Prmt5 gene specifically in adipocytes (Prmt5AKO). The Prmt5AKO mice exhibit sex‐ and depot‐dependent progressive lipodystrophy that is more pronounced in females and in visceral (than subcutaneous) white fat. The lipodystrophy and associated energy imbalance, hyperlipidemia, hepatic steatosis, glucose intolerance, and insulin resistance are exacerbated by high‐fat‐diet. Mechanistically, Prmt5 methylates and releases the transcription elongation factor SPT5 from Berardinelli‐Seip congenital lipodystrophy 2  promoter, and Prmt5AKO disrupts Seipin‐mediated lipid droplet biogenesis. Prmt5 also methylates Sterol Regulatory Element‐Binding Transcription Factor 1a (SREBP1a) and promotes lipogenic gene expression, and Prmt5AKO suppresses SREBP1a‐dependent fatty acid metabolic pathways in adipocytes. Thus, PRMT5 plays a critical role in regulating lipid metabolism and lipid droplet biogenesis in adipocytes.The protein arginine methyltransferase 5 (PRMT5) is an emerging regulator of cancer and stem cells including adipogenic progenitors. Here, a new physiological role of PRMT5 in adipocytes and systemic metabolism is reported. Conditional knockout mice were generated to ablate the  Bscl2 gene, whose mutation causes Bernardinelli‐Seip Congenital Lipodystrophy in humans. Second, PRMT5 methylates SREBP1a to regulate fatty acid metabolism. Mice lacking PRMT5 in adipocytes develop progressive lipodystrophy and insulin resistance, resembling BSCL2‐mutation disease.PRMT5 regulates protein function through methylating arginine residues. This study identifies two new functions of PRMT5 in adipocytes. First, PRMT5 methylates SPT5 to promote transcription of  BAT dissipates glucose and fatty acids to generate heat, and thus it has an important role in cold‐ and diet‐induced thermogenesis. WAT is the main organ responsible for fat storage and serves as an indispensable endocrine organ that regulates systemic energy homeostasis. WAT is located in many distinct depots that arise from different progenitor cell populations, and WAT depots of different origin have distinct metabolic properties and adipokine secretion profiles. The subcutaneous WAT accounts for the majority of total AT in humans and has metabolic protective effects. The visceral WAT is, in contrast, associated with risks of metabolic diseases. White adipocytes are highly plastic and can uptake, esterificate and store excess lipids in the form of triacylglycerols (TAG) within lipid droplets (LDs), as well as undergo lipolysis to provide energy when nutrition is limited. Thus, understanding how lipid storage and utilization are regulated will provide a foundation for the development of therapeutics to overcome obesity and lipodystrophy caused by dysfunction of AT.Adipose tissue (AT) has the ability to store or oxidize excess lipids and is beneficial for the whole body energy homeostasis. Dysfunction of AT can result in insulin resistance and other metabolic complications in patients with too much or too little body fat, referred to as obesity and lipodystrophy, respectively.1 Ther Several human pathologies, such as Type 2 Diabetes (T2D) and non‐alcoholic fatty liver disease, are associated with the dysregulation of the life cycle and physiological functions of LDs. Thus, maintaining the homeostasis of LDs is critical for preventing ectopic lipid deposition and lipotoxicity. A favorite model for LD biogenesis includes several steps. First, neutral lipids (mostly TAG and sterol esters) are synthesized within the bilayer of endoplasmic reticulum (ER) membrane and accumulated to form an oil lens. Next, as TAG accumulates and the lipid lens grows, Seipin and other LD biogenesis factors are recruited to facilitate the budding of the lipid lens from ER into the cytosol, forming a nascent LD. After initial budding, newly formed LDs retain a functional connectivity with the ER, and keep expanding through fusion or local lipid synthesis. Finally, small LDs free from the ER membrane can also coalesce to form large LDs. Seipin is a widely conserved oligomeric ER transmembrane protein that is required for correct LD budding. Seipin deficiency causes delayed LD formation and lower incorporation of proteins and lipids into the LDs, resulting in LDs that are very small and aggregated, and sometimes supersized. Although increasing evidence has shown how Seipin controls LD biogenesis and affects lipid metabolism, little is known about its transcriptional regulatory mechanism.LDs are ubiquitous, highly dynamic cellular organelles responsible for storage and release of neutral lipids. Depending on metabolic demands, LDs undergo fascinating cycles of biogenesis and degradation.7, 8 PRMT enzymes are classified depending on the type of methylation they catalyze: ω‐NG‐monomethylarginine, asymmetric ω‐NG and NG‐dimethylarginine, and symmetric ω‐NG and N′G‐dimethylarginine (sDMA). PRMT5 is a major type II arginine methyltransferase that catalyzes sDMA on multiple substrates. PRMT5 is brought to the forefront for its function in promoting tumor progression, and mounting evidence from mouse models demonstrates that PRMT5 inhibition can decrease the growth of many types of tumors including mantle cell lymphoma, acute myeloid lymphoma, chronic myeloid lymphoma, B‐cell lymphoma, glioma, and breast cancer.Arginine methylation catalyzed by PRMT family enzymes is a post‐translational modification in histone and non‐histone proteins. Arginine methylation is known to affect numerous cellular activities, including RNA processing, DNA repair, protein‐protein interactions and gene regulation.13 PRM but the in vivo physiological function of PRMT5 in adipose tissues is unknown. In the present study, by employing the conditional Prmt5 knockout mouse model, we found that PRMT5 is required for white adipose tissue homeostasis and function. Deletion of Prmt5 disrupts fatty acid metabolism and impairs lipid droplet biogenesis in white adipocytes, which causes age‐dependent fat loss and lipodystrophy, and eventually leads to insulin resistance and systemic metabolic syndrome. Mechanically, PRMT5 controls lipid droplet formation through methylation of SPT5, which transcriptionally regulates Bscl2 expression. Meanwhile, PRMT5 promotes fatty acid biosynthesis through targeting SRBEP1a. Our study for the first time reports the physiological function of PRMT5 in AT and identifies a novel regulatory pathway controlling Bscl2 gene expression and LD biogenesis, SREBP1a and lipid metabolism. These results provide insights into the role of PRMT5 in lipid metabolism, lipodystrophy and non‐obese T2D.Despite the well‐understood oncogenic role of PRMT5, its function outside the cancer field is just beginning to be elucidated. Recent studies have reported that PRMT5 regulates adipogenesis through controlling adipogenic and lipogenic gene expression in 3T3‐L1 cells,18 but22.1Prmt5 knockout mice by crossing the f/fPrmt5 mice with Adipoq‐Cre mice  domain .To investigate the role of PRMT5 in adipocytes, we generated adipocyte‐specific e Figure 1a. In thipocytes.19 We g Figure , but notAKO mice were born at the expected Mendalian ratio, and were morphologically indistinguishable from their WT littermates. However, as the Prmt5AKO mice grew, they began to appear leaner in an age‐dependent manner . Interestingly, the reduction in fat mass gain appeared to be more prominent in female than in male Prmt5AKO mice . The size of eWAT adipocytes was also obviously smaller in 6 and 10 month old Prmt5AKO mice than age‐matched WT mice ‐induced obesity than the WT mice. Indeed, both male and female Prmt5AKO mice were consistently leaner and gained less body weight after being fed with HFD for 2 weeks , nor were the lipid droplet size and the ultrastructure of brown adipocytes altered . These data demonstrate that Prmt5AKO mice exhibit sex‐ and depot‐ dependent resistance to HFD‐induced fat accumulation.In view of their reduced accumulation of WAT, we predicted that Prmt5s Figure 2a,b eveny Figure . EchoMRIs Figure , Specifis Figure . We alsoe Figure . The masAKO mice affects their glucose tolerance and insulin sensitivity. We first conducted glucose tolerance tests (GTT) to determine glucose disposal after intraperitoneal (i.p.) injection of glucose into Prmt5AKO and WT mice at various ages . We next performed insulin tolerance tests (ITT) to determine how blood glucose level changes in response to insulin injection  exhibited normal insulin sensitivity, as manifested by similar rates of insulin‐mediated glucose clearance and identical area above the curve (AAC) in Prmt5AKO and WT , but exhibited a trend to decrease during the development of glucose intolerance . Notably, PRMT5 expression levels are significantly lower in non‐obese T2D than obese T2D patients . These results establish a negative correlation between PRMT5 expression level and non‐obese T2D in humans, as we shown in mice.The lean body mass and insulin resistance phenotype of the Prmt5 studies,21 in 2.3AKO mice using an indirect calorimetry approach. Prmt5AKO mice had lower levels of oxygen (O2) consumption and carbon dioxide (CO2) production than WT mice at 10‐month‐old . After fed with HFD for 12 weeks, male Prmt5AKO mice also had lower rates of O2 consumption and CO2 production than WT mice . Similar results were observed in female Prmt5AKO mice after HFD treatment . The differences in energy expenditure between WT and Prmt5AKO mice were not due to energy intake, as the WT and Prmt5AKO mice consumed comparable amounts of food  of adipocytes differentiated from Prmt5s Figure ,l. These2.4Figure Prmt5 in AT based on the microarray dataset GSE25506 and who are also candidate genes in our cold‐induced thermogenesis  database CITGeneDB. We then validated the expression of the four genes and found that expression of Acsl1, Bscl2, and Fh1 was downregulated in Prmt5AKO AT. By analyzing RNA‐Seq dataset GSE60188, we also found that Bscl2 is down‐regulated when Prmt5 is perturbed. In addition, our Prmt5AKO mice phenocopied the Bscl2 knockout mice in lipodystrophy. The Bscl2 gene encodes Seipin, a critical regulator of LD biogenesis following TAGs biosynthesis in the space between inner and outer membranes of ER  and performed dual luciferase assay with or without Prmt5 overexpression. The relative luciferase activity of cells co‐transfected with Prmt5 was significantly higher than cells without Prmt5 co‐transfection  into LD‐laden adipocytes. Strikingly, Prmt5AKO adipocytes contained LDs that were highly heterogeneous in size . We further examined the ultrastructure of LDs in eWAT adipocytes by transmission electron microscope (TEM). TEM confirmed the abnormal presence of numerous small LDs in the Prmt5AKO adipocytes of 10‐month‐old mice, in contrast to unilocular large LDs normally seen in WT adipocytes . Notably, we also observed dramatic changes in ER structure of Prmt5AKO mice after fed with HFD for 12 weeks . These observations point to a