Growth hormone-secreting pituitary adenoma (GHPA) is an insidious disease with persistent hypersecretion of growth hormone and insulin-like growth factor 1, causing increased morbidity and mortality. Previous studies have investigated the transcription of GHPA. However, the gene regulatory landscape has not been fully characterized. The objective of our study was to unravel the changes in chromatin accessibility and transcription in GHPA.Six patients diagnosed with GHPA in the Department of Neurosurgery at Huashan Hospital were enrolled in our study. Primary pituitary adenoma tissues and adjacent normal pituitary specimens with no morphologic abnormalities from these six patients were obtained at surgery. RNA sequencing (RNA-seq) and assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) were applied to investigate the underlying relationship between gene expression and chromatin accessibility changes in GHPA.Totally, 1528 differential expression genes (DEGs) were identified by transcriptomics analyses, including 725 up-regulated and 803 down-regulated. Further, we obtained 64 significantly DEGs including 10 DEGs were elevated and 54 DEGs were negligibly expressed in tumors tissues. The up-regulated DEGs were mainly involved in terms related to synapse formation, nervous system development and secretory pathway. In parallel, 3916 increased and 2895 decreased chromatin-accessible regions were mapped by ATAC-seq. Additionally, the chromatin accessible changes were frequently located adjacent to transcription factor CTCF and Rfx2 binding site.Our results are the first to demonstrate the landscape of chromatin accessibility in GHPA, which may contribute to illustrate the underlying transcriptional regulation mechanism of this disease. Growth hormone-secreting pituitary adenoma (GHPA) is characterized by increased growth hormone (GH) and insulin-like growth factor I IGF-1) secretion  secretio. NotablyGsα), the stimulatory G protein (GNAS), α-subunit of protein kinase C (PKC) and aryl hydrocarbon receptor-interacting protein (AIP) have been discovered to be associated with GHPA ) for 10 min on ice to prepare the nuclei. Immediately after lysis, nuclei were spun at 500 × g for 5 min to remove the supernatant. Nuclei were then incubated with the Tn5 transposome  and tagmentation buffer at 37 °C for 30 min. After the tagmentation, the stop buffer was added directly into the reaction to end the tagmentation. PCR was performed to amplify the library for 10–12 cycles using the following PCR conditions: 72 °C for 3 min; 98 °C for 30 s; and thermocycling at 98 °C for 15 s, 60 °C for 30 s and 72 °C for 3 min; following by 72 °C 5 min. After the PCR reaction, libraries were purified with Agencourt Ampure XP beads  to remove contaminating primer dimers and quantitated using Qubit 2.0 Fluorimeter . All libraries were sequenced on the Illumina HiSeq X Ten with 150 bp paired-end reads .The ATAC-seq libraries were prepared as previously described , 19. Brihttp://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Reads from RNA-seq experiments were aligned to human genome build hg19 using STAR [p value < 0.05, fold change (FC) > 1.5 and the average FPKM > 5 for at least one of the two groups. The aligned BAM files were converted to bigWig format and normalized by reads per kilobase million (RPKM) with the bamCoverage script in deepTools 2.0 [The quality control of mRNA sequencing data was performed via FastQC (ing STAR . The mRNing STAR . Differeools 2.0 . The deeools 2.0  from R phttp://www.bioinformatics.babraham.ac.uk/projects/fastqc/). All sequencing data were mapped onto the hg19 human genome assembly using Bowtie2 [https://broadinstitute.github.io/picard/) and ggplot2 [The quality of sequenced ATAC-seq data was evaluated with FastQC  enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed using clusterProfiler , which iTo annotate the location of ATAC-seq signal in terms of important genomic features, the annotatePeaks.pl script in HOMER  was usedFor purposes of assessing the alteration of chromatin accessibility genome-wide, deepTools was used to compute the average scores  for transposase hypersensitive sites in each sample. Hyper-accessible regions (hyper) associated with increased ATAC-seq signal were defined if the regions showed fold change (FC) > 1.5 in the tumor group as compared to the normal group. On the contrary, the hypo-accessible regions associated with decreased ATAC-seq signal were defined if the regions showed fold change >1.5 in normal group compared to tumor group. The heatmaps and average profiles of regions expanded to ±1.5 Kb surrounding differentially accessible regions (DARs) center were generated using the plotHeatmap and plotProfile functions in deepTools . The annThe findMotifsGenome function in HOMER v4.9  were usehttps://www.R-project.org/) was used to compute statistics and generate plots. Fisher’s exact test, Student’s t test, Pearson’s correlation coefficient, Wilcoxon signed rank test were used to assess the significance. Fold change, p values and false discovery rate were calculated in analysis. Significant differences for all quantitative data were considered when *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Genome browser images were made using the Integrative Genomics Viewer (IGV) [If not specified, R platform (er (IGV)  with bigq value < 0.05, |log2fold change| ≥2 and average FPKM ≥ 10. As shown in Fig. CBLN1, FHIT and BPIFA1, were elevated in the tumors, and the other 54 DEGs were negligibly expressed, such as ESAM, TGFBR2, ID1 and FSHB. Furthermore, GO enrichment analysis was performed to reveal the probable biological process, cellular components, and specific molecular functions of all DGEs. As shown in Fig. To investigate gene expression profiles of GHPA, mRNA-seq was used to examine genotypic changes in tumors and adjacent normal pituitary tissues Fig. A, B. A t4–105 bp away from TSS, while a shrink of hyper-accessible regions was displayed within 0–103 bp. On the contrary, regions with a distance beyond 104 bp to TSS, which were defined as newly closed, comprised an enormous part of all regions. An increased proportion of hypo-accessible regions was discovered with more than 1000 bp distance from TSS, which indirectly suggested distal cis-regulatory element like enhancer might be deactivated , but the relationship between the hyper-accessible regions and their nearest DEGs was slight , Sterol Regulatory Element Binding Transcription Factor 1 (SREBF1), Wnt Family Member 5B (WNT5B) and Growth Hormone Releasing Hormone Receptor (GHRHR) were significantly upregulated nearby the hyper-accessible regions. On the contrary, restrained chromatin accessibility diminished expression of several genes such as Sorbitol Dehydrogenase (SORD), Dual specificity phosphatase 6 (DUSP6), Laminin subunit alpha-2 (LAMA2), WW Domain Containing Transcription Regulator 1 , Metalloproteinase inhibitor 3 (TIMP3), Yes1 Associated Transcriptional Regulator (YAP1) and Follicle Stimulating Hormone Subunit Beta (FSHB)  Fig. .Fig. 3FiTo deeply insight into the functional role and the latent signaling pathway of the DARs and DEGs, KEGG pathways analysis were performed. The DEGs associated with hyper-accessible regions were enriched in pathways relative to Insulin, Estrogen, GH, ErbB, Axon guidance, and cAMP signaling pathway Fig. . The actSSTR1, WNT5B, and GHRHR  are proteins that regulate gene expression through binding to specific DNA sites in cis-regulatory regions of target genes . Normallpression . AltogetThe genome-wide landscape of GHPA has been investigated in a few studies, but the variation of chromatin state in GHPA has never been investigated , 36. As GHRHR is significantly up-regulated in GHPA compared with normal pituitary tissues. Somatostatin binds to SSTR1-5 to inhibit GH secretion [Here, based on the RNA-seq, 64 significantly DEGs were obtained, including 10 upregulated DEGs and 54 down-regulated DEGs, in GHPA compared with normal pituitary tissues. It is worth noting that most up-regulated DEGs in tumors participate in various hormone-related signaling pathways  and pathways in GH synthesis, secretion and action. GH secretion by the somatotroph cells is mediated by ghrelin and GH-releasing hormone (GHRH) and inhibited by somatostatin and IGF-1 feedback inhibition . The effecretion . Previouecretion . Elevateecretion . Here, oAdditionally, our data reveal that the down-regulated DGEs are enriched in signaling pathways that included focal adhesion pathway. Focal adhesion, a subcellular structure between the cell and the extracellular matrix, plays an important role in cell migration through tissues . PreviouFSHB.Further, ATAC-seq were undertook to reveal the noticeable alterations in transcriptomes and chromatin accessibility of GHPA. Comparing with normal pituitary tissues, there are 64 significantly DEGs identified in GHPA, the proportion of down-regulated DEGs is higher than that of up-regulated DEGs, which is corresponding to previous studies , 56. ComIn conclusion, our study is the first to depicture global changes in chromatin accessibility and transcription in GHPA. Combination of these data can provide relevant insight into the tumor biology, especially in regulatory network that contributes to tumor formation and growth. Our results demonstrated that the decreased chromatin-accessible regions are depleted for TF binding consensus motifs, which will lead the expression decrease in GHPA terminally. However, we are aware of the limitations of this research. Firstly, the sample size of GHPA and control group is small. Secondly, more informative is needed to validate the present findings in vitro and in vivo. The mechanism and functional consequence behind the interactions between TFs and chromatin accessibility change regions is worth further investigation.Supplementary Figure legendsFigure S1Figure S2Supplementary Tables


