of Additional Supplementary FilesSupplementary DataReporting Summary


Auditory neuropathy is an unusual type of hearing loss. At least 40% of patients with this disease have underlying genetic causes. However, in many hereditary auditory neuropathy cases, etiology remains undetermined.We collected data and blood samples from a four-generation Chinese family. After excluding relevant variants in known deafness-related genes, exome sequencing was conducted. Candidate genes were verified by pedigree segregation, transcript/protein expression in the mouse cochlea, and plasmid expression studies in HEK 293T cells. Moreover, a mutant mouse model was generated and underwent hearing evaluations; protein localization in the inner ear was also assessed.XKR8 was identified. Genotyping of 16 family members confirmed the segregation of this variant with the deafness phenotype. Both XKR8 mRNA and XKR8 protein were expressed in the mouse inner ear, predominantly in regions of spiral ganglion neurons; Moreover, this nonsense variant impaired the surface localization of XKR8 in cells. Transgenic mutant mice exhibited late-onset auditory neuropathy, and their altered XKR8 protein localization in the inner ear confirmed the damaging effects of this variant.The clinical features of the family were diagnosed as auditory neuropathy. A novel variant c.710G > A (p.W237X) in apoptosis-related gene XKR8 gene that is relevant to auditory neuropathy. The essential role of XKR8 in inner ear development and neural homeostasis should be explored.We identified a variant in the The online version contains supplementary material available at 10.1186/s12967-023-04139-x. Hearing loss is usually associated with a loss of hair cell function, which can be fully restored by cochlear implantation. In contrast, auditory neuropathy (AN), also referred to as auditory neuropathy spectrum disorder, results from synaptic disorders in afferent nerves with intact outer hair cell function . In AN, OTOF) gene is the most well-studied AN-related gene [DIAPH3 [PJVK [It is estimated that about 40% of AN has a genetic origin . The ototed gene . VariantH3 [PJVK . HoweverXKR8 (NM_018053) gene is located at 1p35.3, and has a total length of 2710 bp and a coding region of 1188 bp. It encodes XK-related protein 8 (XKR8), which contains 395 amino acids and 10 transmembrane regions and is localized at the cell membrane surface. This protein belongs to the XK-related protein family (XKR1–XKR9) [XKR8 have focused on cell apoptosis; there are no reports of a relationship between XKR8 and the inner ear or deafness.The R1–XKR9) . Suzuki R1–XKR9) . Thus faXKR8 as an AN-related gene in a Chinese family. A combination of clinical data, genetic verification, and functional analysis in mice and cells supports our hypothesis that a dominant variant in XKR8 is linked to hereditary AN in this family.In the present study, we report the identification of Informed consent wasobtained from all patients or their guardians. This study was approved by theinstitutional review board of the First Affiliated Hospital, Sun Yat-senUniversity. All animal experiments were performed using protocols approved bythe Animal Care and Use Committee of Sun Yat-sen University.A four-generation family with postlingual hearing loss from Guangxi Province, China, visited our clinic for genetic counseling Fig. . The proGJB2, SLC26A4, and mitochondrial genes) were first sequenced as previously described [Blood samples were collected from 16 members of this family  and two unaffected (II-4 and III-7) family members  was completed using ClustalX2 software. The transcript levels of candidate genes in adult mouse cochlea were evaluated by reverse transcription PCR.After standard data filtering, the target sequences in candidate genes were subjected to Sanger sequencing in 16 family members. Amino acid conservation alignment across different XKR8+/−) were generated from the Jikai Laboratory . Genotypic verification of the transgenic mice was performed according to recommendations from the Jikai Laboratory. For mRNA detection, samples of cochlea were dissolved in TRIzol. The rest of each cochlea was immediately fixed with 4% paraformaldehyde for at least 1 h at room temperature. Frozen cochlear sections were used for immunofluorescence staining.C57BL mice were used in all experiments. Transgenic mice carrying heterozygous variant c.710G > A , resulting in a 444-bp PCR product. The GAPDH gene was also amplified . The resulting products were detected by 2% agarose gel electrophoresis. Data were analyzed using ImageJ software . Each experiment was replicated at least three times.A routine TRIzol method was used for the isolation of postnatal day 30 mouse cochlear RNA; the product was converted to cDNA using the All-in-One First-Strand cDNA Synthesis Kit . Forward and reverse primer pairs for mouse Staining of XKR8 was performed in cross sections of mouse cochlea. Sections were blocked with 1% bovine serum albumin (BSA) and 1% Triton X-100 in 0.1 M phosphate buffer (pH 7.2) for 1 h at room temperature. Next, the sections were incubated overnight at 4 °C with primary antibodies against XKR8 , myosin VIIA , and class III beta-tubulin  diluted in 1% BSA and 10% Triton X-100. Sections were then incubated for 1 h at room temperature with secondary antibodies diluted in 0.1% BSA and 0.1% Triton X-100. An LSM 700 confocal microscope  or Leica microscope  was used to capture images.XKR8 and the XKR8 c.710G > A mutant were inserted into the plasmids. Four types of recombined plasmids were constructed and confirmed by Sanger sequencing: pEGFP-C2-XKR8-c.710G-A, pEGFP-C2-XKR8-WT, pmCherry-C1-XKR8-c.710G-A, and pmCherry-C1-XKR8-WT.The fluorescent plasmids pEGFP-C2 and pmCherry-C1 were obtained from our laboratory. The entire coding sequences of human wild-type (WT) 2 incubator for 48–72 h prior to immunofluorescence. Each experiment was replicated at least three times.Plasmids were then transfected into HEK 293T cells using Lipofectamine 2000 Transfection Reagent . For the heterozygous model, WT and mutant plasmids were mixed at a 1:1 ratio and added to the cell plate. Cells were maintained at 37 °C in a COMouse ABR was conducted using a High Frequency Transducer . The computer generated 4, 8, 16, and 32 kHz click and tone burst sounds as the stimuli, with the following settings: duration of acoustic stimulation, 5 ms; rising and falling time, 0.5 ms; scanning time, 10 ms; stimulation sound repetition rate, 11 times/s, band-pass filtering frequency, 300–3000 Hz; acquisition amplification gain, 100,000 times; and average stacking, 1024 times. Data were analyzed using SmartEP 5.40 software. In mice, DPOAE evaluations were performed using DP-Gram on a Titan system  in our lab according to the manufacturer’s instructions.IBM SPSS Statistics for Windows, version 23.0  was used for statistical analyses. All data are presented as the mean ± standard deviation. Two-tailed, independent Student’s t-tests were performed. P-values < 0.05 were considered significant.This family contained four patients living with hearing loss Fig. A. The afGJB2 or SLC26A4) and the customized gene panel failed to identify any causal variant in the family. We therefore performed exome sequencing, in which 46 variants in 45 genes were filtered  or the production of a truncated XKR8 protein. The amino acids around Trp237 are highly conserved among different species .The placenta . HoweverImmunofluorescence suggested that XKR8 was weakly expressed at the bottom of inner hair cells but strongly expressed in