critical role of PRMT5 in LD biogenesis.We then examined the role of PRMT5 in lipid droplet biogenesis. We cultured SVF preadipocytes from subcutaneous WAT of Prmt5e Figure . Comparee Figure , insets.s Figure . Additios Figure . In adips Figure . Howevers Figure . The def we therefore asked if the defective LD biogenesis in Prmt5AKO adipocytes is due to the reduced level of Seipin. To address the question, we performed Seipin gain‐of‐function studies in SVF cells isolated from the subcutaneous WAT from WT and Prmt5AKO mice. We used adenovirus to express GFP (control) or Bscl2/GFP in the SVF cells, and Seipin protein levels were substantially elevated by the Bscl2‐adenovirus compared to GFP‐adenovirus infection . We then differentiated the SVF cells and used LipidTOX staining to visualize LDs. In WT SVF cells, Bscl2 overexpression (marked by GFP) decreased the numbers of the extra‐large and very small LDs, but slightly increased the average size of LDs . In Prmt5AKO SVF cells, GFP expression had no effects on LD composition, as abnormally large LDs were detected in both GFP+ and GFP− cells  effectively reduced the abundance of cells containing abnormally large LDs, which were mostly found in non‐infected (GFP−) cells , we overlapped known PRMT5 substracts from previous publications and regulators of BSCL2 based on information in GeneCards (https://www.genecards.org), and identified SPT5 as a candidate target of PRMT5. Analysis of previously published chromatin immunoprecipitation (ChIP) data revealed that the transcription start site (TSS) region of Bscl2 is occupied by SPT5 in mouse embryonic stem cell (mES)  at the same TSS region in mouse eWAT . These data reveal association of PRMT5, POL2, and SPT5 at the TSS of Bscl2 gene.To understand how PRMT5 regulates the expression of Seipin , which showed a concomitant reduction of TSS enrichment by SPT5 antibody  promoter region showed almost no enrichment by SPT5 antibody . We also isolated SVF preadipocytes from Prmt5AKO and WT mice. There were no differences between WT and KO cells in Seipin TSS enrichment at D0 by PRMT5 (which has low expression in preadipocytes) or SPT5 antibody . At D6 (after differentiation), the Bscl2 TSS enrichment by SPT5 antibody was significantly higher in Prmt5AKO compared with WT cells . Collectively, these results provide strong evidence that PRMT5 and SPT5 have mutually exclusive binding patterns on Bscl2 TSS, and upregulation of PRMT5 disassociates SPT5 from Bscl2 TSS.We then performed ChIP‐qPCR assay using primers flanking the s Figure . On the n Figure . We theny Figure . As binds Figure . ConsistBscl2 TSS. PRMT5 has been shown to interact with and methylate SPT5. We performed Co‐IP experiments in undifferentiated (D0) and differentiated (D6) 3T3‐L1 cells and found that PRMT5 could be pulled down by SPT5 at D6 , suggesting that Prmt5 KO reduces symmetric demethylated arginine of proteins. We also performed IP with SYM10 antibody and detected a putative SPT5 band between 130 and 170 kDa only in WT but not Prmt5AKO eWAT lysates . SYM10 IP followed by SPT5 blotting using the same samples confirmed a robust symmetrically dimethylated SPT5 band in the WT sample, and the intensity of the sDMA SPT5 band was nearly abolished in Prmt5AKO lysates  to pull down lysates from adipose tissues to check if SPT5 could be detected. Western blotting of total lysates from eWAT of WT and Prmt5s Figure . These d2.6Prmt5 knockout. We performed lipidomics analysis on eWAT from 6‐month‐old Prmt5AKO and WT mice. The analysis identified 191 TAGs whose relative abundance were significantly different, including 137 downregulated and 54 upregulated TAGs in eWAT of Prmt5AKO mice compared to WT mice . Among the top 25 altered TAGs, the downregulated TAGs were mostly TAG of 48–52 carbons, while the up‐regulated were TAG of 52–54 carbons  chain . All the top 25 significantly changed TAGs were downregulated and were TAG of 48–60 carbons with different levels of unsaturation . These data indicate that HFD exacerbates the lipid metabolism deficiency in Prmt5AKO mice.We also performed lipidomics on eWAT after mice were fed with HFD for 12 weeks. The relative abundance of 195 TAGs were altered, including 178 downregulated, and 17 upregulated TAGs in Prmt5n Figure . Five TAT Figure . Interese Figure ,c. In cot Figure . In addide novo lipid synthesis, was significantly increased in KO eWAT , TAG synthesis , and LD biogenesis  were decreased in Prmt5AKO mice  and C18:1 monounsaturated fatty acid (MUFA) were increased in KO eWAT, while other MUFAs and saturated fatty acids were reduced Figure . The ratT Figure . We furtT Figure ,h. The p . Conversely, Prmt5 overexpression elevated the protein level of SREBP1, accompanied by increased mRNA levels of SREBP1 target genes Fasn, Elovl6, and Scd1  after HFD treatment. Depot, age, and gender specific effects of various other genes has also been reported, for example Cited4 has distinct roles in regulating thermogenic gene expression in female and male mice, and different fat depots. Previous in vitro studies showed that knockdown of Prmt5 in 3T3‐L1 cells inhibits adipogenic differentiation and adipogenic gene expression, through a mechanism involving Prmt5 facilitated enhancer–promoter looping to promote expression of Pparγ2. However, we did not observe a very significant decrease in Pparγ expression in Prmt5‐null adipocytes. This discrepancy may be explained by the Adipoq‐Cre model that we used, which drives deletion of Prmt5 in post‐differentiation adipocytes and thus bypass the effect on adipogenesis. This discrepancy suggests different roles of Prmt5 in preadipocytes and mature adipocytes. In our model, we observed a more specific reduction in genes related to fatty acid metabolic pathways. Combining the observation that PRMT5 is expressed at higher level in the late phase of differentiation and its immunofluorescence is more abundant in differentiated adipocytes, these results indicate that PRMT5 plays a more important role in in mature adipocytes than in preadipocytes. The interesting observation that the old Prmt5AKO mice were insulin resistant but glucose tolerant may be due to compensatory glucose disposal by the skeletal muscle, as the lean mass of Prmt5AKO mice was greater than WT mice at 10‐month old. We also found that RER of Prmt5AKO mice were higher at 10‐month old and after HFD treatment, indicating a fuel source switch from fat to carbohydrate in response to Prmt5 knockout. Thus, future studies evaluating the systematic glucose uptake would be warranted to directly address the possibility.We used an adipocyte‐specific  tissues.35 In AKO mice. Tightly controlled LD formation and expansion from ER is important for the lipid homeostasis and adipogenesis. We also observed that the LD structure is disrupted in Prmt5 knockout adipocytes and ER structure defects in Prmt5AKO eWAT. Specifically, the Prmt5‐null adipocytes phenocopy one of the most prominent feature of Seipin‐deficient cells in the formation of “supersized” LDs. Furthermore, Prmt5AKO mice also phenocopy the lipodystrophy and lipidomic profiling phenotypes of the adipose‐specific Seipin KO (ASKO) mice and in vitro behavior of Seipin‐null adipocytes. Consistently, we showed that knockout and pharmacological inhibition of PRMT5 both suppressed Bscl2 expression. Overexpression of Seipin partially restored the sizes of LDs further support PRMT5 acting through Seipin to regulate LD homeostasis. Interestingly, we did not find any obvious defects in BAT of Prmt5AKO mice. This phenotype was different from UCP‐1Cre mice mediated Bscl2 deletion in BAT, which shows reduced BAT mass and supersized unilocular LDs in BAT. The strong phenotypes in WAT, especially eWAT, of Prmt5AKO mice may have masked any subtle phenotypes in BAT. Future study using UCP1Cre driven Prmt5 knockout specifically in brown adipocytes will elucidate the function of PRMT5 in BAT.We found that the expression of LD biogenesis related genes is reduced in Prmt5zed” LDs.39 FurBscl2 after differentiation, supporting a direct connection between PRMT5 and Bscl2 expression. As PRMT5 typically functions through methylation of its substrates, either histone proteins or non‐histone proteins, we speculated that PRMT5 should most likely regulate Bscl2 expression by interacting with another protein. Previous work demonstrates that PRMT5 methylates SPT5 in the region containing the KOW domains and arginine methylation of SPT5 inhibits its ability to associate with promoter‐bound POL2. Consistent with this notion, by analyzing the ChIP‐seq data in mES cells and in mouse eWAT, we identified a common binding region at Bscl2 TSS by SPT5, POL2, and PRMT5. Interestingly, binding affinity of SPT5 to Bscl2 TSS is dramatically decreased after differentiation or Prmt5 overexpression, suggesting that PRMT5 negatively regulate SPT5 binding to the Bscl2 TSS. SPT5 is a subunit of the DSIF (DRB Sensitivity Inducing Factor) complex which can either negatively or positively affect transcription by POL2. DSIF interacts with negative elongation factor (NELF) to promote the pausing of POL2 at some genes. In addition, PRMT5 methyltransferase activity also requires the interaction with its cofactors such as MEP50 or BRG1 to form a complex. In our model, we speculated that SPT5 acts as a transcriptional pausing factor in preadipocytes and arginine methylation by PRMT5 after adipogenic differentiation releases SPT5 from Bscl2 TSS to promote its transcription. However, there are factors other than PRMT5‐SPT5‐POL2 in this complex that are required for maintaining the continuous Bscl2 elongation despite SPT5's dissociation, which needs to be evaluated in further study. PRMT5 preserves a substrate preference to methylate arginine at Arg‐Gly (RG) and Gly‐Arg‐Gly (GRG). Pharmacological and genetic inhibition of PRMT5 demonstrate that 681R and 696R of human SPT5 are critical arginine residues methylated by PRMT5. The 696R (R690 in M. Musculus Spt5) is followed by a Gly, forming a featured PRMT5 targeting RG motif, and this RG motif is conserved across the vertebrates. In addition, the R696 is located closely to the KOW5 domain of SPT5, which is critical for its interaction with POL2. Thus, the arginine methylation of SPT5 at 696R by PRMT5 is probably a specific conserved signal that initiates the LD biogenesis. This is the first example of an evolutionarily conserved genetic regulator of Bscl2 expression. Congenital generalized lipodystrophy type 2, also known as Berardinelli‐Seip congenital lipodystrophy type 2 (BSCL2), is a disease with a wide spectrum of inherited and acquired syndromes characterized by lipodystrophy, hypertriglyceridemia, and insulin resistance. Mutations of four genes have been linked to BSCL2, with BSCL2 mutations representing the most severe form of human BSCL2. Our findings suggest that dysregulation of PRMT5 may also contribute to the development of BSCL2.We used