The physical interactions between enhancers and promoters create chromatin conformations involved in gene regulation. In cancer cells, the chromatin conformations can be altered with uncontrolled deposition of histone marks resulting in varied gene expression. Although it is not entirely comprehensive how chromatin-mediated enhancer–promoter (E–P) interactions with various histone marks can affect gene expression, this proximity has been observed in multiple systems at multiple loci and is thought to be essential to control gene expression. In this review, we focus on emerging views of chromatin conformations associated with the E–P interactions and factors that establish or maintain such interactions, which may regulate gene expression.Eukaryotic gene expression is regulated through chromatin conformation, in which enhancers and promoters physically interact (E–P interactions). How such chromatin-mediated E–P interactions affect gene expression is not yet fully understood, but the roles of histone acetylation and methylation, pioneer transcription factors, and architectural proteins such as CCCTC binding factor (CTCF) and cohesin have recently attracted attention. Moreover, accumulated data suggest that E–P interactions are mechanistically involved in biophysical events, including liquid–liquid phase separation, and in biological events, including cancers. In this review, we discuss various mechanisms that regulate eukaryotic gene expression, focusing on emerging views regarding chromatin conformations that are involved in E–P interactions and factors that establish and maintain them. The spatial organization of the genome into transcriptionally active and silenced chromatin plays a fundamental role in the three-dimensional 3D) architecture required for the regulation of eukaryotic genes ,2. GenomD architecis-elements that contain diverse DNA sequences to which various transcription factors (TFs) and transcriptional co-activators bind and that are enriched with various histone modifications that facilitate gene transcription [cis-element) to transcriptional control.In one key mechanism, gene transcription is regulated through chromatin loops that form between promoters and various regulatory elements, including enhancers ,7. Enhancription ,9,10. InHistone modification is widely used as a means to classify enhancers according to their activity: H3K4me1 and the binding of trithorax-related mixed lineage leukemia (MLL) complex define primed or active enhancers; H3K27me3 is a key marker of poised or inactive enhancers; histone H3 lysine 27 acetylation (H3K27ac) is a hallmark of transcriptionally active enhancers  11,12,1,14. ReceHyperacetylation of histone H4 at its N-terminal tail is essential for normal spermatogenesis ,21 and oH3K27me3 is present at high levels in CpG islands, which are associated with the promoters of developmental genes in mammals ,31. In acis-regulatory elements contain TFs, RNA polymerase II, and chromatin-remodeling and histone-modifying enzymes, whose combined activities induce chromatin accessibility. Specifically, proteins bound to enhancers and promoters may interact with each other non-randomly and preferentially [How do specific enhancers locate and contact promoters to accomplish transcriptional regulation? Remarkable progress has been achieved regarding the molecular principles and combinatorial logic underlying these processes ,7, but centially ,39, leadentially . In cancentially . Here, wHow is the genome regulatory domain formed that facilitates E–P interactions? Several mechanisms participate and involve TFs (particularly pioneer factors) at enhancers to mediate chromatin remodeling ; loop exThe combined activities of TFs, RNA polymerase II, and chromatin-remodeling and histone-modifying enzymes generate cell type-specific accessible chromatin regions; these factors also create marks that serve as binding sites for effector proteins . These tThe binding of pioneer factors to DNA sequence motifs is context-dependent and cell-type–specific. For example, in several human cell lines, including liver carcinoma (Hep2), lung carcinoma A540), and ESC-derived endoderm, FOXA2 show cell-type–specific binding: only 6% to 14% of FOXA2 binding occurs in its recognition motifs in the mentioned cell types . Cell-ty0, and ESIn addition, several other proteins, including yin and yang 1 (YY1), zinc finger protein 143 (ZNF143), and Myc-associated zinc finger protein (MAZ), may contribute to the organization of chromosomal architecture, especially of E–P interactions in mammals . YY1 is Znf143 in adult hematopoietic stem and progenitor cells leads to the loss of CTCF binding on promoters and enhancers and induces changes in gene expression [Znf143-knockout hematopoietic stem and progenitor cells, whereas TAD formation and compartmentalization in the knockout cells are not affected [Like YY1, ZNF143 is a regulator of E–P contact . Conditipression . Importaaffected . This fiaffected .in cis- and in trans- (in sister chromatid) fashion [β-globin locus could form a chromatin loop [CTCF mediates chromatin looping by associating with the cohesin complex ,65. The  fashion . CTCF pl fashion . In addi fashion , and CTCtin loop . In additin loop . Accorditin loop ,71, thustin loop ,73. Howetin loop .The transcriptional co-factor BRD4 facilitates the recruitment of Mediator  to actiPcG complexes and LIM domain binding proteins may contribute to E–P interactions via homotypic contacts . PcG prLIM domain binding protein 1 (LDB1) is a nuclear adapter protein that interacts with the C-terminal LIM domain in numerous LIM domain–containing TFs . In addiLLPS is the formation of membrane-less biomolecular condensates and occurs when overall protein(s) content increases to a critically high point, leading to the formation of phases according to the concentration  of the associated proteins ,46. The LLPS may provide a mechanistic explanation of the classic ‘transcription factory’ model , wherebyHbb) promoter and its enhancer led to strong transcriptional activation of Hbb, even in the absence of the lineage-specific transcription factor GATA1 [HBG) involved in β-thalassemia and sickle cell anemia [Now that we have reviewed the factors and mechanisms that influence the formation of E–P interactions, we focus our discussion on the role of chromatin-mediated E–P contact in transcriptional control. In permissive chromatin, the contact between enhancer and promoter regions appears to be sufficient to induce transcription in the absence of lineage-specific transcription factors . For exaor GATA1 , thus dil anemia ,107. Howl anemia .cis-regulatory sequences devoid of classic enhancer chromatin marks might have the potential to perform enhancer-like functions and provide the E–P contact necessary for transcription. For example, in mouse ESCs, a class of non-canonical enhancers marked by H3K122ac—but lacking H3K27ac—controls the expression of target genes, including key pluripotency genes such as Tbx3 and Sox2 [MYCN and NFIC reduced their expression in human glioblastoma stem-like cells and diminished these cells’ stem-like properties . Together, these data suggest that alternative sites for histone acetylation other than H3K27ac—modified by either p300/CBP or other enzymes—might maintain E–P contact and thus lead to gene activation.In this section, we discuss how repositioning just a few histone modifications—especially in cancer cells—changes local interactions between enhancer and promoter regions. Increased levels or breadth of H3K27ac enrichment at enhancer regions has been associated with oncogenic enhancer activity in human colorectal cancer cells, even in the absence of chromosomal alterations . Althougand Sox2 . Using aRegarding heterochromatin, H3K27me3-rich regions consist of silencers that can repress target genes, such as tumor suppressors in human cancer cells, either in proximity or through long-range chromatin interactions . CRISPR-Along with CTCF and cohesin, post-translational modification of histones is necessary to determine the 3D structural organization of chromatin . The enrIn human leukemic cells, the increased accumulation or distribution of H3K27ac at specific enhancers serves to ‘zip’ together these regions to increase the E–P contact frequency and the number of interacting regions of target gene promoters, thus influencing oncogene expression . ImportaThe high levels of context-specific histone marks, TFs, and pioneer TFs at enhancers relative to promoters proclaim that E–P interactions are likely important to the expression of many genes. In addition, the expression level of a gene and the number of promoters interacting with enhancers are positively correlated ,125, thuWe surmise that, through the combined action of various histone acetyltransferases, diverse histone acetylations, including H4K5acK8ac and H2BNTac, create an acetylation-dependent condensate and recruit regulatory factors to enhancers to establish their interaction with a specific promoter, resulting in transcriptional activation. Given that histone modifications can alter the genomic architecture, the conformation of chromatin loops with variable active and permissive histone marks changes, leading to either a gain or loss of E–P interactions and subsequent up- or down-regulation of the expression of the associated gene. To overcome the difficulty in determining the causal order among architectural proteins, TFs, histone marks, and gene expression with E–P contact, CRISPR-Cas9 genome editing might be applied to assess the role of each factor, especially that of enriched histone marks in maintaining E–P contact. The functional relationship between E–P contact and gene expression is rather complex in terms of chromatin structure, and further mechanistic studies are required to explain this dependence. The integration of rapidly advancing computational models, locus-specific imaging, and functional perturbation through CRISPR–Cas9 genome editing, inducible degron systems, and epigenetic inhibitors hold great promise in addressing such questions.


Cattle-yak, a crossbreed of yak and cattle, which can exhibit obvious heterosis and can adapt to the harsh environmental conditions of the Qinghai Tibet Plateau (QTP). However, F1 cattle-yak were found to be sterile because they were unable to produce sperm, which adversely restricted the fixation of heterosis. Many prior attempts have been made to decipher the mechanism underlying the spermatogenesis stagnation of cattle-yak. However, the open chromatin region (OCR) map of yak and cattle-yak testes has not been generated yet. Here, we have analyzed the OCRs landscape of testicular tissues of cattle-yak and yaks by performing ATAC-seq technology. The OCRs of cattle-yak and yak testes displayed similar genome distribution and showed priority in intergenic regions, introns and promoters. The pathway enrichment analysis indicated that the differential OCRs-related genes were involved in spermatogenesis, involving the cell cycle, as well as Hippo, mTOR, MAPK, Notch, and Wnt signaling pathways. The integration of ATAC-seq and mRNA-seq indicated that the majority of the gene expression levels were positively correlated with chromatin openness. At the same time, we have identified a number of transcription factors (TFs) related to spermatogenesis and the differential expression of these TFs may contribute to the spermatogenesis stagnation of the cattle-yak. Overall, the findings of this study provide valuable information for advancing the research related to yak crossbreeding improvement and sperm production stagnation of cattle-yak. Bos grunniens) is a bovid animal, which possesses strong adaptability to harsh ecological environmental conditions such as extreme cold, hypoxia, and strong ultraviolet radiation. It is an indispensable omnipotent livestock found in the Qinghai-Tibet Plateau (QTP) and serves as an important means of production and living for the local herdsmen [The yak  were silenced due to methylation of the promoter region, which effectively disrupted the production of piRNAs in pachytene and interfered with spermatogenesis in cattle-yak [Anatomical histology has shown that the circumference, diameter, and weight of cattle-yak testes were significantly smaller in comparison to those of yaks. The spermatogonia (SG) in the convoluted seminiferous tubule of the cattle-yak act as the main germ cells, and can produce only a few primary spermatocytes (PSC) and fewer secondary spermatocytes . Cytogenttle-yak . Howeverttle-yak . bta-miRttle-yak .CTCF, BORIS, NFY, DMRT6, EN1, ISL1, and GLI3. Moreover, combined with scRNA-seq data, it was found that TLE3 was specifically expressed in the differentiated sperm, PFN4 participated in the localization of actin cytoskeleton during meiosis, and the unique area upstream of TLE3 and PFN4 exhibited high accessibility [The transcriptome of cattle-yak testis was altered and epigenetic modification plays an important role. However, more research is needed to completely dissect the molecular mechanisms underlying spermatogenesis disorder in cattle-yak. Eukaryotic DNA is not bare, but is packaged into nucleosomes to form a beaded structure and