auditory neurons along the cochlear modiolus. XKR8 was co-localized with TUJ1 and projected from ribbon synapses along the spiral limbus, similar to the location of type I afferent nerve fibers. In contrast, XKR8-positive fluorescence was not observed in hair cells or supporting cells Fig. B. This oIn WT mice, XKR8 was localized at the surface of cell membranes. By contrast, in mice with homozygous or heterozygous variants, the normal protein structure did not form at the cell membrane surface Fig. . InsteadXKR8+/− mice, hearing was within the normal range  or neuroplastin (NPTN), which are type I membrane proteins from the immunoglobin superfamily [XKR8 in AN is yet to be clarified, a molecular correlation among XKR8, NP65, and BSG strongly implicates an essential role of XKR8 in maintaining synaptic homeostasis and neural function in the cochlea. Moreover, dysregulated XKR8 expression may impair BSG or NPTN function in synaptogenesis or the spiral ganglion.The location of XKR8 in spiral ganglion cells and the bottom of the inner ear led us to speculate that erfamily . Intraceerfamily . Furthererfamily ; this paerfamily . AlthougXKR8+/− mutant mice. Hearing in these mice remained stable after the onset of hearing, and our primary animal hearing data were consistent with clinical observations from the four patients in this family. An elevated hearing threshold may be associated with a partial loss of function in XKR8 protein, as observed in the 20-week-old mutant mice. Disrupted synaptogenesis and neural function may in part, or in combination with apoptotic dysfunction in the inner ear, contribute to the AN phenotype observed in both the patients and the mouse model. We are currently investigating the detailed pathomechanism of this XKR8 variant in transgenic mouse.To confirm our results from the family with AN, we generated XKR8 in the inner ear requires further studies. Additionally, the pathomechanism of the identified XKR8 mutation remains unclear. Finally, the prevalence of the XKR8 variant in AN and nonsyndromic hearing loss also needs to be investigated.Our study has some limitations. The physiological function of XKR8 gene is responsible for autosomal dominant AN in this family. The essential role of XKR8 in inner ear development and neural homeostasis should be further investigated.We provided evidence that a variant in the Additional file 1. Table S1.


O-GlcNAcylation, a rather understudied modification that plays critical roles in almost all biological processes and is added and removed by O-linked N-acetylglucosamine transferase and O-GlcNAcase, respectively. This review provides a current overview of our knowledge of how O-GlcNAcylation impacts the histone code both directly and by regulating other chromatin modifying enzymes. This highlights the pivotal emerging role of O-GlcNAcylation as an essential epigenetic marker.Histones display a wide variety of post-translational modifications, including acetylation, methylation, and phosphorylation. These epigenetic modifications can influence chromatin structure and function without altering the DNA sequence. Histones can also undergo post-translational  R. RO-GlcNrocesses . To datee, v1.3) . This reIn eukaryotic nuclei, the DNA is wrapped around a histone octamer , to form a nucleosome, and is locked by histone H1. The broad spectrum of histone PTMs constitutes the “histone code”, which not only modulates the recruitment of key enzymes involved in gene expression, but also impacts the condensation of chromatin. This results in distinct areas of euchromatin, which is only slightly condensed and transcriptionally active, and highly condensed and transcriptionally silent heterochromatin , 20.O-GlcNAcylation was first reported in 2010. Using several biochemical and mass spectrometry (MS) approaches, Sakabe et al. revealed that H2A, H2B, H3, and H4 were O-GlcNAcylated  in HeLa cells . T. T. demoO-GlcNAcylation actively influences gene accessibility by modulating the open/closed state of chromatin and the recruitment of key enzymes. Lercher et al. demonstrated that O-GlcNAcylation of H2A T101 decreased nucleosome stability, favoring an open state and thus promoting the recruitment of proteins involved in nucleosome remodeling  [O-GlcNAcylation are full-fledged players in histone modification.In addition, 2 and 6) . Taken tO-GlcNAcylation are closely linked and can compete against each other [O-GlcNAcylation can positively or negatively regulate the phosphorylation of nearby residues, and OGT/OGA can interact with kinases/phosphatases, creating multi-enzyme complexes that can phosphorylate/O-GlcNAcylate proteins [O-GlcNAcylated, which partially supressed its phosphorylation. They also highlighted that increasing O-GlcNAc levels via glucosamine was associated with decreases in both H3K9ac and H3K4me3—both active marks—and increases in H3 S28 phosphorylation and H3K9me3, which are active and repressive, respectively [. determined that H3 T32 was O-GlcNAcylated. As aurora B, the kinase that phosphorylates H3 S10 and S28, is physically associated with OGT/OGA, they evaluated the impact of O-GlcNAc levels on these phosphosites. Mitotic cells that overexpressed OGT or were treated with OGA inhibitors (PUGNAc or thiamet G) displayed reduced H3 S10, S28, and T32 phosphorylation , phosphorylation and ch other . Moreoveproteins –39. Lownectively . In 2012oduction . RegardlO-GlcNAcylation in histone modification is reinforced by OGA’s C-terminal HAT activity [. demonstrated that mammalian OGA can acetylate all four core histones in synthetic nucleosomes in vitro. They also identified an O-GlcNAcylated site for H4 (K8) and H3 (K14) -induced granulopoiesis . They showed that the three proteins interacted, limiting the ubiquitination and thus the degradation of MLL5. Although the authors identified two O-GlcNAcylated sites on MLL5 (S435 and T440), they did not determine their roles in protein stability. Moreover, as USP7 can be phosphorylated [O-GlcNAcylated and how that would impact its functions [. demonstrated that O-GlcNAcylation of MLL5 on T440 favors the recruitment of MLL5β to the MLL5β-AP-1 complex, which allows the transcription of human papillomavirus E6/E7 oncogenes implicated in the pathogenesis of cervical cancer [MLL can cause MLL-fusion leukemia, in which an MLL N terminus is fused to another protein. AF9, AF10, and ENL, which all interact with the H3K79 methyltransferase DOT1L, are the most common MLL fusion partners [O-GlcNAcylated on S1511, which promotes its stability by protecting it from UBE3C-mediated ubiquitination  is a histone lysine methyltransferase (HKMT) involved in regulating cell cycle progression, spermatogenesis, hematopoiesis, and the maintenance of genomic stability . Fujiki opoiesis . More reorylated , it woulunctions . Finallyunctions , 55. Conl cancer . In addipartners –59. In 2O-GlcNAcylation also controls HKMTs in plants. The Arabidopsis homolog of trithorax (ATX1) is a H3K4 methyltransferase. Xing et al. demonstrated that secret agent (SEC), the OGT in Arabidopsis, regulated both the stability and activity of ATX1 through O-GlcNAcylation of S947. They also demonstrated that O-GlcNAcylation of ATX1 promotes H3K4me3 deposition on FLOWERING LOCUS C, which encodes key negative regulators of flowering  contains male-specific lethal and nonspecific lethal (NSL) complexes. Interestingly, OGT1 is a component of the NSL complex [. demonstrated that OGT1 interacted with and O-GlcNAcylated the NSL complex subunit NSL3, which was associated with increased stability and activity, thereby