ChIP‐sequencing assay to demonstrate that PRMT5 binds to the TSS region of und POL2.30 ConAKO and Bscl2AKO mice, the hepatic steatosis, insulin resistance, and lowever energy expenditure of Prmt5AKO mice were not observed in Bscl2AKO mice. The distinct phenotype may be explained by additional targets of PRMT5 besides Seipin. In this scenario, we found that genes  in the TAG synthesis pathway were inhibited by Prmt5AKO. FASN is a key enzyme that catalyzes de novo synthesis of palmitate, and SCD1 converts saturated fatty acids into monounsaturated fatty acids. ELOVL6 is identified as an SREBP target gene that catalyzes elongation of saturated and monounsaturated acyl‐CoAs with 12–16 carbons. The expression of all these three genes were down‐regulated in Prmt5‐null WAT and elevated by Prmt5 overexpression, indicating that PRMT5 also regulates TAG synthesis. We further show that PRMT5 regulate lipogenic genes through interacting with and methylating SREBP1a. Consistent with this finding, PRMT5 has been reported to methylate SREBP1a at R321 and this modification in turn stabilizes the cleaved nuclear form of SREBP1a, thus upregulating lipogenic gene expression and promoting de novo lipogenesis. Interestingly, the LD size as well as the expression of Bscl2 were not rescued by SREBP1a, suggesting that PRMT5‐SREBP1a pathway mainly regulates fatty acid synthesis without affecting LD biogenesis.Despite the similar lipodystrophy phenotype in Prmt5AKO mice.25 The we found that PRMT5 expression in WAT is positively associated with the glucose tolerance and have lowest expression in T2D. As patients with T2D are usually linked with overweight and obese, we examined if PRMT5 expression is corrected to body weight. Through analyzing a published dataset, we could not establish a correlation between PRMT5 in WAT and body weight status. A study following up 2625 T2D participants for over 10 years showed that 12% diabetic patients with normal body weight have almost twice higher overall mortality rate. We found that PRMT5 expression in WAT is positively correlated with the body weight status in T2D patients by analyzing a published dataset. In our mouse model, especially after HFD treatment, Prmt5 deficiency also leads to a lean but more diabetic phenotype. As SPT5 also bind at human BSCL2 TSS region, a similar functional requirement for the PRMT5‐SPT5‐Seipin pathway in maintaining the homeostasis of WAT may exist in humans. Based on these lines of evidence, it is important to examine how dysregulation of PRMT5 occurs and contributes to the insulin resistant in animal models and non‐obese T2D patients.The development of T2D is accompanied by impaired systemic glucose tolerance. By analyzing previously published data,21 we 4Adipoq‐Cre (stock #01 0803) mice were obtained from Jackson Laboratory. The frozen sperms from mice harboring tm2c(EUCOMM)wtsiPrmt5 were purchased from Wellcome Trust Sanger Institute. The in vitro fertilization, embryo development and implantation in female C57BL/6 were performed in the Purdue University Transgenic and Genomic Editing Facility. PCR‐based genotyping was performed to screen for f/fPrmt5 mice. Request for f/fPrmt5 mice should be directed to Dr. Chang‐Deng Hu at hu1@purdue.edu. The Adipoq‐Cre and f/fPrmt5 mice were intercrossed for several generations to generate the Prmt5AKO mice. Mice were genotyped by PCR of ear DNA using primers listed in Table 1, Supporting Information. The genotypes of experimental KO and associated control animals were as follows: Prmt5AKO (Adipoq‐Cre:: f/fPrmt) and WT (f/fPrmt). Mice were housed in the animal facility with free access to water and standard rodent chow food (control diet) or HFD (TD.06414 Harlan). Food intake assay was calculated by measuring food consumption weekly, the average food consumption of two mice per day from the same cage was considered as one group. Unless otherwise indicated, 8 week, 3–10 month old male mice were analyzed in this study. Mouse maintenance and experimental use were performed according to protocols approved by the Purdue Animal Care and Use Committee .The 2), carbon dioxide production (VCO2), respiratory exchange ratios (RER), and heat production were measured by using indirect calorimetry system , installed under a constant environmental temperature (22 °C) and a 12 h light (06:00–18:00 h), 12 h dark cycle (18:00–06:00 h). Mice in each chamber had free access to food (chow diet or HFD) and water. The raw data were normalized by body muscle mass and the histograms of day (06:00–18:00 h) and night (18:00–06:00 h) values were the mean value of all points measured during the 12 h period.Oxygen consumption , 0.2% Triton X‐100, and 0.1% sodium azide for 1h. Samples were subsequently incubated with diluted primary antibodies  overnight at 4 °C. After washing with PBS, the samples were incubated with secondary antibodies and DAPI for 1 h at room temperature. Fluorescent images were captured with a CoolSnap HQ charge coupled‐device camera (Photometrics) by using a Leica DM6000 microscope (Leica).−1d‐glucose  after overnight fasting for 14 h, and tail blood glucose concentrations were measured by a glucometer  every 30min after injection continuously for 2 h. For ITT, mice were fasted for 5 h before i.p. administration of human insulin (Santa Cruz) (0.75U per kg body weight), and tail blood glucose concentrations were monitored. For both GTT and ITT, mice were caged with blinded cage number and random orders.For GTT, mice were given i.p. injection of 100 mg ml−1 collagenase type I at 37 °C for 45–60 min. The digestion was terminated with DMEM containing 10% FBS, and filtered through 70 µm filters to remove undigested tissues. Cells were then centrifuged at 1700 rpm for 5 min to separate the SVF preadipocytes in the sediment and lipid‐containing adipocytes in the floating layer. The freshly isolated SVF cells were seeded and cultured in growth medium containing DMEM, 20% FBS, 1% penicillin/streptomycin (P/S) at 37 °C with 5% CO2 for 3 days, followed by changing fresh medium every 2 days. The 3T3‐L1 (ATCC) and HEK293T (ATCC) cell line were cultured in DMEM with 10% FBS. For adipogenic differentiation, the cells were induced with induction medium contains DMEM, 10% FBS, 2.85 mm insulin, 0.3 mm dexamethasone (DEXA), 1 mm rosiglitazone, and 0.63mm 3‐isobutyl‐methylxanthine (IBMX) for 4 days on confluence and then differentiated in differentiation medium contains DMEM, 10% FBS, 200nm insulin, and 10 nm T3. To avoid the effect of cell density on adipogenic differentiation, cells were induced to differentiate when they reach 100% confluence. pcDNA3.1‐2xFLAG‐SREBP‐1a plasmid was a gift from Timothy Osborne . For transfection, SVF preadipocytes from WT and KO mice were seeded into six‐well plate and allowed to grow until 90% confluency. Then each well was transfected with 1mg of pcDNA3.1‐2xFLAG‐SREBP‐1a or pcDNA3.1 plasmid together with the induction medium. Samples were collected 6 days after transfection for further protein and RNA analysis. Each transfection was performed in three biological and three technical repeats (three pairs of mice).Primary WAT SVF preadipocytes were isolated using collagenase digestion and followed by density separation. Briefly, the inguinal or epidydimal white adipose were minced and digested in 1.25 mg mL_26 801).51 Form glucose, 2 mm glutamine, and 1mm pyruvate) for 1 h at 37 °C without CO2. The OCR was measured using the XF Extracellular Flux Analyzer (Seahorse Biosciences). Oligomycin (2 mm), FCCP (2 mm), and Antimycin A and Rotenone (0.5 mm) were preloaded into cartridges and injected into XF wells in succession. OCR was calculated as a function of time (picomoles per minute).Primary SVF preadipocytes from inguinal WAT were isolated and cultured for 2 days before being plated in XF cell culture microplates (Seahorse Bioscience). 30 000 cells were seeded in each well, and after differentiated 8 days, cultured adipocytes were washed twice and pre‐incubated in XF medium  at 260 and 280 nm. Ratios of absorption (260/280 nm) of all samples were ≈2.0. Then 3 µg of total RNA were reversed transcribed using random primers and MMLV reverse transcriptase. Real‐time PCR was carried out with a Roche Lightcycler 480 PCR System using SYBR Green Master Mix and gene‐specific primers. Primer sequences are listed in Table S2, Supporting Information. The 2m NaCl, 1% NP‐40, 0.5% sodium Deoxycholate, 0.1% SDS50 mm Tris‐HCl, pH 8.0) that contained a protease inhibitor cocktail (Sigma) and phosphatase inhibitors NaF and Na3VO4. Protein concentration was measured using the BCA protein quantification kit (Pierce). Equal amounts of each protein sample were loaded for electrophoresis (Bio‐Rad). Protein concentrations were determined using Pierce BCA Protein Assay Reagent (Pierce Biotechnology). Proteins were separated by SDS–PAGE, transferred to a polyvinylidene fluoride membrane (Millipore Corporation), blocking in 5% fat‐free milk for 1 h at room temperature (RT), then incubated with primary antibodies in 5% milk overnight at 4 °C. The Prmt5 antibody was from Millipore , β‐Tubulin was from Abcam , Atgl was from Cell Signaling Technology , Srebp1 was from Invitrogen , other antibodies were from Santa Cruz Biotechnology (Santa Cruz), including Pparγ , Glut4 , Fabp4 , C/EBPα , Spt5 , and GAPDH . The horseradish peroxidase (HRP)‐conjugated secondary antibody  were diluted 1:10000 and incubated at RT for 1 h. Immunodetection was performed using enhanced chemiluminescence Western blotting substrate (Santa Cruz) and detected with FluorChem R System (ProteinSimple). Results shown in the Figure are representative results from at least three independent experiments.Protein was extracted from homogenized muscle tissue or muscle cells with RIPA buffer (150 mhttps://en.novogene.com/). Briefly, RNA quality analysis was checked by Agarose Gel Electrophoresis and Agilent 2100. A complementary DNA library was then constructed using mRNA enriched by anti‐polyA beads, and sequencing was performed according to the Illumina HiSeq standard protocol. Raw reads from RNA‐seq libraries are filtered to remove reads containing adapters or reads of low quality. After filtering, statistics analysis of data production and quality was performed to confirm the sequencing quality. Reference genome and gene annotation files were downloaded from a genome website browser (NCBI/UCSC/Ensembl). TopHat2 was used for mapping the filtered reads to the reference genome. For the quantification of gene expression level, HTSeq V0.6.1 was used to analyze the read numbers mapped for each gene. The RPKM of each gene was calculated based on the gene read counts mapped to genes or exons. A differential expression analysis was performed using the DESeq R package (1.10.1) with the threshold of significance set as p‐adjusted <0.05. Heatmap was made by an online tool (http://heatmapper.ca/) based on the Log2RPKM.Total RNA was extracted from