further folded and packaged. Transcription of genes, however, requires unraveling this high-level structure, making DNA a bare state that allows various transcription machines to bind to it, forming open chromatin regions (OCRs). Importantly, the map of OCRs of the testes of yak and cattle-yak has not been generated, and different transcription factors (TFs) can bind to the DNA in OCRs in a sequence-specific manner and regulate the transcription process . These Osibility . ATAC-sesibility . The finn = 3) and yak (n = 3) testicular tissue preparations were sequenced on the Illumina Hiseq 2500 platform, and 150 bp paired-end reads were generated. A total of 78.26 G of data were sequenced from six different samples, raw reads with an average (±SD) per sample of 43,469,756  were initially collected. Thereafter by removing the linkers and low-quality reads, at least 40,851,986 clean reads per sample were obtained. Bowtie2 software was used to compare the clean read with the yak reference genome (version: LU_Bosgru_v3.0), and the comparison rate was about 94.2%. Then the bases were removed with a mass value of less than 30 and repetitive sequences introduced by PCR. Overall, finally, each sample has 19,748,788  valid reads  to obtain the difference in OCRs. The results indicated that there were 49,659 different OCRs, among which 42,236 OCRs in the testicular tissue of the cattle-yak displayed increased openness and 7423 OCRs exhibited decreased openness  and promoter region difference OCRs  was analyzed. The chromatin open level and mRNA level of 2958 genes were found to be significantly different, among which 1696 genes upregulated chromatin open level and mRNA expression level, 1106 genes increased chromatin open level but downregulated mRNA expression level, 13 genes decreased chromatin open level but upregulated mRNA expression level, whereas open chromatin level and mRNA level expression of 143 genes were significantly down-regulated (ODF1 (outer dense fiber of sperm tails 1) and TEKT5 (tektin 5), as well as the cell cycle-related gene TP53 (tumor protein p53) ATAC-seq and RNA-seq signals. The results indicated that the level of ATAC-seq signal in the promoter region of the related genes was significantly higher in comparison to that found in other locations  of the samples in this study for further analysis. According to the FPKM value of the gene expression, we divided the genes into five distinct groups , and calculated the chromatin openness level at the different gene positions. The results showed that the expression levels of most genes were positively correlated with the level of chromatin openness, that is, the higher the gene expression level, the stronger the chromatin openness. In addition, we found that except for the lower group, the chromatin openness level of the other four groups exhibited the highest peak near the TSS a. We paiegulated b. To anaocations c.MYC (myelocytomatosis oncogene), GATA3 (GATA binding protein 3), GATA1, and TFEB (transcription factor EB) were the most connected TFs, and the expression levels of these TFs were observed to be significantly different between cattle-yak and yak groups. They can cooperate closely with several other TFs in the regulatory network and might play an important role in regulating the optimal development of the cattle-yak testis and spermatogenesis.It has been established that TFs and other regulatory proteins are required to identify the specific motifs to combine with DNA to initiate gene expression. We used the MEME Suite tool to perform Motif scanning and enrichment analysis on the differential OCRs in the JASPAR database . Figure The openness of cis-regulatory element promoters and enhancers to DNA-binding proteins is substantially limited by the local structure of chromatin. It has been found that only OCRs without nucleosome encapsulation can bind to TFs and recruit RNA polymerase into the core promoter to initiate transcription . In addiThe peaks in both cattle-yak and yak groups exhibited a higher proportion distribution in the distal intergenic and intron, followed by the promoter, then exor, 5’UTR, downstream, and 3’UTR. This might be probably caused by the high proportion of the distal Intergenic and intron in the yak genome . This reTGFB2 (transforming growth factor-β2) and TGFB3 were significantly enriched in the cell cycle, Hippo, MAPK, and other signaling pathways related to spermatogenesis. They are two of the three subtypes of transforming growth factor-β that have been identified in mammals, and play an important role in regulating the growth, differentiation, and development of the various cells in living things. Transforming growth factor-β can act on the retinoblastoma protein and cyclin-dependent kinases to block the late G1 phase of the mitotic cell cycle and thus inhibit the cell growth [The functional enrichment analysis of the differential OCRs-related genes in the promoter region showed that differential OCRs-related genes can be significantly enriched in the pathways related to spermatogenesis, such as cell cycle, mTOR, Notch, and Wnt signaling pathways. The notch signaling pathway is essential for initiating mitotic arrest and enables the maintenance of male germ cells’ identities. A prior study has reported that the conditional activation of the Notch1 intracellular domain in the germ cells could lead to an increase of germ cell apoptosis, reduction in the sperm counts, and progressive loss of testis weight with age . Anotherl growth , as wellCTCF binding sites are mainly concentrated in intergenic regions as well as introns and overlap with enhancer and promoter sequences. It has been reported that the binding of CTCF with promoter or enhancer regions usually plays an inhibitory role [SP3 factor and active SP1 factor bind to the same site, they can act as passive repressors by preventing the active factors from binding to DNA binding sites, thus preventing activation [Through the joint analysis of ATAC-seq and mRNA-seq data, we further explored the possible interaction between chromatin openness and gene expression levels. We found that the expression level of most genes was positively correlated with the chromatin opening level. Moreover, the genes exhibited the highest level of ATAC-seq peak near TSS, which was consistent with the report in the study of early bovine embryos  and yak ory role . Moreovetivation . These fMYC, TFEB, GATA1, and GATA3. MYC is a member of the helix-loop-helix leucine zipper superfamily, including C-myc, N-myc, and L-myc. C-myc is an androgen-regulatory gene expressed in Sertoli cells. A number of studies have shown that in the transgenic rats expressing C-myc, spermatogenesis at the primary spermatocyte level was blocked, and apoptotic DNA fragments increased significantly [TFEB is a major regulator of lysosomal biogenesis, autophagy, and endocytosis. The regional expression and activation of TFEB were closely related to the retinoic acid signal, which can effectively promote cell migration in the blood-testis barrier and transport along the seminiferous epithelium [GATA family is a kind of transcription regulator with a zinc finger structure, which plays an important role in regulating organ morphogenesis, cell proliferation, and gender differentiation. GATA1 and GATA3 are developmental stage and spermatogenic cycle-specific regulators of gene expression in the Sertoli cells [GATA1 occupied the MYC promoter in vivo, thus suggesting the direct mechanism of gene inhibition. Surprisingly, the forced expression of MYC prevented the cell cycle arrest induced by GATA1 [C-myc is a key target for GATA3 and can promote cell proliferation [MYC interacted with GATA1 and GATA3, which may contribute to the spermatogenesis defect of the cattle-yak, but the specific mode and mechanism of its role in spermatogenesis need further analysis.In this study, we conducted an enrichment analysis on the motif of differential OCRs. The TFs identified with more connections were ficantly . TFEB isithelium . The GATli cells ,38. A prby GATA1 . Anotherferation . In our All experimental procedures involved in this study were reviewed and confirmed by the Animal Administration and Ethics Committee of Lanzhou Institute of Husbandry and Pharmaceutical Sciences, Chinese Academy of Agricultural Sciences (SYXK-2014-0002).Three male Gannan yaks  were obtained from Linxia County, Linxia Hui Autonomous Prefecture. Three male cattle-yaks  were the hybrid offspring of Jersey cattle (♂) and Gannan yak (♀), obtained in Xiahe County, Gannan Tibetan Autonomous Prefecture. All animals were 4 years old. After the yak was euthanized, the testicular tissue was collected, and the testicular tissue of the cattle-yak was collected by veterinary surgery. The samples were sent to the Lanzhou Institute of Husbandry and Pharmaceutical Sciences, Chinese Academy of Agricultural Sciences, for further experiments.TM, Waltham, MA, USA). The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS  according to the manufacturer’s instructions. After the cluster generation, the library preparations were sequenced on an Illumina Hiseq 2500 platform and 150 bp paired-end reads were generated.ATAC-seq was performed as reported previously ,41. BrieThe raw data was first used in Trimmomatic  to removThe peaks co-existing in at least 2 samples within a group were annotated by employing ChIPseeker . Thereaf2FoldChange | ≥ 0.58, p ≤ 0.05. We used the R package clusterProfiler4.0 [http://jaspar.genereg.net, accessed on 12 September 2021) by using MEME Suite [R package DiffBind was used to analyze the differential OCRs. First, an overlap of each sample peak was taken to obtain the summit and expanded 200 bp upstream and downstream with the summit as the center, to redefine the peak, and calculate the reads counts of each sample on the overlapping peak. The reads counts on the peaks of each sample were then normalized for the correction, by using the difference comparison tool DESeq2 , and thefiler4.0  to perfoME Suite .TM, Waltham, MA, USA) was employed for assessing the RNA’s quantification and purity. Agilent 2100 Bioanalyzer  was used for probing RNA integrity. Finally, the library was created as per the manufacturer’s manual by employing the TruSeq Stranded mRNA LT Sample Prep . The sequencing was then performed by using an Illumina HiSeq 2500, generating 150 bp paired-end reads. All the sample raw data used Trimomatic [2FoldChange | ≥ 1 and q ≤ 0.05.Total RNA from samples was extracted using TRIzol reagent. The NanoDrop 2000 Spectrophotometer  according to the quintile threshold of the FPKM value of the gene expression. R/Gviz was used to study the distribution trend of the ATAC-seq signal in the gene body and its upstream and downstream 3 kb regions at the different expression levels. IGV browser was used to display the visualized results at the whole genomic level. STRING database was employed to generate TF tuning network and visualize it in Cytoscape .In summary, our study described the landscape of OCRs in the testes of both yak and cattle-yak. The integration of ATAC-seq and mRNA-seq indicated that most gene expression levels were positively correlated with chromatin openness. Additionally, we have identified several TFs related to spermatogenesis, and the differential expression of these TFs might contribute to the spermatogenesis stagnation of the cattle-yak. The findings of this study provide valuable information related to the research about yak crossbreeding improvement and sperm production stagnation of cattle-yak.