facilitating H4 acetylation on K5, K8, and K16 [O-GlcNAcylation of T755 of NSL3, which increased NSL3 stability by blocking UBE2S-dependent ubiquitination. Even more importantly, O-GlcNAcylated T755 was required to maintain the integrity and holoenzyme activity of the NSL complex. In type II epithelium-like lung carcinoma (A549) cells, NSL3 O-GlcNAcylation promoted proliferation, leading them to conclude that O-GlcNAcylation acts as a link between oncogenic signals and the epigenetic changes that occur in cancer , is an enzyme that asymmetrically dimethylates proteins on arginine residues. It is both a substrate and interactor of OGT [O-GlcNAcylation levels via thiamet G or N-acetyl-glucosamine-thiazoline did not impact H3R17me2 levels or CARM1 phosphorylation, suggesting the direct involvement of OGT [. identified four O-GlcNAcylated sites on CARM1: S595, S598, T601, and T603. They showed that these modifications impacted CARM1’s substrate specificity without affecting its function, cellular localization, stability, or dimerization , as also known as protein arginine r of OGT . In 2010t of OGT . Later, -histone proteins. Humans express 18 HDACs  [O-GlcNAcylated. Zhu et al. showed that OGT interacted with and O-GlcNAcylated HDAC1 on T114 and S263, which suppresses HDAC1 enzymatic activity. They also showed that HDAC1 O-GlcNAcylation regulates the migration, proliferation, and invasion of HepG2 cells, thus identifying a new potential therapeutic strategy for hepatocellular carcinoma [O-GlcNAcylated on S642. In the diabetes mellitus mouse model, this O-GlcNAcylation event counteracted pathological CAMKII signaling and thus was deemed cardioprotective [O-GlcNAcylation impacts the histone acetylation landscape. HDAC6 plays a pivotal role in cilia assembly and is regulated by phosphorylation [O-GlcNAcylation [O-GlcNAcylation. They discovered that OGT interacted with and O-GlcNAcylated HDAC6 in hTERT-RPE1 cells, resulting in ciliary shortening, and demonstrated that treatment with the OGT inhibitors thiamet G or GlcNAcstatin enhanced HDAC6’s deacetylase activity [O-GlcNAcylated HDACs on histone acetylation have not been examined.Histone deacetylases (HDACs) catalyze the removal of acetyl groups from both histones and nonSIRT1–7) . To datearcinoma . More reotective . Considerylation . Considecylation , Tian anactivity . To date-histone proteins . Interestingly, O-GlcNAcylation is also pivotal in the stress response [O-GlcNAcylated on S549, which enhanced its deacetylase activity ) and its substrate affinity . They found that under stress, SIRT1 O-GlcNAcylation allowed some targets to be deacetylated, including p53 and FOXO3, which regulate the decisions governing cell death and survival [. found that SIRT1 O-GlcNAcylation on S549 did not affect its subcellular localization, and a recent study reported complementary results. In fact, Chattopadhyay et al. revealed that the nutrient-dependent SIRT1 O-GlcNAcylation of T160/S161 exerts spatiotemporal control by promoting its localization to the cytosol, where it undergoes ubiquitin-mediated degradation [O-GlcNAcylation impacts the acetylation status of histones would be worth exploring. SIRT7, which catalyzes the selective deacetylation of H3K18, was recently identified as O-GlcNAcylated by OGT at S136, which stabilizes SIRT7 by decreasing its interaction with proteasome activator subunit 3 (PSME3), a core molecule of a new ubiquitin-independent pathway [O-GlcNAcylation has been associated with repressing tumor suppressor genes to promoting tumor progression in nude mice  are nicotine adenine dinucleotide( +)-dependent HDACs that regulate a wide variety of biological processes, such as metabolism, oxidative stress, apoptosis, and inflammation . SIRT1 iresponse . Consistsurvival . Han et radation . Conside pathway . By redu-, di-, and trimethylates H3K27 to form H3K27me1, me2, and me3, respectively. O-GlcNAcylation plays an important role in gene regulation in Drosophila. As proof, OGT is encoded by the PcG gene super sex combs (sxc) [Drosophila, no major features have been identified recently [The polycomb group (PcG) proteins form three complexes known as PRC1, PRC2, and PR DeUBiquitinase (PR-DUB). PRCs play a pivotal role in development by repressing homeotic genes. Mutations in the subunits of PRCs are associated with human neurodevelopmental disorders and cancer , 90. In bs (sxc) , and losbs (sxc) . Althougrecently .O-GlcNAcylated. Using MS coupled to beta-elimination and Michael addition with dithiothreitol, Maury et al. identified S278 as an O-GlcNAcylation site. They also found that T250 and S251 were important for O-GlcNAcylation, but could not discriminate which residue harbored the GlcNAc moiety. Using chromatin immunoprecipitation coupled with sequencing (ChIP-Seq), the authors revealed that RING1B O-GlcNAcylation impacted its target genes: O-GlcNAcylated RING1B was bound to genes involved in neuronal differentiation, while unmodified RING1B was bound to genes related to cell cycle and metabolism. Accordingly, unmodified RING1B decreased throughout human embryonic stem cell (hESC) differentiation [O-GlcNAcylation in differentiation [. demonstrated that OGT interacts with BMI1 and O-GlcNAcylates it on S255. Like phosphorylation, O-GlcNAcylation increased BMI1’s stability by inhibiting its polyubiquitination and proteasomal degradation. They also illustrated the negative role of BMI1 O-GlcNAcylation in prostate cancer tumorigenesis, as it inhibits p53, PTEN, and CDKN1A/CDKN2A signaling, thus favorizing apoptosis, invasion and proliferation, respectively . In contrast to RING1A, RING1B can be ntiation , reinforntiation . RING1B ntiation . BMI1 phntiation . In 2017cylation .Fig. 5ImO-GlcNAcylation levels through its deubiquitinase activity [O-GlcNAcylation are indispensable for the proteolytic maturation of HCFC1 [O-GlcNAcylation was demonstrated only recently, with no evidence of impacts on its expression/stability or its deubiquitinase activity [. demonstrated that HCFC1 recruits OGT to PRGC1 and that O-GlcNAcylation facilitates the binding, thus stabilizing PRGC1 and promoting gluconeogenesis [. identified ASXL1 as an OGT substrate that displayed increased stability after O-GlcNAcylation of S199 [PR-DUB is a complex composed of BAP1, HCFC1, FOXK1/2, either ASXL1, 2, or 3, and interestingly, OGT . In spleactivity . Since Oof HCFC1 , BAP1  interacted with the EZH2/PRC2 complex, (ii) was essential for EZH2—and therefore, PRC2—stability, and (iii) O-GlcNAcylated EZH2 on S76. Finally, the authors demonstrated that the OGT/EZH2 axis downregulated tumor suppressor genes in breast cancer cells, thus identifying a new therapeutic target [®O-GlcNAc enzymatic labeling system, Wong’s group identified five new O-GlcNAcylated sites on EZH2: S73, S84, S87, T313, and S729. After excluding S87, since O-GlcNAcylation at this site was very low, they showed that the N-terminal O-GlcNAcylated sites  increased the stability of EZH2 by limiting its ubiquitination, while the C-terminal O-GlcNAcylated site (S729) stimulates its di- and trimethyltransferase activity. None of the sites altered the affinity of EZH2 for other PRC2 components [O-GlcNAcylated; therefore, it would be relevant to study whether O-GlcNAcylation also impacts SUZ12, EED, and RbAp46/48 , embryonic ectoderm development (EED), and suppressor of zeste 12 (SUZ12). The first evidence of the impact of EZH2 c target . In 2018mponents . EZH2 hamponents . In addi/48 Fig. c.O-GlcNAcylated, evidence for H1 O-GlcNAcylation remains limited. In 2011, an in