Cyclocarya paliurus is an important medical plant owing to the diverse bioactive compounds in its leaves. However, the heterodichogamy with female and male functions segregation within protandry (PA) or protogyny (PG) may greatly affect seed quality and its plantations for medicinal use. To speculate on the factor playing the dominant role in regulating heterodichogamy in C. paliurus, based on phenotypic observations, our study performed a multi comparison transcriptome analysis on female and male buds (PG and PA types) using RNA-seq. For the female and male bud comparisons, a total of 6753 differentially expressed genes (DEGs) were detected. In addition, functional analysis revealed that these DEGs were significantly enriched in floral development, hormone, and GA-related pathways. As the dominant hormones responsible for floral differentiation and development, gibberellins (GAs) in floral buds from PG and PA types were quantified using HPLC-MS. Among the tested GAs, GA3 positively regulated the physiological differentiation (S0) and germination (S2) of floral buds. The dynamic changes of GA3 content and floral morphological features were consistent with the expression levels of GA-related genes. Divergences of GA3 contents at S0 triggered the asynchronism of physiological differentiation between male and female buds of intramorphs (PA-M vs. PA-F and PG-F vs. PG-M). A significant difference in GA3 content enlarged this asynchronism at S2. Thus, we speculate that GA3 plays the dominant role in the formation of heterodichogamy in C. paliurus. Meanwhile, the expression patterns of GA-related DEGs, including CPS, KO, GA20oxOX, GA2, GID1, and DELLA genes, which play central roles in regulating flower development, coincided with heterodichogamous characteristics. These results support our speculations well, which should be further confirmed. Cyclocarya paliurus  Iljinskaja is the sole species in the genus Cyclocarya Iljinskaja (Juglandaceae) [C. paliurus is generally known as the sweet tea tree, and its leaves are used to cure fever, detoxify, and alleviate pain [C. paliurus, including polysaccharides, proteins, minerals, phenolic compounds, vitamins, saponins, sugars, triterpenoids, and amino acid have beneficial effects on antihypertensive activity, hypoglycemic activity, hypolipidemic activity, and antioxidant activity [C. paliurus for leaf production to fulfill the need for its medicinal use.ndaceae) . It is mndaceae) . C. paliate pain . Moreoveate pain . Recentlactivity ,6. ThereC. paliurus, resulting in the low seed setting rate and high seed empty rate in its natural state [Owing to the lack of advancement in asexual reproduction technology, sexual reproduction is still the primary means of reproduction. Nevertheless, asynchronous flowering exists in heterodichogamous al state  to limital state . As a tral state ,10,11,12Agapanthus praecox ssp. orientalis [4/7 spray application affected flowering response in white spruce (Piceaglauca (Moench) Voss) [Carica papaya L. could induce carpel development in the male plant [Cucumis sativus) and spinach (Spinacia oleracea) induced male expression [Malus pumila) [Ginkgo biloba L.) [The synchronism of male and female flowering originates from different processes of floral bud differentiation and development. The complex process of floral development arises in response to the integration of signals from the external environment and internal factors, such as temperature, light, nutrients, and hormones ,14. As tientalis . Cecich h) Voss) . Moreovele plant . Exogenopression . In cont pumila) , ginkgo loba L.) , and rosloba L.) .ginkgo) and monoecism (such as apple). However, the regulating mechanism of GAs remains unknown in asynchronous floral differentiation and developmental processes in dichogamous plants. Heterodichogamy, a special type of dichogamy, was found in Magnoliaceae [Juglans regia L. [Juglans mandshurica Maxim [C. paliurus [C. paliurus [Various studies have stated the effects of GAs on homogamy plants, including dioecism . paliurus . Such a paliurus .C. paliurus, we took this species as an example, aiming to investigate the following puzzles: (1) do GAs  play a role in regulating two sexual floral differentiations and development; and (2) if so, how do GAs regulate the asynchronism of opposite sexual flowers of intramorphs (PA or PG). In this study, we not only describe the effects of GAs on flower bud differentiation in heterodichogamous plants and its regulation mechanism, but also provide an effective method of improving seed (fruit) yield for high-efficiency cultivation.Due to the solid basis and application necessary in C. paliurus were selected for RNA-Seq analysis based on morphological changing. At S0, all flower buds were not visible. At S1, although PA-F buds enclosed by leaf buds were not visible, male and PG-F buds were clearly differentiated. At S2, all the flower buds began to protrude, except PA-F. At S3, feathery stigma formed in PG-F, while this was not observed in PA-F; PA-M turned from turquoise to yellow-green with anther dehiscence, while PG-M still remained turquoise. At S4, the feathery stigma of PG-F opened and the mature pollen of PA-M was released from the dehisced anther; the stigma of PA-F was not fully formed, and the anther of PG-M was slowly dehiscent  at five stages  and leaves were used for transcriptome profiling. In total, more than 64 million raw reads were obtained. After quality evaluation and filtration, the number of clean reads ranged from 64,141,024 to 79,962,072 with an average of 73,855,021 and the average value of Q30 was 94.81%, indicating that the clean reads were of high quality. After alignment with the rubber tree genome sequence, more than 94% of the clean reads were mapped to the genome and paired-end reads were aligned to 35,221 e models .Two comparisons of three floral buds  detected a total of 6753 DEGs that were consistently upregulated or downregulated in at least two stages . ComparaFor the PG-F vs. PG-M comparison, GO functional annotation showed that most of the DEGs during the five stages were enriched in a series of biological processes involved in flower and organ development, hormone biosynthetic and metabolic processes, and GA signaling pathways a. MoreovFor the PA-M vs. PA-F comparison, the functional analysis revealed that these DEGs were also enriched in floral development, hormone, and GA-related pathways c,d. SimiDEGs in two comparisons were both significantly enriched in hormone and gibberellin biosynthesis, metabolic, and signaling pathways. Moreover, a series of ABCE module genes, such as AP3, PI, AG, and SEP, could be effectively activated by GA and play key role in regulating floral stamen and pistil morphogenesis ,37. HencFor the PG-F vs. PG-M comparison, many genes that are involved in GA biosynthesis, such as CPS , KO (ent-kaurene oxidase), GA20ox (GA 20-oxidase), GA2ox (GA 2-oxidase), and GID1 (gibberellin receptor), were highly expressed in PG-M at the S2 stage. However, DELLA genes, which negatively modulate GA signaling via binding to GAs and play important functions in inhibiting vegetative and reproductive growth, were lowly expressed in PG-F at S2 .For the PA-M vs. PA-F comparison, GA2ox and DELLA genes were highly expressed in PA-M at S2, but lowly expressed at S0. However, a series of genes including CPS, KO, KAO (ent-kaurenoic acid oxidase), and GA3ox (GA 3-oxidase) were highly expressed in PA-F at S2 . Taken t1, GA3, GA4 and GA7) contents are depicted in p = 0.049). Among the four GA types, only small contents of GA1, GA4, and GA7 were detected, and their fluctuations changed little across the floral developmental process. Comparatively, the contents of GA4 and GA7 in floral buds were negligible, although the content of GA1 was higher than that of GA4 and GA7. However, the maximum of GA3 in four floral buds was much higher than that of the other three GA types, varying from 40.37 ng/g FW to 116.50 ng/g FW at the S0 and S2 stages, with the minimum decreasing abruptly to trace amounts at the S3 and S4 stages , while the lowest level (<16.09 ng/g FW) was measured at other stages . For male floral buds (PG-M), a similar changing pattern of GA3 content to that in PG-F was observed, with higher values at S0 and S2 but lower values at S1, S3, and S4. Remarkably, the contents in PG-M were higher at S0 (106.33 ng/g FW) and S2 (89.53 ng/g FW) than in PG-F at those stages  and germination of male and female floral buds (S2) in PG, and make no difference at other phases; this was also supported by the significant differences of GA3 contents in male/female flower buds over various stages (p < 0.05). Multiple comparisons revealed no significant differences between males and females at S0, suggesting that GA3 could regulate both sexual floral physiological differentiations. However, the difference of GA3 content between two floral buds at S2 was significant (p < 0.05), showing that a higher GA3 content is needed for the germination of male floral buds than for female ones. All findings were confirmed by their corresponding morphological differences: differentiated male floral buds were visible but differentiated female buds were invisible at S0. Moreover, the onset of germination of male buds was earlier and at a faster rate than that of females at S2 , GAe stages a. These es at S2 .3 content in male and female buds at all stages displayed identical patterns to those in PG, except for a lower value in PA-F at S0. These dynamics correlated well with the characteristics of sexual floral bud differentiation and growth in PA, where the process of male floral buds was prior to that of female buds , CpaF1st20577 (G6PDH), CpaF1st20010 (NPR), CpaF1st42837 (PIF3), CpaF1st05926 (FCA), CpaF1st23696 (FLC), CpaF1st27743 (EARLY), and CpaF1st46181 (FLD). Generally, the relative expression of selected genes was consistent with the RPKM values from RNA-seq, indicating the expression profiles of our transcriptomics results were reliable . Particularly, the PA type is male prior to female and the PG type is female prior to male, and two flowering stages appear during the flowering season. Hence, flowering asynchronization emerges not only between the male and female flower of intramorphs (such as PA-F and PA-M), but also the same sexual flower of intermorphs (such as PG-F and PA-F). This phenomenon has been noticed in many heterodichogamous plants, particularly in Juglandaceae species, such as regia L. , J. manddshurica , Carya ioinensis , Platycaobilacea , and C. paliurus . HoweverC. paliurus female buds grow at the apex of the growing shoot and male buds locate at the lateral short branch; the expression of GA-related DEGs in male and female buds may be triggered under the genetic control depending on the mating type. Based on the analysis of differentially expressed genes, 371 enriched DEGs in PG-F vs. PG-M and 893 DEGs in PA-M vs. PA-F related to gibberellin biosynthesis and signal transduction pathways further confirmed the GA3 role in regulating floral differentiation in C. paliurus. For the differentiation and germination of floral buds at S0 and S2, a series of GA-related genes, including CPS, KO, KAO, GA20ox, GA2ox, GA3ox, GID1, DELLA, and SCF, were differentially expressed, demonstrating the importance of GA3 in floral bud differentiation and germination. For instance, as the positive key enzyme genes, CPS, KO, KAO, and SCF were significantly down-regulated in the comparison of PA-M vs. PA-F  Standl and Luffa cylindrica L. (Roem) [Malus domestica Borkh.) [3 content showed significantly higher values with apparent variations over five stages  , and sig Borkh.) . In thise stages , suggestds in PA  is in acds in PG  was also3 contents in male over female buds in both the PA and PG types at two stages infers that the activity of male buds could be more dependent on high GA3. Collectively, these results from C. paliurus are consistent with previous studies , that GA3 promoted floral induction and germination and even affected the number of days taken to flower [3 in controlling the physiological differentiation in male buds and germination in C. paliurus was also verified in Arabidopsis thaliana, whose stamen development required higher GA contents than other floral organs  [3 in male floral buds at stage 8 (mature pollen) in Diospyros kaki Thunb. was considered to promote the development of stamen primordia [3 content with GA-related DEGs expressions, we deduced that gibberellin response genes potentially play crucial roles in adjusting the asynchronization of the male and female flowers of intramorphs in C. paliurus.In addition, higher GAo flower ,48,49. Qd calyx) . Moreoverimordia . CombiniC. paliurus was established in Hongya Mountain Forest Farm, Chuzhou, Anhui Province, China  in 2013, containing 800 seedlings from a germplasm bank, which were planted in the Baima Experimental Base of Nanjing Forestry University. Voucher