eWAT of 6 month old WT and KO mice, and subjected to RNA‐seq analysis performed by Novel Bioinformatics Co., Ltd.  beads , and 500 µL of ultrapure water was added to homogenize the sample using the Precellys tissue homogenizer  at three cycles of 6200 rpm for 20 s. Next, 200 µL of homogenized tissue was transferred to a new microtube and mixed with 250 µL of chloroform and 450 µL of methanol. This solution was incubated at room temperature for 15 min. After that, 250 µL of chloroform and 250 µL of water were added and the sample was centrifuged for 10 min at 16 000 × g, forming a two‐phase solution where the bottom phase contained the lipids (organic phase). The organic phase was transferred to a new tube and dried using a speedvac centrifuge , and samples were stored at −80 °C until mass spectrometry analysis.For MS analysis, eWAT samples (50mg per mouse) from WT and Prmt5n method.53 In  Specifically, for sample preparation, dried lipid extracts were diluted in 500 µL of methanol/chloroform 3:1 (v/v) (stock solution). The stock solution was further diluted 50× in injection solvent (aceto‐ nitrile/methanol/ammonium acetate 300 mm 3:6.65:0.35 (v/v)) and and 8µL of this solution was used for the profiling analysis of each lipid class using a micro‐autosampler (G1377A) to the ESI source of an Agilent 6410 triple quadrupole mass spectrometer . A capillary pump was connected to the autosampler and operated at a flow rate of 7 µL min−1 and pressure of 150 bar. Capillary voltage on the instrument was 3.5–5 kV and the gas flow 5.1 L min−1 at 300 °C.Targeted lipid profiling was performed using discovery MRM‐profiling methods and instrumentation as described.54 Spehttps://www.metaboanalyst.ca). Uploaded data was auto‐scaled and volcano plots , principal component analyses (PCAs), and heatmaps were plotted. Fold change was calculated by dividing values of ion intensities for each of the MRMs measured in each sample by the ion intensity of the corresponding MRM in the blank. Average fold change was graphed for significantly different lipid species using GraphPad Prism Software Version 8.0.1. Significant changes between groups were analyzed by students’ t‐test and p values < 0.05 were considered statistically significant.For the MS analysis, relative amounts of ion abundances were used for statistics. Values of ion intensities for each of the MRMs monitored were normalized by total ion intensity of all MRMs in the method for a given sample. Further statistical analysis was then performed using Metaboanalyst 4.0 software  and 500 ng pcDNA‐Myc‐Prmt5 plasmid were co‐transfected following the manufacturer's instructions. After transfected for 48 h, cells were harvested and analyzed with the Dual‐Luciferase Reporter Assay System (Promega).HEK293A cells were seeded into 24‐well plates 1 day before Lipofectamine 2000‐mediated transfection. The pGL3‐Bscl2‐1kb promoter luciferase plasmid was generated by cloning the 1kb promoter region of m cacodylate buffer. After washing, the blocks were dehydrated in a graded ethanol series and then embedded in Epon Generic Resin. The sections with a thickness of about 90 nm were prepared with uranyl acetate and lead citrate stain and examined with a transmission electron microscope .The eWAT from 6, 10 month old, and 12 week HFD treated WT and KO mice were cut into 1 × 2 mm blocks in a 6 cm culture dish immediately after euthanizing the mice, and then fixed in 2.5% glutaraldehyde for 30 min. The blocks were continued to be fixed in 2.5% glutaraldehyde for 1 h, followed by fixation in 2% osmium tetroxide for 1 h. All the fixatives were made with 0.1 −1 Oil red O stock solution in isopropanol, 4 mL ddH2O, filtered for three times) for 30 min. After staining, the cells were washed with 60% isopropanol and pictured.Cultured cells were washed with PBS and fixed with 4% paraformaldehyde (PFA) for 15 min at RT. Then the cells were stained using the Oil red O working solutions (6 mL 5 g L−1).The blank adenovirus (pAd‐GFP) and adenovirus with Bscl2 insertion (pAd‐Seipin‐GFP) were gifts from Dr. Weiqin Chen from Medical College of Georgia at Augusta University, Augusta, Georgia, USA. For retroviral overexpression of Prmt5, pBABE‐puro‐Prmt5, pCL‐Eco, and pCMV‐VSV‐G plasmids were co‐transfected into HEK‐293T cells seeded in six‐well plates. After 48 h, the retrovirus was collected and 3T3‐L1 cell line (20%–30% confluent) was infected by control or Prmt5‐OE retrovirus, and then the stable expressing cell were selected by puromycin  and differentiated (D1) 3T3‐L1 cells were cross‐linked with 1% formaldehyde  for 10 min at room temperature. After quenching of the formaldehyde with 125 mm glycine for 5 min, fixed cells were washed twice with ice‐cold phosphate‐buffered saline (PBS) supplemented with protease cocktail inhibitor (PCI)  and resuspended in cell lysis buffer , then were incubated on ice for 10 min. The nuclei were pelleted, washed with MNase digestion buffer , and incubated for 20 min at 37 °C in the presence of 2000 gel units of micrococcal nuclease  per mL of MNase digestion buffer. The reaction was stopped by adding equal volume sonication buffer . Samples were sonicated for 10–12 min  in a Bioruptor Pico Sonicator  at 4 °C, and centrifuged at 21000 × g for 5 min. The length of the fragmented chromatin was between 200 and 500 bp as analyzed on agarose gels. Chromatin concentrations were measured using a Qubit 3 fluorometer (Invitrogen). 5 µg chromatin was subjected to immunoprecipitation with rabbit polyclonal Prmt5 antibody  or with anti‐IgG as a negative control at 4 °C overnight. Immunocomplexes were recovered by incubation with protein A‐agarose magnetic beads (Invitrogen) for 3–4 h at 4 °C. Sequential washes of 5 min each were performed with ChIP buffer , ChIP buffer with 0.5 m NaCl, Tris/LiCl buffer  and Tris/EDTA buffer . Immune complexes were eluted from the Protein A magnetic beads by incubation in 100 µL of elution buffer  for 30 min at 65 °C. Eluates were then reverse cross‐linked overnight at 65 °C. Following this, samples were treated with 1µL RNase A  for 1 h at 37 °C and 1 µL of Proteinase K  for 2 h at 37 °C. DNA was purified using a ChIP DNA Clean and Concentrator kit .Chromatin immunoprecipitation (ChIP) was performed as previously described55 wit Reads with a mapq score of 20 or greater were retained using Samtools. PCR duplicates were filtered out using picard. Peak calling was performed using macs2.ChIP was performed in duplicates and sequencing libraries were prepared using the ThruPLEX DNA‐seq kits (Rubicon Genomics). Libraries were prepared with 0.1–33.2 ng of ChIP DNA or input and individually barcoded. qPCR library amplification was performed for five to nine cycles. Libraries were sequenced using an Illumina NextSeq High Output instrument with paired‐end 75‐bp reads. Read depth ranged between 37 and 52 million reads per replicate. Fastq files were aligned to the 10 mm reference genome with bowtie2.56 Ream glycine for 5 min at room temperature, after which samples were washed twice with cold PBS and placed in ChIP cell lysis buffer containing 20 mm Tris, 0.1% SDS, 1% Triton‐100, 150 mm NaCl, 1 Mm EDTA and protease inhibitor on ice for 10 min. After centrifugation, the nuclei were resuspended in muclear lysis buffer and sonicated. Polyacrylamide gel electrophoresis was used to confirm the size of DNA fragment. Then the lysis was diluted for IP with the indicated antibodies  and incubation at 4 °C overnight. The immunoprecipitants were eluted and reverse crosslinked overnight at 65 °C. DNA fragments were purified using the Using Phenol–Chloroform method and quantitative PCR was performed. Primer sequences are listed in Table S2, Supporting Information. Each ChIP‐qPCR were performed in three biological and two technical repeats.Culture blank and PRMT5‐overexpressed 3T3‐L1 cells or SVF preadipocytes from the WT and KO mice were crosslinked using fixed with 3.7% formaldehyde for 10 min at room temperature with shaking, followed by the addition of 125 mHEK293T cells were transfected with pcDNA3.1‐2xFLAG‐SREBP‐1a or pcDNA3.1‐2xFLAG‐SREBP‐1a +pcDNA‐Myc‐Prm5, and total protein was extracted after 48 h. The lysate was precleared with protein A/G agarose at 4 °C for 3 h. Then 2 µg of primary antibody anti‐FLAG or anti‐PRMT5 was added into lysate contains 500 µg total protein and rotating at 4 °C overnight. The next morning added the protein A/G agarose and rotating for 3 h.Total protein was extracted from HEK293T cells, transfected with pcDNA‐Myc‐Prm5 after 48 h, undifferentiated/differentiated 3T3‐L1 cell line. The lysate was precleared with protein A/G agarose at 4 °C for 3 h. Then 2 µg of primary antibody anti‐Spt5 was added into lysate contains 500 µg total protein and rotating at 4 °C overnight. The next morning added the protein A/G agarose and rotating for 3 h. The samples were washed with cold PBS for three times and collected for Western blot.m Tris‐HCl, pH 7.5, 150 mm NaCl, and 1% Nonidet P‐40 with 1 × protease inhibitor mixture) on ice. After another 10 min on ice, tissue lysate was centrifuged with 14 000 rpm at 4 °C, and the middle layer (containing 4 mg proteins determined by the Pierce bicinchoninic acid assay) was transferred to 50 µL BSA blocked Protein A/G agarose. After incubation on a rotator for 3 h in a cold room (4 °C), the beads were washed out and then 2 µg of anti‐Spt5 or anti‐SYM10 were added, anti‐mouse IgG or anti‐rabbit IgG were added as controls, respectively. After incubation on a rotator for overnight in a cold room (4 °C), 50 µL BSA blocked Protein A/G agarose was added and kept incubating for another 3 h. The beads were then washed using high and low salt lysis buffer for six times. Beads captured proteins were eluted with 50 µL of low salt lysis buffer and equal volume of 2× protein loading buffer on a heater . Proteins were separated by 12% SDS–PAGE. For silver staining, the gel was kept in a fixing solution  under constant shaking at 4 °C overnight. Then the gel was washed for six times with ddH2O (5 min for each time). The gel was sensitized in 0.02% sodium thiosulfate solution for 2 min and then washed with ddH2O for three times (5 min for each time). The gel was kept in staining solution  for 20 min in dark with rotation at room temperature. The gel was rinsed and then kept in a developing solution  until the bands were visible. Stopping solution (50% methanol and 12% acetic acid) was poured on the gel to stop the reaction. The gel was imaged using a FluorChem R System (ProteinSimple).Total protein was extracted from eWAT of Prmt5 KO and WT mice. Briefly, the 200 mg of fat tissue was collected from each mouse and homogenized with 1 mL lysis buffer (50 mn ≥ 3) and statistical comparisons were based on Student's t‐test with a two‐tail distribution. Comparisons with p values <0.05 or <0.01 were considered statistically significant. Graphs were generated by GraphPad prism software.Trial experiments or experiments done previously were used to determine sample size with adequate statistical power. The researchers involved in the in vivo treatments were not completely blinded, but all ITT and GTT were conducted blindly. All images were randomly captured from sample and analyzed in a blinded manner. No data were excluded from statistical analysis. All experimental data are represented as mean ± s.e.m (The authors declare no conflict of interest.Z.J. and F.Y. contributed equally to this work. S.K. conceived the project. Z.J., F.Y., and S.K. designed the experiments and prepared the manuscript. Z.J., F.Y., X.C., N.N., J.Q., and S.S. performed the experiments and analyzed the data. P. Y. performed data mining and gene expression analysis. A.I., M.D., and C.H. provided key reagents and all authors revised the manuscript.Supporting InformationClick here for additional data file.