silico study proposed O-GlcNAcylation of H1’s serine and threonine residues; however, this study relied on YinOYang 1.2 predictive tools, which remain controversial [O-GlcNAcylation in this context is warranted.While many studies over the past decade have established that H2A, H2B, H3, and H4 are oversial , 104. Asoversial , furtherO-GlcNAcylation goes beyond histone modifying enzymes. The different TET isoforms , which catalyze the oxidation of 5-methylcytosine to remove DNA methylation [O-GlcNAcylation of TET proteins affects their stability, phosphorylation, and DNA binding, and thus ability to remove DNA methylation [O-GlcNAcylation is well documented, the first evidence of O-GlcNAcylation of a DNA writer, DNA methyltransferase (DNMT), only emerged in 2020 [DNMT3B, which encodes one of two enzymes regulating de novo methylation, suggesting that its expression might be controlled [O-GlcNAcylation of DNMT1, the enzyme that ensures the transmission of DNA methylation patterns during replication, was recently shown to reduce its methyltransferase activity [O-GlcNAcylation regulates these proteins could lead to promising research avenues [O-GlcNAcylation offers a promising avenue for exploring how external stimuli modulate gene expression and cellular responses via epigenetic regulation  potentially unlocking new insights into the mechanisms of cellular adaptation and homeostasis during early development [The influence of hylation , also inhylation , 107. Thhylation . While T in 2020 . Interesntrolled . Moreove avenues –113. Froelopment –116.O-GlcNAcylation as an epigenetic mark is a major challenge in the field. Currently, only two antibodies against O-GlcNAcylated histones are available (for H2BS112 and H2AS40) [O-GlcNAcylation antibodies have different selectivity and poor specificity [O-GlcNAcylated histone residues would allow better mapping of the modification using ChIP-Seq- or MS-based approaches. Current strategies to understand the role of O-GlcNAcylation include tissue-specific knockouts, RNA interference, and OGT and OGA inhibitors [O-GlcNAcylation in pathophysiological situations , pairing these strategies with advanced epigenetic methods could clarify how O-GlcNAcylation interacts with other histone and DNA marks in various contexts [O-GlcNAcylated sites on histones and epigenetic enzymes using MS, then preventing their modification via mutation, will enhance our understanding of the broad implications of O-GlcNAcylation [Developing tools and approaches to enhance our understanding of  H2AS40) , 30. Altcificity . Thus, ehibitors . Considecontexts , 118. Ficylation .O-GlcNAcylation on core histones and its cross talk with the other nucleosomal PTMs. It is very likely that known O-GlcNAcylated proteins represent only a fraction of the broader role it plays in epigenetics. By developing more refined methods, we will identify more proteins influenced by this modification. Notably, some studies have taken a more critical look at histone O-GlcNAcylation. For instance, Gambetta et al. emphasized the need to treat previous results on the role of O-GlcNAcylation in epigenetics with caution [O-GlcNAcylation on histones as well as presence of other factors for efficient O-GlcNAcylation [O-GlcNAcylation’s roles in epigenetic regulation and determine how these roles can be targeted to improve human health, underscoring the importance of continued research in this area.In this review, we have highlighted the important roles of  caution . Moreovecylation , 121. Th


In this review, we consider various aspects of enhancer functioning in the context of the 3D genome. Particular attention is paid to the mechanisms of enhancer-promoter communication and the significance of the spatial juxtaposition of enhancers and promoters in 3D nuclear space. A model of an activator chromatin compartment is substantiated, which provides the possibility of transferring activating factors from an enhancer to a promoter without establishing direct contact between these elements. The mechanisms of selective activation of individual promoters or promoter classes by enhancers are also discussed. Enhancers were discovered in experiments on the transfection of expression constructs with a reporter gene into eukaryotic cells. It turned out that the inclusion of some fragments of viral genomes in such constructs significantly increases the level of reporter gene expression ,2,3,4. DAn analysis of the positions of enhancers in the genome showed that they can be located at a considerable distance (up to 1 Mb or more) from the target promoters. However, most enhancers are located at a relatively short distance  from target promoters . In someAlthough the number of enhancers in mammalian genomes can be overestimated, activation of a gene or gene locus by multiple enhancers is quite common . In someRecent advances in the study of the 3D genome have shown that the spatial organization of the genome plays an important role in controlling promoter activation by enhancers. Key observations were made using experimental procedures based on the proximity ligation protocol ,29 and kAlthough enhancers were discovered more than 40 years ago and have been actively studied since then, the mechanism of their action remains elusive. In eukaryotic cells, transcription occurs in cycles ,51,52. TThe ability of enhancers to initiate transcription raises the problem of the functional distinction between enhancers and promoters ,71. InitRegardless of the nature of the factors transferred from the enhancer to the regulated promoter, some transferring channel should exist. The creation of such a channel is usually referred to as “enhancer-promoter communication” (E-P communication). Three main models for E-P communication were proposed: tracking, linking, and looping. The first model assumes that factors initially bound to the enhancer are somehow moved along the chromatin fibril and reach the target promoter. This model is illustrated by the movement of various enzymes that are held in complex with an elongating RNA polymerase that performs intergenic transcription A 77]..77].Shh gene during neuronal differentiation of mouse embryonic stem cells is accompanied by a decrease in the spatial proximity between Shh and the SBE6 enhancer that controls its work [The linking model postulates that certain protein factors bind to the fragment of the chromatin fibril separating the enhancer and promoter and, interacting with each other, provide compression of this fragment. This results in spatial proximity between enhancer and promoter. As an example of this mechanism, oligomerization of the Drosophila architectural protein Chip between the enhancer and the promoter can be mentioned ,79. Withits work . Howeverits work .Mechanisms ensuring the retention of enhancers and promoters in spatial proximity remain the subject of discussion. Several models are being considered. The most developed is the model with the participation of architectural elements (insulators) and proteins associated with them. A significant number of architectural proteins are encoded in the Drosophila genome. In model experiments, it was convincingly shown that contacts between these proteins can modulate the spatial organization of the genome, including keeping enhancers close to promoters ,91,92. ICTCF binding sites are often found adjacent to promoters and enhancers. Accordingly, the extrusion of chromatin loops will contribute to the convergence of promoters and enhancers. Binding of CTCF to the recognition motives on DNA can be regulated