Leishmania tarentolae. These experiments show that two histone variants (H2A.Z and H2B.V) and three histone H3 PTMs  are enriched at transcription start sites (TSSs); while a histone variant (H3.V) and the trypanosomatid-specific hyper-modified DNA base J are located at transcription termination sites (TTSs). Reduced nucleosome density was observed at all TTSs and TSSs for RNA genes transcribed by RNA polymerases I (RNAPI) or RNAPIII; as well as (to a lesser extent) at TSSs for the PTUs transcribed by RNAPII. Several PTMs  and base J were enriched at centromeres, while H3K50ac was specifically associated with the periphery of these centromeric sequences. These findings significantly expand our knowledge of the epigenetic markers associated with transcription, DNA replication and/or chromosome segregation in these early diverging eukaryotes and will hopefully lay the groundwork for future studies to elucidate how they control these fundamental processes.Eukaryotes use histone variants and post-translation modifications (PTMs), as well as DNA base modifications, to regulate DNA replication/repair, chromosome condensation, and gene expression. Despite the unusual organization of their protein-coding genes into large polycistronic transcription units (PTUs), trypanosomatid parasites also employ a “histone code” to control these processes, but the details of this epigenetic code are poorly understood. Here, we present the results of experiments designed to elucidate the distribution of histone variants and PTMs over the chromatin landscape of  Leishmania are transmitted by sand flies, in which they proliferate as procyclic promastigotes and differentiate into non-dividing metacyclic promastigotes. After transmission to the vertebrate host, metacyclics infect macrophages and differentiate into amastigotes, which grow and divide within the phagolysosome and ultimately lyse the host cell to serially infect other macrophages [Trypanosomatid parasites cause widespread suffering and death in humans and animals, resulting in a substantial economic burden in many countries around the world . Leishmarophages . Infectirophages , rangingLeishmania and other trypanosomatids) is packaged into chromatin by interaction with four core histone  homodimers to form heteromeric nucleosomes that each have ~150 bp of DNA wrapped around them. The nucleosomes are joined by a linker histone (H1) to form higher-order structures, compacting the DNA and protecting it from damage. DNA base modification and post-translational modifications (PTMs) on the histones (and their variants) are used to control diverse biological processes, including DNA replication/repair, chromosome condensation, and temporal regulation of gene expression. Lysine and arginine residues in N-terminal “tails” of the core histones protruding from the nucleosome are covalently modified by methylation, acetylation, or ubiquitination; while serine or threonine residues are phosphorylated. These PTMs can change the local nucleosome density, thereby controlling the rate of transcription initiation, elongation, and termination. The number of PTMs is large, and our understanding of the functional significance of this epigenetic “histone code” is still in its infancy, even for humans and model organisms [The nuclear genome of all eukaryotes  located between PTUs, suggesting that J may also have a role in regulating transcription initiation in some species [In most eukaryotes, each gene normally has its own transcription start site (TSS) and termination site (TTS), allowing for regulation of mRNA production on an individual basis. In contrast, trypanosomatid genomes are organized into polycistronic transcription units (PTUs) containing tens-to-hundreds of protein-coding genes on the same strand of DNA . The pri at TTSs ; but in g siRNAs . In cont species .Leishmania H1 contains a single domain that corresponds to the lysine-rich C-terminus of H1 in higher eukaryotes, but few of the residues with PTMs in human H1 are conserved [Leishmania H2A and H2A.Z both appear to represent a sister branch to the H2A.Z variant of higher eukaryotes. Most of the lysines that have PTMs in human H2A/H2A.Z are conserved, but Leishmania H2A.Z contains a larger N-terminal extension with more PTMs than in other organisms [Leishmania H2B has a shorter N-terminal tail than in mammals, with only two of the modified lysines being conserved . However, H2B.V in Leishmania  has a longer tail containing additional lysines, which may be the functional equivalent of H2B residues that are acetylated in actively transcribed human chromatin [Leishmania and Trypanosoma contains a longer N-terminal tail , although there are differences between the two species [T. brucei has an H4 variant (H4.V), but Leishmania appears to lack an obvious orthologue [While trypanosomatids have orthologues for all the canonical histones, they are quite divergent from those in other organisms ,20,21,22onserved . Leishmarganisms . Leishmahromatin . H3 and  species . H4 is tthologue , althougT. brucei [T. cruzi [T. brucei, including trimethylation of H3K4, H3K10 and H3K11, acetylation of H3K23, and acetylation of several lysines on H2A.Z, H2B.V and H4. Interestingly, at least two different histone acetyltransferases (HATs) are involved in acetylation of T. brucei histones; HAT1 is primarily responsible for acetylation of H2A.Z and H2B.V, while HAT2 acetylates H4 [Leishmania, but H3K9ac  has been shown enriched at TSSs [L. major; while H4K4ac, H4K10ac and H4K14ac are enriched at TSSs in L. donovani [L. tarentolae.Eukaryotes have evolved a panoply of “readers” and “writers” that recruit chromatin modification complexes and alter local nucleosome density, respectively, thereby controlling its accessibility to RNA polymerase and transcription factor complexes ,28. The . brucei  and ~200T. cruzi . A more T. cruzi  identifilates H4 . There h at TSSs ,30,31 an at TSSs  in L. madonovani ,30,31. Hdonovani  to signiL. tarentolae, we employed Cleavage Under Targets and Tagmentation (CUT & Tag) [L. tarentolae genome using a short-read aligner and the read density plotted for each of the 36 chromosomes in the genome. Leishmania chromosomes, although some minor differences were occasionally seen, as outlined below.To map the location of histone variants and PTMs across the chromatin landscape of T & Tag) , in whicT & Tag)  that shoH2A and H2B (to a lesser extent) were depleted at all TSSs, irrespective of whether they were bi-directional, unidirectional or telomeric B, consisL. tarentolae chromosomes were identified by chromatin confirmation capture followed by next generation sequencing (Hi-C) and confirmed by synteny with L. major [The centromeres of all 36 L. major . The cenIn agreement with our previous results , base J Leishmania is similar to that previously reported for T. brucei [T. brucei [Leishmania TSSs  found in T. brucei [Leishmania and Trypanosoma [In h base J . As expeishmania . However. brucei , providipanosoma .L. major have shown that TTSs have a low occupancy of nucleosomes [Previous studies using leosomes , suggestL. tarentolae  is used to generate more rigid nucleosomes at centromeres , althougrentolae . Two (H3L. tarentolae Parrot TarII [7 cells/mL.A clone (M2) of ot TarII  was growot TarII . In geneL. tarentolae Parrot TarII using the BluePippinTM System with a 7.5 kb size-cut (https://pacbio.gs.washington.edu/ (accessed on 30 July 2022)). The library was sequenced using P2/C2 chemistry on 14 SMRTCells to generate 2,104,088 reads with an average read length of 3241 nt. Filtering for duplicates reduced this to 913,321 reads (average length = 5.408 nt), which were assembled using HGAP v3.0 [L. major and L. tarentolae reference genomes using ABACAS [L. tarentolae reference genome were prepared by synthesis of ~11 amino acid peptides with the modification at the central position, conjugation to carrier protein, and immunization of rabbits. Antisera were evaluated for specificity by Western blotting against modified or unmodified peptides . Antibodies against T. brucei H3 and H3K76me3 were supplied by Christian Janzen [A previously published protocol  was usedrom H3.V . Antisern Janzen . After oL. tarentolae genome, we used a modification of the previously described protocol [To localize base J within the protocol . DNA  described above, using Bowtie2 [The Illumina libraries described above were sequenced by Novogene Corporation Inc., Sacramento, CA, USA, using their NovaSeq PE150-30G-WOBI protocol. Reads were aligned against the  Bowtie2  with the


Dynamic changes in epigenetic landscape reflect a critical command of lineage-specific gene expression. In an effort to discern the epigenetic regulatory networks of myogenic differentiation, we have used systematic and integrative approaches to explore multi-omics datasets on global myogenic gene expression, histone acetylation and acetyltransferase occupancy in view of distinct chromatin states. In this brief report, we discuss experimental design and provide a comprehensive assessment regarding data quality control, filtering and processing. We also define a gene-level overlap between RNA-seq and ChIP-seq datasets through integrative analyses to offer strategies for future use of the data. Furthermore, our analyses generate a blueprint on chromatin state distribution of residue-specific histone acetylation and concomitant association with histone acetyltransferase p300 in committed skeletal myoblasts and differential histone acetylation signatures at the onset of myoblast differentiation. These datasets can be further utilized to delineate the function of muscle-specific regulatory elements governed by other muscle myogenic regulators or signaling molecules. Epigenetic regulatory mechanisms affect global gene expression patterns underpinning lineage commitment and stem cell differentiation, and thus impact tissue homeostasis including skeletal muscle –4. RecenDifferent regulatory elements are marked by distinct patterns of chromatin modifications, in that H3K9ac, H3K18ac, and H3K27ac are often found near transcription start sites (TSS), while H4K8ac is mostly associated with promoters and gene bodies that are actively transcribed –13. In aPartaking in transcription control of a vast number of genes, p300 is specifically required for the function of muscle-specific transcription factors and essential for myogenic differentiation , 23. UndCell type specific chromatin states can be defined by large-scale analyses of co-occurrence of different histone marks , 27. We Since epigenetic modifiers and transcriptional regulators are essential for myogenic differentiation and their dysfunction is implicated in pathogenesis of skeletal muscles, a better grasp of epigenetic regulatory mechanisms of the myoblast genome is paramount. In this report, we provide technical validation and comprehensive analyses of total of 14 genomic datasets which profile global gene expression, histone acetylation, and p300 occupancy for an unbiased and genome-wide view of chromatin states associated with myoblast differentiation. For an in-depth usage of the datasets, we provide detailed information on the experimental design, data quality control, and integrative analyses. Our objective is to offer strategies for future integrative analyses of the datasets to advance the knowledge of epigenetic regulatory networks on myoblast genome in a specific signaling pathway or pathological context. Moreover, comprehensive analyses of epigenomes and transcriptomes in differentiating myoblasts also provide a platform to identify additional histone acetylation signatures of myoblasts genome.Total RNA was extracted from C2C12 myoblasts using RNeasy kit (Qiagen) according to the manufacturer’s instruction. RNA samples from two biological replicates were processed by the McGill University Genome Quebec Innovation Centre for cDNA library construction and deep sequencing using an Illumina HiSeq 2000 sequencer as single-end 50 base pair reads as previously described . Salmon ChIP-seq library preparation and deep sequencing were performed by the McGill University Genome Quebec Innovation Centre with Illumina HiSeq 2000 as single-end 50 bp reads . The seqThe resulting peak files were used as input into HOMER for gene annotation. ChIP enrichment and p300-associated sites was calculated using ngs.plot, which calculates the coverage vectors for each region based on specified ChIP-seq alignment files. Following normalization and transformation on the coverage, a heatmap as well as an average profile is plotted from the mean of all regions, normalized to the total number of mapped reads in millions. Integrative Genomics Viewer (IGV) was used for visualization of the ChIP-seq read signals. Model-based Analysis of ChIP-seq (MACS) was used to determine the scores of Fraction of Reads in Peaks (FRiP) and to call broad peaks of histone acetylation for chromatin states distribution.We examined myogenic expression and histone acetylation profiles on a genome-wide scale using C2C12 skeletal myoblasts as a system of differentiation. These myoblasts are myogenic precursors of mesenchymal origin and represent a well-established model of myogenic differentiation. They switch from proliferation to differentiation under low mitogen conditions, followed by a fusion process to form multinucleated myotubes and have been a model of choice for many genome-wide studies due to a high correlation with primary myoblasts , 29. TheIn our studies, the C2C12 myoblasts were differentiated for 24 h (Fig. To assess the quality of RNA-seq datasets for differentiating and proliferating myoblasts, base call scores were analyzed using the FastQC program Fig. A. The quNext, the RSeQC gene body coverage tool was used to examine uniformity of read coverage across the gene body for indications of any 5ʹ/3ʹ biases. Figure In RNA-seq, distribution of the reads across known gene features is an additional important measure of experimental validity. Most reads are expected to map to exons compared to introns and intergenic regions . As showIn addition to detecting novel transcripts, a principal benefit of RNA-sequencing has been replicability between samples, which was measured as the correlation of gene expression between samples Fig. F. SpearmThe average base quality scores for all ChIP-seq datasets in the present study fell within the high confidence range Fig. A and disFollowing the confirmation of satisfactory alignment metrics, we also examined the genome coverage and the strength of ChIP-seq simultaneously through cumulative enrichment analysis Fig. D. IntersWe have previously established a model of 14-chromatin states in committed skeletal myoblasts , with muHere, we analyzed ChIP-seq of histone acetylation marks including H4K8ac, H3K9ac, H3K18ac, and H3K27ac after quality verification of the raw sequencing data, since transcriptomes are controlled by epigenetic regulatory networks. PCA plots were first used to assess the clustering of histone acetylation marks into their respective biological groups for chromatin modification and differentiation status (Fig. Next, we examined the chromatin state distribution of residue-specific histone acetylation in early differentiation utilizing our established chromatin state model. As shown in Fig. Tbc1d1, a glucose regulator in skeletal muscle [We also delineated epigenetic networks involved in p300-mediated gene expression, since p300 is a critical HAT in myogenic differentiation , 24, 39.l muscle .Fig. 5HiIntegrative analyses of p300 and histone acetylation ChIP-seq data with corresponding chromatin state and transcriptome offer an invaluable avenue to identify chromatin states distribution of residue-specific histone acetylation and discern the distinct function of HATs in epigenetic landscape and gene regulatory networks within a specific biological milieu. The high throughput sequencing datasets presented here are biologically validated and in high quality, and thus can be used for future studies to determine the roles of muscle regulatory factors, residue-specific histone acetylation and p300 function in early myogenic differentiation, particularly in terms of their impact on chromatin dynamics.