Skin wounding is very common and may be slow to heal. Increasing evidence shows that exosomes derived from mesenchymal stem cells (MSCs) dramatically enhance skin wound healing in a paracrine manner. However, the mechanism underlying this phenomenon has not yet been elucidated. Thus, the objective of the present study was to identify the signaling pathways and paracrine factors by which MSC-derived exosomes promote de novo skin tissue regeneration in response to wound healing.2O2) and excising full-thickness mouse skin, respectively. Exosomes were extracted from human umbilical cord Wharton’s jelly MSCs (hucMSC-Ex) by ultracentrifugation of cell culture supernatant.In vitro and in vivo skin wound healing models were created by treating immortalized human keratinocytes (HaCaT) with hydrogen peroxide (H2O2 by inhibiting nuclear translocation of apoptosis-inducing factor (AIF) and upregulating poly ADP ribose polymerase 1 (PARP-1) and poly (ADP-ribose) (PAR). The animal experiments showed that relative to hucMSCs, hucMSC-Ex attenuated full-thickness skin wounding by enhancing epidermal re-epithelialization and dermal angiogenesis.The hucMSC-Ex treatment significantly increased HaCaT cell proliferation and migration in a time- and dose-dependent manner, suppressed HaCaT apoptosis induced with HThese findings indicated that direct administration of hucMSC-Ex may effectively treat cutaneous wounding and could be of great value in clinical settings. The skin is the largest tissue in the body, and its main function is to protect the subcutaneous tissue. Wound healing is a complex process, and successful skin wound healing requires a series of steps, including inflammation, new tissue formation, and remodeling . In addiAt present, it is believed that excessive apoptosis of cells with injury repair function, namely, dermal fibroblasts and epidermal keratinocytes, is one of the main reasons for the difficulty in healing or delayed healing of diabetic skin wounds , 6. DiabMesenchymal stem cells (MSCs) are a rich, readily accessible source of somatic stem cells. They are easily acquired, highly expanded, and efficiently differentiated into numerous lineages of specialized cells. They are used to engineer functional organs or for in situ tissue repair and regeneration . A previExosomes are nanoscale microvesicles sprouting from various types of cells by the budding way . The exo2O2) and by excising full-thickness skin from the backs of C57 mice, respectively. We isolated exosomes from hucMSCs (hucMSCs-Ex) by ultracentrifugation and compared them with hucMSCs-conditioned medium (hucMSCs-CM), exosome-free hucMSCs-CM (hucMSCs-dp-Ex), and hucMSCs in terms of skin wound healing efficacy. Our results showed that hucMSCs-Ex significantly inhibited the nuclear translocation of apoptosis-inducing factor (AIF), the activation of poly (ADP-ribose) polymerase-1 (PARP1), and the induction of HaCaT cell apoptosis by H2O2. It also enhanced cutaneous wound healing in full-thickness skin defect animal models as did hucMSCs and hucMSCs-CM.In this study, we hypothesized that hucMSCs-Ex can promote cutaneous wound healing via the PARP-1/AIF apoptosis pathway.To this end, we created cellular and animal models of skin defects by treating the HaCaT epidermal cell line with hydrogen peroxide  and 10 ng mL−1 basic fibroblast growth factor  at 37 °C under a 5% CO2 atmosphere. Immunofluorescence staining and flow cytometry were performed on hucMSCs to evaluate the expression levels of the surface markers CD34, CD45, CD44, CD73, CD90, and CD105 . For the multipotency assay, the hucMSCs were cultured in adipogenic medium consisting of DMEM, 10% (w/v) FBS, 1 μM dexamethasone , 0.5 mM isobuty1 methylxanthine , 200 μM indomethacin , and 10 μM insulin  in osteogenic medium consisting of DMEM, 10% (w/v) FBS, 10 mM β-glycerophosphate , 0.1 μM dexamethasone, and 50 μM ascorbate-2-phosphate  and chondrogenic medium consisting of DMEM, 10% (w/v) FBS, 6.25 μg mL−1 insulin, 10 ng mL−1 transforming growth factor-beta 1 , and 50 nM ascorbate-2-phosphate . Three weeks after adipogenic, osteogenic, and chondrogenic induction, intracellular lipid droplets, mineralized bone nodules, and sulfated proteoglycans were detected by Oil Red O staining, Alizarin Red-S staining, and Alcian Blue staining  according to the manufacturers’ instructions.The present study was approved by the Ethics Committee of the School of Public Health, Jilin University, Jilin, China. The umbilical cords were obtained from healthy patients at the Affiliated Hospital of Jilin University. The donors had granted prior informed written consent. The isolation, phenotype characterization, and multipotency assay of mesenchymal stem cells (hucMSCs) from umbilical cord Wharton’s jelly were performed as previously described . Briefly−1 bFGF and 10% (w/v) FBS deprived of exosomes by centrifugation at 120,000×g overnight at 4 °C. When the hucMScs reached 80% confluency, they were cultivated in DMEM containing 2% (w/v) exosome-free fetal bovine serum (FBS) for 24 h. The culture medium was then collected and centrifuged at 300×g at 4 °C for 10 min to pelletize the cells. The supernatant was collected, centrifuged at 16,500×g  at 4 °C for 20 min then passed through a 0.22-μm filter to remove cell debris. This medium was designated conditioned medium (hucMSCs-CM). The filtrate was centrifuged at 120,000×g at 4 °C for 90 min. The exosomes were collected and designated hucMSCs-derived exosomes (hucMSCs-Ex). The hucMSCs-Ex were resuspended in phosphate-buffered saline (PBS) and stored at − 80 °C. The protein concentration in the hucMSCs-Ex was measured with bovine calf albumin (BCA) kit . The size distribution and concentration of exosomes were analyzed by nanoparticle tracking analysis using a ZetaView particle tracker from ParticleMetrix (Germany), Each NTA measurement for the different protocols for each subject was repeated in triplicate. The morphology of the hucMSCs-Ex was examined by transmission electron microscopy . The expression levels of CD9 and CD63  and Alix  and TSG101  and HSP70  in the exosomes were determined by western blot assay. The exosome-free medium was designated exosomes-deprived hucMSCs-conditioned media (hucMSCs-dp-Ex).Exosome isolation from MSCs was described elsewhere . Briefly−1) and incubated at room temperature (20–25 °C) for 25 min. Then, 1 mL of 1% (w/v) bovine serum albumin  was added to the incubation mixture to terminate labeling. PKH26-labeled hucMSCs-Ex were collected by centrifugation at 100,000×g at 4 °C for 2 h, washed by PBS for once, then used as a supplement in the HaCaT cell culture. The HaCaT cells were cultured with PKH26-labeled hucMSCs-Ex for 24 h, fixed with 4% (w/v) paraformaldehyde, counterstained with Hoechst 33342 , observed under a fluorescence microscope , and photographed with a microscope-mounted digital camera .The hucMSCs-Ex were labeled with PKH26  as previously described -1(2H)-isoquinolinone (DPQ)  or the broad-spectrum caspase inhibitor Z-VAD.fmk  at the indicated dosages or at 1000 ng mL−l hucMSCs-Ex for 4 h. Then propidium iodine-Annexin V staining and flow cytometry were performed to detect HaCaT cell apoptosis.HaCaT cells were seeded in a 24-well plate at a density of 5 × 104 cm−2 and cultured overnight in DMEM containing 10% (w/v) FBS. The next day, the culture medium was aspirated and the cells were washed thrice in PBS and cultured for 4 h in DMEM containing 2% (w/v) exosome-free FBS with or without 1 mM H2O2. The cells were then washed thrice in PBS and cultured for another 24 h in DMEM containing 2% (w/v) exosome-free FBS (control), hucMSCs-CM, hucMSCs-dp-Ex, or hucMSCs-Ex at the indicated concentrations or time points. Then, a CCK8 assay was run to detect HaCaT proliferation under the aforementioned culture conditions.HaCaT cells were seeded in a 96-well plate at a density of 5 × 104 cm−2 and cultured overnight in DMEM containing 10% (w/v) FBS. The next day, the cells were washed thrice in PBS and cultured for 4 h in DMEM containing 2% (w/v) exosome-free FBS with or without 1 mM H2O2. Then, the HaCaT cells cultured in the 6-well plates were scratched with a 200-μL micropipette tip. The HaCaT cells cultured in the Transwell and 6-well plates were washed thrice in PBS and cultured for 24 h in DMEM containing 2% (w/v) exosome-deprived FBS, hucMSCs-CM, hucMSCs-dp-Ex, or hucMSCs-Ex in 6-well plates or in 100 μL DMEM containing 2% (w/v) exosome-deprived FBS in the upper Transwell chamber. The lower Transwell chamber was filled with 600 μL DMEM containing 2% exosome-free FBS, hucMSCs-CM, hucMSC-dp-Ex, and 1000 μg hucMSCs-Ex for 24 h. Then, the Transwells were removed from the tissue culture plate, the culture medium in the upper Transwell chamber was aspirated, and the HaCaT cells in the Transwells were fixed with methanol, dried under a laminar flow hood, and stained with Hoechst 33342 . A cotton tab was used to remove the HaCaT cells retained in the upper Transwell chamber and the cells were washed thrice in PBS. The HaCaT cells retained in the lower Transwell chamber were photographed under a fluorescent microscope . The HaCaT cells in five randomly selected view fields were analyzed by ImageJ software . The HaCaT cells cultured in 6-well plates were photographed. ImageJ was used to measure their migration under the aforementioned culture conditions. Migration rates were calculated as follows: (distance of cells at scratching time point−distance of cells 24 h post-scratching)/(distance of cells at scratching time point).Transwell and cell scratching assays were implemented to determine cell migration. Briefly, HaCaT cells were seeded in the upper chamber of a Transwell  according to the manufacturer’s instructions. The HaCaT cells were also seeded in 6-well plates at a density of 5 × 104 cm−2 and cultured overnight in DMEM containing 10% (w/v) FBS. Next day, the cells were washed thrice in PBS and cultured in DMEM containing 2% (w/v) exosome-free FBS with or without 1 mM H2O2 for the indicated time intervals. Then