specimens were identified by Xin Chen (a taxonomist of Nanjing Forestry University) and deposited in the Silviculture Lab of Nanjing Forestry University . Nine protogynous (PG) and protandrous (PA) individuals, respectively, were randomly selected and marked for phenological observation and sampling. An experimental plantation of Leaves and floral buds in various protandry (PA) and protogyny (PG) individuals were collected at five different stages of floral differentiation and development including (1) S0, the physiological differentiation period (26 May 2019); (2) S1, the dormancy period (23 December 2019); (3) S2, the germination period (15 March 2020); (4) S3, the inflorescence elongation period (18 April 2020); and (5) S4, the maturation period (28 April 2020). Among them, only male and female floral buds from two mating types were collected as experiment materials at S1, S2, and S3, because of their deciduous characteristic. However, during the other two phases, floral buds and leaves were harvested for GAs content determination. Leaves at S4 and floral buds at five stages were collected for transcriptome sequencing. All flower buds were invisible during physiological differentiation (S0), so the flower buds were harvested based on last year’s position on the branch and the duration of morphological observation was from the dormancy stage (S1) to the maturation period (S4). Samples of complementary gender in each mating type were harvested from three individuals and combined as one independent biological replicate, and the total of nine trees were grouped as three replicates. Collectively, more than 15 male flower buds and at least 45 female flower buds were collected for each replicate, respectively. All samples were preserved in liquid nitrogen and stored at −80 °C for further analysis.260/OD280 within 1.8–2.0 were reverse transcribed to cDNA using a PrimeScript™ RT reagent kit with gDNA Eraser , according to the instructions of the manufacturer. Then, the cDNAs were diluted at 1:10 with RNase-free water, and stored at −20 °C for qRT-PCR analyses. All cDNA libraries were loaded onto an Illumina HiSeqTM2000 system (2 × 100 bp read length). Finally, 6 Gb of RNA-seq raw data for each sample was generated, and the quality of raw reads was checked by FastQC [Total RNA was extracted from various tissues using an E.Z.N.A Plant RNA Isolation Kit . RNA concentration and purity were determined by a NanoDrop spectrophotometer 2000C , and RNA integrity was confirmed by 1.0% agarose gel electrophoresis. The RNAs with the values of ODy FastQC . Clean rC. paliurus genome using Hisat2 [p-value was calculated by the false discovery rate (FDR). If the FDR was less than 0.05, and the absolute value of log2 (fold change) was more than 1, the gene expression differences were considered to be significant.Paired-end short reads were aligned to the PG-dip g Hisat2 . StringTg Hisat2  was usedg Hisat2 , which wg Hisat2 . Moreoveg Hisat2  was applThe Gene Ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed using the clusterProfiler R package .1, GA3, GA4, and GA7) at different developmental stages. A UPLC-MS/MS system  was used to determine the content of four types of GAs (bioactive GA3) were purchased from Sigma-Aldrich , while MeOH and ACN were purchased from Merck . C. paliurus vacuum freeze-dried floral buds and leaves were ground into powder , and 500 mg of powder was extracted with 1.5 mL of methanol/water/methanoic acid solution . The extract was vortexed every 30 min and stored at 4 °C for one night. After centrifugation , the supernatants were filtered with a microporous membrane (0.22 µm pore size) and collected for UPLC-MS/MS analysis.Standards  and the mobile phase consisted of ultrapure water (A) and acetonitrile (B), which both contained 0.1% formic acid. Gradient elution was as follows: the original B ratio was 5%; the B ratio linearly increased to 70% within 3 min and was maintained for 3 min; and the B ratio decreased to 5% from 6.1 to 8 min and was maintained for 1.9 min. The flow rate was kept at 0.2 mL/min, temperature at 40 °C, and injection volume was 2 µL.® qPCR Master Mix  were used for RT-qPCR reaction. The reaction system was as follows: 2× MasterMix 10 µL, Primer F 1 µL(10 μM), Primer R 1 µL(10 μM), cDNA 1 µL, and nuclease-free water up to 20 µL. The reaction procedure and methods of relative gene expression calculation were presented by Chen [Eight unigenes related to floral bud differentiation and development, as well as the hormone-mediated signaling pathway, were selected for qRT-PCR analysis. Moreover, 18S ribosomal RNA as an internal control and the cDNA Synthesis Kit for reverse transcription were selected according to Chen . Proprie by Chen .3, may play an important role in contributing to the heterodichogamy morphs with asynchronous flowering in C. paliurus. The DEGs during five stages were significantly enriched in a series of biological processes involved in flower and organ development, hormone biosynthetic and metabolic processes, and GA signaling pathways. GA3 content showed a significant role in controlling flowering bud differentiation and development. Our results can provide the basis for further understanding the molecular mechanisms for gibberellin regulating floral bud differentiation, development, and their asynchronism in heterodichogamous plants.This study reveals that hormones, especially GA


BSCL2) gene, is famous for its key role in the biogenesis of lipid droplets and type 2 congenital generalised lipodystrophy (CGL2). BSCL2 gene mutations result in genetic diseases including CGL2, progressive encephalopathy with or without lipodystrophy , and BSCL2-associated motor neuron diseases. Abnormal expression of seipin has also been found in hepatic steatosis, neurodegenerative diseases, glioblastoma stroke, cardiac hypertrophy, and other diseases. In the current study, we comprehensively summarise phenotypes, underlying mechanisms, and treatment of human diseases caused by BSCL2 gene mutations, paralleled by animal studies including systemic or specific Bscl2 gene knockout, or Bscl2 gene overexpression. In various animal models representing diseases that are not related to Bscl2 mutations, differential expression patterns and functional roles of seipin are also described. Furthermore, we highlight the potential therapeutic approaches by targeting seipin or its upstream and downstream signalling pathways. Taken together, restoring adipose tissue function and targeting seipin-related pathways are effective strategies for CGL2 treatment. Meanwhile, seipin-related pathways are also considered to have potential therapeutic value in diseases that are not caused by BSCL2 gene mutations.Seipin, a protein encoded by the Berardinelli-Seip congenital lipodystrophy type 2 ( BSCL2) gene, is found to locate in the endoplasmic reticulum (ER) [Seipin, an evolutionarily conserved protein encoded by the Berardinelli-Seip congenital lipodystrophy type 2 (lum (ER) ,2. SEIPIlum (ER) . Seipin lum (ER) . Therefolum (ER) ,6,7.BSCL2 damage different organs, such as the brain [BSCL2-associated motor neuron diseases . Both CGL2 and PELD are inherited as autosomal recessive, whereas BSCL2-associated motor neuron diseases are almost all autosomal dominant. Furthermore, the expression levels of seipin are found to be closely related with the progress of more common diseases not caused by BSCL2 mutations, implying that seipin may have potential prognostic and therapeutic values in these diseases [In recent years, numerous findings have been unravelled by studying the role of seipin in diverse diseases. Mutations of he brain ,9,10, mahe brain ,12, panche brain , and teshe brain ,15, whicdiseases ,19,20,21The N-terminal and C-terminal of the ER-located seipin protein are exposed to the cytoplasm, and the two transmembrane helices wrapped in the ER lumen are connected by a large, highly conserved cavity ring . MultiplPhysiologically, seipin has tissue-specific functions. In the adipose tissue, seipin serves as an adipocyte safeguard that maintains normal neutral lipid storage. In the liver, seipin is crucial for hepatic intracellular TAG accumulation . InteresBSCL2 usually induce a variety of serious clinical consequences .SEIPIN is widely expressed in human tissues, and the highest levels of this protein are found in the adipose tissue, brain, and testis ,57. Striequences , includiAGPAT2, BSCL2, CAV1, and PTRF) induce CGL1, CGL2, CGL3, and CGL4, respectively. All subtypes of CGL lose metabolically active adipose tissue, but each of the subtypes has their unique features in addition to genetic testing (gold criteria) for differential diagnosis: (1) CGL1 patients usually suffer from focal lytic lesions in long bones after puberty; (2) CGL2 patients lose their mechanical adipose tissue except for metabolically active adipose tissue; (3) CGL3 patients are characterised by short stature, functional megaoesophagus, and hypocalcaemia; (4) CGL4 patients may progressively lose their adipose tissue during infancy, and face the challenge of congenital myopathy with high serum levels of creatine kinase [CGL is a metabolic disease characterised by almost complete loss of adipose tissue and severe metabolic disorders, including early-onset diabetes mellitus, insulin resistance, hypertriglyceridaemia, and hepatic steatosis . Variante kinase . CGL2 ace kinase ,72. Up te kinase , but a fe kinase ,74. SincCompared with other types of CGL, the loss of adipose tissue in patients with CGL2 is more serious ,76. In aCGL2 patients often have many physical changes, mainly including acanthosis nigricans, hirsutism, muscular hypertrophy, and acromegaly ,90,93. AApart from the typical features of metabolic disturbances, CGL2 patients also have a greatly increased probability of developing mild to moderate mental disability ,61,79,95CGL2 patients also suffer from impairment of the reproductive system. Effects on male patients are more severe, mainly manifested as enlarged external genitalia and infertility ,86. TeraSome studies suggest that CGL2 patients are prone to inflammation, including upper respiratory tract infection ,99, skinBSCL2 gene, and the mutation can be homozygous or in compound heterozygosity with c.507_511del, c.538G > T, or c.1004A > C [PELD is a fatal paediatric neurodegenerative disease manifested as severe progressive neurodegeneration and concomitant premature death caused by status epilepticus or pneumonia . PELD is004A > C . Recentl004A > C . The mutBSCL2 causes a broad spectrum of motor neuron diseases, not CGL2 [LMNA) gene mutations, all diseases caused by BSCL2 mutations  should be called “seipinopathies” [BSCL2-associated motor neuron diseases” as per a dyadic nomenclature in this review. The main manifestations of patients with BSCL2-associated motor neuron diseases are distal limb muscle weakness and atrophy, hypertonia, abnormal reflex, gait abnormalities, and so on [BSCL2 [BSCL2 variants are associated with epileptic encephalopathy, which have been reported in three patients [BSCL2 variant including p.N88T [Single heterozygous, compound heterozygous, or homozygous missense mutation (N88S or S90L) in not CGL2 ,67. Thesnot CGL2 ,67, whicnot CGL2 . Howeverpathies” ,105. Thend so on . There in [BSCL2 ,107, butn [BSCL2 . Two dispatients . Furtherg p.N88T , p.S141Ag p.N88T , or p.R9g p.N88T  may alsoBSCL2 mutations is mainly directed towards symptoms [The clinical treatment for diseases caused by symptoms , and we symptoms ,112. In symptoms ,117,118.symptoms . Finallysymptoms ,120.Bscl2 ablation through homologous recombination in embryonic stem cells [Bscl2 in 2020 [In 2011, Cui et al., established the first CGL2 animal model in mice, also named seipin knockout (SKO) mice, by em cells . The SKOem cells . Howeverem cells ,122. Carem cells  and renaem cells  induced em cells , depressem cells ,125, spaem cells , and impem cells . For repem cells ,15, whilem cells . The abo in 2020 . Since the expression pattern of seipin in the organs of rats is more similar to humans than in mice, Ebihara et al., established the first CGL2 rat model by using the method with ENU (N-ethyl-N-nitrosourea) mutagenesis for SKO in 2015 . It has Metabolic disorders are the predominant characteristics of SKO mice and attract numerous researchers investigating the underlying mechanisms and potential therapeutic targets . Magré aBscl2 deletion on mature adipose tissue [In the review, Magré and Prieur described that seipin deficiency causes severe lipodystrophy by impairing adipogenesis, accelerating lipolysis, damaging LD homeostasis and other unclear mechanisms . Here, we tissue ; (3) trae tissue ; (4) seie tissue ,52,140. e tissue ,136; (2)e tissue .Ldlr double knockout mice compared with SKO mice (300 mg/dL) or Ldlr knockout mice (1000 mg/dL) alone [Additionally, accumulating evidence also puts forward that seipin also participates in other physiological functions: (1) Seipin may play an important role in insulin synthesis and secretion via regulating the expression of PPARγ in pancreatic islets, and this function is affected by oestradiol . (2) SeiL) alone . (3) SeiL) alone . ResearcL) alone . HoweverL) alone . (4) SeiL) alone . Bruder-L) alone ,146. AddL) alone .Bscl2 gene expression impairs excitatory post-synaptic currents via reducing levels of surface AMPAR [212/Ser214 and Ser202/Thr205 through increasing protein kinase B–mammalian target of rapamycin (AKT-mTOR) signalling, which not only suppresses autophagy, but also elevates the glycogen synthase kinase-3β (GSK3β) activity, eventually leading to axonal atrophy [396 induced by the c-Jun N-terminal kinase (JNK) pathway [Seipin deficiency can result in neurodegenerative phenotypes through a variety of mechanisms, which can be summarised as the following aspects : (1) Neuce AMPAR . In the ce AMPAR . In hipp atrophy . In dopa atrophy . (2) Met pathway , and low pathway ,150,151.BSCL2 mutations, the potential effects on the reproductive system are proposed  cells (but not in primary liver cells) [Studies in SKO, germ-cell-specific seipin knockout (seipin-gKO), and seipin-aKO mice show that the absence of seipin protein in germ cells (SKO and seipin-gKO), but not in adipose tissue, leads to infertility and teratozoospermia . The undr cells) . Seipin r cells) . Additior cells) .According to previous studies, phenotypes involving the female reproductive system induced in SKO mice mainly include abnormal mammary gland development, insufficient milk production, and defective parturition. Female SKO mice have longer and more dilated mammary gland ducts at 5 weeks old , and theBSCL2-associated motor neuron diseases damage upper motor neurons, lower motor neurons, and peripheral motor axons differently [BSCL2-associated motor neuron diseases, including N88S mutant seipin transgenic (N88S seipin Tg) mice [BSCL2-associated motor neuron diseases, including the progressive spastic motor deficit, reactive gliosis in the spinal cord, and neurogenic muscular atrophy [ferently . To dateTg) mice , N88S seTg) mice , and N88Tg) mice . Both th atrophy ,160. The atrophy .Bscl2 inhibit the glycosylation of seipin, which leads to the misfolded and aggregated proteins and the excessive accumulation of misfolded proteins, resulting in ER stress in neurons [BSCL2-associated motor neuron diseases. (2) N88S/S90L seipin expression results in the formation of inclusion bodies in the cytoplasm. Compared with normal cells, N88S/S90L seipin induces the formation of inclusion bodies in the cytoplasm in different cell types, including motor neurons at a 25–30 percent level in vitro [The potential molecular mechanisms are summarised as follows: 1) Whether ER stress should be considered as a predominant mechanism is a subject of debate. Mutations of N88S and S90L in  Whether  neurons ,162. N88 neurons . In cont neurons ,160. Thein vitro ,162. N88in vitro ,160. Thein vitro . Howeverin vitro . (3) Seiin vitro ,164. Seiin vitro . AdditioBSCL2 mutations has attracted more and more attention in recent years. At present, the relationships between seipin and metabolic [The role of seipin in diseases not caused by etabolic , neurodeetabolic ,20,166, etabolic ,167,168 BSCL2 mutations affect the levels of seipin expression, and the abnormal seipin expression would in turn affect disease progression  restoration of adipose tissue itself or its function to treat metabolic complications resulting from adipocyte seipin deficiency; (2) tackling seipin or seipin upstream or downstream targets to alleviate the effects of seipin deficiency in organs in which seipin works in a cell-autonomous way.To date, the main treatments for diseases caused by  options ,59, and The restoration of adipose tissue function, which includes improving TAG storage capacity and maintaining adipokine levels, is the main principle to treat metabolic disorders and complications in CGL2 . The pot25-35/1-42-induced Alzheimer’s disease [In organs where seipin works in a cell-autonomous way, tackling seipin-related PPARγ pathways is the main principle to ameliorate disorders of these organs in CGL2. In SKO mice, rosiglitazone treatment ameliorates anxiety , depress disease . In seip disease . Rosigli disease . The dow disease ,125; a G disease ,148; andIn addition, some treatments that cannot be concluded by these two strategies also ameliorate CGL2 phenotypes. In SKO mice, the sodium-glucose cotransporter 2 inhibitor dapagliflozin successfully reduces the O-GlcNAcylated protein levels and blocks the development of hypertrophic cardiomyopathy ; JNK inhBSCL2 mutations, but also in more common diseases that are not related to BSCL2 mutations. Limitations exist in studies focusing on the relationships between seipin and diseases: (1) studies on differential expression patterns of seipin in different diseases and the underlying mechanisms require exploration; (2) the precise mechanism of seipin in the brain and testis where it works in a cell-autonomous way is still unclear; and (3) there are rare seipin signalling-pathway-based treatment options for relevant diseases. Therefore, further studies to address these limitations are awaited. Moreover, similar to seipin, other ER-located proteins involved in LD biogenesis such as fat storage-inducing FIT2, Atlastin, REEP1 and so on are also closely associated with human diseases. For example, FIT2 has been certified to play a key role in lipodystrophy [In the current review, the summarised evidence suggests that seipin plays an important role not only in rare diseases induced by ystrophy , diabeteystrophy ,174, andystrophy , as wellystrophy , and the


VvARF4-2 might be the major factor in the modulation of GA-induced parthenocarpy via the crosstalk of IAA, CTK, SA, and ethylene signaling, followed by VvARF6-1 and VvARF9 involved in SA and ABA signaling pathways, respectively. Finally, we developed a VvARFs-mediated regulatory network by responding to GA-mediated multi-hormone crosstalk during grape parthenocarpy. Collectively, our findings provided novel insights into the regulatory network of VvARFs in GA-guided multi-hormone signaling to modulate grape parthenocarpy, which has great implications for the molecular breeding of high quality seedless grape berries.Exogenous gibberellin (GA) was widely used to improve berry quality through inducing parthenocarpic seedless berries in grapes. We revealed that auxin response factors (ARFs), the key transcription factors in response to auxin, might respond to GA involving modulation of grape parthenocarpy. However, the underlying molecular mechanism in this process remains yet unclear. Here, a total of 19 VvARF members were identified in the ovaries during GA-induced grapes’ parthenocarpy. Interestingly, almost all members were GA-responsive factors, of which 9 could be classified in plant hormone signal transduction (KO04075) and involved in the tryptophan metabolic pathway (K14486). Moreover, VvARFs were predicted to have 310 interacted proteins involved in 19 KEGG pathways. Of them, 32 interacted proteins participated in the KO04075 pathway, including auxin (IAA), salicylic acid (SA), abscisic acid (ABA), cytokinin (CTK), and ethylene signaling pathways by responding to GA-mediated multi-hormone crosstalk. Further analysis demonstrated that  Gibberellin (GA) is an essential phytohormone, which plays important roles in multiple aspects of plant growth and development, such as seed germination, stem hypocotyl elongation, leaf elongation, cell expansion, epidermal hair development, controlling flowering time, and flower and fruit development, especially in parthenocarpy . NowadaySolanum lycopersicum (tomato), miR167/ARF8 and miR159/GAMYB act synergistically to regulate tomato ovule development and produce parthenocarpic seedless fruits [ARF8 stimulates parthenocarpy in Arabidopsis thaliana and tomato [SlDELLA and auxin-signaling components SlIAA9 and SlARF7 repress the parthenocarpy in tomato [VvARF10(VIT_208s0040g01810)/16(VIT_213s0019g04380)/17(VIT_218s0001g04180) accompanied with the induction of grape parthenocarpy [The understanding of molecular mechanisms underlying auxin-regulated processes in collaboration with GA-signaling cascades is required for creating seedless fruits. Previous studies showed that several members of the auxin response factors (ARFs) family could be involved in parthenocarpy . Moreoves fruits . The expd tomato , while tn tomato . Similarenocarpy ,15 and gOryza sativa, Arabidopsis thaliana, Populus trichocarpa, Malus domestic, and Solanum lycopersicum, respectively [ARF2 gene is induced and expressed in senescent leaves, regulating the time of leaf senescence, flowering, and abscission in Arabidopsis thaliana [SlARF6 and SlARF8 mainly regulate the development process of tomato flowers and fruits [AtARF8 can also regulate the elongation of Arabidopsis thaliana hypocotyls [Arabidopsis thaliana can delay flowering through increasing indole-3-butyric acid and suppressing the transcription of ARF6, ARF8, and flowering-related genes FT, SOC1, AP1, and LFY [SlARF not only mediates part of auxin signaling, but also acts as part of gibberellin signaling during the development of tomato fruits [There is a different number of ARFs members among plant species. With the improvement of genome sequencing technology, 25, 23, 37, 29, and 21 members of ARF gene’s family have been identified in ectively ,18,19,20ectively ,23. For thaliana . In addid fruits , and AtA and LFY . Moreoveo fruits ,28. The o fruits . All theIt seems that GA-induced parthenocarpy in grape is more typical than other fruit trees. Grape, as an important economical fruit crop in the world, can be used in human diets such as fresh berry, juice, and wine. Therefore, seedless grapes are very popular with consumers. However, due to the shortage of seedless grape varieties, at present, GA-induced parthenocarpy is the main approach of producing seedless berries in the grape industry. Nevertheless, the mechanism by which GA modulates parthenocarpy in collaboration with the ARF gene family is poorly understood. In this study, we characterized the ARF gene family at the whole genome level and investigated the ARFs-mediated regulatory network via multi-hormone signal crosstalk during the GA-induced parthenocarpy process in grapes. Our findings provide important insights into how VvARFs’ regulatory network promotes the parthenocarpic process in grape inflorescences, leading to significant implications for the development of high-quality grape seedless berries.3 treatment. After 10 DAT (Days after treatment) with GA3, the development of their seeds was repressed drastically and led to seedless with a rate of 99.6%, while their berry brushes had no obvious changes  at the 20, 30, 50, 70, and 95 DAT, respectively . The types of Arabidopsis thaliana ARF proteins were used to determine the grouping of VvARF proteins; results , participated in tryptophan metabolism pathway (K14486), and affected auxin biosynthesis in plants . Among t process , indicatTo more comprehensively recognize the potential functions of the VvARFs family, we further investigated the molecular interaction, reaction, and relation networks of their interacted proteins. The results showed that a total of 310 interacted proteins were identified from STRING v10.0, of which VvARF4-2 had the most interacted proteins with 179 ones, followed by VvARF18-1 with 122 proteins. Moreover, through KEGG analysis, we revealed that these interacted proteins contributed to 19 KEGG pathways. The pathways with the most interacted proteins were plant hormone signal transduction (ko04075) with 32 members A. FurtheVvARF2-2 had the highest expression level, followed by VvARF2-1, VvARF6-1, and VvARF9. Meanwhile, VvARF17 was not expressed, as additionally, VvARF4-1/4-2/5-1/18-1/18-3 were basically in the low expression levels, while the remaining genes were at a moderate level of expression in the control group. In the treated samples with GA, based on the definition of DEGs [VvARF18-1 and VvARF4-2) were significantly up-regulated, another 4 members  were slightly up-regulated, conversely, VvARF18-3, VvARF9 and VvARF1-1 were slightly down-regulated, respectively, while VvARF17 was not examined in corresponding tissues , VIT_219s0090g01740 (VvPAT1), VIT_219s0085g01030 (VvAMC5), and VIT_213s0019g04390(VvGATA8) were highly expressed in the control. During GA-induced parthenocarpy, the expression of VvPAT1, VvAMC5, and VvGATA8 increased by twice as much, but that of VvMYB44-1 was distinctly down-regulated. Therefore, these genes strongly contributed to GA signaling during the modulation of parthenocarpy. As analyzed above, VvPAT1 was predicted to interact with VvARF2-1 and VvARF2-2. Similarly, both VvAMC5 and VvGATA8 were interacted with VvARF4-2 and VvARF18-1, while VvMYB44-1 was associated with VvARF5-1. Therefore, GA also affects the expression