The Hippo pathway regulates tissue homeostasis in normal development and drives oncogenic processes. In this review, we extensively discuss how YAP/TAZ/TEAD cooperate with other master transcription factors and epigenetic cofactors to orchestrate a broad spectrum of transcriptional responses. Even though these responses are often context- and lineage-specific, we do not have a good understanding of how such precise and specific transcriptional control is achieved—whether they are driven by differences in TEAD paralogs, or recruitment of cofactors to tissue-specific enhancers. We believe that emerging single-cell technologies would enable a granular understanding of how the Hippo pathway influences cell fate and drives oncogenic processes, ultimately allowing us to design better pharmacological agents against TEADs and identify robust pharmacodynamics markers of Hippo pathway inhibition. Drosophila ortholog Yorkie, Yki) . LATS1/2 is regulated by kinase, Mammalian STE20-like 1/2 , and their adaptor proteins SAV1  and MOB1 (Drosophila homolog Mats) [Hippo signaling pathway is a key modulator of tissue growth with widespread implications in organ development, cell growth, regeneration, and stem cell function . It is aie, Yki) . YAP/TAZog Mats) .Drosophila ortholog Scalloped, Sd), TEAD-independent gene regulation networks have also been documented [YAP/TAZ lack a DNA-binding domain, and primarily rely on their interaction with additional transcription factors (TFs) to exert transcriptional activity ,9 Figur. YAP/TAZcumented .Tumor heterogeneity, functional degeneracy, and lineage plasticity are factors that reduce drug efficacy and lead to acquired resistance. Recent studies have established the role of Hippo signaling network at the epicenter of cancer-associated transcriptional addiction and drug resistance. Thus, mechanistic understanding of cross talks between YAP/TAZ/TEAD with other TFs and chromatin remodelers may reveal gene regulatory networks that drive tumor invasion and growth. In this review, we focus on the nuclear effectors of YAP/TAZ that play a role in YAP-mediated transcriptional addictions and drug resistance in cancer . Our priDrosophila to humans. They regulate developmental processes transcending a wide variety of tissue types, from the formation of neural tubes to the development of heart, brain, and skeletal muscle. Several chromatin immunoprecipitation (ChIP) studies reveal that TEADs serve as the primary effectors of the YAP/TAZ signaling: (1) 78% of the TEAD4-bound promoters and enhancers are co-occupied by YAP/TAZ in NF2-null breast cancer cell line MDA-MB-231 [TEAD TFs (TEAD1-4) are evolutionarily conserved from A-MB-231 ; (2) YAPA-MB-231 ; and (3)A-MB-231 .TEADs bind to the DNA but are barely known to exert any transcriptional activity by themselves . They foPreviously, it was thought that YAP/TAZ are coactivators for promoter driven transcription of a limited number of target genes . HoweverChIP data from various cell lines of different lineages and genetic backgrounds—such as glioblastoma line SF268 (YAP amplification), malignant mesothelioma line NCI-H2052 (NF2 mutation and LATS2 deletion), and non-transformed cells (IMR90)—has revealed that only a small set of YAP1/TEAD peaks are found within the gene transcription start sites (TSSs) while the majority are located at the distal active enhancers marked by histone post-translational modifications, including H3K27ac. The fact that YAP is required to maintain H3K27ac levels and TEAD occupancy at the YAP1-bound enhancer signifies its importance in establishing proper chromatin structures via positive feedback loop. Thus, YAP/TEAD distal binding is a general characteristic feature that tightly regulates transcription of a myriad of genes in both normal and cancer cells .YAP further recruits mediator complexes to active enhancer sites, which can in turn regulate transcription via recruitment of Cyclin-Dependent Kinase 9 (CDK9), instilling “super enhancer”-like characteristics. Recruitment of CDK9 releases paused RNA Polymerase II (RNAPolII) at the promoters and induces elongation of YAP/TAZ targets to drive tumorigenesis in liver cancer cells. In the model liver cancer cell line HuCCT1, YAP occupies only 7% of the sites bound by TEADs, indicating that these super enhancer-like sites form a small subset of modules that are heavily overloaded with cofactors and activating histone marks. They play an important role in driving rapid gene expression in response to physiological and oncogenic cues and may play a role in YAP/TAZ-dependent cancer addiction as well .In agreement with these studies, overexpression of the constitutively active form of YAP (YAP5SA) has been shown to reprogram cardiomyocytes into a fetal-like state by engaging a subset of global enhancers that are enriched for TEAD and AP-1 motifs. YAP overexpression promotes chromatin looping, which in turn increases enhancer–promoter contacts to access new target genes involved in developmental processes . In flieIt still remains a major challenge to correlate target gene expression with a specific set of enhancers. New technological advances in single cells can address this issue (covered in later section) and answer questions in this emerging field of enhancer deregulation in YAP driven cancer and their role in therapeutic resistance.Activator protein-1 (AP-1) is a well-characterized heterodimeric transcription factor, comprising two families of oncoproteins, namely, FOS and Jun. FOS family of proteins are early gene products and are rapidly induced in response to many cellular and extracellular cues, including cellular stress, developmental cues, and growth factors, as well as mitogens such as serum and lysophosphatidic acid (LPA) phorbol esters, which are also known to activate YAP activity . The FOSIn the NF2-null breast cancer cell line (MDA-MB-231), 70% of the YAP/TAZ/TEAD-occupied enhancers also contained AP-1-binding motifs, making AP-1 the second most abundant motif after TEAD. Sequential ChIP seq analysis for YAP followed by JUN suggested that both TEAD and AP-1 can bind to the cis-regulatory elements bearing TEAD and AP-1 composite sites at the same time and can physically interact with each other. AP-1 synergizes with YAP to increase oncogenic growth in mammary cells via activating target genes that control S-phase entry and mitosis. AP-1 has elevated activity in skin tumorigenic induced by chemical carcinogenesis. YAP/TAZ-deficient mice failed to produce tumors when subjected to chemical carcinogenesis, underscoring the importance of YAP/TAZ in AP-1-mediated tumorigenesis .Since YAP/TAZ rapidly translocate to the nucleus upon serum or LPA stimulation, they act as immediate sensors regulating early gene expression. Recent studies have shown that AP-1 is a direct transcriptional target of YAP/TAZ and is induced in response to mitogenic signals. Knocking out YAP in HEK293 cells severely reduces the expression of AP-1 upon LPA treatment, whereas overexpressing a constitutively active form of YAP (5SA-YAP) induces AP-1 expression under serum starvation. In addition, the induction of FOS expression is dependent on TEAD binding to YAP, which can then synergize with YAP/TAZ to drive target gene expression. A mutant version of YAP with defective TEAD binding (S94AYAP) fails to rescue AP-1 expression in YAP/TAZ KO cells. Consistent with these observations, inhibition of AP-1 significantly rescued YAP-mediated liver overgrowth in mice, highlighting a previously unknown role of AP-1/YAP/TAZ in the regulation of immediate early gene expression and organ growth . There hDrosophila. Activating transcription factor 3 (Atf3) is a direct transcriptional target of Sd, which is significantly upregulated in Ras driven tumor formation. Tumor specific gene expression in Drosophila is tightly regulated by a few key transcription factors, and AP-1 forms one of the major regulatory nodes. Loss of AP-1 or STATS can break this regulatory network by reducing the expression of tumor signature genes [YAP transcriptional regulation of AP-1 factors is also evolutionarily conserved in re genes . In the re genes . Furtherre genes .A recent study from our lab focused on an integrated strategy combining machine learning with chemicogenomics, to identify lineage-independent gene signatures for Hippo pathway in pan-cancer cell lines. We observed crosstalk between Hippo and MAPK signaling, converging at the level of AP-1 and serving as a common node to regulate gene expression. FOSL1 can interact with TEADs in the presence of YAP1. ATAC-seq under conditions of YAP depletion combined with MEK inhibition revealed decreased chromatin accessibility at TEAD and AP-1 binding sites . SeveralYAP/TAZ also act as a transcriptional coactivator for JUNB and STAT, promoting cellular transformation. YAP/TAZ, but not JUNB, are required for STAT3 phosphorylation during breast epithelial cell transformation. Along with TEAD/AP-1, YAP/TAZ target genes are also associated with STAT3, albeit to a lesser extent. YAP/TAZ are recruited by different forms of TEAD/STAT3/AP-1 complex depending on the cis-recruiting motifs, which can then regulate different sets of YAP/TAZ target genes. Interaction with these transcription factors is cell-type-dependent, causing specific gene expression profiles with distinct functions .There is limited evidence on whether expression of YAP is regulated by other AP-1-like TFs. A recent study showed that YAP1 is a downstream effector of MAPK signaling activated by the FGFR axis, which promotes gastric cancer (GC) progression. JUN physically interacts with YAP1/TEAD4 complex and, consequently, knockdown of JUN impairs YAP1/TEAD4 complex formation, leading to reduced cell proliferation in model cell lines. FGFR activation promotes cJUN phosphorylation, and knockdown of FGFR significantly decreases YAP mRNA expression; however, the exact molecular mechanism is still unclear . FurtherEstrogen Receptor α (ERα) is a TF that controls cell proliferation and survival during tumor growth, and is elevated in 70% of breast