through DNA methylation and other ways  ). ChangeOnce spatial contacts between enhancers and promoters are established, they can be maintained by mechanisms that do not rely on DNA loop extrusion. Current results strongly suggest that the demixing of enhancer- and promoter-associated proteins results in the assembly of a phase condensate that keeps the enhancer and promoter in spatial proximity ,98,99. TThe results of the initial studies using C-methods were interpreted in terms of the establishment of direct enhancer-promoter contacts through the formation of protein bridges between enhancers and promoters. It was assumed that the DNA fragments crosslinked by such bridges could be solubilized, after which proximity ligation could be carried out in a dilute solution . HoweverDespite some uncertainty about how close an enhancer should be to an activated promoter, the loop model for establishing E-P communication is currently the most reasonable. Stimulation of promoter activity by forced looping with an enhancer is a strong argument in favor of this model ,110,111.Although cohesin extrudes DNA loops symmetrically , additioIt should be noted that the juxtaposition of enhancer and promoter is not always sufficient to activate the promoter. Spatial reconfiguration of the genome usually occurs during cell differentiation and does not always directly lead to the activation of transcription. Enhancers that control the transcription of many inducible genes are often located in close spatial proximity to these genes in the nuclear space, even in the absence of an inducer ,125. HowShh gene and its ZRS (zone of polarizing activity regulatory sequence) enhancer are located about 1 Mb apart [dpp, Slh, and oaf genes in Drosophila melanogaster. These genes are located within a 70-kilobase segment of the genome. In the same segment of the genome, there are several enhancers that selectively activate only the dpp gene promoter [Pcdhα) gene cluster, each promoter contains a CTCF binding site capable of looping with a convergent CTCF binding site on the enhancer. However, the CTCF sites on the promoters are methylated, preventing CTCF binding and looping. Selective demethylation of the CTCF binding site on a particular promoter allows that promoter to establish contact with the enhancer, resulting in activation of the expression of that particular Pcdhα gene [Enhancers are often located at a considerable distance from target promoters. For example, in the mouse genome, the Mb apart . During Mb apart ,128,129.Mb apart ,130,131.Mb apart . Some enMb apart . There aMb apart . In somepromoter . Analysipromoter . The autpromoter . In globpromoter . In the promoter . In the dhα gene ,141.Cell identity in multicellular organisms is determined by transcription profiles, which are largely controlled by enhancers. Although enhancers were discovered about 40 years ago and have been actively studied since then, many questions regarding both the mechanism of enhancer action and the mechanisms that ensure the specificity of activation of certain promoters remain open. One of the common problems in studying the regulatory systems of the eukaryotic genome is that most of the observations are made in experiments with cell populations. At present, a whole range of experimental approaches have emerged that allow studying individual cells, which should significantly expand our knowledge of the mechanisms of transcription regulation. Currently, single-cell Hi-C studies confirm the presence of TADs in individual cells but demonstrate significant variability in their shape  ,144,145.


Aegilops tauschii, the D genome donor of bread wheat. We found that TEs contributed to the complex but orderly epigenetic landscape as chromatin states showed diverse distributions on TEs of different orders or superfamilies. TEs also contributed to the chromatin state and openness of potential regulatory elements, affecting the expression of TE-related genes. Some TE superfamilies, such as hAT-Ac, carry active/open chromatin regions. In addition, the histone mark H3K9ac was found to be associated with the accessibility shaped by TEs. These results suggest the role of diversiform TEs in shaping the epigenetic landscape and in gene expression regulation in Aegilops tauschii. This has positive implications for understanding the transposon roles in Aegilops tauschii or the wheat D genome.Transposons (TEs) account for more than 80% of the wheat genome, the highest among all known crop species. They play an important role in shaping the elaborate genomic landscape, which is the key to the speciation of wheat. In this study, we analyzed the association between TEs, chromatin states, and chromatin accessibility in  Aegilops tauschii is the ancestor of the D genome of hexaploid wheat [Ae. tauschii. Transposons (TEs) are important markers of speciation and the main reason for genome amplification [Ae. tauschii are repetitive sequences, with TEs accounting for 85.9% of the genome, higher than any other known crop species [Common wheat is the most widely distributed staple crop in the world . It is aid wheat . The addid wheat ,7, and tid wheat . The stufication ,10. The  species . A growi species ,12,13. F species ,15. In a species .Studies involving epigenome profiling of hexaploid wheat ,18,19 maAe. tauschii in hexaploid wheat resulted in an overall decrease in gene expression in this chromosome arm [The chromatin states and accessibility are dynamic during wheat polyploidization. TEs in the ancestral species made different contributions to hexaploid wheat. The convergent and divergent regulation of wheat subgenomes is attributed to ancient TE expansions before the divergence of the diploid ancestors and the emerging DNA expansions following the subgenome divergence, respectively, affecting the plasticity of polyploid wheat regulation . Changessome arm . In addisome arm ,31.Triticum urartu , the A-genome progenitor [Ae. tauschii  are still unclear. The Ae. tauschii epigenome exploring TEs will shed light on studies involving the wheat D epi-subgenome and provide important insights into the relationship between the formation of the epigenetic landscape and the TEs in wheat.Histone modifications and chromatin accessibility are important components of the epigenetic landscape. Studies involving polyploid plants have shown that parental inheritance is dominant in the differentiation of polyploid subgenomes . Wheat gogenitor , where TAe. tauschii AL8/78 and investigated the chromatin states and open regions on TEs. TE-associated chromatin states accounted for 92.0% of the genome, with a strongly diverse state composition of TE superfamilies. In addition, TE superfamilies were found related to the formation of chromatin states of genes and regulatory elements, which has a positive significance for gene expression regulation.Here, we used data from chromatin immunoprecipitation and sequencing (ChIP-seq) and micrococcal nuclease digestion and sequencing (MNase-seq) to profile the histone modification and chromatin accessibility maps of Ae. tauschii   to detern states a. The din states b,c. We fn states .Chromatin states 1–9, which are TE-associated states that maintain genome stability, mainly enriched in the heterochromatin regions and accounted for 92.0% of the genome . These sCRWs and Quintas, the two types of LTR [We first calculated the enrichment of all chromatin states in TE orders a. We fous of LTR . A substWe further divided TEs into superfamilies, the classification under TE orders, and calculated the fold enrichment