of these interacted VvARF genes and modulates grape parthenocarpy. On the other hand, we exhibited another 10 interacted genes with VvARFs that possess the significant variation at their expression levels during GA-induced grape parthenocarpy, even though they indicated the medium expressions in control. Among them, VIT_211s0016g01190 (At1g04910), VIT_205s0029g00330 (VvSAHH), and VIT_218s0001g11630 (VvCYP74A) were up-regulated by GA. On the contrary, VIT_207s0129g01050 (VvMYB44-2), VIT_203s0063g00380 (VvCYP707A4), VIT_209s0002g08370 (VvTPL), VIT_205s0062g01060 (VvSPBC2A9.03), VIT_201s0010g03780 (VvAHK3), VIT_218s0001g06650 (VvBHLH96), and VIT_218s0001g08090(VvIAA9) were down-regulated in response to GA, while these genes were predicted to mainly interact with VvARF4-2, VvARF6-1, and VvARF18-1, implying that among the VvARF family, they might be the main regulatory factors during GA-induced grape parthenocarpy.According to the expression profiles of the interacted genes with VvARFs B,C, we rVvARF genes  were randomly selected to detect their expression levels in corresponding tissues using qRT-PCR. As shown in VvARF6-1 was up-regulated significantly and VvARF2-1 was down-regulated by exogenous GA, which were consistent with that in RNA-Seqdata, demonstrating that they might be the important factors of responding to GA-mediated grape parthenocarpy.To validate the result of RNA-seq, 8 VvARF6-1 obviously increased, while the corresponding interacted gene VvIAA9, an important repressor in the auxin signaling pathway, reduced. Similarly, VvYUC10-2, a key synthesis enzyme in IAA metabolism was down-regulated, and thus we presumed GA promotes the VvARF6-1 level potentially by inhibiting IAA9, while VvARF6-1 and VvYUC10-2 might maintain the dynamic balance of auxin signaling. In the ABA signaling pathway, VvARF9 was predicted to interact with VvPYR1, a receptor protein of the ABA signaling pathway, while VvPYR1 was down-regulated during GA-induced parthenocarpy, indicating it might possess the negative regulatory effect in ABA signaling; and VvNCED1-2, one key ABA synthesis enzyme also decreased. Meanwhile, we revealed that VvARF4-2 was involved in both the CTK and ETH hormone signaling pathways through interacting genes VvAHK-1 and VvCTR1, respectively, during this process. Exogenous GA repressed the expression of VvAHK-1 and VvCTR1 and reduced the levels of VvIPT and VvACS/VvACO1-2 as key enzyme genes in CTK and ETH synthesis. Further analysis demonstrated that both VvARF4-2 and VvARF6-1 might participate in the regulation of the SA signaling pathway via interacting with the VvTGA2.2 gene. Exogenous GA inhibited the expression levels of the VvTGA2.2 gene and VvPAL in the SA synthesis pathway. From these results, we discovered VvARF4-2 might respond to GA involved in the regulation of 4 hormone signaling pathways including IAA, CTK, SA, and ethylene during parthenocarpy , including IAA, CTK, SA, ABA, and ethylene hormone signaling pathway . Of themenocarpy , followePopulus trichocarpa, Malus domestic, and Solanum lycopersicum, although the ARF family possessed some evolutionary changes across diverse plant species. ARF family members were divided into 4 till 5 subfamilies, of which the cluster relationship was close to the subfamily members with similar gene functions [Vitis vinifera, Oryza sativa, Arabidopsis thaliana, Populus trichocarpa, Malus domestic, and Solanum lycopersicum were clustered into 5 branches from Class I to V, of which VvARFs family was distributed in each branch. Both VvARF6(VvARF6-1/2) and AtARF6 were clustered in Class V, implying they might have similar functions. AtARF6 was identified to interact with transcription factors BZR1 and PIF4 in response to GA, BR, IAA, and other hormone in the signaling pathways [Arabidopsis thaliana [VvARF family members possess higher conservation with those in Arabidopsis thaliana [Oryza sativa [Solanum lycopersicum [With the continuous development of biological information technology and the improvement of sequencing technology, it is possible to identify and characterize a certain gene family and its mediating regulatory network at the whole genome level. As a class of ARF genes encoding important transcription factors, they participate in multiple processes of plant growth and development. In this study, based on transcriptome data, 19 ARF genes were identified in the ‘Wink’ grape, which were similar to the ARF family members of unctions ,34. The pathways , while Bthaliana , rice [1thaliana , tomato thaliana , longan thaliana , and sugthaliana . Furtherthaliana , Oryza spersicum  than theArabidopsis thaliana and Solanum lycopersicum described above. In this study, we revealed that VvARFs had differential expression profiles in response to GA treatment during grape inflorescence development, of which VvARF6-1 and VvARF2-2 showed the higher expression than other members, and up-regulated and down-regulated respectively, might be the important factors of responding to GA-mediated grape parthenocarpy. Together with the analysis of the VvARFs interacted network, we revealed that the VvARF family was involved in the regulation of 5 hormone signaling pathways including IAA, CTK, SA, ABA, and ethylene. Of them, VvARF4-2 might participate with four hormone signaling pathways through interacted genes, and thus might mediate the network of multiple hormone signals to regulate the development of grape flowers. For instance, the arf3 arf4 double mutant Arabidopsis plants have narrow petals and deformed stamens, while Arabidopsis miR390 positively directs the biosynthesis of trans-acting siRNA3 (tasiRNA3), while tasiRNA3 negatively regulates the expression of ARF3/ARF4 to affect lateral root development, leaf polarity, and flowering time [IAA9 gene in tomatoes causes leaf deformation and plant parthenocarpy [IAA9 could form a complex with ARF8, which prevents ARF8 from combining with AuxRE region in the promoter of auxin-responsive genes, whereby inhibiting the expression of the auxin response gene and finally preventing fruit setting and development [VvIAA9 was repressed by exogenous GA, and thus it might participate in the modulation of GA-induced grape parthenocarpy through releasing auxin signaling.Analysis of VvARF family promoters showed that the cis-acting elements related to hormone responses were identified, of which six VvARF family members contained cis-elements that respond to auxin as well as gibberellin. This implies VvARFs might respond to multi-hormone signal’s crosstalk to be involved in the modulation of plant growth and development, similar to the previous reports on manipulating plant flower development, formation, and bloom ,40,41, bing time ,43,44,45enocarpy , while Ielopment , and alselopment . SimilarVvARF10/16/17 were down-regulated during the fruit-set stage of GA-induced grape parthenocarpy; and GA obviously repressed the level of VvARF18 at the grape seed development period, indicating VvARFs could respond to the GA signal to be involved in manipulation of parthenocarpy. Together with the fact that GA induces plant parthenocarpy through multi-hormones interactions [SlARF7 formed parthenocarpic fruits with the increased auxin and GA responses [SlARF8a was up-regulated in parthenocarpy 159-OE tomatoes [ARF8 also induced parthenocarpic fruit-set in eggplants independently pollinated/fertilized; while arabidopsis arf6/arf8 flowers arrest as closed buds with short petals, short stamens, and indehiscent anthers, as well as defect in the growth of the pollen tube [ARF6/8 to be unable to fruit-set [VvARF6-1 were up-regulated during GA-induced grape parthenocarpy, and it might be involved in modulation of parthenocarpy through responding to both SA and IAA signals. These findings indicate ARFs could participate in modulation of plant parthenocarpic fruit-set.Many research studies confirmed GA was one of the important factors in the efficient induction of plants ,50,51,52ractions ,54, we pesponses . Recent tomatoes , and thelen tube . Similarruit-set . In thisVvPAL-5 was up-regulated by GA to promote SA synthesis; conversely, VvPYR1, VvACO1-2, and VvYUC10-2 were mainly down-regulated by GA to repress the ABA, ETH, and IAA synthesis, respectively, while genes in CTK synthesis had almost no change after GA application . All collected samples were frozen rapidly in liquid nitrogen and stored at −80 °C for further analysis.The horizontal and vertical lengths of the berries were measured using an automatic vernier caliper. Total soluble solids were determined using a portable hand-held dialyzer , while the content of soluble sugar was assayed according to the anthrone reagent method described by Cao et al. . The tithttp://genomes.cribi.unipd.it/grape/index.php, accessed on 1 August 2021). We constructed the phylogenetic tree using the Maximum Likelihood method against the target species genome and the reference genome. The genes in gene families that were single copies in the whole genome of sequencing species and reference species were used, and the evolutionary tree was constructed using MEGA11.0 to study the evolutionary relationship between species. Based on the results of clustering analysis of a homologous gene family, a single copy of homologous gene was selected for multi-sequence alignment  and then a phylogenetic tree was constructed based on the single copy gene method. Similarly, one more phylogenetic tree was prepared using all the available ARF protein sequences from grapes and the other five species .The software MEGA version 11.0 and ClustalX2 were employed to conduct the phylogenetic analysis. The theoretical gene and protein sequences of grape VvARFs family were obtained from CRIBI database .The exon/intron organization of VvARF genes was determined by comparing predicted coding sequences with their corresponding genomic sequences using the GSDS software  and Grape Genome Browser . For cis-acting element finding, we used PLANTCARE online database .The sequence of 1500 bp upstream of the transcription initiation site (ATG) of VvARFs was obtained from the grape genome database CRIBI  were extracted using our modified CTAB method . RNA quap-value) according to the model; and (3) perform multiple hypothesis testing and correction to obtain the false discovery rate (FDR). Based on this analysis, transcripts with FDR < 0.01 and |log2FC| = >1 (where FC is fold-change) were screened as significantly different and classed as differentially expressed genes (DEG). Transcripts with |log2FC| < 0.25 were assumed to no change in expression levels. Other transcripts (0.25 < |log2FC| < 1) were considered as “up-regulated slightly” or “down-regulated slightly”.Reads count data was used as input data for the analysis of differentially expressed genes in the software package DESeq2. The analysis was divided into three parts: (1) standardize on read Count; (2) calculate the hypothesis-testing probability  and Gene Ontology (GO) , we performed enrichment analysis and unscrambled the interaction network of VvARFs. TBtools was used for heat map analysis.Using Kyoto Encyclopedia of Genes and Genomes database (KEGG)  was used to predict the interaction proteins of the VvARF family based on our RNA-seq data. For this analysis, we performed as follows: Select “multiple proteins “, input VvARF family member gene number at “the protein name”; enter Vitaceae at “Organism”; then, the results of the interacted proteins for VvARFs were predicted in this work.The online software STRING (Actin gene was used as a reference gene in the qRT-PCR detection of VvARFs. The primers were presented in ® Premix Ex TaqTM II kit from TaKaRa Company according to the instructions. All cDNAs were stored at −80 °C for the next analysis. All experiments were performed with 3 biological replicates and relative expression was calculated according to the 2−ΔΔCt method.To evaluate the reliability of the sequencing results, 8 VvARFs were selected randomly for qRT-PCR verification. The VvARF4-2 might be the major factor in the modulation of GA-induced parthenocarpy via the crosstalk of IAA, CTK, SA, and ethylene signaling, followed by VvAFR6-1 and VvARF9 involved in SA and ABA signaling pathways, respectively. These findings provide novel insight into the GA-mediated multi-hormone signaling networks to regulate the parthenocarpy process via guiding the VvARF family, which has implications for the molecular breeding of high-quality seedless grape berries.In summary, a total of 19 VvARF members were identified in the ovaries during GA-induced grapes’ parthenocarpy, and almost all VvARF members were GA-responsive genes, of which 8 members were up-regulated, and another 10 were down-regulated by exogenous GA. Furthermore, VvARFs were predicted to have 310 interacted proteins involved in the 19 KEGG pathways and participated in IAA, ABA, CTK, SA, and ethylene signaling pathways by responding to GA-mediated multi-hormone crosstalk.