cancer cases. The binding of estrogen promotes the localization of ERα from cytoplasm to nucleus, where it predominantly binds to distal enhancer regions to regulate target gene expression. ERα, in association with forkhead box A1 (FOXA1) is known to recruit other TFs that act as co-activators in-trans to form a stable enhancer activating machinery. Proximity labeling techniques in MCF7 breast cancer cell lines have identified YAP and TEAD4 as cofactors for ERα/FOXA1 on active estrogen-regulated enhancers. Instead of binding to its canonical cis-binding sites, TEAD4 can be recruited to the ERα enhancers in-trans to regulate expression of E2-induced ERα targets in addition to non-coding enhancer RNA (eRNA) transcription from estrogen-regulated enhancers. YAP1 and TEAD4 can also recruit mediator complex subunit 1 (MED1), an important enhancer activating machinery, to facilitate breast cancer cell growth . This unBesides YAP/TAZ, LATS has also been implicated in the regulation of ER expression. In one study, LATS is required to maintain ER expression via YAP and TAZ inhibition. Contrary to its conventional role as a tumor suppressor, knockout of LATS inhibits growth of ERα+ breast cancer cells and mouse breast organoids by reducing the expression of ERα mRNA . A diffeRecently, it has been shown that multiple TFs can interact with mediator subunits to form phase separated condensates. This interaction is facilitated by their activation domains in the TFs, which are intrinsically disordered, while their DNA-binding domains cooperate to anchor to their target sites. ER contains a C-terminal ligand dependent activation domain that facilitates formation of phase separated particles with MED1 upon estrogen stimulation . ConformBromodomain-containing protein 4 (BRD4) is one of the well characterized histone acetyltransferases (HATs), belonging to the bromodomain and extra-terminal domain (BET) family. Recent studies have shown that YAP/TAZ can also contribute to transcriptional addiction by over engaging the chromatin reader BRD4 at YAP/TAZ/TEAD-bound promoters and enhancers and thereby exhibiting super enhancer-like properties.In the breast cancer cell line (MDA-MB-231), YAP/TAZ recruit BRD4 to target enhancers and promoters to augment H3K122 acetylation, which is essential for loading of Pol II. YAP/TAZ targets are overloaded with BRD4 and are highly vulnerable to BET inhibitors. BET confers YAP/TAZ-mediated drug resistance in vemurafenib-treated BRAF mutant melanoma cells, which can be re-sensitized by JQ1 . BRD4 anApart from its role in transcription activation, BET can also suppress TAZ activity. Loss of function in LATS2, TAOK1, or NF2 in lung cancer cells results in nuclear accumulation of TAZ, leading to higher transcriptional activity of targets even in the presence of JQ1. According to this study, accumulation of TAZ is more likely to confer resistance to BET inhibitor than YAP. This supports the notion that YAP and TAZ serve tissue specific functions . TAZ hasMammalian SWI/SNF  complexes belong to the class of ATP-dependent chromatin remodelers, which contain a core ATPase that binds nucleosomes and disrupts DNA–histone interactions . BAFs enSWI/SNF complex has been shown to inhibit YAP-mediated transcription . The ARIOn the contrary, recruitment of ACTL6A to SWI/SNF (ACTL6A-BAF complexes) can increase chromatin accessibility at YAP/TEAD-bound enhancers. ACTL6A gene amplification plays a crucial role in squamous cell carcinoma (SCC) development by inducing an open chromatin landscape. ACTL6A reduction decreases TEAD accessibility, suggesting that it can regulate YAP/TAZ/TEAD-dependent oncogenic signaling. ACTL6A promotes TEAD-YAP binding to the chromatin, which can further recruit additional BAF complexes at the enhancer. This creates a positive feedback loop to maintain accessibility, and high transcriptional activity at TEAD-specific enhancer sites . ConsistBiomolecular condensates are membrane-less subcellular bodies, formed by liquid-liquid phase separation (LLPS). They can regulate multiple intracellular processes including cooperative cellular events such as transcription initiation and elongation by compartmentalizing and concentrating known transcription hub components at super-enhancer-regulated genes. YAP/TAZ are known to physically recruit BRD4 and MED1 to super-enhancers. Interestingly, the intrinsically disordered regions (IDRs) of BRD4 and MED1 have been shown to facilitate formation of phase separated super-enhancer regions . MultiplIn this review, we have extensively discussed how YAP/TAZ/TEAD cooperate with other master TFs and epigenetic cofactors to orchestrate a broad spectrum of transcriptional responses. This elaborate transcriptional network regulates the expression of key target genes in developmental and tumorigenic processes and is itself tightly modulated through feedback loops. Some pertinent questions remain: Are there additional master regulators that fuel transcriptional addiction in cancer? How is the context- and lineage-specific regulation of the Hippo pathway achieved? How does YAP/TAZ/TEAD specifically regulate gene expression under stress and external signaling cues? Is this specificity orchestrated through the recruitment of specific epigenetic and transcription factors to enhancer elements?Our knowledge about the differential regulation of the four TEAD paralogs remains limited. Interaction of TEADs with their cofactors is highly context specific. Even though TEADs have redundant roles in certain developmental scenarios , very liGiven the importance of the Hippo pathway, there is tremendous interest in drugging YAP/TAZ/TEAD in oncology. Targeting YAP/TAZ still remains a major challenge, owing to the lack of structural rigidity and tolerability concerns. These challenges point towards drugging the YAP/TAZ transcriptional coactivators and TEADs. As reviewed elsewhere ,62, therHigh YAP/TAZ activity has also been implicated as a resistance mechanism to several therapeutic interventions against other oncogenic pathways. Drug-tolerant cancer cells can revert back to the sensitive state upon drug removal, or they undergo transcriptional and epigenetic reprogramming to become drug resistant cells. A pertinent question in the field will be to figure out how the downstream effectors of YAP/TAZ drive therapy resistance. Efforts should be made towards gaining insights into molecular mechanisms that cancer cells can exploit to activate bypass mechanisms and transcriptional reprogramming against small molecule inhibitors targeting TEAD or any of its cofactors. A comprehensive mechanistic understanding of transcriptional dysregulation in the context of tumor heterogeneity is imperative to developing effective combination therapeutic strategies.All the questions outlined above require the examination of transcriptional control in the context of cell identity and lineage plasticity. Given the highly heterogeneous and dynamic nature of tumor cells when exposed to treatment, traditional bulk assays may be insufficient to resolve resistance mechanisms that are present only in small subsets of cells. Therefore, there is a pressing need to apply the state-of-art, single-cell genomics technologies  to betteIncreasing evidence suggests that the Hippo pathway can have profound effects on specific cell populations in both normal developmental and tumorigenic processes. Applying single-cell ATAC-seq technologies, Little et al. discovered the specific localization of YAP/TAZ expression to the AT-1 cells of the lung and their role in recruiting lineage factor NKX2-1 to lineage-specific regions . SubsequSingle-cell RNA-seq alone may not be sufficient to assess changes in cell states. Increasing evidence has demonstrated that other modalities such as protein expression level, DNA accessibility, and histone/TF binding could dovetail nicely with transcriptome readout. These single-cell multiomic technologies can better resolve rare cell populations and provide mechanistic details of the cofactors associated with each lineage and/or each TEAD paralog. In terms of protein expression assay, CITE-seq is one of the earliest efforts to enable multiomic readout in single-cell genomics assays . Using aAs mentioned earlier, DNA accessibility is another important feature that provides unique insights into gene transcription regulation. Several methods have been developed to simultaneously detect the transcriptome and open chromatin information from the same cells, including sci-CAR , SNARE-sIn addition, a few groups have been trying to obtain all three modalities  in the same single-cell experiment, which resulted in newer technologies coupled to existing 10× Genomics Multiome system, such as NEAT-seq , TEA-seqWith the emergence of various modalities targeting TEADs, it is of high interest to compare the mechanisms of action amongst these pharmacological agents, as well as to understand how they may differ from genetic ablation of YAP/TAZ/TEAD. It is thus important to develop technologies to multiplex genetic and compound perturbation experiments. Traditionally, these experiments are limited by the numbers of perturbations and the types of readout in a single experiment. Several approaches have been introduced to overcome these issues by coupling single-cell genomics assays with the CRISPR-based genome editing technologies. In these assays, CRISPR is introduced to the samples in a pooled manner. Within every single cell, the sgRNA can be captured together with the transcriptome, which links the perturbation targets to their direct functional consequences. These methods include CRISP-seq , PerturbWe are hopeful that the era of single-cell technologies will usher in a granular understanding of the Hippo pathway—how YAP/TAZ/TEAD and master regulators cooperate to orchestrate lineage- and context-specific gene regulation, determine lineage fates, and confer treatment resistance. This finer resolution of regulatory control will ultimately enable us to design more precise therapeutic strategies, aimed at the right cellular populations.