of 15 chromatin states on TE superfamilies whose repeat numbers were greater than 100 see . The relWe also found that TE superfamilies may contribute to the chromatin states of regulatory elements and genes . For exacis regulatory elements or the binding sites of trans factors, that is, the open chromatin regions. We herein identified the MNase HSs in Ae. tauschii AL8/78 genome using the MNase-seq . We analyzed the openness of the chromatin states and the expression of state-related genes and found that states 11–13 showed the highest openness, while the openness of states for gene bodies and TEs was basically low a. The geAe. tauschii genome.Although the MNase HSs were highly enriched near AL8/78 genes, especially in gene promoter regions , 18.6% a. Based −20). These motifs were found enriched in GCC/GGC, consistent with the characteristics of open regions on promoters reported in wheat [Arabidopsis [p-value = 6.58 × 10−4).We found that 57.9% of the hAT-Ac elements with HSs were distributed within the 5 kb gene promoters and 76.3% within the 10 kb gene promoters, substantially higher than the expected ratios of 22.7% and 33.0% for the total hAT-Ac, indicating that hAT-Ac was more likely to contribute to the openness in the promoters. The sequence set of HSs on these hAT-Ac was used to predict the possible transcription factor-binding motifs and found that the three motifs significantly enriched on these HSs  of the HSs were distal, 81.2% (2441/3006) of the TE-HSs were distributed distally. We found that distal TE-HSs were predominant in Unknown, LTR/Gypsy, LTR/Copia, and LINE/L1, while proximal TE-HSs were mostly present in LINE/L1 and DNA/HAT-Ac, followed by LTR/Gypsy and LTR/Copia . This inGO enrichment analysis showed that the genes with proximal TE-HSs were mostly enriched in binding and catalytic activity functions . We thencis regulatory elements. Together, these results suggest a close correlation between the activated chromatin states and the formation of open regions on TEs.The distal HSs usually indicate remote regulatory elements, such as enhancers, which act in long-distance gene regulation . We focuWe further profiled the distributions of H3K9ac and H3K9m2, which are representative marks of state 12/13 and state 7, respectively, around distal LTR/Gypsy c. ComparAe. tauschii genome revealed that TEs are significantly associated with other genomic features such as gene and pseudogene density, miRNA levels, and gene expression and recombination, highlighting the multiple effects of TEs on Ae. tauschii genomic landscape [Ae. tauschii genome contributed to approximately 92.0% of the chromatin states, which is very similar to that in maize, but much higher than that in Arabidopsis and rice [Ae. tauschii. Our results further highlight the contribution of TEs to Ae. tauschii as well as to the wheat D genome.Wheat provides nearly 20% of the total dietary calories and proteins worldwide and feeds almost 35% of the world’s population ,43. The andscape . In thisand rice  . The accession was previously selected for reference genome sequencing for its genetic proximity to the wheat D genome and as it has been extensively characterized genetically [etically ,66. SeedWe referred to a previous experimental protocol used for Chip-seq and used IgG as a negative control . Sampleshttps://sourceforge.net/projects/samtools/, accessed on 17 April 2023) [For ChIP-seq, we evaluated the data quality using FastQC v0.11.5 software  and cleail 2023) . The bamil 2023) . The maxhttps://www.encodeproject.org/software/bedtools/, accessed on 17 April 2023). The heavy-digestion coverage depths were subtracted from the light-digestion ones, and differential nuclease sensitivity sites (DNSs) were obtained. The positive DNSs were defined as MNase hypersensitive sites (HSs), and the negative DNSs were defined as MNase hyper-resistant sites (HRs). Regions with highly credible HSs were further defined using the Bayes factor criterion. Highly credible MNase HSs located within 1 kb upstream and 1 kb downstream of TSSs were defined as proximal HSs, and highly credible MNase HSs within 3 kb upstream of TSSs were defined as distal HSs.We used the same quality control method as the ChIP-seq and aligned the MNase-seq clean data to the AL8/78 genome reference using Bowtie2 v2.3.4 software with parameters ‘--no-mixed --no-discordant --no-unal --dovetail’. Alignments with MAPQ < 20 as well as duplicate reads resulting from PCR amplification were identically removed. The depth of read coverage in 10 bp bins was calculated using the coverage function in the Bedtools software  . were used to calculate and plot histone modification enrichment around the TEs.TEs were classified according to order or superfamily, and we used the OverlapEnrichment function in ChromHMM v1.19 software to compute the fold enrichment of each state on TEs of the superfamily. The intersection in the BEDtools software was used to obtain the TEs or HSs overlapped with the chromatin states. The computeMatrix and plotProfile in the deepTools software  /(B/D) and (G/E)/(F/H), respectively.https://meme-suite.org/meme/tools/meme-chip, accessed on 25 July 2022) was used for motif prediction, and the JASPAR (NON-REDUNDANT) CORE plants database [https://meme-suite.org/meme/tools/tomtom, accessed on 25 July 2022) was used to predict the similarity to the known motifs. Uniprot Website [https://www.uniprot.org/, accessed on 25 July 2022) was used to view the function of the predicted transcription factors.The MEME-ChIP website  . We identified the TE-associated chromatin states of the three species based on fold enrichment and preferential location of the TEs. These data were then compared with our data in Ae. tauschii (The chromatin state data used in this study for Database  (PCSD, htauschii .Ae. tauschii genome are associated with most of the chromatin states, with a diverse state distribution across TE orders and superfamilies. TEs were associated with chromatin accessibility and related gene expression. Furthermore, hAT-Ac was enriched substantially in open regions at higher levels than the expected value and was concentrated in the proximal gene promoters. We identified motifs highly similar to the transcription factor-binding motifs of the Arabidopsis ERF/DREB family. The enrichment of active histone modifications such as H3K9ac and activated chromatin states on gene-distal TEs were highly correlated with the formation of open regions. The results provide insight into the role of TEs in shaping the epigenetic landscape and highlight the importance of TEs in Ae. tauschii or wheat D genome.In conclusion, our study reveals that TEs in the