PACS1, MCHR1, DCLK3, HAPLN4, LMAN2L, TMEM258, GNL3, LRRC57, CACNA1C, CACNA1D, and NOVA2 and their potential biological role in the pathogenesis of bipolar disorder. Molecular mechanisms under control of these genes do not translate into a unified picture and substantially more research is needed to fill the gaps in knowledge and to solve current limitations in prognosis and treatment of bipolar disorder. In conclusion, the genetic and functional studies confirm the complex nature of bipolar disorder and indicate future research directions to explore possible targeted treatment options, eventually working toward a personalized approach.The lifetime prevalence of bipolar disorder is estimated to be about 2%. Epigenetics defines regulatory mechanisms that determine relatively stable patterns of gene expression by controlling all key steps, from DNA to messenger RNA to protein. This Mini Review highlights recent discoveries of modified epigenetic control resulting from genetic variants associated with bipolar disorder in genome-wide association studies. The revealed epigenetic abnormalities implicate gene transcription and post-transcriptional regulation. In the light of these discoveries, the Mini Review focuses on the genes  Bipolar disorder (BD) is viewed by clinicians as a spectrum of related, but differentiable conditions, spanning from BD type 1, to BD type 2, to cyclothymic disorder, to other specified or unspecified BD  to protein. These regulatory mechanisms, found to be implicated in the pathogenesis of many complex disorders , 3, inclGenetic epidemiology studies estimated heritability of BD being in the range of 60–85% , 15. Thein silico analyses indicate that the largest contribution to SNP heritability in complex traits comes from genetic variants in regulatory regions  genetic variants, traditionally referred to as single-nucleotide polymorphisms (SNPs), typed throughout the human genome to reveal associations between particular alleles of the variants and phenotypes in groups of unrelated individuals . The SNP regions . GWASs o regions , 29. An  regions , the varp-value ≤ 5 × 10–8 that reflects the Bonferroni correction for testing associations with one million SNPs). A fraction of the associated SNPs were studied for their role in gene transcription and in post-transcriptional regulation. See The genetic studies mainly report results for BD type 1 and BD type 2 e.g., , 22]. Th. Th22)].BMPR1B, DCLK3, HAPLN4, HLF, LMAN2L, MCHR1, UBE2Q2L, SNAP91, TTC39A, TMEM258, and VPS45 is regulated by eQTLs (identified using human brain tissues) that are SNPs associated with BD in a GWAS reported by the Psychiatric Genomics Consortium (PGC)   [samplesum (PGC) ]. These um (PGC) , in addium (PGC) . In addium (PGC) . AuthorsIn a further investigation by the PsychENCODE Consortium the genome-wide chromosome conformation capture technique Hi-C  was applcis-regulatory domains that are defined by histone epigenetic marks (peaks) and represent 3D organization of chromatin implicating various interacting cis-regulatory elements. The study used MAGMA  using adult brain tissues from the prefrontal cortex of individuals with schizophrenia and BD, compared with controls, to investigate neuronal acetylation and methylation landscapes . The auted MAGMA  to disco with BD . The res with BD .STK4, ADD3, FURIN, HAPLN4, LMAN2L, PACSIN2, DCLK3, TMEM258, GNL3, PACS1, LRRC57, HTR6, and MCHR1.The most recent and the largest PGC3 GWAS of BD that included 41,917 cases and 371,549 controls also deployed FUSION to conduct a TWAS, followed by SMR, to reveal a transcriptomic impact of the BD-associated genomic loci . The rescis-regulatory elements associated with these disorders  regulate the metabolism of RNAs, including the transcription, stability, splicing, localization, translation, and decay . MountinNDUFA13, RBX1, NEK4, CACNA1D, and CHDH (Another epigenetic component miRNAs regulate the stability of mRNAs through the mechanism of RNA interference and thus are implicated in post-transcriptional regulation , 9. Theiand CHDH .This Mini Review highlighted recent discoveries of modified epigenetic control resulting from genetic variants associated with BD. Eight coding genes revealed by at least two studies and other three coding genes with functional connections in multiple studies deserve further discussion of their possible biological roles in the pathogenesis of BD.PACS1 reported in three studies ( studies , 41, 43  studies . It inte studies . PASC1 h studies . OverallMCHR1, DCLK3, HAPLN4, LMAN2L, and TMEM258 (GNL3 and LRRC57 (MCHR1 codes for Melanin Concentrating Hormone Receptor 1 that is a G protein-coupled receptor mainly expressed in the hypothalamus (DCLK3 that codes for Doublecortin Like Kinase 3 is expressed in the striatum and hippocampus; it is involved in regulation of transcription and has a neuroprotective role (GNL3 codes for G Protein Nucleolar 3, a protein that promotes cell proliferation in neural stem and progenitor cells and prevents subsequent neuronal differentiation (GNL3’s expression levels are increased in BD (GNL3 in the pathogenesis of BD could be linked to the neurodevelopmental aspects of BD ( TMEM258 , 33, as d LRRC57 , 41 werethalamus . This rethalamus . All thethalamus . DCLK3 tive role . Reducedive role . In addiive role . Finallyntiation , 56. GNLHAPLN4, LMAN2L, TMEM258, and LRRC57 in the pathogenesis of BD are less obvious. HAPLN4 codes for Hyaluronan and Proteoglycan Link Protein 4 that secures proper neuronal communication in the cerebellum (LMAN2L encodes Lectin, Mannose Binding 2 Like that guides certain glycoproteins from the endoplasmic reticulum to extracellular secretion (TMEM258, Transmembrane Protein 258, is involved in N-linked protein glycosylation and regulation of stress responses in the endoplasmic reticulum (LRRC57, coding for Leucine Rich Repeat Containing 57, is still obscure.The roles of ecretion . Pathogeecretion . The proeticulum . The funCACNA1C (CACNA1D (2+ binds to calmodulin attached to the cytoplasmic portion of the channel (CACNA1C and CACNA1D are associated with syndromic intellectual disability (CACNA1C  and CACN(CACNA1D  code for(CACNA1D . Importa(CACNA1D  and neur(CACNA1D , 64. Ent channel . These f channel , which c channel  or by ab channel . In suppsability  and commsability , 67.NOVA2 reported by Gandal et al. (Finally, l et al.  codes fol et al. . In the l et al. , regulatIn conclusion, functional investigations of the regulatory genetic variants associated with BD confirm the substantially complex nature of this psychiatric disorder. Both pre- and post-synaptic components, as well as neurodevelopmental and neuroprotective aspects contribute to the pathogenesis. These distinct elements do not translate into a clear unified picture and substantially more research is needed to fill the gaps in knowledge and to solve current limitations in prognosis and treatment of BD. Multiple avenues should be pursued, including further efforts in the study of epigenetic consequences of BD-associated genetic variants. For example, to increase the rate of discovery of functional common genetic variants, larger cohorts with more detailed phenotypes should be used to perform GWASs. Detailed phenotyping will allow identifying more homogeneous groups of patients with the hope that similarities in phenotypes are a reflection of similar pathogenic mechanisms involved. Likewise, more attention should be given to rare, high impact genetic variants associated with BD, such as single nucleotide substitutions, indels, or copy number variants , 25, witAL contributed to the conception of the mini-review and prepared the first draft. AL and MP wrote sections of the manuscript. Both authors contributed to the manuscript revision, read, and approved the submitted version.