The histone variant H3.3 is enriched at active regulatory elements such as promoters and enhancers in mammalian genomes. These regions are highly accessible, creating an environment that is permissive to transcription factor binding and the recruitment of transcriptional coactivators that establish a unique chromatin post-translational landscape. How H3.3 contributes to the establishment and function of chromatin states at these regions is poorly understood.We perform genomic analyses of features associated with active promoter chromatin in mouse embryonic stem cells (ESCs) and find evidence of subtle yet widespread promoter dysregulation in the absence of H3.3. Loss of H3.3 results in reduced chromatin accessibility and transcription factor (TF) binding at promoters of expressed genes in ESCs. Likewise, enrichment of the transcriptional coactivator p300 and downstream histone H3 acetylation at lysine 27 (H3K27ac) is reduced at promoters in the absence of H3.3, along with reduced enrichment of the acetyl lysine reader BRD4. Despite the observed chromatin dysregulation, H3.3 KO ESCs maintain transcription from ESC-specific genes. However, upon undirected differentiation, H3.3 KO cells retain footprinting of ESC-specific TF motifs and fail to generate footprints of lineage-specific TF motifs, in line with their diminished capacity to differentiate.H3.3 facilitates DNA accessibility, transcription factor binding, and histone post-translational modification at active promoters. While H3.3 is not required for maintaining transcription in ESCs, it does promote de novo transcription factor binding which may contribute to the dysregulation of cellular differentiation in the absence of H3.3.The online version contains supplementary material available at 10.1186/s13059-023-02867-3. In eukaryotic cells, DNA is wrapped around histone proteins to form nucleosomes, the fundamental repeating unit of chromatin , 2. WhilH3.3 differs from replication-coupled H3 by only 4–5 amino acids, yet this is sufficient to drive dedicated chaperone association and deposition at specific regions of the genome . H3.3 waSeveral studies suggest that H3.3 may influence the local chromatin environment by recruiting specific complexes to chromatin –29. For In this study, we performed genomic analyses to determine the effect of H3.3 deposition on regulatory element architecture and downstream transcription in ESCs. We find that promoter-proximal regulatory elements become less accessible in the absence of H3.3. Reduced accessibility is accompanied by reduced transcription factor footprinting and binding, attenuation of chromatin states thought to be downstream of transcription factor binding, and decreased RNA polymerase II engagement at affected promoters. While ESCs appear quite tolerant to these changes in genome regulation, they are unable to respond to cellular differentiation cues and show perdurance of the regulatory landscape associated with pluripotency. Thus, we propose that H3.3 is a necessary upstream component of transcriptional activation that becomes dispensable for the maintenance of established gene regulatory networks.n = 12,903) showed higher correlation with H3.3 deposition (rho = 0.463) compared to active promoter-distal regulatory elements . We next asked whether regulatory element accessibility is dependent upon H3.3 by comparing ATAC-seq data from WT and H3.3 KO ESCs . Nuclei were collected by centrifugation, washed once with 1 mL Lysis Buffer, and resuspended in 500 μL of Freezing Buffer  and stored at −80 °C.ESCs were lysed in Hypotonic Lysis Buffer  for 1 h at room temperature. Beads were washed once with binding buffer, low-salt wash buffer , and high-salt wash buffer , followed by two washes in TET buffer (TE + 0.05 mM Tween). RNA was eluted 4× with elution buffer  and extracted with acid phenol:chloroform. The resulting RNAs were reverse transcribed, size-selected, and amplified. The quality of the libraries was assessed using a D1000 ScreenTape on a 2200 TapeStation (Agilent) and quantified using a Qubit dsDNA HS Assay Kit (Thermo Fisher). Libraries with unique adaptor barcodes were multiplexed and sequenced on an Illumina NextSeq 500.For nuclear run-on, 5×10Quality control for the GRO-seq data was performed using the FastQC tool. GRO-seq reads were trimmed to remove adapter contamination and poly(A) tails using the default parameters of Cutadapt software (Martin 2011). Reads >32 bp long were retained for alignment to the mouse reference genome. Transcript calling was performed using groHMM.p<0.05.RNA-seq datasets used for this study were obtained from the GEO (GSE114549). Quality of raw RNA-seq reads was assessed using the FastQC tool. Reads were aligned to the mouse reference genome (mm10) with STAR . After n2 fold change of read counts in each gene were determined using DESeq2. These values were plotted on the x-axis and y-axis respectively for all transcripts detected. To visualize changes in expression of genes thought to play a role in pluripotency, mouse homologs of genes included in the core ESC-like gene module [For each comparison, the mean of normalized counts and the loge module , or Plure module  were hig2, 0.2% Triton X-100, and 5 mM sodium butyrate. Proteins from whole cell lysate were separated in Laemmli buffer by SDS-PAGE and transferred to PVDF membranes (Millipore). Membranes were blocked in 5% milk in Tris-buffered saline with 0.1% Tween-20 (TBST) and incubated with primary antibodies overnight at 4 °C or for 2 h at room temperature. Membranes were washed with TBST, incubated with HRP-conjugated secondary antibodies for 1 h, incubated with HRP substrate (Fisher), and imaged using a ChemiDoc MP Imaging System (BioRad).ESCs were lysed with micrococcal nuclease (Worthington) in 50 mM Tris pH 7.5, 1 mM CaClAdditional file 1: Fig. S1. Loss of H3.3 deposition modestly reduces chromatin accessibility at enhancers. Fig. S2. HIRA facilitates H3.3 deposition at enhancers and promoters. Fig. S3. Loss of HIRA-mediated H3.3 deposition reduces chromatin accessibility at promoters. Fig. S4. HIRA-dependent loss of H3.3 deposition alters promoter architecture. Fig. S5. H3.3-dependent promoters display hallmarks of active chromatin. Fig. S6. ATRX/DAXX-dependent loss of H3.3 deposition increases accessibility at IAPEz. Fig. S7. Reduced ATAC-seq footprinting in the absence of H3.3. Fig. S8. Loss of HIRA phenocopies promoter dysregulation. Fig. S9. Altered H3.3 deposition influences TF binding at repeat elements. Fig. S10. Changes in promoter architecture are not correlated with transcriptional changes in associated proteins. Fig. S11. Chromatin Landsacpe Is Dysregulated with H3.3 Loss. Fig. S12. Loss of H3.3 alters RNAPII activty. Fig. S13. Transcriptional changes associated with loss of H3.3 in ESCs and during differentiation. Fig. S14. Uncropped blots from Fig. Fig. S15. Exogenously expressed TFs bind at both open and closed chromatin in WT ESCs.Additional file 2: Table S1. Pairwise comparison of TF binding activity between WT and H3.3 KO, HIRA KO, ATRX KO and DAXX KO ESCs.Additional file 3: Table S2. Summary of pairwise comparisons of differential expression and binding activity of TFs between WT and H3.3 KO or HIRA KO ESCs.Additional file 4: Table S3. Pairwise comparison of TF binding activity between WT ESCs and EBs or between WT and H3.3 KO EBs.Additional file 5: Table S4. Summary of datasets generated or used in this study.Additional file 6. Review history.


Ciona robusta DYRK1 (CrDYRK1) showed transcriptional activation activity independent of its kinase function. The transcriptional activation activity of CrDYRK1 could be autoinhibited by a repression domain in the N-terminal. More excitingly, both activation and repression domains were retained in HsDYRK1A in humans. The genes, activated by the activation domain of HsDYRK1A, are mainly involved in ion transport and neuroactive ligand–receptor interaction. We further found that numerous mutation sites relevant to the DYRK1A-related intellectual disability syndrome locate in the C-terminal of HsDYRK1A. Then, we identified several specific DNA motifs in the transcriptional regulation region of those activated genes. Taken together, we identified a conserved transcription activation domain in DYRK1 in urochordates and vertebrates. The activation is independent of the kinase activity of DYRK1 and can be repressed by its own N-terminal. Transcriptome and mutation data indicate that the transcriptional activation ability of HsDYRK1A is potentially involved in synaptic transmission and neuronal function related to the intellectual disability syndrome.Dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 1 (DYRK1) encodes a conserved protein kinase that is indispensable to neuron development. However, whether DYRK1 possesses additional functions apart from kinase function remains poorly understood. In this study, we firstly demonstrated that the C-terminal of ascidian  DYRK1A has been reported to be constitutively expressed at a high level in the central nervous system (CNS) of Rattus [DYRK1A is detected in neuronal progenitors and differentiated neurons [DYRK1A locates on the DS critical region (DSCR) of chromosome 21. Therefore, the extra copy increases DYRK1A expression level by approximately 1.5-fold in DS brains [DYRK1A has been demonstrated to cause morphological and cognitive defects in mouse DS models [DYRK1A regulates brain size in a dose dependent manner in mouse, Xenopus, and Drosophila [DYRK1 might be regulated rigidly for maintaining the normal development and function of brain. However, the molecular mechanisms of how DYRK1A regulates the downstream genes remains elusive.Dual specificity tyrosine-(Y)-phosphorylation-regulated kinase (DYRK), as a protein kinase, plays vital regulatory roles in development and disease ,2,3,4. Tf Rattus . In chic neurons ,10. DYRK neurons  and has  neurons . DS is t neurons . The maiS brains . OverexpS models . AdditioS models . Previouosophila ,16,17, iDYRK1A participates in transcriptional regulation. DYRK1A shows transcriptional activation activity that is dependent on kinase activity of itself [DYRK1A could increase Tau mRNA stability in a kinase-independent manner, thereby enhancing the expression level of Tau [DYRK1 regulates the gene expression through some new mechanisms. Whether DYRK1 possesses an authentic transcription activation domain independent of the kinase domain remains unrevealed.DYRK1 phosphorylates a broad set of proteins involved in diverse cellular processes, such as cell cycle, cell differentiation, and cell apoptosis ,19,20. Df itself . DYRK1A l of Tau . These fCiona’s genome does not duplicate and remains much ancestral single-copy gene [Ciona and proved that the C-terminal of CrDYRK1 possessed the transcriptional activation activity that was regulated by the repression domain localizing in the N-terminal. We then demonstrated that the transcriptional activation and repression mechanism were retained in HsDYRK1A but not in HsDYRK1B. Using RNA-seq, we further revealed that the transcriptional activation ability of HsDYRK1A was mainly involved in ion transport and neuroactive ligand–receptor interaction. Taken together, we demonstrated that, apart from kinase activity, CrDYRK1 possessed transcriptional activation activity independent of kinase activity and regulated by the repression domain of itself. The on–off mechanism of transcriptional activation activity was conserved in HsDYRK1A, and the transcriptional activation ability might be responsible for neuronal development and function.As a branch of chordate, ascidian opy gene ,27. It iAnimals were acquired from the eastern coast of Qingdao . Mature eggs and sperm were collected and fertilized in seawater at room temperature; dechorionation and electroporation were performed as previously described . After ePCR was performed with the Phanta Max Super-Fidelity