Clostridioides difficile. Four potential TcdR-dependent promoters in the pathogenicity locus of C. difficile showed different activities. In this study, we constructed a heterologous system in Bacillus subtilis to investigate the molecular basis of TcdR-dependent promoter activity. The promoters of the two major enterotoxins showed strong TcdR-dependent activity, while the two putative TcdR-dependent promoters in the upstream region of the tcdR gene did not show detectable activity, suggesting that the autoregulation of TcdR may need other unknown factors involved. Mutation analysis indicated that the divergent -10 region is the key determinant for different activities of the TcdR-dependent promoters. Analysis of the TcdR model predicted by AlphaFold2 suggested that TcdR should be classified into group 4, i.e., extracytoplasmic function, σ70 factors. The results of this study provide the molecular basis of the TcdR-dependent promoter recognition for toxin production. This study also suggests the feasibility of the heterologous system in analyzing σ factor functions and possibly in drug development targeting these factors.The alternative σ factor TcdR controls the synthesis of two major enterotoxins: TcdA and TcdB in  Clostridioides difficile is an anaerobic Gram-positive spore-forming bacterium, and C. difficile infection (CDI) is a leading cause of worldwide antimicrobial-associated diarrhea in hospitalized elderly patients [Clostridioides difficile produces two enterotoxins, TcdA and TcdB, which are the major virulence factors in CDI pathogenesis, while about 20% of C. difficile strains produce an additional toxin CDT whose role in the infection is less clear [The human enteropathogen patients ,2. Clostss clear ,4. TcdA ss clear . TcdA anss clear ,7.70 family of RNA polymerase σ-factors and it is the only positive regulator of toxin production in C. difficile [tcdA and the tcdB genes contain a TcdR-dependent promoter, while the upstream region of the tcdR gene also contains two putative TcdR-dependent promoters as well as σD-dependent and σA-dependent promoters [TcdR is a σ-factor belonging to the σifficile ,8,9. Tcdifficile , but recifficile . The upsromoters ,13,14. Tromoters ,14.tcdR promoter region to a β-glucuronidase gene in C. difficile or Clostridium perfringens and two putative promoters P1tcdR and P2tcdR were proposed for TcdR recognition [tcdR and P2tcdR did not show detectable activity using alkaline phosphatase as a reporter in C. difficile, while the promoters of tcdA and tcdB showed high and low activities with about a twenty-fold difference [Bacillus subtilis as the host. Using this system, we analyzed the activities of different TcdR-dependent promoters and investigated the key determinants in the promoter for the transcriptional activity. We further analyzed the structural basis of promoter recognition using the structure of TcdR predicted by AlphaFold2. The results in this study provide the molecular basis of the TcdR-dependent promoter activity for toxin production, and the method used in this study provides a new tool for studying C. difficile toxin regulation.A previous study showed that TcdR can stimulate the transcription of fusion of the ognition . Howeverfference . It has fference , but theB. subtilis is a safe model bacterium with many available genetic tools. It has been widely used in protein heterologous expression and functional studies [B. subtilis, the bacterium should be suitable for studying promoter activity controlled by TcdR. By referring to a previous report [B. subtilis reporter system that contains two parts , while the -10 elements of P1tcdR and P2tcdR are more different from PtcdA and PtcdB. Therefore, we suspected that the -10 element is the key to determining the activity of different promoters. We constructed several mutant promoters by exchanging the -10 regions (6 bp) of the four TcdR-dependent promoters. As expected, the PtcdA-sR1 and PtcdA-sR2 showed no activity in the reporter system, while P1tcdR-sA showed high activity (about 30% activity of wild-type PtcdA-s)  B. This rtcdA-s and P1tcdR-s  from Mycobacterium tuberculosis (PDB 5ZX2) [H contain a helix-turn-helix motif which could bind to the major groove of -35 promoter DNA , which may be proposed to recognize the crucial T base at the second position of the -10 region of the TcdR-dependent promoter because the same T bases exist in the alignment of the TcdR- and σH-dependent promoters  is the key part in the active site cleft of the RNAP core enzyme. All these features are consistent with the structural features of ECF σ factors, suggesting that TcdR is also an ECF σ factor, i.e., a group 4 σ70 factor.TcdR showed very low homology with known group 1–4 σ group 5 . Current group 5 . CompareDB 5ZX2) , the Tcdoter DNA B. The suoter DNA C. The spromoters D. The roromoters E. SingleC. difficile by recognizing the promoters of their genes [tcdR gene, but a later study showed no detectable activity of these putative promoters [Bacillus heterologous system, confirmed that the two promoters do not have TcdR-dependent activity. The reason, elucidated by mutation analysis, is the -10 region in these promoters.TcdR controls the expression of the major toxins in ir genes . TcdR waromoters ,14. This70 factors because of the low sequence identity compared with group 1–4 σ70 factors [70 factors are mainly classified according to the domain organization [70 factors are alternative σ factors with fewer domains. The group 4 σ factors  are most functionally divergent but with the simplest domain organization containing only the σ2 and σ4 domains by a linker (σ3.2). Our analysis in this study showed that the structure, domain organization, and promoter recognition of TcdR are consistent with ECF σ factors, suggesting that it should be classified into ECF σ factors. A previous study showed that the expression of TcdR is controlled by temperature [TcdR and homologous σ factors in several pathogens for toxin regulation were considered to be a new group (group 5) of σ factors ,22. Howenization ,24. Grouperature , conformBacillus heterologous system constructed in this study has several advantages in the study of the TcdR function. The genetic manipulation of Bacillus subtilis as a safe model microorganism is more convenient than C. difficile. This heterologous system can avoid the manipulation of pathogens; therefore, it can be easily operated in more complex and large-scale equipment. B. subtilis neither contains TcdR nor enterotoxins; therefore, the interference of other factors in the original host in the results is reduced. A similar heterologous system has been constructed to study a distinct σ factor σI which regulates the expression of a multienzyme complex, cellulosome, for lignocellulose degradation [Bacillus heterologous system can be used in the study for more σ factors specific in pathogens and other bacteria that are difficult to genetically manipulate. For example, besides TcdR, C. difficile contains distinct σ factors CsfT and CsfU, which also play important roles in the pathogenesis [Bacillus system. Furthermore, RNAP and σ factors are also important targets for drug development [Bacillus system could be used to screen inhibitors more conveniently with high-throughput equipment. It should be noted that the heterologous system may also have disadvantages in these potential studies and applications. For example, the studied promoters may contain binding sites for some Bacillus-specific transcription factors which may cause results not relevant to C. difficile. Careful setup of control experiments should be conducted when using the heterologous system. Ultimately, important findings from the heterologous system should be assessed in the native organism.The radation ,26,27,28ogenesis ,30. The elopment ,32. The Bacillus heterologous system to study the TcdR-dependent promoter activity of TcdR for toxin expression in C. difficile, and the results revealed the molecular basis of the different promoter activities. Structural analysis suggested that TcdR has a domain organization and promoter recognition mechanism consistent with ECF σ factors and thus it should be classified into the ECF σ factors. The heterologous system is valuable in future studies for σ factor functions and drug development targeting these factors.In conclusion, this study constructed a Escherichia coli and B. subtilis were routinely grown in LB media at 37 °C. SM1 and