the HaCaT cells were washed thrice in PBS and either harvested by trypsin digestion and centrifugation or left for another 24 h in DMEM containing 2% (w/v) exosome-free FBS, hucMSCs-CM, hucMSCs-dp-Ex, or hucMSCs-Ex at the indicated concentrations. Then, the HaCaT cells were washed twice in cold PBS and harvested by trypsin digestion as described above. Total, cytoplasmic, nuclear, and mitochondrial proteins from the HaCaT cells were extracted using kits  according to the manufacturer’s instructions. Briefly, the HaCaT cells were lysed by ultrasound in ice-cold radioimmunoprecipitation assay (RIPA) buffer containing 1 mM phenylmethylsulfonyl fluoride (PMSF)  on ice. The lysates were centrifuged at 12,000×g for 20 min at 4 °C. The supernatants contained the total protein. For cytoplasmic and nuclear protein extraction, the HaCaT cells were resuspended in protein extraction buffer A  then buffer B was added and cell lysates were centrifuged at 16,000×g and 4 °C for 5 min. The supernatant was collected, an equal volume of buffer C was added to it, and the mixture was centrifuged at 15,000×g for 10 min at 4 °C. The supernatant was collected and designated the cytoplasmic protein. The nuclear pellet was washed with buffer A, resuspended in buffer C , and centrifuged at 16,000×g for 10 min at 4 °C. The supernatant was then isolated. For mitochondrial protein separation, the post-centrifugation cytoplasm fraction was transferred to a new tube and the mitochondrion pellet was resuspended in buffer D (20% (w/v) glycerol, 20 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.5 mM PMSF, 8 μg/mL aprotinin, 2 μg/mL leupeptin) and centrifuged at 15,000×g for 10 min at 4 °C. The pellet contained the mitochondrial protein.HaCaT cells were seeded in tissue culture plates at a density of 5 × 10Equal amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electronically transferred to a polyvinylidene fluoride (PVDF) membrane . The membranes were blocked in 5% (w/v) non-fat milk at room temperature for 1 h and incubated overnight at 4 °C with the antibodies listed in Table 2O2. Alternatively, the HaCaT cells were pretreated for 1 h with DPQ (30 μM mL−1) and Z-VAD.fmk (100 μM mL−1) or hucMSCs-CM, hucMSCs-dp-Ex, and hucMSCs-Ex then cultured with 1 mM H2O2 for another 4 h. The cells were then washed twice in PBS, fixed in 4% (w/v) PFA/PBS, and permeabilized in 1% (w/v) Triton X-100 followed by incubation with anti-PAR, anti-AIF, and anti-cyt C antibodies (Table Immunofluorescence staining was performed on HaCaT cells as previously described . Briefly6 hucMSCs 100 μL−1 PBS (hucMSCs group), 100 μg hucMSCs-Ex 100 μL−1 PBS (hucMSCs-Ex group), 100 μL PBS (PBS group); 100 μL hucMSCs-CM (hucMSCs-CM group), 100 μL hucMSC-dp-Ex (hucMSCs-dp-Ex group), or nothing (sham group). The wounds were wrapped with a single layer of oil gauze covered by three layers of cotton gauze as previously described.All animal experiments were conducted in compliance with the guidelines approved by the China Association of Laboratory Animal Care and the Institutional Animal Care Committee. Balb/C mice  were purchased from Fukang Animal Breeding Center, Beijing, China, and maintained at the Institutional Animal Center of Jilin University, Jilin, China. The mice were acclimated to their environment for 1 week. After acclimation, a full-thickness excisional wound (0.8 cm × 0.8 cm) was made with surgical scissors on the dorsal skin of each mouse. The mice were randomly divided into six groups, each of which comprised 10 mice. The corners of each wound margin were subcutaneously injected with 10At days 7 and 14 after wounding, photographs of the wound sites were taken for gross inspection of wound closure. The outline along the wound margin was depicted with transparent film and the wound closure rate was calculated as follows:/original wound area) × 100.The mice were sacrificed by anesthetic overdose and skin specimens including wounds and neighboring tissues were collected, fixed with 10% (w/v) buffered formaldehyde/PBS, embedded in paraffin, sectioned at a 5-μm thickness in the center of the wound, and stained with hematoxylin and eosin (H&E). The tissue sections were observed and photographed under a microscope. Epidermal tissue subtended by hair follicle-free dermis was designated newly formed epidermis. The histological wound healing rate was calculated as follows:(length of newly formed epidermis)/ Immunofluorescence staining was performed to detect epidermal formation, dermal angiogenesis, and scar tissue in the wounded skin. Briefly, skin section slides were deparaffinized, rehydrated, and blocked in 1% (w/v) BSA/PBS at room temperature for 30 min. The slides were incubated with rat anti-mouse primary antibody against CD31 , cytokeratin 10 , or α-SMA  at 4 °C overnight then washed thrice in PBS. The slides were incubated with Alexa Fluor-488/555-conjugated anti-mouse secondary antibody at room temperature for 30 min and counterstained with Hoechst 33342  to track the nuclei. The slides were observed and photographed under a fluorescence microscope fitted with a digital camera . Five randomly selected fields per tissue section were observed at × 400. Three sections were chosen from three mice per group. Image-Pro Plus  was used to determine the average optical densities for CD31 and α-SMA expression. Five randomly selected fields were examined per group at each time point and used to calculate the average optical density per unit area.t test. P < 0.05 was considered statistically significant.Statistical analysis was performed using SPSS v. 17.0 . Data are means ± standard deviation (SD) for ≥ 3 independent experiments. Multiple group comparisons were made by one-way ANOVA. Paired group comparisons were made with Student’s HucMSCs were obtained from the culture of human Wharton’s jelly tissues. They were spindle-shaped and highly expressed the MSC surface markers CD44, CD73, CD90, and CD105  and 26.7 ± 3.08% after 6 h (P < 0.01) following treatment with 1 mM H2O2  and from 3.32 ± 0.86% at time 0 to 21.23 ± 2.84% at 4 h    and cytochrome C significantly decreased in HaCaT cell mitochondria over time (P < 0.01)  Fig. a, b. AIF01) Fig. c, d. Cyt01) Fig. e–g. A whime Fig. h, i. The2O2-induced HaCaT cell death, we pretreated HaCaT cells with the broad-spectrum caspase inhibitor Z-VAD.fmk and the PARP-specific inhibitor DPQ. Annexin V/PI staining combined with flow cytometry disclosed that 30 mM DPQ significantly reduced H2O2-induced HaCaT cell apoptosis. The reductions were 15.23 ± 2.84% vs. 29.32 ± 0.86% . And 13.45 ± 2.85% vs. 23.68 ± 0.8% . In contrast, 1–100 μM Z-VAD-fmk had no significant protective effects against H2O2-induced HaCaT cell apoptosis   as did hucMSCs-CM . On the other hand, hucMSCs-dp-Ex exhibited no significant efficacy in enhancing HaCaT cell proliferation (P > 0.05)  . Similar results were obtained for the hucMSCs-CM group  , in the hucMSCs-Ex group was significantly lower than that of the H2O2 group . The percentage of apoptotic cells for Annexin V-and PI positive cell (AV+ and PI+), in the hucMSCs-Ex group significantly reduced . This finding was comparable to that determined for the hucMSCs-CM group .Paracrine factors play important roles in stem cell-based regenerative medicine. To explore the mechanism by which paracrine factors promote cutaneous wound healing, we exposed HaCaT cells to H2O2 then incubated with hucMSC-CM, hucMSC-dp-Ex, or hucMSC-Ex for 24 h. The Transwell assay revealed that incubation with hucMSC-Ex or hucMSC-CM significantly increased the numbers of migrated cells relative to the hucMSC-Ex-dp (3.26 ± 0.26 (hucMSC-CM) vs. 3.05 ± 0.22 (control) (P < 0.05); (4.5 ± 0.2 (hucMSC-Ex) vs. 3.05 ± 0.22 (control) (P < 0.05); 1.9 ± 0.02 (hucMSC-Ex-dp) vs. 21 ± 5.2 (control) (P < 0.05)) ; 76 ± 4 (hucMSC-Ex) vs. 21 ± 5 (control) (P < 0.05); 35 ± 6 (hucMSC-Ex-dp) vs. 21 ± 5.2 (control) (P < 0.05))  but increased nuclear AIF and PARP/1 expression (P < 0.01). Compared with H2O2 group, hucMSC-Ex or hucMSCs-CM significantly reduced nuclear AIF and PARP/1 expression (P < 0.01). However, similar to H2O2 group, hucMSCs-dp-CM did not significantly change mitochondrial AIF expression and nuclear AIF and PARP/1 expression   Fig. c, d.Fig2O2, the percentage of tunnel positive cells abruptly increased, significantly higher than the control group . However, pretreatment of HaCaT cells with hucMSC-Ex or hucMSCs-CM significantly reduced H2O2 induced HaCaT cell death , in contrast to hucMSCs-dp-Ex in that hucMSCs-dp-Ex did not show any effects in reducing H2O2 induced HaCaT cell death . Immune fluorescence staining showed that after treatment with H2O2, the percentage of nuclear AIF-positive cells abruptly increased, significantly higher than the control group , with remarkable PAR accumulation in the nucleus. However pretreatment with hucMSC-Ex or hucMSCs-CM, the number of nuclear AIF-positive cells abruptly decreased, being significantly lower than the H2O2 group  and with less PAR accumulation in the nucleus. In agreement with tunnel staining, hucMSCs-dp-Ex did not show any effects in reducing the percentage of nuclear AIF-positive cells  and nuclear PAR accumulation in H2O2-treated HaCaT cells  vs. 22.35 ± 3.6% (PBS); P < 0.01; 47.23 ± 4.9% (hucMSC) vs. 22.35 ± 3.6% (PBS), P < 0.01; 26.31 ± 2.3% (hucMSCs-dp-Ex) vs. 22.35 ± 3.6% (PBS), P > 0.01. At day 14: 76.23 ± 8.1% (hucMSCs-CM) vs. 62.41 ± 2.6% (PBS), P < 0.05; 98.41 ± 1.9% (hucMSC-Ex) vs. 62.41 ± 2.6% (PBS); P < 0.01; 92.23 ± 2.6% (hucMSC) vs. 62.41 ± 2.6% (PBS); P < 0.01; 68.42 ± 3.5% (hucMSCs-dp-Ex) vs. 62.41 ± 2.6% (PBS); P > 0.01  vs. 22.35 ± 3.6% (PBS); P < 0.05; 39.3 ± 2.6% (hucMSC-Ex-dp) vs. 32.63 ± 3.5% (PBS); P > 0.05. At day 14: 94 ± 4.2% (hucMSC-Ex) vs. 75.3 ± 3.5% (PBS); 87.91 ± 5.2% (hucMSC) vs. 75.3 ± 3.5% (PBS); P < 0.05; 79.63 ± 2.6% (hucMSC-Ex-dp) vs. 75.3 ± 3.5% (PBS); P > 0.05  vs. 32.63 ± 3.5% (PBS); 57.91 ± 5.2% (hucMSC) vs. 32.63 ± 3.5% (PBS); Immunohistological staining showed that hucMSC-Ex and hucMSC injection significantly upregulated CK10, CD31 and downregulated α-SMA 14d post-skin wounding Fig. a–e. The In this study, we demonstrated that subcutaneous hucMSC-Ex injection promoted rapid wound closure, re-epithelialization, and new blood vessel formation, and reduced scar formation. Thus, it significantly enhanced skin wound healing.2O2. Peroxide induced HaCaT cell death through mitochondrially mediated caspase-dependent- and caspase-independent apoptosis signaling pathways. Previous reports indicated that the caspase-independent apoptosis pathway is called PARP-1-dependent cell death or parthanatos [2O2-induced HaCaT released ROS which injures DNA and activates PARP-1 [2O2 induced AIF nuclear translocation, PARP-1 hyperactivation, and PAR accumulation. H2O2 decreases mitochondrial membrane permeability, releases cyt C, activates caspase-3, and stimulates cell apoptosis. When the broad-spectrum caspase inhibitor