RUNX1-RUNX1T1 and one donor-derived MDS with deletion 5q. A review of the cytogenetic profiles of previously reported DDMN indicates a significant contribution of therapy-related myeloid neoplasms. Cases with direct evidence of donor- or recipient-dependent factors are rare; a role of direct transfer of leukemic cells, genomic instability of the donor, abnormal gene methylation in donor cells, proleukemic potential of abnormal stromal niche, and the role of immunological surveillance after transplantation has been observed. The role of additional potential pathogenetic factors that are without clinically observed evidence are also reviewed.Donor-cell derived myeloid neoplasm (DDMN), a rare complication after allogeneic hematopoietic cell transplantation (HCT), is of interest for its potential to reveal donor-derived and host-derived factors that contribute to the pathogenesis of leukemia. The accurate diagnosis of donor-derived leukemias has been facilitated by the more frequent use of molecular techniques. In this study, we describe three additional cases of DDMN; the first reported case of donor-derived chronic myelomonocytic leukemia (CMML), one acute myeloid leukemia (AML) with t;  Donor-derived myeloid neoplasm (DDMN) is an infrequent complication encountered after allogeneic hematopoietic cell transplantation (HCT) and occurs as a result of the oncogenic transformation of apparently normal donor hematopoietic cells in the transplant recipient. Since the first report of donor-derived leukemia in 1971, an increasing number of cases have been reported in the literature over the past six decades, though a complete catalog of these cases does not exist. Reporting of this intriguing entity has accelerated in recent years due to the more frequent use of molecular techniques. DDMN is the subject of considerable interest for providing unique insights into the mechanisms of donor-derived and host-derived factors of leukemogenesis. In this study, we describe three new cases of DDMN. We analyze the cytogenetic spectrum of previously reported DDMN and discuss individual cases that provide an insight into contributing factors from either the donor’s or the recipient’s perspective. We review the current understanding of DDMN pathogenesis.Three cases of donor-derived myeloid neoplasm (DDMN) were identified in the Pathology Archives of the Mayo Clinic in Arizona. The diagnosis and subclassification were based on the 2016 World Health Organization Classification of Myeloid Neoplasms [Peripheral blood (PB) smears were stained with Wright stain. Bone marrow (BM) aspirate smears and biopsy touch imprint preparations were stained with Wright-Giemsa. BM biopsies were fixed in zinc-formalin and stained with hematoxylin eosin (H and E). Evaluation of morphologic dysplasia and blast count was performed on PB and BM aspirate smears. BM cellularity was evaluated on BM core biopsy specimens.RPN1), 3q26.2 (MECOM) dual color/double fusion; 3q25.32(MLF1), 5q35.1(NPM1) dual color/double fusion; 5p15.2(D5S630), 5q31 (EGR1), region gain or loss; 6p23(DEK), 9q34(NUP214) dual color/double fusion; 7CEN(D7Z1), 7q31(D7S486) region gain and loss; 8p11.2(KAT6A), 16p13.3 (CREBBP) dual color/double fusion; 8q22(RUNX1T1), 21q22(RUNX1), dual color/double fusion; 8CEN(D8Z2), 8q24.1(MYC), region gain and loss; 9q34(ABL1), 22q11.2(BCR), dual color/double fusion; 11q23, 13q34(LAMP1), region gain and loss; 15q24.1 (PML), 17q21(RARA), dual color/double fusion; 16p13(MYH11), 16q22(CBFB), dual color/double fusion; 17p13(TP53), 17CEN(D17Z1), region gain and loss; 20q12(D20S108), 20qter, region gain and loss. FISH of sex chromosomes was performed using the X centromere (CEPX) and Y centromere (CEPY) probe set. Cytogenetic abnormalities were classified according to the International System for Human Cytogenetic Nomenclature [Karyotype analysis was performed in duplicates on fresh anticoagulated BM aspirate specimens in a complete tissue culture medium. Chromosome preparations, including harvesting and GTW banding, were performed using standard methods. FISH was performed using the Mayo Clinic MDS/AML panel including the following probes and probe strategies: 3q21.ASXL1, BRAF, BCOR, CALR, CBL, CEBPA, CSF3R, DNMT3A, ETV6, EZH2, FLT3, GATA1, GATA2, IDH1, IDH2, JAK2, KIT, KRAS, MPL, MYD88, NOTCH1, NPM1, NRAS, PHF6, PTPN11, RUNX1, SETBP1, SRSF2, TERT, TET2, TP53, U2AF1, WT1, and ZRSR2. This NGS panel has a sensitivity of 5–10% variant allele fraction (VAF) with a minimum depth coverage of 250x for single-base substitutions and for insertion/deletion events.DNA was extracted from PB or BM aspirate sample and, following library preparation by hybrid capture, subjected to NGS with post-sequencing analysis of tumor-associated mutations, including the following mutations: TP53 mutation. He was treated with a 7 + 3 induction and mitoxantrone, etoposide, and cytarabine (MEC) chemotherapy, followed by a matched sibling donor PB hematopoietic cell transplantation (HCT) from his brother. He received post-transplant azacitidine and was well until 28 months after transplantation when he developed macrocytic anemia and thrombocytopenia. BM morphologic examination showed 23% small to intermediate-sized blasts with occasional Auer rods and subtle morphologic dysplasia. The cytogenetic study demonstrated t. The donor, the patient’s 64-year-old brother, was asymptomatic at the time of the onset of DDMN. The donor’s BM biopsy was normal, with a normal male karyotype and no mutations detected by NGS myeloid mutation panel. A 71-year-old man was diagnosed with an AML with myelodysplasia-related changes (AML-MRC) characterized by a complex hypodiploid karyotype including deletions of 3p, 5q, and 17p, with one additional metaphase representing a hypo-tetraploid subclone, and a known TP53 mutation. He was treated with decitabine and venetoclax, but soon venetoclax had to be withheld due to infectious complications. He died 5 months after the relapsed MDS/AML (58 months after his transplantation). The patient received 7 + 3 induction and four cycles of high-dose cytarabine chemotherapy. He achieved remission at 7 months after the onset of donor-derived AML, with a normal karyotype and 100% donor chimerism detected in a bone marrow sample. Two years after his donor-derived AML, he developed a relapse of his original AML-MRC, with recurrent complex cytogenetics and A 59-year-old man with a history of T-lymphoblastic leukemia/lymphoma (T-ALL), status post an allogeneic HSCT from his brother, presented with leukocytosis and splenomegaly 8 years after allo-HSCT. Complete blood counts (CBC) showed hemoglobin of 12.0 g/dL, total leukocyte count of 275,000 cells/μL and platelet count of 316,000/μL. The peripheral blood smear was notable for absolute monocytosis , neutrophilia, basophilia (1%), and granulocytic left shift with 2% circulating blasts. BM examination showed hypercellular marrow with myeloid predominance, 2% blasts, increased monocytes, and small monolobated megakaryocytes. Flow cytometric analysis was performed on the diagnostic bone marrow aspirate and showed 2% myeloid blasts with an aberrant immunophenotype as follows: CD5, CD13, CD33, CD34, CD117, and HLA-DR positive. Monocytes were increased, comprising 25% of the total analyzed cells and expressed CD13, CD14, CD15, CD33, CD64, HLA-DR, and aberrant CD56. Lymphoid populations showed no immunophenotypic abnormalities.aureus infection, and he died 12 months after the diagnosis of DDMN  was rendered. The donor was reported healthy at the onset of DDMN. The patient was treated with hydroxyurea. He had a complicated clinical course with polyneuropathy, chronic alcohol dependence, and methicillin-resistant Staphylococcus  of DDMN .A 59-year-old male with a history of an allogeneic HSCT 24 years prior to AML presented with macrocytic anemia. The donor was an unrelated matched female. BM showed normocellular bone marrow with many small round megakaryocytes and dyserythropoiesis without an increase in blasts. Flow cytometry performed on the bone marrow aspirate revealed a small myeloid blast population with an unremarkable immunophenotype. Lymphocytes included polyclonal B-cells and T-cells without immunophenotypic abnormalities.A cytogenetic study performed on a bone marrow sample showed that 20 metaphases were 46, XX, and fourteen of them had additional 5q deletion. The 5q region had a segment of unidentified additional material on it, resulting in the effective deletion of 5q. The diagnosis of a donor cell-derived MDS with deletion 5q was rendered. He was treated with lenalidomide and responded well. The patient is currently alive and well, 32 months after the diagnosis of DDMN. At present, he is off lenalidomide and continues to have mild macrocytic anemia .A review of the literature identified 82 cases of DDMN, including our current three cases. The demographic, clinicopathological features, and transplant characteristics are summarized in A review of the cytogenetic results revealed that the most common cytogenetic finding in DDMN was a normal karyotype (28.0%), and the most common abnormal karyotype was monosomy 7 (17.2%). Complex karyotypes, MLL rearranged, and t were represented in 6.1% of cases each, respectively, followed by t (4.9%), deletion 5q (4.9%), and the remaining abnormalities were rarely observed, represented by either one or two cases in the cohort , t, inv(3), inv(16), t, t, t, add 21, del(20q). In some cases, a cytogenetic result was not reported. Our three new cases represent an AML with t, a CMML with a normal karyotype, and an MDS with deletion 5q abnormality. (Cytogenetic findings of 82 cases of DDMN are shown in Few cases reported findings that provide direct insight into the pathogenesis of DDMN . The traDonor cell-derived leukemia (DDL) was first recognized in 1971, but for many years, the paucity of reported cases suggested it to be a rare phenomenon. The majority of evidence in the literature is still anecdotal, and the exact incidence is not known. Based on several reviews, the incidence of DDL has been estimated to be between 0.12% and 5% ,17,18,19In this paper, we report three new cases of DDMN, including a donor cell-derived chronic myelomonocytic leukemia, a subtype that has not been reported before. The other two cases, an AML with t8;21) and a case of MDS with deletion of 5q, has been reported previously. Only one of the three cases had an opposite-sex transplant. Chimerism analysis has confirmed the donor cell origin of all three cases. Discussion of the myeloid donor cell-derived neoplasms separated from their lymphoid counterparts is justified because myeloid disorders share a cytogenetic classification and often occupy a continuum of disease progression. Since myeloid neoplasms are subclassified along the cytogenetic guidelines, it is informative to review the spectrum of cytogenetic abnormalities in the donor cell-derived cohort. Notably, the single most common cytogenetic affiliation is represented by monosomy 7, an abnormality known to be associated with therapy-related myeloid neoplasm (t-MN)  and a ca. The various hypotheses postulated for the mechanism leading to leukemia usually separate along the lines of donor-derived and recipient-derived factors. Several cases of donor-cell leukemia provide evidence that undetected leukemia cells in the donor at the time of the donation are capable of establishing leukemia in the recipient environment. Accidental transplantation of AML, MDS, CML, cutaneous T-cells lymphoma, and various B-cell lymphomas have been reported ,22,23,24CEBPA and RUNX1 gene mutations [TP53 defect), increase the lifetime risk of MDS/AML [TEL-AML1, and 0.2% AML-ETO [Alternatively, healthy donor HSCs may carry genetic defects predisposing to malignant potential. Since 80% of donor cell leukemias involve related donors, familial cancer predisposition syndromes characterized by germline mutations of tumor suppressor or DNA repair genes, genomic instability, and oncogenic mutations, are of great interest. Hereditary predisposition to MDS/AML has been described with utations . Chromos MDS/AML . In thes MDS/AML . Another MDS/AML . With an MDS/AML , and lat MDS/AML . Potenti AML-ETO , raising AML-ETO .Recently there has been a shift in emphasis moving away from the role of the stem cells and toward the microenvironment in understanding the pathogenesis of donor-cell leukemia . Due to In addition, impaired immune surveillance in the recipient microenvironment may promote the evolution of a donor cell neoplasm. T-cells remain quantitatively and qualitatively suppressed for long periods after HSCT . The T-cTelomere shortening may also have implications in donor-cell leukemia. Telomeres, the noncoding regions of DNA flanking the end of chromosomes, play a critical role in chromosome integrity. Several studies have documented significant telomere shortening following HSCT ,35,36. TDue to the sporadic nature of reported cases, lack of sufficient follow-up information in many of the cases, and the variations in treatments, it is difficult to assess the prognosis of DDMN in comparison with de novo leukemia. Most cases are reported by case reports of small case series, and at the time of the report, the patient is alive. Overall, the prognosis is predicted to be poor, in line with other secondary leukemias. Based on observations of the case series of European Blood and Marrow Transplantation (EBMT), six of nine patients entered remission after chemotherapy . Long-laDonor-derived leukemias pose a set of ethical questions in relationship to the physician’s responsibility to inform recipients. The afterlife of stem cell donation is not known for unrelated donors, and even sibling donors may not be aware of the clinical outcome in the recipient. Though feedback of adverse events in recipients is encouraged to donor registries, this may be practically not feasible due to the absence of centralization and due to international donations. Further complicating the issue is the observation that most donors do not develop leukemia themselves . The feeDDMN is a rare complication of allo-HCT and holds promise as a model to study in vivo leukemogenesis. With the evolution of new molecular techniques for chimerism analysis, the detectability of DDMN became easier. Donor cell-derived neoplasms should be considered for myeloid diseases that develop in the post-transplant period, especially if clinically different from the original disease. The etiology is multifactorial, and the relative contribution of each of the genetic and transplant-related factors is still largely unknown. Further molecular and immunological investigations would help clarify the pathogenesis. There is a need for large multicentric studies to ensure more inclusive reporting and comprehensive analysis of cases.