SM2 media were used for the transformation of B. subtilis [The bacterial strains used in this study are listed in subtilis . MCSE mesubtilis . When neThe plasmids and primers used in this study are listed in C. difficile in B. subtilis. The tcdR gene was amplified from the genomic DNA of C. difficile 630 by PCR using the primers T01 and T02. The PCR product was ligated with the linearized pAX01 vector, which was prepared by PCR using primers T03 and T04, forming the plasmid pAT01. The plasmid pAT01 contains the tcdR gene controlled by the xylose-inducible promoter PxylA and can integrate the TcdR expression cassette and the erythromycin resistance gene into the B. subtilis lacA chromosomal locus by homologous recombination. The plasmids for the expression of TcdR mutants were constructed by the QuikChange mutagenesis method using pAT01 as the template.Plasmid pAT01 was constructed from the vector pAX01 ,35 to exlacZ from E. coli as a reporter gene, a spectinomycin resistance gene for screening, and homologous regions for integration in the amyE locus of B. subtilis. The promoter fragments of PtcdA, PtcdB, P1tcdR, P2tcdR, PtcdR, and P2tcdR-s were amplified from the genomic DNA of C. difficile 630 by PCR using the primers U01-F/R, U03-F/R, U05-F/R, U07-F/R, U11-F/R, and U16-F/R, respectively, and the pUC19 vector was linearized using the corresponding primers U02-F/R, U04-F/R, U06-F/R, U08-F/R, U12-F/R, and U17-F/R, respectively. The PCR products of the promoters, the lacZ gene, the spectinomycin resistance gene, and the homologous regions of amyE were ligated to the linearized pUC19 vectors, obtaining the plasmids pUT04, pUT07, pUT08, pUT09, pUT11, and pUT15. The reporter plasmids pUT12, pUT13, and pUT14 for the short-version promoters PtcdA-s, PtcdB-s, and P1tcdR-s were constructed by the deletion of the 5′ and 3′ regions of the long-version promoters using two rounds of PCR for the plasmid. The first round of PCR was performed using the primers U13-F/R, U14-F/U13-R, and U15-F/U13-R with the plasmids pUT04, pUT07, and pUT08, respectively, as the template. The PCR products were directly transformed into E. coli Top10 and were cyclized in the cells. The second round of PCR was performed using the primers U13-2-F/R, U14-2-F/R, and U15-2-F/R with the plasmids obtained in the first round as the template. The second-round PCR products were transformed into E. coli Top10, obtaining the plasmids pUT12, pUT13, and pUT14. To investigate the key regions and nucleotides recognized by TcdR, the pUT plasmids containing different length regions and mutants of TcdR-dependent promoters were constructed by PCR or the QuikChange mutagenesis method.A series of plasmids of pUT were constructed from the vector pUC19 and each contains a TcdR-dependent promoter and the β-galactosidase B. subtilis as the reported protocol [B. subtilis 168, obtaining the strain 168R01 after screening the double-crossover in the lacA locus. Then, each of the pUT series plasmids was transformed into TR01, obtaining the final strain  after screening the double-crossover in the amyE locus for reporting the activity of the TcdR-dependent promoter. For the study of TcdR mutants, each of the plasmids pUT04 and pUT07 containing the reporter cassette of PtcdA and PtcdB, respectively, was first transformed into the B. subtilis 168, and then the plasmids containing the expression cassette of TcdR mutant were transformed into each of these strains.The natural competence method was used for the transformation of protocol . After tB. subtilis mutant strain was inoculated and cultivated in the MCSE medium, with shaking and 250 rpm at 37 °C, until the optical density at 600 nm (OD600nm) reached 0.4–0.5. Briefly, 1% xylose was supplemented to induce the expression of the downstream gene under the control of the promoter PxylA for 3 h at 37 °C. Then, 3 mL of the bacterial cells were collected by centrifugation at 15,493× g for 10 min. The cell pellets were washed twice with Z-buffer  and resuspended with 600 μL of the working buffer . In total, 50 μL of suspension was used to determine the value of OD600nm, and the rest was lysed by ultrasonication. The lysate was centrifugated at 15,493× g for 10 min. The reaction was initiated by adding 10 μL of ortho-nitrophenyl-β-galactoside  into 90 μL of the supernatant, and the mixture was incubated at 37 °C for 4–60 min. Then, the reaction was terminated by adding 200 μL of 1 M Na2CO3 to 40 μL of the sample. The released 2-nitrophenol (ONP) was measured by determining the absorbance at 420 nm (A420nm). All the β-galactosidase activities were normalized with the OD600nm, and one unit of enzyme activity was defined as the amount of β-galactosidase that releases 1 nmol of ONP per minute.The measurement of the β-galactosidase activity was performed as previously described with minor modifications . The B.


Dear Editor,Cells enter senescence, or irreversible growth arrest, when exposed to stressors such as DNA damage, epigenetic alterations and chronic inflammation . In aginThe transport of macromolecules between the cell’s nucleus and cytoplasm is a regulated mechanism essential for eukaryotic organisms. It requires a sophisticated barrier system with channels  embedded in the nuclear envelope, and shuttling nuclear transport receptors (NTRs) that transfer cargoes. The NPCs consist of a cytoplasmic ring, the inner pore ring, the nuclear ring and peripheral elements (nuclear basket and cytoplasmic filaments) . NTRs coNTCs) (WRN-deficient) and HGPS  hMSCs; the latter two of which are human stem cell models of premature aging  screening library (NTSAG library), which contained 208 sgRNAs targeting 32 nucleoporins and 34 NTRs with three sgRNAs per gene and 10 non-targeting controls (sg-NTCs)  and S1A.re aging . In the nsporter , as the nsporter . To confnsporter . Consistnt hMSCs  and 1P. nt hMSCs . Thus, oXPO7−/− hESCs) by CRISPR/Cas9-based gene editing   . In addi/− hESCs  and S2F.XPO7+/+ and XPO7−/− hESCs into hMSCs  and increased IL-6 secretion  and with reduced H3K9me3  to elevate XPO7 expression in early-passage WT hMSCs (young hMSCs)  and S5B.ecretion . Additio H3K9me3  and S5I. H3K9me3 . Further H3K9me3 . CollectHDAC2 mRNA level in XPO7-deficient hMSCs as revealed by RT-qPCR , we observed that XPO7 deficiency led to increased protein stability of HDAC2  with FLAG antibody followed by mass spectrometry analysis (IP-MS). Within our dataset of XPO7-interacting proteins, we observed a subset that was associated with chromosome organization and histone modification . Among t RT-qPCR  and S6G.of HDAC2 . To confof HDAC2 , suggestof HDAC2 . TogetheXPO7−/− hMSCs, such as decreased proliferative ability , increased SA-β-gal positive cells, repressed expression of Lamin B1 and LAP2 and increased the expression of p16 , DNA damage response and SASP related genes via epigenetic mechanisms  , while tchanisms . Post-trchanisms . FurtherCollectively, we performed a CRISPR/Cas9-based LOF screening for nuclear transport system-related genes in regulating human MSC senescence and identified XPO7 as a new mediator of hMSC senescence. Our analyses also provide a potential intervention target for aging-related diseases using new therapeutic strategies, such as gene delivery and small molecular inhibitors.pwad012_suppl_Supplementary_MaterialsClick here for additional data file.pwad012_suppl_Supplementary_Table_S1Click here for additional data file.pwad012_suppl_Supplementary_Table_S2Click here for additional data file.pwad012_suppl_Supplementary_Table_S3Click here for additional data file.