Z-VAD.fmk and the PARP-1 inhibitor DPQ were added to the HaCaT cells, the apoptosis rate in the DPQ group was significantly lower than that of the Z-VAD.fmk group. Moreover, whereas Z-VAD.fmk could not inhibit AIF translation to the nuclei, DPQ was able to do so. Caspase activation is a hallmark of apoptotic cell death but does not appear to play any role in parthanatos as broad-spectrum caspase inhibitors could not protect the cells [2O2-induced HaCaT apoptosis might be mediated via the PARP-1 cell death pathway or parthanatos.To explore the mechanism by which hucMSC-Ex stimulated the healing of skin wounds, a skin cell injury model was created by exposing HaCaT cells to Hthanatos –29. It ithanatos , 31. H2Os PARP-1 , 33. Ourhe cells , 35. ThuOver the years, MSC-based therapies  have bee2O2-induced cell death in a time- and dose-dependent manner. Other studies reported that hucMSC-Ex inhibited H2O2-induced cell death by suppressing AIF translation to the nuclei and subsequent PARP-1 hyperactivation [Exosomes are membrane-bound vesicles produced by MSCs. In cell culture, they are released into the ambient medium. They may be critical messengers for cell-to-cell communication . Studiestivation . Thus, htivation ; therefotivation ; thus, wtivation , 48; fortivation , MALAT1 tivation , or Wnt4tivation  protein There were several limitations to the present study. First, it was not established whether it is more advantageous to heal skin wounds by using large volumes of simultaneously injected exosomes or small volumes of exosomes administered over a long period of time. Second, the changes in AIF or PARP-1 expression in wound tissues after hucMSC-Ex transplantation and the mechanism by which AIF/PARP-1 regulates target gene expression were not evaluated in the present study. Third, it was not determined which exosome components regulate the caspase-independent apoptosis pathway in wound healing stimulation. We went further and established the exosomal mechanism of action by tracking exosomal interaction with cells in the real time. We then delved deeper and identified the abundant proteins within the exosomes cargo using a proteomics approach and to look for the key protein for wound healing.2O2-induced HaCaT cell death in vitro. In hucMSC-Ex-dependent regulation of HaCaT cell function, the caspase-independent PARP-1 and AIF apoptosis pathway play vital roles as hucMSC-Ex markedly reduces AIF translocation and PARP-1 hyperactivation. Our findings suggest that hucMSC-Ex is a new therapeutic tool for tissue wound healing by inhibiting deep apoptosis of repair cells. Next, maybe we will choose the big animals, such as pig, to experiment that whether the hucMSCs-Ex can also enhance diabetic skin wound healing. After elucidating the mechanism of hucMSCs-Ex for tissue repair, it may contribute to the treatment of clinical patient-related diseases in the future.In summary, our research indicated that hucMSC-Ex effectively enhances cutaneous wound healing in mice possibly by HaCaT cell activation in the skin. The hucMSC-Ex promoted HaCaT cell proliferation and migration and inhibited H2O2-induced HaCaT cell apoptosis in vitro via a caspase-independent pathway in which PARP-1 activation signals AIF release from the mitochondria to the nuclei.The hucMSc-Ex effectively enhances cutaneous wound healing possibly by activating HaCaT cells in the skin. HucMSC-Ex promotes HaCaT cell proliferation and migration, and inhibits H


PRMT5 participates in various cellular processes, including transcription regulation, signal transduction, mRNA splicing, and DNA repair; however, its mechanism of regulation is poorly understood. Here, we demonstrate that PRMT5 is phosphorylated at residue Y324 by Src kinase, a negative regulator of its activity. Either phosphorylation or substitution of the Y324 residue suppresses PRMT5 activity by preventing its binding with the methyl donor S-adenosyl-L-methionine. Additionally, we show that PRMT5 activity is associated with non-homologous end joining (NHEJ) repair by methylating and stabilizing p53-binding protein 1 (53BP1), which promotes cellular survival after DNA damage. Src-mediated phosphorylation of PRMT5 and the subsequent inhibition of its activity during the DNA damage process blocks NHEJ repair, leading to apoptotic cell death. Altogether, our findings suggest that PRMT5 regulates DNA repair through Src-mediated Y324 phosphorylation in response to DNA damage. Hwang et al. show that the activity of PRMT5 methyltransferase is regulated by Src kinase-mediated phosphorylation at Y324 in response to DNA damage. They also show that PRMT5 participates in NHEJ repair by regulating 53BP1 protein levels and is critical for cellular survival after DNA damage. Many studies have focused on the epigenetic role of PRMT5 that is executed through the symmetric dimethylation of histone proteins at H3R8, H4R3, and H2AR3 sites, resulting in suppression or activation of gene expression6. In addition, several studies have shown that PRMT5 also targets various non-nucleosomal proteins, including components of spliceosome, PIWI proteins, Rad9, REL-A, EGFR, CRAF, E2F-1, and p5315, thereby implicating it in multiple cellular signaling events. Furthermore, it has been reported that upregulation of PRMT5 is closely associated with tumor progression and poor prognosis in several human cancers, including breast, gastric, colorectal, ovarian, leukemia, and lymphoma16. Although the cellular functions of PRMT5 have been extensively studied, little is known about how its methyltransferase activity is regulated. A single report shows that PRMT5 is phosphorylated and inhibited by the constitutively active Janus Kinase 2 (JAK2) mutant JAK2V617F, but not by wild type JAK217. In this case, JAK2V617F-mediated phosphorylation of PRMT5 disrupts its association with methylosome protein 50 (MEP50)/WDR77, a well-known co-activator required for PRMT5 activity. JAK2V617F-dependent regulation of PRMT5 has provided an understanding of the molecular pathogenesis of myeloproliferative neoplasm; however, the regulatory mechanism for PRMT5 activity in normal physiological processes is yet to be unraveled.Arginine methylation is an abundant post-translational modification (PTM), which is catalyzed by protein arginine methyltransferases (PRMTs). To date, nine PRMTs have been characterized that produce three types of methylarginines: monomethylarginine, asymmetric dimethylarginine (ADMA), and symmetric dimethylarginine (SDMA). PRMT5 is a type II methyltransferase that catalyzes mono- or symmetric dimethylation of arginine residues on its substrate proteins19. Defects to DNA repair mechanism lead to mutagenic diseases, including cancer and neurological dysfunction20. DSB repair is accomplished through two main pathways: homologous recombination (HR) and non-homologous end joining (NHEJ). HR is error-free, but requires a sister chromatid as a template and, therefore, only occurs during late S/G2, while NHEJ is error-prone and can occur throughout the cell cycle22. There is evidence that an increase in error-prone NHEJ is linked to chromosomal instability in human cancers, which is considered to be an attractive therapeutic target25. The NHEJ-mediated repair pathway is primarily controlled by a protein complex containing p53 binding protein 1 (53BP1), Rif1, PTIP, and Rev721. After DSBs, 53BP1 is rapidly recruited into damaged DNA lesions, where it forms nuclear foci and transduces DNA repair processes. The disruption of signaling pathways that regulate the targeting of 53BP1 may be a good way to sensitize cancer cells to radiotherapy.DNA double-strand break (DSB) repair processes are critical for the maintenance of genomic stability, and they protect cells against exogenous and endogenous DNA damage stressesIn this study, we show that PRMT5 activity is regulated by Src kinase-mediated phosphorylation at Y324, a critical residue for its enzymatic activity. This phosphorylation occurs in response to DNA damage stresses. PRMT5 participates in the NHEJ repair process by regulating 53BP1 protein levels, and consequently, it is critical for cellular survival after DNA damage. Thus, our findings demonstrate the importance of PRMT5 activity in the DNA repair process, which is regulated by Src kinase-mediated Y324 phosphorylation.http://scansite3.mit.edu), which predicts protein motifs that are likely to be phosphorylated by specific protein kinases, we found that several kinases, including Src kinase, PDGFR, AKT, and ATM, are predicted as the responsible enzymes for phosphorylation of PRMT5 at Y324, T634, and S446 residues, respectively ) was overexpressed in HEK293T cells  with the intact Y324 residue  and 3[H]-AdoMet. PRMT5 (co-expressed with an empty vector) strongly methylated MBP  and slightly reduced CHK2 phosphorylation  regulated by PRMT5 in DNA damage responses. 53BP1 protein is a key regulator of non-homologous end joining (NHEJ) DNA repair process, which has a glycine-arginine rich (GAR) motif that is methylated by PRMT1tro Fig. , which wtro Fig. . Moreovetro Fig. , howevertro Fig. , indicattro Fig. . This wa34, we investigated whether the protein stability of 53BP1 is controlled by PRMT5. Upon treatment with cycloheximide, the levels of 53BP1 protein in resting condition were rapidly diminished in PRMT5 inhibitor-treated or Y324F-expressing cells without affecting cell viability or cell cycle progression  and DR-GFP  reporter assays41  repeats, the tandem Tudor domains, an oligomerization domain (OD), a GAR motif, an ubiquitination-dependent recruitment (UDR) motif, and 28 amino-terminal Ser/Thr-Gln (S/T-Q) sites22. It has been reported that the GAR motif is asymmetrically dimethylated by PRMT1 and this methylation is required for DNA binding affinity of 53BP1 rather than its oligomerization49. However, this notion has been challenged by other observations that the localization of 53BP1 into foci is driven by physical interaction between the tandem Tudor domain of 53BP1 and lysine-methylated histones, not by asymmetric dimethylation of the GAR motif52. In this study, we show that the GAR motif can also be symmetrically dimethylated by PRMT5, which leads to an increase in stability of 53BP1. It has been reported that ubiquitin-conjugating enzyme H7 53, ring finger protein RNF8/RNF168 (both E3 ligases)35 and Nudix hydrolase NUDT1654 are involved in proteosomal degradation of 53BP1, respectively. Nevertheless, the mechanisms that regulate protein stability of 53BP1 are still not well understood. Further studies are needed to clarify the correlation between PRMT5-mediated arginine methylation and RNF8/RNF168-mediated ubiquitination or NUDT16-mediated ADP-ribosylation. Because accumulating evidence support that upregulation of 53BP1 enhances mutagenic NHEJ repair and radioresistance, which is correlated with reduced survival rate and poor prognosis in several tumor56, a precise regulation of 53BP1 