YAP1 is the major effector of the pathway. In this study, we assessed the role of YAP1 in prognostic value, immunomodulation, and drug response from a pan-cancer perspective.Dysregulation of the Hippo signaling pathway has been implicated in multiple pathologies, including cancer, and YAP1 expression between normal and cancerous tissues and among different pathologic stages survival analysis and gene set enrichment analysis were performed. Additionally, we performed correlation analyses of YAP1 expression with RNA modification-related gene expression, tumor mutation burden (TMB), microsatellite instability (MSI), immune checkpoint regulator expression, and infiltration of immune cells. Correlations between YAP1 expression and IC50s  of drugs in the CellMiner database were calculated.We compared YAP1 was aberrantly expressed in various cancer types and regulated by its DNA methylation and post-transcriptional modifications, particularly m6A methylation. High expression of YAP1 was associated with poor survival outcomes in ACC, BLCA, LGG, LUAD, and PAAD. YAP1 expression was negatively correlated with the infiltration of CD8+ T lymphocytes, CD4+ Th1 cells, T follicular helper cells, NKT cells, and activated NK cells, and positively correlated with the infiltration of myeloid-derived suppressor cells (MDSCs) and cancer-associated fibroblasts (CAFs) in pan-cancer. Higher YAP1 expression showed upregulation of TGF-β signaling, Hedgehog signaling, and KRAS signaling. IC50s of FDA-approved chemotherapeutic drugs capable of inhibiting DNA synthesis, including teniposide, dacarbazine, and doxorubicin, as well as inhibitors of hypoxia-inducible factor, MCL-1, ribonucleotide reductase, and FASN in clinical trials were negatively correlated with YAP1 expression.We found that YAP1 is aberrantly expressed in various cancer types and regulated by its DNA methylation and post-transcriptional modifications. High expression of YAP1 is associated with poor survival outcomes in certain cancer types. YAP1 may promote tumor progression through immunosuppression, particularly by suppressing the infiltration of CD8+ T lymphocytes, CD4+ Th1 cells, T follicular helper cells, NKT cells, and activated NK cells, as well as recruiting MDSCs and CAFs in pan-cancer. The tumor-promoting activity of YAP1 is attributed to the activation of TGF-β, Hedgehog, and KRAS signaling pathways. AZD2858 and varlitinib might be effective in cancer patients with high YAP1 expression.In conclusion,  The Hippo signaling pathway is an evolutionarily conserved pathway with a biological role in cell fate determination, organ size control, and tissue regeneration in most tissues and organs –4. DysreYAP1 overexpression has been reported to be oncogenic in bile duct, breast, colon, lung, and liver cancers  complex, YY1, or EZH2, a polycomb repressive complex member  in several cancer types  and regulate tumor progression . Recentler types . HoweverYAP1 may act in a TEAD-independent manner , which is derived from TCGA resources , batch effects normalized mRNA data], genome-wide DNA methylation levels (DNA Methylation 450K), and clinical characteristics  of samples related to 33 cancer types were downloaded from UCSC Xena  and survival R package (version 3.3.1). p-values of the Kaplan–Meier method and hazard ratio (HR) with a 95% confidence interval (95% CI) were calculated for each cancer type and presented as forest plots using the forestplot R package (version 2.0.1).Survival information, including overall survival (OS), progression-free interval (PFI), disease-free interval (DFI), and disease-specific survival (DSS), was also downloaded from the UCSC Xena database. The Kaplan–Meier model and univariate Cox regression were then used to assess the prognostic value of YAP1 expression and infiltration of immune cells or immune score were calculated using the ggpubr R package (version 0.4.0), and scatter plots were visualized using ggplot2 (version 3.3.6).The proportions of 22 immune cell types and immune scores of all samples of 33 cancer types were downloaded from the supplementary data of the published paper . InfiltrYAP1 expression above the median level were grouped as high YAP1 expression, and the others were grouped as low YAP1 expression to compare the difference in their hallmark gene sets. Fifty hallmark gene sets were downloaded from the Molecular Signatures Database , and gene set enrichment analysis (GSEA) was performed using GSEA software (version 4.2.3). One thousand times was set as the number of permutations and phenotypes was set as permutation type. Normalized enrichment score (NES) and nominal p value for each biological process were calculated for each cancer type. Hallmark gene sets with nominal p value < 0.05 were presented as a bubble plot using the ggplot2 R package.For each cancer type, samples with YAP1 expression and TMB or MSI levels for each cancer type were calculated using ggpubr R package and displayed as radar charts using fmsb R package (version 0.7.3).Tumor mutation burden (TMB) andmicrosatellite instability (MSI) levels for each sample in pan-cancer were downloaded from the supplementary data of the published paper . PearsonCRC was induced in C57BL/6J mice by azoxymethane (AOM)/dextran sulfate sodium (DSS) as previously described . Colon s50s  of drugs and gene expression of cancer cell lines were downloaded from the CellMiner database (https://discover.nci.nih.gov/cellminer/home.do). Only drugs with FDA approval or in clinical trials were included in further analyses. Pearson correlation coefficients between YAP1 expression and the IC50z score of each drug were calculated using the ggpubr R package and represented as bubble plots or scatter plots using the ggplot2 R package.IC4 cells/well in 96-well plates. When cells reached 60% confluence, drugs were added into the wells and incubated for 24 h or 48 h. Medium containing 10% Cell Counting Kit-8 (CCK-8) reagent  was then added into the cells and incubated for another 1.5 h at 37°C. The light absorbance was measured at 450 nm on the microplate reader . Each group was performed in sextuplicate. Verteporfin, AZD-2858 were purchased from TargetMol, USA, and varlitinib was purchased from MCE, USACells were seeded at a density of 1 × 10Cellular proteins were extracted with protein extraction reagent (Beyotime Biotechnology) and quantified by BCA protein assay (Beyotime Biotechnology). A total of 12 μg of protein per sample was added to SDS-PAGE gels for electrophoresis , followed by constant flow membrane transfer . The transferred polyvinylidene fluoride (PVDF) membranes were blocked with 5% BSA-containing Tris-buffered saline with Tween (TBST) for 2 h at room temperature, and incubated with primary antibodies overnight at 4°C. Then, membranes were washed three times with TBST and incubated with secondary antibody at room temperature for 2 h. Membranes were examined with a gel imager . Antibodies of Vimentin, E-Cadherin, smad2, ERK, p-ERK, CREB, and GAPDH were purchased from Cell Signaling Technology (CST); YAP1, p-YAP1, and PD-L1 were purchased from Proteintech. All primary antibodies were used at 1:1,000 dilution. Secondary antibodies were purchased from Beyotime Biotechnology .https://www.ncbi.nlm.nih.gov/tools/primer-blast/) and synthesized by Sangon Biological Engineering Co., Ltd. . The sequences of the primers are listed in 2O, and 5 µl of 2× SYBR buffer. The reaction was performed with 1 cycle of 30 s at 95°C and 50 cycles of 10 s at 95°C, 30 s at 60°C, and 15 s at 60°C. Actin was used as the reference mRNA. The qPCR reaction was performed in triplicate.Primers were designed using the Primer-Blast tool at NCBI , colon adenocarcinoma (COAD), and thyroid carcinoma (THCA), and was downregulated in bladder urothelial carcinoma (BLCA), breast invasive carcinoma (BRCA), head and neck squamous cell carcinoma (HNSC), kidney chromophobe (KICH), kidney renal clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), pheochromocytoma and paraganglioma (PCPG), prostate adenocarcinoma (PRAD), and uterine corpus endometrial carcinoma (UCEC) , LUAD, LUSC, and UCEC , rectum adenocarcinoma (READ), skin cutaneous melanoma (SKCM), uveal melanoma (UVM), and testicular germ cell tumors (TGCT)  and univariate Cox regression. High YAP1 expression was a risk factor for the OS in seven cancer types, PFI in eight cancer types, DFI in six cancer types, and DSS in eight cancer types. In general, high YAP1 expression was risky in adrenocortical carcinoma (ACC), BLCA, COAD, brain lower grade glioma (LGG), LUAD, and pancreatic adenocarcinoma (PAAD), but it is a protective factor in esophageal carcinoma (ESCA), KIRC, PRAD, and mesothelioma (MESO)  than that in the early stage (AJCC pathologic stages I and II)  T cells, CD4+ Th1 cells, and myeloid dendritic cells in most cancer types, but positively correlated with the infiltration of MDSCs, cancer-associated fibroblasts (CAFs), and neutrophil cells (YAP1 expression and infiltration of CD4+ T cells in a few cancer types. The correlation of YAP1 expression with infiltration of regulatory T cells (Tregs), plasma B cells, NK cells, and macrophage was contradictory when using different programs , indicating its immunosuppressive role in TME . However, it was positively correlated with the immune score in BRCA, DLBC, GBM, LGG, PAAD, PCPG and PRAD , and PD-1 (PDCD1) are key checkpoint regulators that suppress immune response. They all positively correlated with YAP1 expression in LGG, PAAD, and PRAD, but negatively correlated with YAP1 expression in MESO, SARC, TGCT, and UCEC (CD274 (PD-L1), NRP1, and TNFSF15 were positively correlated with YAP1 expression. In PAAD, PRAD and PCPG, YAP1 expression was highly correlated with the expression of most immune checkpoint genes, suggesting that YAP1 may help cancer cells to evade immune destruction  in 10 cancer types, and angiogenesis in 7 cancer types, indicating that YAP1 is involved in promoting cell proliferation, cancer cell stemness, invasiveness, and migration processes  topped the list, which might be effective in treating cancer patients with high YAP1 expression. Interestingly, an antipsychotic medication, fluphenazine, may also have an effect. Inhibitors of hypoxia-inducible factor (IDF-11774), MCL-1 , ribonucleotide reductase (imexon), FASN (JNJ-54302833), WNT signaling (CCT251545), STAT/STAT3 (CPD-401), and CHK (rabusertib) were among the top 10 drugs in clinical trial that may work in patients with high YAP1 expression, whereas inhibitors of EGFR (TAS6417), Bruton’s agammaglobulinemia tyrosine kinase (BTK) (spebrutinib), SYK (entospletinib), RET (Blu667), VEGFR2 , and αvβ3 integrin  may not work   , 90, whi (NSCLC) Figure 3rocesses . We alsopression . In our r grades , 93. TheYAP activity has been found to be prognostic for poor outcome in four datasets of CRC patients  and fibrosis , and PD-1 (PDCD1), which are key checkpoint regulators that suppress immune reaction. In most cancer types, CD274 (PD-L1), NRP1, and TNFSF15 were positively correlated with YAP1 expression. YAP1 has been reported to induce PD-L1 expression , the Project of Shanghai Science and Technology Committee (19YF1449900), the Shanghai Frontier Research Base of Disease and Syndrome Biology of Inflammatory Cancer Transformation (2021KJ03–12), the “Shu Gguang” project supported by Shanghai Municipal Education Commission and Shanghai Education Development Foundation (21SG43), the Clinical Research Plan of SHDC (SHDC2020CR4043), and the Shanghai Youth Talent Support Program.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