DNA Polymerase . DNA was purified using a GeneJET Gel Extraction Kit . All DNA Fragments were ligated by the One Step Cloning Kit .CrDYRK1 were amplified from C. robusta cDNA separately using PCR and ligated to pGBTK7 (BD) vector , respectively. The HsDYRK1A (residues 485–763) and HsDYRK1B (residues 437–629) fragments were amplified separately from human cDNA by PCR and ligated to BD vector for the transcriptional activation activity analysis. The CsDYRK1 (residues 393–626) fragment was amplified from C. savignyi cDNA by PCR and ligated to BD vector for the transcriptional activation activity analysis. The DmMNB (residues 629–1047) fragment was amplified from Drosophila melanogaster cDNA by PCR and ligated to BD vector for the transcriptional activation activity analysis.To identify the transcriptional activation activity, the isoform I, isoform II, and truncated isoform I of CrNFAT5 (residues 77–979) and CrMyc (residues 1–681) fragments were amplified and ligated to BD vector to serve as a positive control. CrDYRK1 (isoform II) (residues 1–168) fragment was amplified and subcloned into EcoRI restriction sites of the CrNFAT5 (residues 77–979)-BD vector. CrDYRK1 (isoform II) (residues 1–168) and CrMyc (residues 1–681) fragments were amplified separately and ligated to BD vector to construct an expression vector CrDYRK1 (isoform II) (residues 1–168)-CrMyc (residues 1–681)-BD. Truncated versions of CrDYRK1 (isoform II) were amplified separately and ligated to BD vector to construct expression vector CrDYRK1 (isoform II) (residues 43–815)-BD and CrDYRK1 (isoform II) (residues 85–815)-BD. HsDYRK1A-N (1–69) fragment was amplified and subcloned into EcoRI restriction sites of the HsDYRK1A (residues 485–763)-BD vector to construct expression vector HsDYRK1A-N (residues 1–69)-C (residues 485–763)-BD.In the repression assay, HsDYRK1A (residues 485–763) fragment was amplified and ligated to pEGFP-N1 vector . HsDYRK1A-N (1–69)-C (485–763) was amplified from HsDYRK1A-N (residues 1–69)-C (residues 485–763)-BD vector and ligated to pEGFP-N1 vector.In the RNA-seq assay, CrDYRK1 were amplified and ligated to pEGFP-C1 vector driven by Brachyury promoter. HsDYRK1A (residues 485–763) fragment was amplified by PCR and ligated to pEGFP-N1 vector. CsDYRK1 (residues 393–626) and CrDYRK1 (isoform I) (residues 427–647) fragments were amplified by PCR and ligated to pEGFP-N1 vector driven by Epi I promoter, respectively.In the assay of subcellular localization observation, the isoform I and isoform II of All primers are listed in CrDYRK1 were downloaded from the Ghost Database (http://ghost.zool.kyoto-u.ac.jp/cgi-bin/gb2/gbrowse/kh/) (accessed on 10 October 2019) and used for analyzing gene structure in the GSDS-2.0 (http://gsds.gao-lab.org/) (accessed on 10 October 2019). The amino acids sequence of the DYRK1 from different species were acquired from the NCBI, and the sequences of kinase domains were used to build phylogenetic tree by MEGA-X software. The phylogenetic tree was built using the maximum likelihood method with 1000 bootstraps, and the JTT model was selected. The protein 3D structure was predicted using online tools ALPHAFOLD . The conserved domain analysis of DYRK1 was performed by NCBI CD-Search (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) (accessed on 18 December 2021) and SMART (http://smart.embl-heidelberg.de/) (accessed on 18 December 2021). To reveal the preference of HsDYRK1A-activating genes, DNA motif analysis was performed using online tool MEME (https://meme-suite.org/meme/doc/meme.html) (accessed on 19 November 2022). The upstream 1000 bp of translation start sites of 96 upregulated genes were obtained for motif analysis.The genomic sequence and coding sequence (CDS) of DYRK1 from different species were collected from the NCBI. To examine whether these duplicated DYRK1 members diversified, synonymous (Ks) substitution of each duplicated DYRK1 were calculated using KaKs_Calculator 2.0  [The alignment of the amino acids sequence from different species was performed by using DNAMAN software. The coding sequence of the rge.net) .The vectors of BD and pGADT7 (AD), the Y2H Gold strain, and all reagents for Y2H assay were purchased from Takara. Different combinations of bait with AD empty vectors were co-transformed into the yeast strain Y2H Gold and cultured on synthetic complete medium lacking leucine and tryptophan . The transcription activation ability was determined by measuring the growth of serial dilutions on synthetic complete medium lacking leucine and tryptophan synthetic supplemented with Aureobasidin A (AbA) for 2 to 3 d.HeLa cells were cultured in DMEM supplemented with 10% fetal bovine serum  at 37 °C with 5% CO2. The plasmids were transfected into cultured cells according to the manufacturer’s instructions (Lipo3000 from Thermofisher).ftp://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/001/405/GCF_000001405.25_GRCh37.p13/GCF_000001405.25_GRCh37.p13_genomic.fna.gz) (accessed on 25 October 2022). EdgeR was used for differential expression analysis. The resulting p-values were adjusted using Benjamini and Hochberg’s approach for controlling the false discovery rate. Genes with log2 (fold change) > 1 and p value < 0.05 were assigned as differentially expressed. GO and KEGG enrichment analysis of differentially expressed gene sets were implemented using the topG (http://www.bioconductor.org/packages/release/bioc/html/topGO.html) (accessed on 9 November 2022) and KOBAS package, respectively.Sixty hours post-transfection, we extracted the total RNA from HeLa cells overexpressing HsDYRK1A (residues 485–763)-eGFP or HsDYRK1A -eGFP, respectively. Transcriptome sequencing was performed by the Berry Genomics company. Genes were annotated according to genome Homo sapiens-NCBI-hg19  at room temperature for 2 h and then were washed thrice with phosphate-buffered saline (PBS) containing 0.1% Triton X-100  (PBST) for 20 min each time. To identify the cell boundary, fixed embryos were stained with 1/200 TRITC phalloidin  in PBST overnight at 4 °C. Embryos were then washed thrice with PBST and mounted using Vectashield mounting medium with 4′,6-diamidino-2-phenylindole (DAPI)  to label the cell nucleus.HeLa cells were fixed with 4% formaldehyde for 1 h at RT. The fixed cells were rinsed thrice with PBST for 10 min each time. To identify the cell boundary, fixed cells were stained with 1/500 TRITC phalloidin in PBST overnight at 4 °C. HeLa cells were then washed thrice with PBST and mounted using Vectashield mounting medium with DAPI to label the cell nucleus.Images were taken using a Zeiss LSM900 Confocal Microscope .Danio rerio, Mus musculus, and Homo sapiens. However, only one homolog of DYRK1 was identified in Urochordata ascidian Ciona, suggesting that DYRK1 gene underwent duplication during the evolution  and endows the resistance of Y2H for AbA. The AD and BD vectors contain the encode sequence of leucine (Leu) and tryptophan (Trp), respectively. Then, the coding sequences of isoform I and isoform II were amplified from C. robusta and fused to the GAL4 DNA-binding domain to generate CrDYRK1 (isoform I)-BD and CrDYRK1 (isoform II)-BD fusion plasmids. The plasmids were co-transformed into the yeast strain Y2H with an AD empty vector, respectively. The transformants were evaluated for their abilities to activate reporter gene AUR1-C transcription. All transformants could grow on the medium lacking leucine and tryptophan, indicating that AD and BD were transformed into Y2H strains. Excitingly, the strains transformed with CrDYRK1 (isoform I) grew on medium supplemented with AbA but not the strains transformed with CrDYRK1 (isoform II)  B, suggesform II) C. CollecCiona notochord cells driven by the Brachyury promoter. The results showed the different subcellular localization of CrDYRK1 with and without the repression domain. The GFP signal was detected at the nucleus in the isoform I-expressing cells  (residues 1–168) contained a repression domain to inhibit the transcriptional activation activity. To verify this idea, CrDYRK1 (isoform II) (residues 1–168) was fused with two transcription factor CrNFAT5 and CrMyc for function analysis, respectively. The results showed that the transcriptional activation activity of CrNFAT5 or CrMyc was presented; however, their transcriptional activation activities were obviously suppressed after fused with CrDYRK1 (isoform II) (residues 1–168), which indicates that residues 1–168 of CrDYRK1 (isoform II) functioned as a repression domain D,E. Thenng cells . Meanwhing cells . CollectC. savignyi CsDYRK1 (residues 393–626) also showed the transcriptional activation activity. However, the homologous region (residues 629–1047) of DYRK1A in Drosophila  did not show the transcriptional activation activity either  activation domain (residues 427–647) from different species were amplified and used for function analysis in yeast system, respectively . The resy either A. Then, y either B. CollecCiona epidermis cells driven by Epi I promoter. The GFP signal was detected obviously at the nucleus in both the CsDYRK1 (residues 393–626)-expressing cells and CrDYRK1 (isoform I) (residues 427–647)-expressing cells  fused with eGFP was expressed in HeLa cells, and the GFP signal was detected at the nucleus in the HsDYRK1A (residues 485–763)-expressing cells . Then, wng cells . These rp-value < 0.05) including 105 upregulated genes and 130 downregulated ones  involved in ion transport or neuroactive ligand–receptor interaction were highly expressed in the brain  . The result showed that there were many mutations locating in the HsDYRK1A activation domain that might be involved in DYRK1A-related intellectual disability syndrome . Five most representative motifs were identified from the upstream 1000 bp of translation start site of 96 upregulated genes  or APL-like transcription factor has dual localization in mitochondria and the nucleus via alternative splicing, which generates different transcript-coding variant proteins in plants [In this study, we found that the full-length CrDYRK1 (isoform I) rather than CrDYRK1 (isoform II) possessed transcriptional activation activity. CrDYRK1 (isoform II) is 168 amino acids longer than CrDYRK1 (isoform I) at the N-terminal. We further demonstrated that N-terminal (residues 1–168) of CrDYRK1 (isoform II) contained a repression domain, which could suppress the transcriptional activation activity. Then, we found that the on–off mechanism of transcriptional activation activity was retained in activity . This inactivity , which rn plants ,39,40. TATP1B2, TTYH1, ENKUR, SLC8A1, and SLC8A2), enriched by GO analysis, are highly expressed in the brain and crucial for ion transport [2+ transmembrane transport. The communication between neurons is pivotal to cognition, including learning and memory. The process of synaptic transmission is regulated by ion transport [2+ signaling exists in all types of neurons [2+ dyshomeostasis disrupts synaptic transmission [DYRK1A caused the motor abnormalities in a murine model of DS [In this study, some of DEGs . The result also suggests that the transcriptional activation ability of HsDYRK1A was implicated in neuronal functions. Thus, the roles of HsDYRK1A in neuron development and function could be based on two distinct pathways: one is that HsDYRK1A phosphorylates downstream substrates relying on kinase function; another is that HsDYRK1A promotes gene expression depending on the transcriptional activation activity. Our finding provides novel understanding on the function of DYRK1A and suggests that the transcriptional activation domain is a potential target for the therapy of DYRK1A-related neurological diseases.The KEGG analysis showed that DEGs were mainly enriched in neuroactive ligand–receptor interaction terms. Some of DEGs, related to neuroactive ligand–receptor interaction, are also highly expressed in the brain (for example,