levels is critical for tumor development and therapeutic outcome57. In this point of view, our proposed mechanisms on 53BP1 stabilization could provide valuable information for cancer prevention and treatment.53BP1 is a key regulator of NHEJ repair processes by forming large foci near DNA lesions where ATM- or ATR-mediated DNA damage signaling is inducedCollectively, our findings report a previously unidentified regulatory mechanism for PRMT5 methyltransferase activity through Src kinase-induced Y324 phosphorylation, and the involvement of PRMT5 in NHEJ repair pathway through regulation of 53BP1 stability, which can be targeted for cancer treatment.TM site-directed Mutagenesis kit  according to the manufacturer’s protocol. Plasmid containing GFP tagged-human 53BP1 was purchased from Origene  and then subjected to site-directed mutagenesis to generate an arginine methylation-deficient mutant  in GAR motif). GST-53BP1 deletion mutants (GST-53BP1-Tudor (1220–1711) -WT, -5RK, -∆GAR (deletion from 1381 to 1480) were cloned using pGEX vector and GFP-53BP1 plasmid. Etoposide and camptothecin were obtained from Enzo Life Sciences . EGF, dasatinib, and doxorubicin were obtained from Sigma-Aldrich , while PDGF-AA was from PROSPEC . The following peptides were chemically synthesized by AbClone  for antibody production in rabbit and dot blot assays: Unmodified peptide (NH2-PLMDNLESQTYEVFEKDPIKY-COOH) and phosphorylated peptide (NH2-PLMDNLESQT(pY)EVFEKDPIKY-COOH). Antibodies against pY100 (1:5000), pY416-Src kinase , γH2AX , histone H3 , H4 (1:5000), p-CHK1 (1:5000), CHK1 (1:5000), p-CHK2 (1:5000), CHK2 (1:2000), and cleaved caspase-3 (1:1000) were purchased from Cell Signaling Technology . Anti-PRMT5 (1:5000), anti-cSrc kinase (1:5000), anti-SDMA , and anti-ADMA  were purchased from Millipore . Antibodies against c-myc , β-actin , and PARP  were from Santa Cruz . Anti-GFP  was purchased from Invitrogen , and anti-53BP1 , anti-MEP50 , Alexa594-conjugated goat anti-mouse (1:2000), and FITC-conjugated goat anti-rabbit (1:2000) secondary antibodies were purchased from Bethyl Laboratories . Anti-H3R8me2s , anti-H4R3me2s , anti-H3R17me2a  and anti-H4K20me2  were from Abcam . HRP-conjugated secondary antibodies  were purchased from Jackson ImmunoResearch Laboratories .Myc-PRMT5 plasmids were obtained from M.T. Bedford and were sub-cloned into a pcDNA3 vector containing N-terminal FLAG-tag and a pGEX vector containing N-terminal glutathione S-transferases (GST) tag for the purification of recombinant protein. PRMT5 Y324F, Y324A, Y324E, Y324Q, Y324S, Y324T, and PRMT5-G367A-R368A mutants were generated using the Muta-direct2 in a humidified chamber. For siRNA transfection, cells were transfected with TransIT-X2  according to the manufacturerʼs instructions. All siRNA sequences were synthesized by IDT  and all experiments were performed at least 2 days after knockdown. For overexpression from ectopic mammalian expression plasmids, cells were transfected with TransIT-2020 (Mirus Bio).HEK293T (Human Embryonic Kidney 293T cells), U2OS (human osteosarcoma cells), MCF7 (human breast cancer cells), A549 , and NIH3T3 (mouse fibroblast cells) were obtained from American Type Culture Collection . All cell lines were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum  and 100 units/ml penicillin-streptomycin (HyClone Laboratories). Cells were maintained at 37 °C under 5% COWhole-cell extracts were obtained using lysis buffer  supplemented with 1X protease and phosphatase inhibitor cocktails . Cell extracts were incubated for 20 min in an ice bath, followed by sonication and then centrifuged to clarify lysates. Protein concentration was determined by Bradford Assay according to the manufacturer’s instructions . After boiling in Laemmli sample buffer for 5 min, equal amounts of protein lysates were resolved by SDS-PAGE, and then transferred onto PVDF. The membranes were blocked with 5% skim milk/0.1% Tween 20/TBS  for 1 h at room temperature and incubated with primary antibody overnight, followed by HRP-linked secondary antibody for 1 h at room temperature. The signal was detected using ECL western blotting substrate . The band intensity of western band was analyzed by Image Studio version 5.0 .For preparation of lysates for immunoprecipitation (IP), cells were washed three times in ice-cold phosphate-buffered saline (PBS) and lysed with NP40 lysis buffer  supplemented with 1X protease and phosphatase inhibitor cocktails. After centrifugation, appropriate antibodies were added at a concentration of 1 mg/ml of lysate and incubated overnight at 4 °C, followed by antibody-protein complex capture using Protein A/G Sepharose beads  for at least 1 h at 4 °C. After washing twice with NP40 lysis buffer, the complexes were eluted and analyzed by SDS-PAGE and immunoblotting.Cells were plated on coverslips for 24 h prior to transfection with siRNAs or DNA plasmids. After experimental treatment, cells were fixed for 30 min in 4% PFA, washed with PBS three times, and permeabilized and blocked with 0.25% Triton X-100 and 10% BSA in PBS for 1 h. Cells were then incubated with primary antibody overnight at 4 °C and with fluorescence-conjugated secondary antibody for 1 h at room temperature. After DAPI  staining for 5 min, cells were mounted onto glass slides. Staining was assessed using a Zeiss LSM 710 immunofluorescence microscope and ZEN software . Cells with ≥5 foci per cell were classified as foci-positive. A minimum of 300 cells were counted for each experimental repeat. Representative images were obtained at ×100 magnification. The fluorescence intensity of pictures was analyzed by Image Studio version 5.0. Antibodies used include γH2AX , 53BP1 , and PRMT5 .TM  and cDNA synthesized using the SensiFASTTM cDNA synthesis kit (Bioline). The synthesized cDNA was used as template for real-time PCR which was performed using the Eco Real-Time PCR System  and the SensiFAST SYBRTM No-ROX Kit (Bioline). Reaction parameters were as follows: cDNA synthesis at 37 °C for 60 min, transcriptase inactivation at 95 °C for 5 min, PCR cycling at 95 °C for 10 s, 58 °C for 30 s, and 72 °C for 20 s, for 40 cycles. The primer sets for PRMT5 were 5′-TTTCCCATCCTCTTCCCTATTAAG-3′ and 5′-CCCACTCATACCACACCTTC-3′; the primer sets for 53BP1 were 5′-GGCTACGCATTTCTCCTTACC-3′ and 5′-AAGCTGGGATTCTGTATACTGC-3′.Total cellular RNA was extracted using TRIsure3VO4, 10 mM MgCl2) supplemented with 1X protease and phosphatase inhibitor cocktail. Reactions were incubated at 37 °C for 1 h, and proteins were then resolved by gel electrophoresis.In vitro Src kinase assay was performed using immunoprecipitated Src kinase from vSrc-transfected A549 cells, purified myc-PRMT5 from HEK293T cells, and 1 mM ATP in kinase buffer (25 mM Tris-HCl (pH 7.5), 150 mM NaCl, 2 mM DTT, 0.1 mM Na3[H]-AdoMet  at 37 °C for 1 h. The reaction was stopped by adding SDS loading buffer, and the proteins were resolved on SDS-PAGE gels. Proteins were transferred onto PVDF membranes, and the tritium signal was amplified by treating membranes with EN3HANCE (PerkinElmer) spray. Membranes were exposed to autoradiography film for at least 1 week at 80 °C.Myc-PRMT5 was purified from transfected HEK293T cells by anti-myc immunoprecipitation. Immobilized myc-PRMT5 proteins were then incubated with 50 μl of reaction buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 2 mM EDTA, 1 mM PMSF, 1 mM DTT) supplemented with 1 μg of recombinant histone  or 1 μg MBP  and 1 μCi HEK293T cells were co-transfected with myc-PRMT5 and vSrc plasmid for 2 days, and then myc-PRMT5 proteins were immunoprecipitated with anti-myc antibody. Immunoprecipitated PRMT5 was used in CIP assay (New England Biosciences) according to the manufacturerʼs method. In brief, beads were incubated with or without 1 unit of CIP in NEB buffer at 37 °C for 60 min, and the reaction was stopped by adding SDS loading buffer. The samples were analyzed by SDS-PAGE and western blotting.U2OS stable cell lines harboring DR-GFP and EJ5-GFP construct were kind gifts from Prof. Yonghwan Kim . U2OS-DR-GFP or U2OS-EJ5-GFP cells were seeded into six-well plates and co-transfected with 1 μg of I-SceI vector and the indicated siRNA or DNA plasmid, using TransIT-2020 (Mirus Bio). After three days, cells were harvested by trypsinization and washed with PBS. The GFP signal arising from the recombination event was measured by flow cytometry . Fluorescence was detected in the FL1-H channel . Each data point represents the average ± standard deviation from three independent experiments.3[H]-AdoMet, and 1 mM DTT were mixed in 1× PBS and exposed to UV light  at a distance of 1 cm for 30 min at 4 °C using UVGL-58 UV cross-linker . After cross-linking, samples were boiled with Laemmli sample buffer, separated on 7% SDS/PAGE, transferred to PVDF membrane, Coomassie-stained, destained, and dried. The amount of cross-linked 3[H]-labeled GST-PRMT5 was quantified by Liquid scintillation counting.One microgram of GST-PRMT5, 3 μCi MCF7 cells were plated at low density 24 h prior to transfection with siCON, siPRMT5, empty vector, WT-, or Y324F-FLAG PRMT5. Cells were treated with 10 μM etoposide for 2 h, followed by supplementation with fresh complete media. After 14 days, colonies were fixed with 4% PFA and stained with 0.05% crystal violet. Stained cells were washed three times with deionized water and then entirely dried. Data were normalized to respective non-treatment control.The molecular docking analysis was done using Discovery Studio 2016 software  and implementing the CHARMM force field. X-ray crystal structure of PRMT5 (Protein Data Bank code 4GQB) was obtained from the RCSB protein databank, and phospho-Y324 PRMT5 was modified from the structure of the WT form of PRMT5. The protein structures of WT and phospho-Y324 PRMT5 were optimized by smart minimizer algorithm to the energy minimized state. To dock the ligands, the Ligandfit docking method was used. The parameters of Ligandfit were validated using ligands from the PRMT5 crystal structure. The crystallographic ligand -2,5-diamino-6-hexanoic acid (PDB ID: 0XU) was used to determine the binding site. AdoMet was docked to the binding site of both proteins and 30 poses were generated for each. Based on the docking results, various scoring functions  were determined and used to calculate the predicted binding energies.t-test for independent samples and P-value < 0.05 were considered statistically significant. *P < 0.05 and **P < 0.01. For experiments that are difficult to analyze statistically , they were repeated at least three times. The exact number of replicates are presented in individual figure legends.The data are presented as mean ± SD from three independent experiments. Comparison between data from two groups was performed using a Further information on research design is available in the Supplementary figure 1Supplementary Data 1Supplementary informationReporting SummaryPeer review