In recent years, emerging evidence has shown that SUMOylation regulates the development and homeostasis of the skeletal system, with its dysregulation causing skeletal diseases, suggesting that SUMOylation pathways may serve as a promising therapeutic target. In this review, we summarize the current understanding of the molecular mechanisms by which SUMOylation pathways regulate skeletal cells in physiological and disease contexts. The modification of proteins by  The emergence of a skeletal system was a leap forward in evolution, for it created a strong framework for the body, protecting vital organs, facilitating movement, establishing niches for hematopoiesis, and serving as a mineral reservoir. The skeletal system develops from mesenchymal cells originating from the ectoderm and mesoderm through one of two types of ossifications processes: intramembranous or endochondral ossification. In intramembranous ossification, mesenchymal cells directly differentiate into osteoblasts to generate the flat bones of the skull and lateral clavicles . EndochoThe development and homeostasis of the skeletal system require diverse and responsive signaling and cell–cell communication, which heavily rely on dynamic posttranslational modification (PTM) systems. PTMs expand the proteome size without needing de novo protein synthesis, allowing cells to regulate complex cellular processes dynamically and efficiently. PTMs participate in every aspect of cell homeostasis, and their dysregulation often leads to disease . PTM patSUMOylation is a highly dynamic and reversible PTM that attaches SUMO proteins onto target proteins. Five SUMO paralogues  have been identified in mammals, each exhibiting unique expression patterns and levels of homology ,14,15,16SUMO modifications are attached to a single or multiple lysine residue(s) of target proteins . SUMO2 and 3 contain several lysine residues that are themselves SUMOylated, allowing for polymeric and branched SUMO chain formation (polySUMOylation) ,19,20,21sentrin-specific proteases (SENPs), a family of SUMO-specific C-terminal hydrolases, to expose its C-terminal di-glycine (GG) motif. This mature SUMO is then activated by the E1 complex, which consists of SUMO activating enzyme subunit 1 (SAE1) and SAE2 (UBA1), by forming a thioester bond at the cysteine of SAE2 via an ATP-dependent reaction [SUMOylation involves a series of enzymatic reactions with E1, E2, and E3 ligases  Figure . First, reaction . Next, treaction ,30. SUMOreaction ,29,31. Ureaction ,34,35,36In addition to proteolyzing the SUMO precursor, SENPs can also remove SUMO proteins from their targets, a process known as deSUMOylation . Seven SThe effects of SUMO modifications on their target proteins are diverse and are mainly classified into three categories : first, Osteoblasts, chondrocytes, and osteoclasts are the major cell types of the skeletal system and cooperate seamlessly to regulate bone development and homeostasis ,47. SUMOylation regulates key signaling pathways and transcription factors of osteogenesis and osteoblasts; the requirement for this PTM is demonstrated by the dysregulation of bone development and homeostasis when SUMOylation is disrupted  [Ubc9 knockdown can elevate BMP signaling and enhance osteogenic differentiation in C2C12 mouse myoblasts and ST2 mouse bone-marrow derived stromal cells (BMSCs) [Importantly, SUMOylation regulates TGF-β/BMP signaling, a fundamental and diverse signaling network that controls embryonic skeletal development and postnatal bone homeostasis ,50,51,52and Osx) . In cont (BMSCs) . Mutatio (BMSCs) . It is uHormones and their receptors, especially the androgen receptor (AR), are important regulators of skeletal development. AR knockout dramatically reduces trabecular and cortical bone mass . SUMOylaEssential transcription factors for osteoblast differentiation, including the RUNX family members RUNX1, 2, 3, and Osterix ,60,61,62ginkgolic acid, a SUMOylation inhibitor that binds to E1 ligase to prevent the formation of the SAE1-SUMO intermediate, inhibits the expression of RUNX2 and Osterix while promoting the expression of the adipogenic transcription factors PPARγ and CE/BPα [Our own studies have shown that inhibition of SUMOylation can yield profound effects on BMSC fate determination between osteogenesis and adipogenesis. We reported that d CE/BPα . Consistd CE/BPα . When std CE/BPα .DLX3 (an osteogenic transcription factor) and a subset of other HOX genes, thus enhancing osteogenic differentiation [SUMOylation is also implicated in the epigenetic regulation of osteogenesis. In human dental follicle stem cells, SENP3 binds to and deSUMOylates RBBP5 , an impontiation .The above studies demonstrate that SUMOylation has crucial roles in regulating osteogenesis, osteoblast homeostasis, and bone mass via broad mechanisms, including regulation of growth factor signaling, hormone receptors, transcription factors, and epigenetic mechanisms. Chondrocytes of healthy cartilage are formed by the differentiation of skeletal progenitor/stem cells (SSCs) into GPCs through an intermediate and bipotent osteochondroprogenitor, or into ACCs via a multipotent joint progenitor ,73,74. GShp2 [Acan and Col2a1 [SUMOylation also regulates the function of chondrogenic transcription factors. SOX9, the master regulator of chondrogenesis and cartilage development ,78,79, iShp2 , a proteShp2 ,86. The Shp2 . Total Sd Col2a1 . This suUBC9 knockdown or loss of function mutations, or SENP2 overexpression, SOX6 transcriptional activity increases [SOX6 and NKX3.2 are two other chondrogenic transcription factors regulated by SUMOylation ,88. SOX6ncreases . NKX3.2 SUMOylation also likely regulates the maintenance of heterochromatin structure in articular cartilage. For instance, DGCR8 – which maintains heterochromatin through interactions with TRIM28 and HP1γ – is stabilized to prevent its degradation via the ubiquitin-proteasome pathway by SUMO1 modification at the K707 residue ,90. DGCRSENP6 locus, as one of the most strongly OA-associated SNPs [2+-binding S100 proteins that modulates p53 transcriptional activity), resulting in S100A4 nuclear translocation. Nuclear S100A4 binds to the MMP13 promoter region and increases expression of a major OA-associated protease that degrades cartilage, MMP13 [Osteoarthritis (OA) is a disease that is characterized by progressive loss of cartilage, the formation of bone spurs, and chronic synovial inflammation . OA seveted SNPs . SENP6 eted SNPs . Furthere, MMP13  , iNOS (inducible nitric oxide synthase), and AGG1 (aggrecanase-1) [MMP3 and MMP13 [In contrast, several studies have found that SUMOylation decreases OA marker expression. A high-throughput screen of primary human ACCs identified -OA gene . SENP3 oanase-1)   . This mond MMP13   expression [c-Fos and Nfatc1 expression in RAW264.7 cells, thereby blunting RANKL-induced osteoclastogenesis [Recent studies revealed the regulatory role of SUMOylation in osteoclast formation and function. For instance, SENP3 suppresses osteoclastogenesis. Mice with the one mass . These kpression   indepenRunx2 and Mint [SHFM is a rare limb malformation characterized by clefts in the middle of the hands and feet, as well as syndactyly and aplasia/hypoplasia of phalanges, metacarpals, and metatarsals . P63α muand Mint , were deSUMO1 haploinsufficiency was identified in a patient with isolated cleft lip and palate [Craniofacial disorders are one of the most common human birth defects. Cleft lip and palate are the most frequent types of craniofacial disorders , and sevd palate   is a chronic systemic, inflammatory disease characterized by joint stiffness and destruction ,126. SynMMP1 and MMP3. Mechanistically, SUMO1 deficiency suppresses the activity of the Rac1/PAK1 pathway, which normally promotes cell motility [MMP3, MMP9, and MMP13 [A direct relationship between the SUMOylation pathway and RA was first reported in 2000 . SUMO1 mmotility . PIAS3 pmotility . Decreasnd MMP13   mice, a common RA model . TNF-α t pathway   [Several studies have linked increased SUMOylation to osteosarcoma. For example, ll lines . UBC9 kn-TK/GCV) . The int-TK/GCV) . Thus, U-TK/GCV)   and a decrease in the expression of the cell cycle gene, cyclin B1 [In contrast to what was presented above, several studies suggest that SUMOylation can have  tissues . Silenci tissues . The inhyclin B1  , is an anti-cancer drug that induces osteosarcoma cell differentiation, which is used as a prognostic indicator of weakened osteosarcoma malignancy and tumor progression . SUMO1 dHIF1α and VEGF . This reduction in HIF1α normalizes hypoxia-induced SENP1 expression [Bcl-2 expression while increasing Bax expression. The ultimate effect of this is the reduction of MG-63 cell invasiveness by suppressing epithelial-mesenchymal transition (EMT) genes [In a recent study, higher expression of SENP1 was found in human osteosarcoma tissue than in adjacent normal tissue (53/60 vs. 28/60) ; and levpression . SENP1 kT) genes . These fIn summary, changes in both SUMOylation and deSUMOylation enzymes are related to osteosarcoma. UBC9, the only SUMO E2 enzyme, promotes osteosarcoma, suggesting that a global increase in SUMOylation favors osteosarcoma development. However, several SENP deSUMOylases show diverse roles in osteosarcoma, reflecting that the SUMOylation status of their specific targets are the determinants of osteosarcoma development. While we could not make a simple generalization whether SUMOylation is osteosarcoma-promoting or -inhibiting, these studies demonstrate SUMOylation as a crucial PTM in osteosarcoma tumor initiation and progression. SUMOylation is also associated with malignant tumors that form from bone cartilage, known as conventional chondrosarcoma ,153. SUMPTM by SUMOylation regulates signaling pathways and transcription factors that are crucial for skeletal cell differentiation, development, and homeostasis . DysreguTargeting SUMOylation for disease treatments is in a more preliminary state than that of targeting ubiquitination, but some basic studies suggest that this is a promising pursuit. For example, the SUMO E1 inhibitor ML792 reduces proliferation, viability, and colony formation of chondrosarcoma cell lines . The SUMFinally, SUMOylation is involved in regulating stress response, epigenetics, and senescence, all of which are closely associated with aging. Future studies dissecting the relationship between SUMOylation and aging and tissue rejuvenation will likely also bring forth new approaches to promote skeletal health.